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Fischer NG, de Souza Araújo IJ, Daghrery A, Yu B, Dal-Fabbro R, Dos Reis-Prado AH, Silikas N, Rosa V, Aparicio C, Watts DC, Bottino MC. Guidance on biomaterials for periodontal tissue regeneration: Fabrication methods, materials and biological considerations. Dent Mater 2025; 41:283-305. [PMID: 39794220 DOI: 10.1016/j.dental.2024.12.019] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2024] [Accepted: 12/26/2024] [Indexed: 01/13/2025]
Abstract
Regeneration of the multiple tissues and interfaces in the periodontal complex necessitates multidisciplinary evaluation to establish structure/function relationships. This article, an initiative of the Academy of Dental Materials, provides guidance for performing chemical, structural, and mechanical characterization of materials for periodontal tissue regeneration, and outlines important recommendations on methods of testing bioactivity, biocompatibility, and antimicrobial properties of biomaterials/scaffolds for periodontal tissue engineering. First, we briefly summarize periodontal tissue engineering fabrication methods. We then highlight critical variables to consider when evaluating a material for periodontal tissue regeneration, and the fundamental tests used to investigate them. The recommended tests and designs incorporate relevant international standards and provide a framework for characterizing newly developed materials focusing on the applicability of those tests for periodontal tissue regeneration. The most common methods of biofabrication (electrospinning, injectable hydrogels, fused deposition modelling, melt electrowriting, and bioprinting) and their specific applications in periodontal tissue engineering are reviewed. The critical techniques for morphological, chemical, and mechanical characterization of different classes of materials used in periodontal regeneration are then described. The major advantages and drawbacks of each assay, sample sizes, and guidelines on specimen preparation are also highlighted. From a biological standpoint, fundamental methods for testing bioactivity, the biocompatibility of materials, and the experimental models for testing the antimicrobial potential are included in this guidance. In conclusion, researchers performing studies on periodontal tissue regeneration will have this guidance as a tool to assess essential properties and characteristics of their materials/scaffold-based strategies.
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Affiliation(s)
- Nicholas G Fischer
- Minnesota Dental Research Center for Biomaterials and Biomechanics, School of Dentistry, University of Minnesota, Minneapolis, MN 55455, USA
| | - Isaac J de Souza Araújo
- Department of Bioscience Research, College of Dentistry, University of Tennessee Health Science Center, Memphis, TN 38163, USA
| | - Arwa Daghrery
- Department of Restorative Dental Sciences, School of Dentistry, Jazan University, Jazan 82943, KSA; Department of Cariology, Restorative Sciences and Endodontics, University of Michigan, School of Dentistry, Ann Arbor, MI 48109, USA
| | - Baiqing Yu
- Faculty of Dentistry, National University of Singapore, Singapore
| | - Renan Dal-Fabbro
- Department of Cariology, Restorative Sciences and Endodontics, University of Michigan, School of Dentistry, Ann Arbor, MI 48109, USA
| | - Alexandre H Dos Reis-Prado
- Department of Cariology, Restorative Sciences and Endodontics, University of Michigan, School of Dentistry, Ann Arbor, MI 48109, USA; Department of Restorative Dentistry, School of Dentistry, Federal University of Minas Gerais (UFMG), Belo Horizonte 31270-901, Brazil
| | - Nikolaos Silikas
- Dental Biomaterials, Dentistry, The University of Manchester, Manchester, United Kingdom
| | - Vinicius Rosa
- Faculty of Dentistry, National University of Singapore, Singapore; ORCHIDS: Oral Care Health Innovations and Designs Singapore, National University of Singapore, Singapore
| | - Conrado Aparicio
- BOBI-Bioinspired Oral Biomaterials and Interfaces, UPC-Universitat Politènica de Catalunya, Barcelona 08010, Spain; Catalan Institute for Research and Advanced Studies (ICREA), Barcelona 08010, Spain; SCOI - Study and Control of Oral Infections, Faculty of Odontology, UIC Barcelona-Universitat Internacional de Catalunya, Sant Cugat del Vallès, Spain; IBEC - Institute for Bioengineering of Catalonia, Barcelona, Spain
| | - David C Watts
- School of Medical Sciences and Photon Science Institute, University of Manchester, United Kingdom
| | - Marco C Bottino
- Department of Cariology, Restorative Sciences and Endodontics, University of Michigan, School of Dentistry, Ann Arbor, MI 48109, USA; Department of Biomedical Engineering, College of Engineering, University of Michigan, Ann Arbor, MI 48109, USA.
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Pandur E, Pap R, Sipos K. Activated THP-1 Macrophage-Derived Factors Increase the Cytokine, Fractalkine, and EGF Secretions, the Invasion-Related MMP Production, and Antioxidant Activity of HEC-1A Endometrium Cells. Int J Mol Sci 2024; 25:9624. [PMID: 39273575 PMCID: PMC11395051 DOI: 10.3390/ijms25179624] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2024] [Revised: 08/27/2024] [Accepted: 08/30/2024] [Indexed: 09/15/2024] Open
Abstract
Endometrium receptivity is a multifactor-regulated process involving progesterone receptor-regulated signaling, cytokines and chemokines, and additional growth regulatory factors. In the female reproductive system, macrophages have distinct roles in the regulation of receptivity, embryo implantation, immune tolerance, and angiogenesis or oxidative stress. In the present study, we investigated the effects of PMA-activated THP-1 macrophages on the receptivity-related genes, cytokines and chemokines, growth regulators, and oxidative stress-related molecules of HEC-1A endometrium cells. We established a non-contact co-culture in which the culture medium of the PMA-activated macrophages exhibiting the pro-inflammatory phenotype was used for the treatment of the endometrial cells. In the endometrium cells, the expression of the growth-related factors activin and bone morphogenetic protein 2, the growth hormone EGF, and the activation of the downstream signaling molecules pERK1/2 and pAkt were analyzed by ELISA and Western blot. The secretions of cytokines and chemokines, which are involved in the establishment of endometrial receptivity, and the expression of matrix metalloproteinases implicated in invasion were also determined. Based on the results, the PMA-activated THP-1 macrophages exhibiting a pro-inflammatory phenotype may play a role in the regulation of HEC-1A endometrium cells. They alter the secretion of cytokines and chemokines, as well as the protein level of MMPs of HEC-1A cells. Moreover, activated THP-1 macrophages may elevate oxidative stress protection of HEC-1A endometrium cells. All these suggest that pro-inflammatory macrophages have a special role in the regulation of receptivity-related and implantation-related factors of HEC-1A cells.
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Affiliation(s)
- Edina Pandur
- Department of Pharmaceutical Biology, Faculty of Pharmacy, University of Pécs, 7624 Pécs, Hungary; (R.P.); (K.S.)
- National Laboratory of Human Reproduction, University of Pécs, 7624 Pécs, Hungary
| | - Ramóna Pap
- Department of Pharmaceutical Biology, Faculty of Pharmacy, University of Pécs, 7624 Pécs, Hungary; (R.P.); (K.S.)
- National Laboratory of Human Reproduction, University of Pécs, 7624 Pécs, Hungary
| | - Katalin Sipos
- Department of Pharmaceutical Biology, Faculty of Pharmacy, University of Pécs, 7624 Pécs, Hungary; (R.P.); (K.S.)
- National Laboratory of Human Reproduction, University of Pécs, 7624 Pécs, Hungary
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Miyara SJ, Shinozaki K, Hayashida K, Shoaib M, Choudhary RC, Zafeiropoulos S, Guevara S, Kim J, Molmenti EP, Volpe BT, Becker LB. Differential Mitochondrial Bioenergetics in Neurons and Astrocytes Following Ischemia-Reperfusion Injury and Hypothermia. Biomedicines 2024; 12:1705. [PMID: 39200170 PMCID: PMC11352110 DOI: 10.3390/biomedicines12081705] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2024] [Revised: 07/09/2024] [Accepted: 07/11/2024] [Indexed: 09/02/2024] Open
Abstract
The close interaction between neurons and astrocytes has been extensively studied. However, the specific behavior of these cells after ischemia-reperfusion injury and hypothermia remains poorly characterized. A growing body of evidence suggests that mitochondria function and putative transference between neurons and astrocytes may play a fundamental role in adaptive and homeostatic responses after systemic insults such as cardiac arrest, which highlights the importance of a better understanding of how neurons and astrocytes behave individually in these settings. Brain injury is one of the most important challenges in post-cardiac arrest syndrome, and therapeutic hypothermia remains the single, gold standard treatment for neuroprotection after cardiac arrest. In our study, we modeled ischemia-reperfusion injury by using in vitro enhanced oxygen-glucose deprivation and reperfusion (eOGD-R) and subsequent hypothermia (HPT) (31.5 °C) to cell lines of neurons (HT-22) and astrocytes (C8-D1A) with/without hypothermia. Using cell lysis (LDH; lactate dehydrogenase) as a measure of membrane integrity and cell viability, we found that neurons were more susceptible to eOGD-R when compared with astrocytes. However, they benefited significantly from HPT, while the HPT effect after eOGD-R on astrocytes was negligible. Similarly, eOGD-R caused a more significant reduction in adenosine triphosphate (ATP) in neurons than astrocytes, and the ATP-enhancing effects from HPT were more prominent in neurons than astrocytes. In both neurons and astrocytes, measurement of reactive oxygen species (ROS) revealed higher ROS output following eOGD-R, with a non-significant trend of differential reduction observed in neurons. HPT after eOGD-R effectively downregulated ROS in both cells; however, the effect was significantly more effective in neurons. Lipid peroxidation was higher after eOGD-R in neurons, while in astrocytes, the increase was not statistically significant. Interestingly, HPT had similar effects on the reduction in lipoperoxidation after eOGD-R with both types of cells. While glutathione (GSH) levels were downregulated after eOGD-R in both cells, HPT enhanced GSH in astrocytes, but worsened GSH in neurons. In conclusion, neuron and astrocyte cultures respond differently to eOGD-R and eOGD-R + HTP treatments. Neurons showed higher sensitivity to ischemia-reperfusion insults than astrocytes; however, they benefited more from HPT therapy. These data suggest that given the differential effects from HPT in neurons and astrocytes, future therapeutic developments could potentially enhance HPT outcomes by means of neuronal and astrocytic targeted therapies.
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Affiliation(s)
- Santiago J. Miyara
- Elmezzi Graduate School of Molecular Medicine, Manhasset, NY 11030, USA
- Feinstein Institutes for Medical Research, Manhasset, NY 11030, USA
| | - Koichiro Shinozaki
- Feinstein Institutes for Medical Research, Manhasset, NY 11030, USA
- Department of Emergency Medicine, Northwell Health, Manhasset, NY 11030, USA
| | - Kei Hayashida
- Feinstein Institutes for Medical Research, Manhasset, NY 11030, USA
- Department of Emergency Medicine, Northwell Health, Manhasset, NY 11030, USA
| | - Muhammad Shoaib
- Feinstein Institutes for Medical Research, Manhasset, NY 11030, USA
| | | | | | - Sara Guevara
- Feinstein Institutes for Medical Research, Manhasset, NY 11030, USA
| | - Junhwan Kim
- Feinstein Institutes for Medical Research, Manhasset, NY 11030, USA
| | - Ernesto P. Molmenti
- Feinstein Institutes for Medical Research, Manhasset, NY 11030, USA
- Department of Surgery, Renown Health, Reno, NV 89502, USA
| | - Bruce T. Volpe
- Elmezzi Graduate School of Molecular Medicine, Manhasset, NY 11030, USA
- Feinstein Institutes for Medical Research, Manhasset, NY 11030, USA
| | - Lance B. Becker
- Elmezzi Graduate School of Molecular Medicine, Manhasset, NY 11030, USA
- Feinstein Institutes for Medical Research, Manhasset, NY 11030, USA
- Department of Emergency Medicine, Northwell Health, Manhasset, NY 11030, USA
- Department of Emergency Medicine, Kindai University Faculty of Medicine, Osaka 589-8511, Japan
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Kang S, Oh YJ, Kim MR, Jung YN, Song E, Lee H, Hong J. Development of a Convenient and Quantitative Method for Evaluating Photosensitizing Activity Using Thiazolyl Blue Formazan Dye. Molecules 2024; 29:2471. [PMID: 38893346 PMCID: PMC11173384 DOI: 10.3390/molecules29112471] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2024] [Revised: 05/17/2024] [Accepted: 05/21/2024] [Indexed: 06/21/2024] Open
Abstract
Photosensitizers cause oxidative damages in various biological systems under light. In this study, the method for analyzing photosensitizing activity of various dietary and medicinal sources was developed using 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (thiazolyl blue formazan; MTT-F) as a probe. Significant and quantitative decolorization of MTT-F was observed in the presence of photosensitizers used in this study under light but not under dark conditions. The decolorization of MTT-F occurred irradiation time-, light intensity-, and photosensitizer concentration-dependently. The decolorized MTT-F was reversibly reduced by living cells; the LC-MS/MS results indicated the formation of oxidized products with -1 m/z of base peak from MTT-F, suggesting that MTT-F decolorized by photosensitizers was its corresponding tetrazolium. The present results indicate that MTT-F is a reliable probe for the quantitative analysis of photosensitizing activities, and the MTT-F-based method can be an useful tool for screening and evaluating photosensitizing properties of various compounds used in many industrial purposes.
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Affiliation(s)
- Smee Kang
- Department of Food Science and Technology, College of Science and Convergence Technology, Seoul Women’s University, 621, Hwarangro, Nowon-gu, Seoul 01797, Republic of Korea; (S.K.); (M.-R.K.); (Y.N.J.); (E.S.); (H.L.)
| | - Yeong Ji Oh
- Major in Food Science & Biotechnology, Institute of Bio Engineering, College of Future Convergence, Eulji University, Seongnam 13135, Republic of Korea;
| | - Mi-Ri Kim
- Department of Food Science and Technology, College of Science and Convergence Technology, Seoul Women’s University, 621, Hwarangro, Nowon-gu, Seoul 01797, Republic of Korea; (S.K.); (M.-R.K.); (Y.N.J.); (E.S.); (H.L.)
| | - Yu Na Jung
- Department of Food Science and Technology, College of Science and Convergence Technology, Seoul Women’s University, 621, Hwarangro, Nowon-gu, Seoul 01797, Republic of Korea; (S.K.); (M.-R.K.); (Y.N.J.); (E.S.); (H.L.)
| | - Eiseul Song
- Department of Food Science and Technology, College of Science and Convergence Technology, Seoul Women’s University, 621, Hwarangro, Nowon-gu, Seoul 01797, Republic of Korea; (S.K.); (M.-R.K.); (Y.N.J.); (E.S.); (H.L.)
| | - Hyowon Lee
- Department of Food Science and Technology, College of Science and Convergence Technology, Seoul Women’s University, 621, Hwarangro, Nowon-gu, Seoul 01797, Republic of Korea; (S.K.); (M.-R.K.); (Y.N.J.); (E.S.); (H.L.)
| | - Jungil Hong
- Department of Food Science and Technology, College of Science and Convergence Technology, Seoul Women’s University, 621, Hwarangro, Nowon-gu, Seoul 01797, Republic of Korea; (S.K.); (M.-R.K.); (Y.N.J.); (E.S.); (H.L.)
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Doppegieter M, van Leeuwen TG, van Weert A, Aalders MCG, Bakker ENTP. Subminute thermal damage to cell types present in the skin. Int J Hyperthermia 2024; 41:2354435. [PMID: 38754976 DOI: 10.1080/02656736.2024.2354435] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2024] [Revised: 04/29/2024] [Accepted: 05/06/2024] [Indexed: 05/18/2024] Open
Abstract
INTRODUCTION Psoriasis is characterized by an increase in the proliferation of keratinocytes and nerve fiber activity, contributing to the typical skin lesions. Pulsed Dye Laser (PDL) treatment is effective for the treatment of psoriatic lesions but its mechanism remains unclear. One hypothesis is that PDL causes thermal damage by the diffusion of heat to neighboring structures in lesional skin. There is limited information on the thermal sensitivity of these neighboring skin cells when exposed to hyperthermia for durations lasting less than a minute. Our study aimed to investigate the cell-specific responses to heat using sub-minute exposure times and moderate to ablative hyperthermia. MATERIALS AND METHODS Cultured human endothelial cells, smooth muscle cells, neuronal cells, and keratinocytes were exposed to various time (2-20 sec) and temperature (45-70 °C) combinations. Cell viability was assessed by measuring intracellular ATP content 24 h after thermal exposure and this data was used to calculate fit parameters for the Arrhenius model and CEM43 calculations. RESULTS Our results show significant differences in cell survival between cell types (p < 0.0001). Especially within the range of 50-60 °C, survival of neuronal cells and keratinocytes was significantly less than that of endothelial and smooth muscle cells. No statistically significant difference was found in the lethal dose (LT50) of thermal energy between neuronal cells and keratinocytes. However, CEM43 calculations showed significant differences between all four cell types. CONCLUSION The results imply that there is a cell-type-dependent sensitivity to thermal damage which suggests that neuronal cells and keratinocytes are particularly susceptible to diffusing heat from laser treatment. Damage to these cells may aid in modulating the neuro-inflammatory pathways in psoriasis. These data provide insight into the potential mechanisms of PDL therapy for psoriasis and advance our understanding of how thermal effects may play a role in its effectiveness.
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Affiliation(s)
- Meagan Doppegieter
- Department of Biomedical Engineering and Physics, Amsterdam University Medical Centers, Amsterdam, the Netherlands
- Amsterdam Cardiovascular Sciences, Microcirculation, Amsterdam, the Netherlands
- Amsterdam Public Health, Personalized Medicine, Amsterdam, the Netherlands
| | - Ton G van Leeuwen
- Department of Biomedical Engineering and Physics, Amsterdam University Medical Centers, Amsterdam, the Netherlands
- Amsterdam Cardiovascular Sciences, Microcirculation, Amsterdam, the Netherlands
- Cancer Center Amsterdam, Imaging and Biomarkers, Amsterdam, the Netherlands
| | - Angela van Weert
- Department of Biomedical Engineering and Physics, Amsterdam University Medical Centers, Amsterdam, the Netherlands
| | - Maurice C G Aalders
- Department of Biomedical Engineering and Physics, Amsterdam University Medical Centers, Amsterdam, the Netherlands
- Amsterdam Public Health, Personalized Medicine, Amsterdam, the Netherlands
| | - Erik N T P Bakker
- Department of Biomedical Engineering and Physics, Amsterdam University Medical Centers, Amsterdam, the Netherlands
- Amsterdam Cardiovascular Sciences, Microcirculation, Amsterdam, the Netherlands
- Amsterdam Neuroscience, Neurovascular Disorders, Amsterdam, the Netherlands
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Amin ML, Mawad D, Dokos S, Sorrell CC. Comparative Bioactivities of Chemically Modified Fucoidan and λ-Carrageenan toward Cells Encapsulated in Covalently Cross-Linked Hydrogels. Biomacromolecules 2024; 25:3131-3140. [PMID: 38554085 DOI: 10.1021/acs.biomac.4c00228] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/01/2024]
Abstract
The sulfated marine polysaccharides, fucoidan and λ-carrageenan, are known to possess anti-inflammatory, immunomodulatory, and cellular protective properties. Although they hold considerable promise for tissue engineering constructs, their covalent cross-linking in hydrogels and comparative bioactivities to cells are absent from the literature. Thus, fucoidan and λ-carrageenan were modified with methacrylate groups and were covalently cross-linked with the synthetic polymer poly(vinyl alcohol)-methacrylate (PVA-MA) to form 20 wt % biosynthetic hydrogels. Identical degrees of methacrylation were confirmed by 1H NMR, and covalent conjugation was determined by using a colorimetric 1,9-dimethyl-methylene blue (DMMB) assay. Pancreatic beta cells were encapsulated in the hydrogels, followed by culturing in the 3D environment for a prolonged period of 32 days and evaluation of the cellular functionality by live/dead, adenosine 5'-triphosphate (ATP) level, and insulin secretion. The results confirmed that fucoidan and λ-carrageenan exhibited ∼12% methacrylate substitution, which generated hydrogels with stable conjugation of the polysaccharides with PVA-MA. The cells encapsulated in the PVA-fucoidan hydrogels demonstrated consistently high ATP levels over the culture period. Furthermore, only cells in the PVA-fucoidan hydrogels retained glucose responsiveness, demonstrating comparatively higher insulin secretion in response to glucose. In contrast, cells in the PVA-λ-carrageenan and the PVA control hydrogels lost all glucose responsiveness. The present work confirms the superior effects of chemically modified fucoidan over λ-carrageenan on pancreatic beta cell survival and function in covalently cross-linked hydrogels, thereby illustrating the importance of differential polysaccharide structural features on their biological effects.
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Affiliation(s)
- Md Lutful Amin
- School of Materials Science and Engineering, UNSW Sydney, Sydney, NSW 2052, Australia
- Graduate School of Biomedical Engineering, UNSW Sydney, Sydney, NSW 2052, Australia
| | - Damia Mawad
- School of Materials Science and Engineering, UNSW Sydney, Sydney, NSW 2052, Australia
- Australian Centre for NanoMedicine, UNSW Sydney, Sydney, NSW 2052, Australia
| | - Socrates Dokos
- Graduate School of Biomedical Engineering, UNSW Sydney, Sydney, NSW 2052, Australia
| | - Charles C Sorrell
- School of Materials Science and Engineering, UNSW Sydney, Sydney, NSW 2052, Australia
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Miškovská A, Michailidu J, Kolouchová IJ, Barone L, Gornati R, Montali A, Tettamanti G, Berini F, Marinelli F, Masák J, Čejková A, Maťátková O. Biological activity of silver nanoparticles synthesized using viticultural waste. Microb Pathog 2024; 190:106613. [PMID: 38484919 DOI: 10.1016/j.micpath.2024.106613] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2023] [Revised: 03/05/2024] [Accepted: 03/09/2024] [Indexed: 03/21/2024]
Abstract
This research paper presents a novel approach to the green synthesis of silver nanoparticles (AgNPs) using viticultural waste, allowing to obtain NP dispersions with distinct properties and morphologies (monodisperse and polydisperse AgNPs, referred to as mAgNPs and pAgNPs) and to compare their biological activities. Our synthesis method utilized the ethanolic extract of Vitis vinifera pruning residues, resulting in the production of mAgNPs and pAgNPs with average sizes of 12 ± 5 nm and 19 ± 14 nm, respectively. Both these AgNPs preparations demonstrated an exceptional stability in terms of size distribution, which was maintained for one year. Antimicrobial testing revealed that both types of AgNPs inhibited either the growth of planktonic cells or the metabolic activity of biofilm sessile cells in Gram-negative bacteria and yeasts. No comparable activity was found towards Gram-positives. Overall, pAgNPs exhibited a higher antimicrobial efficacy compared to their monodisperse counterparts, suggesting that their size and shape may provide a broader spectrum of interactions with target cells. Both AgNP preparations showed no cytotoxicity towards a human keratinocyte cell line. Furthermore, in vivo tests using a silkworm animal model indicated the biocompatibility of the phytosynthesized AgNPs, as they had no adverse effects on insect larvae viability. These findings emphasize the potential of targeted AgNPs synthesized from viticultural waste as environmentally friendly antimicrobial agents with minimal impact on higher organisms.
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Affiliation(s)
- Anna Miškovská
- Department of Biotechnology, University of Chemistry and Technology Prague, Prague, Czech Republic.
| | - Jana Michailidu
- Department of Biotechnology, University of Chemistry and Technology Prague, Prague, Czech Republic
| | | | - Ludovica Barone
- Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy
| | - Rosalba Gornati
- Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy
| | - Aurora Montali
- Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy
| | - Gianluca Tettamanti
- Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy; Interuniversity Center for Studies on Bioinspired Agro-Environmental Technology, University of Naples Federico II, Portici, Italy
| | - Francesca Berini
- Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy; Interuniversity Center for Studies on Bioinspired Agro-Environmental Technology, University of Naples Federico II, Portici, Italy
| | - Flavia Marinelli
- Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy; Interuniversity Center for Studies on Bioinspired Agro-Environmental Technology, University of Naples Federico II, Portici, Italy
| | - Jan Masák
- Department of Biotechnology, University of Chemistry and Technology Prague, Prague, Czech Republic
| | - Alena Čejková
- Department of Biotechnology, University of Chemistry and Technology Prague, Prague, Czech Republic
| | - Olga Maťátková
- Department of Biotechnology, University of Chemistry and Technology Prague, Prague, Czech Republic
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Chuang YT, Yen CY, Shiau JP, Chang FR, Duh CY, Sung PJ, Chen KL, Tsai YH, Tang JY, Jeng JH, Sheu JH, Chang HW. Demethoxymurrapanine, an indole-naphthoquinone alkaloid, inhibits the proliferation of oral cancer cells without major side effects on normal cells. ENVIRONMENTAL TOXICOLOGY 2024; 39:1221-1234. [PMID: 37921086 DOI: 10.1002/tox.24002] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/15/2023] [Revised: 09/23/2023] [Accepted: 10/07/2023] [Indexed: 11/04/2023]
Abstract
Antioral cancer drugs need a greater antiproliferative impact on cancer than on normal cells. Demethoxymurrapanine (DEMU) inhibits proliferation in several cancer cells, but an in-depth investigation was necessary. This study evaluated the proliferation-modulating effects of DEMU, focusing on oral cancer and normal cells. DEMU (0, 2, 3, and 4 μg/mL) at 48 h treatments inhibited the proliferation of oral cancer cells (the cell viability (%) for Ca9-22 cells was 100.0 ± 2.2, 75.4 ± 5.6, 26.0 ± 3.8, and 15.4 ± 1.4, and for CAL 27 cells was 100.0 ± 9.4, 77.2 ± 5.9, 57.4 ± 10.7, and 27.1 ± 1.1) more strongly than that of normal cells (the cell viability (%) for S-G cells was 100.0 ± 6.6, 91.0 ± 4.6, 95.0 ± 2.6, and 95.8 ± 5.5), although this was blocked by the antioxidant N-acetylcysteine. The presence of oxidative stress was evidenced by the increase of reactive oxygen species and mitochondrial superoxide and the downregulation of the cellular antioxidant glutathione in oral cancer cells, but these changes were minor in normal cells. DEMU also caused greater induction of the subG1 phase, extrinsic and intrinsic apoptosis (annexin V and caspases 3, 8, and 9), and DNA damage (γH2AX and 8-hydroxy-2-deoxyguanosine) in oral cancer than in normal cells. N-acetylcysteine attenuated all these DEMU-induced changes. Together, these data demonstrate the preferential antiproliferative function of DEMU in oral cancer cells, with the preferential induction of oxidative stress, apoptosis, and DNA damage in these cancer cells, and low cytotoxicity toward normal cells.
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Affiliation(s)
- Ya-Ting Chuang
- Department of Biomedical Science and Environmental Biology, PhD Program in Life Sciences, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan
| | - Ching-Yu Yen
- School of Dentistry, Taipei Medical University, Taipei, Taiwan
- Department of Oral and Maxillofacial Surgery, Chi-Mei Medical Center, Tainan, Taiwan
| | - Jun-Ping Shiau
- Division of Breast Oncology and Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan
| | - Fang-Rong Chang
- Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan
| | - Chang-Yih Duh
- Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan
| | - Ping-Jyun Sung
- Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan
- National Museum of Marine Biology and Aquarium, Pingtung, Taiwan
| | - Kuan-Liang Chen
- Department of Oral and Maxillofacial Surgery, Chi-Mei Medical Center, Tainan, Taiwan
| | - Yi-Hong Tsai
- Department of Pharmacy and Master Program, College of Pharmacy and Health Care, Tajen University, Pingtung, Taiwan
| | - Jen-Yang Tang
- School of Post-Baccalaureate Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
- Department of Radiation Oncology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan
| | - Jiiang-Huei Jeng
- School of Dentistry, College of Dental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
- Department of Dentistry, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
- Department of Dentistry, National Taiwan University Hospital, Taipei, Taiwan
| | - Jyh-Horng Sheu
- Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan
- Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, Taiwan
| | - Hsueh-Wei Chang
- Department of Biomedical Science and Environmental Biology, PhD Program in Life Sciences, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan
- Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan
- Center for Cancer Research, Kaohsiung Medical University, Kaohsiung, Taiwan
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Yang KH, Yen CY, Wang SC, Chang FR, Chang MY, Chan CK, Jeng JH, Tang JY, Chang HW. 6- n-Butoxy-10-nitro-12,13-dioxa-11-azatricyclo[7.3.1.0 2,7]trideca-2,4,6,10-tetraene Improves the X-ray Sensitivity on Inhibiting Proliferation and Promoting Oxidative Stress and Apoptosis of Oral Cancer Cells. Biomedicines 2024; 12:458. [PMID: 38398060 PMCID: PMC10887088 DOI: 10.3390/biomedicines12020458] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2024] [Revised: 02/09/2024] [Accepted: 02/16/2024] [Indexed: 02/25/2024] Open
Abstract
This in vitro study examines the anti-oral cancer effects and mechanisms of a combined X-ray/SK2 treatment, i.e., X-ray and 6-n-butoxy-10-nitro-12,13-dioxa-11-azatricyclo[7.3.1.02,7]trideca-2,4,6,10-tetraene (SK2). ATP cell viability and flow cytometry-based cell cycle, apoptosis, oxidative stress, and DNA damage assessments were conducted. The X-ray/SK2 treatment exhibited lower viability in oral cancer (Ca9-22 and CAL 27) cells than in normal (Smulow-Glickman, S-G) cells, i.e., 32.0%, 46.1% vs. 59.0%, which showed more antiproliferative changes than with X-ray or SK2 treatment. Oral cancer cells under X-ray/SK2 treatment showed slight subG1 and G2/M increments and induced high annexin V-monitored apoptosis compared to X-ray or SK2 treatment. The X-ray/SK2 treatment showed higher caspase 3 and 8 levels for oral cancer cells than other treatments. X-ray/SK2 showed a higher caspase 9 level in CAL 27 cells than other treatments, while Ca9-22 cells showed similar levels under X-ray and/or SK2. The X-ray/SK2 treatment showed higher reactive oxygen species (ROS) generation and mitochondrial membrane potential (MMP) depletion than other treatments. Meanwhile, the mitochondrial superoxide (MitoSOX) and glutathione levels in X-ray/SK2 treatment did not exhibit the highest rank compared to others. Moreover, oral cancer cells had higher γH2AX and/or 8-hydroxy-2-deoxyguanosine levels from X-ray/SK2 treatment than others. All these measurements for X-ray/SK2 in oral cancer cells were higher than in normal cells and attenuated by N-acetylcysteine. In conclusion, X-ray/SK2 treatment showed ROS-dependent enhanced antiproliferative, apoptotic, and DNA damage effects in oral cancer cells with a lower cytotoxic influence on normal cells.
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Affiliation(s)
- Kun-Han Yang
- Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 80708, Taiwan; (K.-H.Y.); (F.-R.C.)
| | - Ching-Yu Yen
- School of Dentistry, Taipei Medical University, Taipei 11031, Taiwan;
- Department of Oral and Maxillofacial Surgery, Chi-Mei Medical Center, Tainan 71004, Taiwan
| | - Sheng-Chieh Wang
- Department of Biomedical Science and Environmental Biology, PhD Program in Life Sciences, College of Life Science, Kaohsiung Medical University, Kaohsiung 80708, Taiwan;
| | - Fang-Rong Chang
- Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 80708, Taiwan; (K.-H.Y.); (F.-R.C.)
| | - Meng-Yang Chang
- Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung 80708, Taiwan;
| | - Chieh-Kai Chan
- Department of Chemistry, University of Illinois Urbana, Champaign, IL 61820, USA;
| | - Jiiang-Huei Jeng
- School of Dentistry, College of Dental Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan;
- Department of Dentistry, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan
- Department of Dentistry, National Taiwan University Hospital, Taipei 100225, Taiwan
| | - Jen-Yang Tang
- School of Post-Baccalaureate Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
- Department of Radiation Oncology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
| | - Hsueh-Wei Chang
- Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 80708, Taiwan; (K.-H.Y.); (F.-R.C.)
- Department of Biomedical Science and Environmental Biology, PhD Program in Life Sciences, College of Life Science, Kaohsiung Medical University, Kaohsiung 80708, Taiwan;
- Center for Cancer Research, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
- Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan
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Pierce L, Anderson H, Sarkar S, Bauer SR, Sarkar S. Experimental and computational approach to establish fit-for-purpose cell viability assays. Regen Med 2024; 19:27-45. [PMID: 38247346 DOI: 10.2217/rme-2023-0154] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/23/2024] Open
Abstract
Aim: Cell viability assays are critical for cell-based products. Here, we demonstrate a combined experimental and computational approach to identify fit-for-purpose cell assays that can predict changes in cell proliferation, a critical biological response in cell expansion. Materials & methods: Jurkat cells were systematically injured using heat (45 ± 1°C). Cell viability was measured at 0 h and 24 h after treatment using assays for membrane integrity, metabolic function and apoptosis. Proliferation kinetics for longer term cultures were modeled using the Gompertz distribution to establish predictive models between cell viability results and proliferation. Results & conclusion: We demonstrate an approach for ranking these assays as predictors of cell proliferation and for setting cell viability specifications when a particular proliferation response is required.
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Affiliation(s)
- Laura Pierce
- Biosystems & Biomaterials Division, National Institute of Standards & Technology, Gaithersburg, MD 20899, USA
| | - Hidayah Anderson
- Division of Cellular & Gene Therapies, CBER, FDA, Silver Spring, MD 20993, USA
| | - Swarnavo Sarkar
- Biosystems & Biomaterials Division, National Institute of Standards & Technology, Gaithersburg, MD 20899, USA
| | - Steven R Bauer
- Division of Cellular & Gene Therapies, CBER, FDA, Silver Spring, MD 20993, USA
| | - Sumona Sarkar
- Biosystems & Biomaterials Division, National Institute of Standards & Technology, Gaithersburg, MD 20899, USA
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11
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Pore AA, Kamyabi N, Bithi SS, Ahmmed SM, Vanapalli SA. Single-Cell Proliferation Microfluidic Device for High Throughput Investigation of Replicative Potential and Drug Resistance of Cancer Cells. Cell Mol Bioeng 2023; 16:443-457. [PMID: 38099214 PMCID: PMC10716102 DOI: 10.1007/s12195-023-00773-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2022] [Accepted: 07/10/2023] [Indexed: 12/17/2023] Open
Abstract
Introduction Cell proliferation represents a major hallmark of cancer biology, and manifests itself in the assessment of tumor growth, drug resistance and metastasis. Tracking cell proliferation or cell fate at the single-cell level can reveal phenotypic heterogeneity. However, characterization of cell proliferation is typically done in bulk assays which does not inform on cells that can proliferate under given environmental perturbations. Thus, there is a need for single-cell approaches that allow longitudinal tracking of the fate of a large number of individual cells to reveal diverse phenotypes. Methods We fabricated a new microfluidic architecture for high efficiency capture of single tumor cells, with the capacity to monitor cell divisions across multiple daughter cells. This single-cell proliferation (SCP) device enabled the quantification of the fate of more than 1000 individual cancer cells longitudinally, allowing comprehensive profiling of the phenotypic heterogeneity that would be otherwise masked in standard cell proliferation assays. We characterized the efficiency of single cell capture and demonstrated the utility of the SCP device by exposing MCF-7 breast tumor cells to different doses of the chemotherapeutic agent doxorubicin. Results The single cell trapping efficiency of the SCP device was found to be ~ 85%. At the low doses of doxorubicin (0.01 µM, 0.001 µM, 0.0001 µM), we observed that 50-80% of the drug-treated cells had undergone proliferation, and less than 10% of the cells do not proliferate. Additionally, we demonstrated the potential of the SCP device in circulating tumor cell applications where minimizing target cell loss is critical. We showed selective capture of breast tumor cells from a binary mixture of cells (tumor cells and white blood cells) that was isolated from blood processing. We successfully characterized the proliferation statistics of these captured cells despite their extremely low counts in the original binary suspension. Conclusions The SCP device has significant potential for cancer research with the ability to quantify proliferation statistics of individual tumor cells, opening new avenues of investigation ranging from evaluating drug resistance of anti-cancer compounds to monitoring the replicative potential of patient-derived cells. Supplementary Information The online version contains supplementary material available at 10.1007/s12195-023-00773-z.
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Affiliation(s)
- Adity A. Pore
- Department of Chemical Engineering, Texas Tech University, Lubbock, TX USA
| | - Nabiollah Kamyabi
- Department of Chemical Engineering, Texas Tech University, Lubbock, TX USA
- Present Address: 10x Genomics, Pleasanton, CA USA
| | - Swastika S. Bithi
- Department of Chemical Engineering, Texas Tech University, Lubbock, TX USA
- Present Address: College of Engineering, West Texas A&M University, Canyon, TX USA
| | - Shamim M. Ahmmed
- Department of Chemical Engineering, Texas Tech University, Lubbock, TX USA
- Present Address: Manufacturing Integration Engineer, Intel Corporation, Hillsboro, OR USA
| | - Siva A. Vanapalli
- Department of Chemical Engineering, Texas Tech University, Lubbock, TX USA
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12
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Lyles RDZ, Martinez MJ, Sherman B, Schürer S, Burnstein KL. Automation, live-cell imaging, and endpoint cell viability for prostate cancer drug screens. PLoS One 2023; 18:e0287126. [PMID: 37815978 PMCID: PMC10564233 DOI: 10.1371/journal.pone.0287126] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/03/2023] [Accepted: 05/30/2023] [Indexed: 10/12/2023] Open
Abstract
Androgen deprivation therapy (ADT) is the standard of care for high risk and advanced prostate cancer; however, disease progression from androgen-dependent prostate cancer (ADPC) to lethal and incurable castration-resistant prostate cancer (CRPC) and (in a substantial minority of cases) neuroendocrine prostate cancer (NEPC) is common. Identifying effective targeted therapies is challenging because of acquired resistance to established treatments and the vast heterogeneity of advanced prostate cancer (PC). To streamline the identification of potentially active prostate cancer therapeutics, we have developed an adaptable semi-automated protocol which optimizes cell growth and leverages automation to enhance robustness, reproducibility, and throughput while integrating live-cell imaging and endpoint viability assays to assess drug efficacy in vitro. In this study, culture conditions for 72-hr drug screens in 96-well plates were established for a large, representative panel of human prostate cell lines including: BPH-1 and RWPE-1 (non-tumorigenic), LNCaP and VCaP (ADPC), C4-2B and 22Rv1 (CRPC), DU 145 and PC3 (androgen receptor-null CRPC), and NCI-H660 (NEPC). The cell growth and 72-hr confluence for each cell line was optimized for real-time imaging and endpoint viability assays prior to screening for novel or repurposed drugs as proof of protocol validity. We demonstrated effectiveness and reliability of this pipeline through validation of the established finding that the first-in-class BET and CBP/p300 dual inhibitor EP-31670 is an effective compound in reducing ADPC and CRPC cell growth. In addition, we found that insulin-like growth factor-1 receptor (IGF-1R) inhibitor linsitinib is a potential pharmacological agent against highly lethal and drug-resistant NEPC NCI-H660 cells. This protocol can be employed across other cancer types and represents an adaptable strategy to optimize assay-specific cell growth conditions and simultaneously assess drug efficacy across multiple cell lines.
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Affiliation(s)
- Rolando D. Z. Lyles
- Sheila and David Fuente Graduate Program in Cancer Biology, University of Miami Miller School of Medicine, Miami, Florida, United States of America
- Sylvester Comprehensive Cancer Center, University of Miami, Miami, Florida, United States of America
| | - Maria J. Martinez
- Sylvester Comprehensive Cancer Center, University of Miami, Miami, Florida, United States of America
- Department of Molecular & Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, United States of America
| | - Benjamin Sherman
- Sylvester Comprehensive Cancer Center, University of Miami, Miami, Florida, United States of America
- Department of Molecular & Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, United States of America
| | - Stephan Schürer
- Sylvester Comprehensive Cancer Center, University of Miami, Miami, Florida, United States of America
- Department of Molecular & Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, United States of America
| | - Kerry L. Burnstein
- Sylvester Comprehensive Cancer Center, University of Miami, Miami, Florida, United States of America
- Department of Molecular & Cellular Pharmacology, University of Miami Miller School of Medicine, Miami, Florida, United States of America
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13
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Yang CY, Lee MY, Chen YL, Shiau JP, Tsai YH, Yang CN, Chang HW, Tseng CH. Synthesis and Anticancer Evaluation of 4-Anilinoquinolinylchalcone Derivatives. Int J Mol Sci 2023; 24:ijms24076034. [PMID: 37047007 PMCID: PMC10094048 DOI: 10.3390/ijms24076034] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/02/2023] [Revised: 03/19/2023] [Accepted: 03/21/2023] [Indexed: 04/14/2023] Open
Abstract
A series of 4-anilinoquinolinylchalcone derivatives were synthesized and evaluated for antiproliferative activities against the growth of human cancer cell lines (Huh-7 and MDA-MB-231) and normal lung cells (MRC-5). The results exhibited low cytotoxicity against human lung cells (MRC-5). Among them, (E)-3-{4-{[4-(benzyloxy)phenyl]amino}quinolin-2-yl}-1-(4-methoxyphenyl) prop-2-en-1-one (4a) was found to have the highest cytotoxicity in breast cancer cells and low cytotoxicity in normal cells. Compound 4a causes ATP depletion and apoptosis of breast cancer MDA-MB-231 cells and triggers reactive oxygen species (ROS)-dependent caspase 3/7 activation. In conclusion, it is worth studying 4-anilinoquinolinylchalcone derivatives further as new potential anticancer agents for the treatment of human cancers.
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Affiliation(s)
- Cheng-Yao Yang
- Department of Medicinal and Applied Chemistry, College of Life Science, Kaohsiung Medical University, Kaohsiung City 80708, Taiwan
| | - Min-Yu Lee
- Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
| | - Yeh-Long Chen
- Department of Medicinal and Applied Chemistry, College of Life Science, Kaohsiung Medical University, Kaohsiung City 80708, Taiwan
| | - Jun-Ping Shiau
- Division of Breast Oncology and Surgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
| | - Yung-Hsiang Tsai
- Department of Seafood Science, National Kaohsiung University of Science and Technology, Kaohsiung 811213, Taiwan
| | - Chia-Ning Yang
- Institute of Precision Medicine, National Sun Yat-sen University, Kaohsiung 80424, Taiwan
| | - Hsueh-Wei Chang
- Department of Biomedical Science and Environmental Biology, College of Life Science, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
- Center for Cancer Research, Kaohsiung Medical University, Kaohsiung 80708, Taiwan
| | - Chih-Hua Tseng
- Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, Kaohsiung City 80708, Taiwan
- School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung City 80708, Taiwan
- Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung City 80708, Taiwan
- Department of Pharmacy, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung City 80145, Taiwan
- College of Professional Studies, National Pingtung University of Science and Technology, Pingtung County 912301, Taiwan
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Oxidative-Stress-Mediated ER Stress Is Involved in Regulating Manoalide-Induced Antiproliferation in Oral Cancer Cells. Int J Mol Sci 2023; 24:ijms24043987. [PMID: 36835397 PMCID: PMC9965613 DOI: 10.3390/ijms24043987] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2023] [Revised: 02/06/2023] [Accepted: 02/15/2023] [Indexed: 02/18/2023] Open
Abstract
Manoalide provides preferential antiproliferation of oral cancer but is non-cytotoxic to normal cells by modulating reactive oxygen species (ROS) and apoptosis. Although ROS interplays with endoplasmic reticulum (ER) stress and apoptosis, the influence of ER stress on manoalide-triggered apoptosis has not been reported. The role of ER stress in manoalide-induced preferential antiproliferation and apoptosis was assessed in this study. Manoalide induces a higher ER expansion and aggresome accumulation of oral cancer than normal cells. Generally, manoalide differentially influences higher mRNA and protein expressions of ER-stress-associated genes (PERK, IRE1α, ATF6, and BIP) in oral cancer cells than in normal cells. Subsequently, the contribution of ER stress on manoalide-treated oral cancer cells was further examined. ER stress inducer, thapsigargin, enhances the manoalide-induced antiproliferation, caspase 3/7 activation, and autophagy of oral cancer cells rather than normal cells. Moreover, N-acetylcysteine, an ROS inhibitor, reverses the responses of ER stress, aggresome formation, and the antiproliferation of oral cancer cells. Consequently, the preferential ER stress of manoalide-treated oral cancer cells is crucial for its antiproliferative effect.
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15
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Hazra S, Li R, Vamesu BM, Jilling T, Ballinger SW, Ambalavanan N, Kandasamy J. Mesenchymal stem cell bioenergetics and apoptosis are associated with risk for bronchopulmonary dysplasia in extremely low birth weight infants. Sci Rep 2022; 12:17484. [PMID: 36261501 PMCID: PMC9582007 DOI: 10.1038/s41598-022-22478-5] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2022] [Accepted: 10/14/2022] [Indexed: 01/12/2023] Open
Abstract
Oxidant stress contributes significantly to the pathogenesis of bronchopulmonary dysplasia (BPD) in extremely low birth weight (ELBW) infants. Mitochondrial function regulates oxidant stress responses as well as pluripotency and regenerative ability of mesenchymal stem cells (MSCs) which are critical mediators of lung development. This study was conducted to test whether differences in endogenous MSC mitochondrial bioenergetics, proliferation and survival are associated with BPD risk in ELBW infants. Umbilical cord-derived MSCs of ELBW infants who later died or developed moderate/severe BPD had lower oxygen consumption and aconitase activity but higher extracellular acidification-indicative of mitochondrial dysfunction and increased oxidant stress-when compared to MSCs from infants who survived with no/mild BPD. Hyperoxia-exposed MSCs from infants who died or developed moderate/severe BPD also had lower PINK1 expression but higher TOM20 expression and numbers of mitochondria/cell, indicating that these cells had decreased mitophagy. Finally, these MSCs were also noted to proliferate at lower rates but undergo more apoptosis in cell cultures when compared to MSCs from infants who survived with no/mild BPD. These results indicate that mitochondrial bioenergetic dysfunction and mitophagy deficit induced by oxidant stress may lead to depletion of the endogenous MSC pool and subsequent disruption of lung development in ELBW infants at increased risk for BPD.
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Affiliation(s)
- Snehashis Hazra
- Department of Pediatrics, University of Alabama at Birmingham School of Medicine, 1700 6th Avenue South, Birmingham, AL, 35233, USA
| | - Rui Li
- Department of Pediatrics, University of Alabama at Birmingham School of Medicine, 1700 6th Avenue South, Birmingham, AL, 35233, USA
| | - Bianca M Vamesu
- Department of Pediatrics, University of Alabama at Birmingham School of Medicine, 1700 6th Avenue South, Birmingham, AL, 35233, USA
| | - Tamas Jilling
- Department of Pediatrics, University of Alabama at Birmingham School of Medicine, 1700 6th Avenue South, Birmingham, AL, 35233, USA
| | - Scott W Ballinger
- Department of Pathology, University of Alabama at Birmingham School of Medicine, Birmingham, USA
| | - Namasivayam Ambalavanan
- Department of Pediatrics, University of Alabama at Birmingham School of Medicine, 1700 6th Avenue South, Birmingham, AL, 35233, USA
- Department of Pathology, University of Alabama at Birmingham School of Medicine, Birmingham, USA
| | - Jegen Kandasamy
- Department of Pediatrics, University of Alabama at Birmingham School of Medicine, 1700 6th Avenue South, Birmingham, AL, 35233, USA.
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Dahl E, Villwock S, Habenberger P, Choidas A, Rose M, Klebl BM. White Paper: Mimetics of Class 2 Tumor Suppressor Proteins as Novel Drug Candidates for Personalized Cancer Therapy. Cancers (Basel) 2022; 14:cancers14184386. [PMID: 36139547 PMCID: PMC9496810 DOI: 10.3390/cancers14184386] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2022] [Revised: 09/01/2022] [Accepted: 09/07/2022] [Indexed: 11/21/2022] Open
Abstract
Simple Summary A concept is presented for a new therapeutic approach, still in its early stages, which focuses on the phenotypic mimicry (“mimesis”) of proteins encoded by highly disease-relevant class 2 tumor suppressor genes that are silenced by DNA promoter methylation. Proteins derived from tumor suppressor genes are usually considered control systems of cells against oncogenic properties. Thus they represent the brakes in the “car-of-life.” Restoring this “brake function” in tumors by administering mimetic drugs may have a significant therapeutic effect. The proposed approach could thus open up a new, hitherto unexploited area of research for the development of anticancer drugs for difficult-to-treat cancers. Abstract The aim of our proposed concept is to find new target structures for combating cancers with unmet medical needs. This, unfortunately, still applies to the majority of the clinically most relevant tumor entities such as, for example, liver cancer, pancreatic cancer, and many others. Current target structures almost all belong to the class of oncogenic proteins caused by tumor-specific genetic alterations, such as activating mutations, gene fusions, or gene amplifications, often referred to as cancer “driver alterations” or just “drivers.” However, restoring the lost function of tumor suppressor genes (TSGs) could also be a valid approach to treating cancer. TSG-derived proteins are usually considered as control systems of cells against oncogenic properties; thus, they represent the brakes in the “car-of-life.” Restoring these tumor-defective brakes by gene therapy has not been successful so far, with a few exceptions. It can be assumed that most TSGs are not being inactivated by genetic alteration (class 1 TSGs) but rather by epigenetic silencing (class 2 TSGs or short “C2TSGs”). Reactivation of C2TSGs in cancer therapy is being addressed by the use of DNA demethylating agents and histone deacetylase inhibitors which act on the whole cancer cell genome. These epigenetic therapies have neither been particularly successful, probably because they are “shotgun” approaches that, although acting on C2TSGs, may also reactivate epigenetically silenced oncogenic sequences in the genome. Thus, new strategies are needed to exploit the therapeutic potential of C2TSGs, which have also been named DNA methylation cancer driver genes or “DNAme drivers” recently. Here we present a concept for a new translational and therapeutic approach that focuses on the phenotypic imitation (“mimesis”) of proteins encoded by highly disease-relevant C2TSGs/DNAme drivers. Molecular knowledge on C2TSGs is used in two complementary approaches having the translational concept of defining mimetic drugs in common: First, a concept is presented how truncated and/or genetically engineered C2TSG proteins, consisting solely of domains with defined tumor suppressive function can be developed as biologicals. Second, a method is described for identifying small molecules that can mimic the effect of the C2TSG protein lost in the cancer cell. Both approaches should open up a new, previously untapped discovery space for anticancer drugs.
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Affiliation(s)
- Edgar Dahl
- Institute of Pathology, Medical Faculty, RWTH Aachen University, D-52074 Aachen, Germany
- Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf (CIO ABCD), D-52074 Aachen, Germany
- Correspondence:
| | - Sophia Villwock
- Institute of Pathology, Medical Faculty, RWTH Aachen University, D-52074 Aachen, Germany
- Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf (CIO ABCD), D-52074 Aachen, Germany
| | - Peter Habenberger
- Lead Discovery Center GmbH (LDC), Otto-Hahn-Straße 15, D-44227 Dortmund, Germany
| | - Axel Choidas
- Lead Discovery Center GmbH (LDC), Otto-Hahn-Straße 15, D-44227 Dortmund, Germany
| | - Michael Rose
- Institute of Pathology, Medical Faculty, RWTH Aachen University, D-52074 Aachen, Germany
- Center for Integrated Oncology Aachen Bonn Cologne Duesseldorf (CIO ABCD), D-52074 Aachen, Germany
| | - Bert M. Klebl
- Lead Discovery Center GmbH (LDC), Otto-Hahn-Straße 15, D-44227 Dortmund, Germany
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Azqueta A, Stopper H, Zegura B, Dusinska M, Møller P. Do cytotoxicity and cell death cause false positive results in the in vitro comet assay? MUTATION RESEARCH. GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 2022; 881:503520. [PMID: 36031332 DOI: 10.1016/j.mrgentox.2022.503520] [Citation(s) in RCA: 30] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/07/2021] [Revised: 06/10/2022] [Accepted: 06/21/2022] [Indexed: 10/17/2022]
Abstract
The comet assay is used to measure DNA damage induced by chemical and physical agents. High concentrations of test agents may cause cytotoxicity or cell death, which may give rise to false positive results in the comet assay. Systematic studies on genotoxins and cytotoxins (i.e. non-genotoxic poisons) have attempted to establish a threshold of cytotoxicity or cell death by which DNA damage results measured by the comet assay could be regarded as a false positive result. Thresholds of cytotoxicity/cell death range from 20% to 50% in various publications. Curiously, a survey of the latest literature on comet assay results from cell culture studies suggests that one-third of publications did not assess cytotoxicity or cell death. We recommend that it should be mandatory to include results from at least one type of assay on cytotoxicity, cell death or cell proliferation in publications on comet assay results. A combination of cytotoxicity (or cell death) and proliferation (or colony forming efficiency assay) is preferable in actively proliferating cells because it covers more mechanisms of action. Applying a general threshold of cytotoxicity/cell death to all types of agents may not be applicable; however, 25% compared to the concurrent negative control seems to be a good starting value to avoid false positive comet assay results. Further research is needed to establish a threshold value to distinguish between true and potentially false positive genotoxic effects detected by the comet assay.
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Affiliation(s)
- Amaya Azqueta
- Department of Pharmacology and Toxicology, University of Navarra, C/Irunlarrea 1, 31009 Pamplona, Spain and IdiSNA, Navarra Institute for Health Research, Pamplona, Spain.
| | - Helga Stopper
- Institute of Pharmacology and Toxicology, University of Würzburg, Versbacher Str. 9, 97078 Würzburg, Germany
| | - Bojana Zegura
- Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia
| | - Maria Dusinska
- Health Effects Laboratory, Department of Environmental Chemistry, NILU-Norwegian Institute for Air Research, Instituttveien 18, 2002 Kjeller, Norway
| | - Peter Møller
- Department of Public Health, Section of Environmental Health, University of Copenhagen, Øster Farimagsgade 5A, DK-1014 Copenhagen, Denmark
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18
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Recent Advances in the Application of Essential Oils as Potential Therapeutic Candidates for Candida-Related Infections. Appl Microbiol 2022. [DOI: 10.3390/applmicrobiol2020030] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Candidiasis (oral, vulvovaginal, or systemic bloodstream infections) are important human fungal infections associated with a high global prevalence in otherwise healthy adults but are also opportunistic infections in immunocompromised patients. With the recent discovery of the multidrug resistant—and often difficult to treat—Candida auris, as well as the rising costs associated with hospitalisations and the treatment of infections caused by Candida species, there is an urgent need to develop effective therapeutics against these pathogenic yeasts. Essential oils have been documented for many years as treatments for different ailments and are widely known and utilised in alternative and complementary therapies, including treating microbial infections. This review highlights knowledge from research on the effects of medicinal plants, and in particular, essential oils, as potential treatments against different Candida species. Studies have been evaluated that describe the experimental approaches used in investigating the anticandidal effects of essential oils (in vivo and in vitro), the established mode of action of the different compounds against different Candida species, the effect of a combination of essential oils with other compounds as potential therapies, and the evidence from clinical trial studies.
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Xie SC, Metcalfe RD, Dunn E, Morton CJ, Huang SC, Puhalovich T, Du Y, Wittlin S, Nie S, Luth MR, Ma L, Kim MS, Pasaje CFA, Kumpornsin K, Giannangelo C, Houghton FJ, Churchyard A, Famodimu MT, Barry DC, Gillett DL, Dey S, Kosasih CC, Newman W, Niles JC, Lee MC, Baum J, Ottilie S, Winzeler EA, Creek DJ, Williamson N, Parker MW, Brand SL, Langston SP, Dick LR, Griffin MD, Gould AE, Tilley L. Reaction hijacking of tyrosine tRNA synthetase as a new whole-of-life-cycle antimalarial strategy. Science 2022; 376:1074-1079. [PMID: 35653481 PMCID: PMC7613620 DOI: 10.1126/science.abn0611] [Citation(s) in RCA: 31] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
Aminoacyl transfer RNA (tRNA) synthetases (aaRSs) are attractive drug targets, and we present class I and II aaRSs as previously unrecognized targets for adenosine 5'-monophosphate-mimicking nucleoside sulfamates. The target enzyme catalyzes the formation of an inhibitory amino acid-sulfamate conjugate through a reaction-hijacking mechanism. We identified adenosine 5'-sulfamate as a broad-specificity compound that hijacks a range of aaRSs and ML901 as a specific reagent a specific reagent that hijacks a single aaRS in the malaria parasite Plasmodium falciparum, namely tyrosine RS (PfYRS). ML901 exerts whole-life-cycle-killing activity with low nanomolar potency and single-dose efficacy in a mouse model of malaria. X-ray crystallographic studies of plasmodium and human YRSs reveal differential flexibility of a loop over the catalytic site that underpins differential susceptibility to reaction hijacking by ML901.
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Affiliation(s)
- Stanley C. Xie
- Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC 3010, Australia
| | - Riley D. Metcalfe
- Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC 3010, Australia
| | - Elyse Dunn
- Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC 3010, Australia
| | - Craig J. Morton
- Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC 3010, Australia
| | - Shih-Chung Huang
- Takeda Development Center Americas, Inc., Cambridge, Massachusetts 02139, USA
| | - Tanya Puhalovich
- Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC 3010, Australia
| | - Yawei Du
- Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC 3010, Australia
| | - Sergio Wittlin
- Swiss Tropical and Public Health Institute, 4051 Basel, Switzerland,University of Basel, 4003 Basel, Switzerland
| | - Shuai Nie
- Melbourne Mass Spectrometry and Proteomics Facility, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC 3010, Australia
| | - Madeline R. Luth
- Department of Pediatrics, School of Medicine, University of California, San Diego, La Jolla, California 92093, USA
| | - Liting Ma
- Takeda Development Center Americas, Inc., Cambridge, Massachusetts 02139, USA
| | - Mi-Sook Kim
- Takeda Development Center Americas, Inc., Cambridge, Massachusetts 02139, USA
| | | | - Krittikorn Kumpornsin
- Parasites and Microbes Programme, Wellcome Sanger Institute, Hinxton, CB10 1SA, United Kingdom
| | - Carlo Giannangelo
- Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia
| | - Fiona J. Houghton
- Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC 3010, Australia
| | - Alisje Churchyard
- Department of Life Sciences, Imperial College London, London SW7 2AZ, UK
| | | | - Daniel C. Barry
- Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC 3010, Australia
| | - David L. Gillett
- Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC 3010, Australia
| | - Sumanta Dey
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, United States
| | - Clara C. Kosasih
- Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC 3010, Australia
| | - William Newman
- Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC 3010, Australia
| | - Jacquin C. Niles
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, United States
| | - Marcus C.S. Lee
- Parasites and Microbes Programme, Wellcome Sanger Institute, Hinxton, CB10 1SA, United Kingdom
| | - Jake Baum
- Department of Life Sciences, Imperial College London, London SW7 2AZ, UK
| | - Sabine Ottilie
- Department of Pediatrics, School of Medicine, University of California, San Diego, La Jolla, California 92093, USA
| | - Elizabeth A. Winzeler
- Department of Pediatrics, School of Medicine, University of California, San Diego, La Jolla, California 92093, USA
| | - Darren J. Creek
- Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia
| | - Nicholas Williamson
- Melbourne Mass Spectrometry and Proteomics Facility, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC 3010, Australia
| | - Michael W. Parker
- Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC 3010, Australia,St. Vincent’s Institute of Medical Research, Fitzroy, VIC 3065, Australia
| | - Stephen L. Brand
- Medicines for Malaria Venture, PO Box 1826, 20, Route de Pré-Bois, 1215, Geneva 15, Switzerland
| | - Steven P. Langston
- Takeda Development Center Americas, Inc., Cambridge, Massachusetts 02139, USA
| | - Lawrence R. Dick
- Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC 3010, Australia,Seofon Consulting, 30 Tucker Street, Natick, Massachusetts 01760, USA
| | - Michael D.W. Griffin
- Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC 3010, Australia
| | - Alexandra E. Gould
- Takeda Development Center Americas, Inc., Cambridge, Massachusetts 02139, USA,For correspondence. Alexandra E. Gould, Takeda Development Center Americas, Inc., Cambridge, Massachusetts 02139, USA, (Chemistry) and Leann Tilley, Department of Biochemistry and Pharmacology, Bio21 Institute, The University of Melbourne, Melbourne, VIC 3010, Australia. (Biology)
| | - Leann Tilley
- Department of Biochemistry and Pharmacology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC 3010, Australia,For correspondence. Alexandra E. Gould, Takeda Development Center Americas, Inc., Cambridge, Massachusetts 02139, USA, (Chemistry) and Leann Tilley, Department of Biochemistry and Pharmacology, Bio21 Institute, The University of Melbourne, Melbourne, VIC 3010, Australia. (Biology)
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Inhibition of Aldose Reductase by Novel Phytocompounds: A Heuristic Approach to Treating Diabetic Retinopathy. EVIDENCE-BASED COMPLEMENTARY AND ALTERNATIVE MEDICINE 2022; 2022:9624118. [PMID: 35356240 PMCID: PMC8959960 DOI: 10.1155/2022/9624118] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/03/2022] [Accepted: 03/03/2022] [Indexed: 01/05/2023]
Abstract
Aldose reductase (ALR2) activation in the polyol pathway has been implicated as the primary mechanism for the progression of diabetic retinopathy. Most of the aldose reductase inhibitors (ARIs), used for the treatment of diabetic complications, were withdrawn due to ineffective treatment and adverse side effects caused by nonspecificity. Epalrestat, a carboxylic acid inhibitor, is the only ARI used for the treatment of diabetic neuropathy, though associated with minor side effects to 8% of the treated population. Our study exploited the interactions of Epalrestat-ALR2 crystal structure for the identification of specific phytocompounds that could inhibit human lens ALR2. 3D structures of plant compounds possessing antidiabetic property were retrieved from PubChem database for inhibition analysis, against human lens ALR2. Among the shortlisted compounds, Agnuside and Eupalitin-3-O-galactoside inhibited lens ALR2 with IC50 values of 22.4 nM and 27.3 nM, respectively, compared to the drug Epalrestat (98 nM), indicating high potency of these compounds as ALR2 inhibitors. IC50 concentration of the identified ARIs was validated in vitro using ARPE-19 cells. The in silico and in vitro approaches employed to identify and validate specific and potent ALR2 inhibitors resulted in the identification of phytocompounds with potency equal to or better than the ALR2 inhibiting drug, Epalrestat.
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Mihajlovic M, Vinken M. Mitochondria as the Target of Hepatotoxicity and Drug-Induced Liver Injury: Molecular Mechanisms and Detection Methods. Int J Mol Sci 2022; 23:ijms23063315. [PMID: 35328737 PMCID: PMC8951158 DOI: 10.3390/ijms23063315] [Citation(s) in RCA: 31] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2022] [Revised: 03/16/2022] [Accepted: 03/17/2022] [Indexed: 12/12/2022] Open
Abstract
One of the major mechanisms of drug-induced liver injury includes mitochondrial perturbation and dysfunction. This is not a surprise, given that mitochondria are essential organelles in most cells, which are responsible for energy homeostasis and the regulation of cellular metabolism. Drug-induced mitochondrial dysfunction can be influenced by various factors and conditions, such as genetic predisposition, the presence of metabolic disorders and obesity, viral infections, as well as drugs. Despite the fact that many methods have been developed for studying mitochondrial function, there is still a need for advanced and integrative models and approaches more closely resembling liver physiology, which would take into account predisposing factors. This could reduce the costs of drug development by the early prediction of potential mitochondrial toxicity during pre-clinical tests and, especially, prevent serious complications observed in clinical settings.
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Mishra R, Mishra PS, Varshney S, Mazumder R, Mazumder A. In Vitro and In Vivo Approaches for Screening the Potential of Anticancer Agents: A Review. Curr Drug Discov Technol 2022; 19:e060122200071. [PMID: 34994330 DOI: 10.2174/1570163819666220106122811] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2021] [Revised: 10/08/2021] [Accepted: 11/01/2021] [Indexed: 06/14/2023]
Abstract
BACKGROUND Anticancer drug development is a tedious process, requiring several in vitro, in vivo, and clinical studies. In order to avoid chemical toxicity in animals during an experiment, it is necessary to envisage toxic doses of screened drugs in vivo at different concentrations. Several in vitro and in vivo studies have been reported to discover the management of cancer. MATERIALS AND METHODS This study focused on bringing together a wide range of in vivo and in vitro assay methods developed to evaluate each hallmark feature of cancer. RESULT This review provides detailed information on target-based and cell-based screening of new anticancer drugs in the molecular targeting period. This would help in inciting an alteration from the preclinical screening of pragmatic compound-orientated to target-orientated drug selection. CONCLUSION Selection methodologies for finding anticancer activity have importance for tumor- specific agents. In this study, advanced rationalization of the cell-based assay is explored along with broad applications of the cell-based methodologies considering other opportunities.
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D'Souza A, Burch A, Dave KM, Sreeram A, Reynolds MJ, Dobbins DX, Kamte YS, Zhao W, Sabatelle C, Joy GM, Soman V, Chandran UR, Shiva SS, Quillinan N, Herson PS, Manickam DS. Microvesicles transfer mitochondria and increase mitochondrial function in brain endothelial cells. J Control Release 2021; 338:505-526. [PMID: 34450196 PMCID: PMC8526414 DOI: 10.1016/j.jconrel.2021.08.038] [Citation(s) in RCA: 98] [Impact Index Per Article: 24.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/20/2021] [Revised: 07/31/2021] [Accepted: 08/21/2021] [Indexed: 12/13/2022]
Abstract
We have demonstrated, for the first time that microvesicles, a sub-type of extracellular vesicles (EVs) derived from hCMEC/D3: a human brain endothelial cell (BEC) line transfer polarized mitochondria to recipient BECs in culture and to neurons in mice acute brain cortical and hippocampal slices. This mitochondrial transfer increased ATP levels by 100 to 200-fold (relative to untreated cells) in the recipient BECs exposed to oxygen-glucose deprivation, an in vitro model of cerebral ischemia. We have also demonstrated that transfer of microvesicles, the larger EV fraction, but not exosomes resulted in increased mitochondrial function in hypoxic endothelial cultures. Gene ontology and pathway enrichment analysis of EVs revealed a very high association to glycolysis-related processes. In comparison to heterotypic macrophage-derived EVs, BEC-derived EVs demonstrated a greater selectivity to transfer mitochondria and increase endothelial cell survival under ischemic conditions.
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Affiliation(s)
- Anisha D'Souza
- Graduate School of Pharmaceutical Sciences and School of Pharmacy, Duquesne University, Pittsburgh, PA, USA
| | - Amelia Burch
- Department of Anesthesiology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA
| | - Kandarp M Dave
- Graduate School of Pharmaceutical Sciences and School of Pharmacy, Duquesne University, Pittsburgh, PA, USA
| | | | - Michael J Reynolds
- Heart, Lung, Blood Vascular Institute, University of Pittsburgh Medical School, PA, USA
| | - Duncan X Dobbins
- Graduate School of Pharmaceutical Sciences and School of Pharmacy, Duquesne University, Pittsburgh, PA, USA
| | - Yashika S Kamte
- Graduate School of Pharmaceutical Sciences and School of Pharmacy, Duquesne University, Pittsburgh, PA, USA
| | - Wanzhu Zhao
- Graduate School of Pharmaceutical Sciences and School of Pharmacy, Duquesne University, Pittsburgh, PA, USA
| | - Courtney Sabatelle
- Graduate School of Pharmaceutical Sciences and School of Pharmacy, Duquesne University, Pittsburgh, PA, USA
| | - Gina M Joy
- Graduate School of Pharmaceutical Sciences and School of Pharmacy, Duquesne University, Pittsburgh, PA, USA
| | - Vishal Soman
- Department of Biomedical Informatics, University of Pittsburgh Medical School, PA, USA
| | - Uma R Chandran
- Department of Biomedical Informatics, University of Pittsburgh Medical School, PA, USA
| | - Sruti S Shiva
- Heart, Lung, Blood Vascular Institute, University of Pittsburgh Medical School, PA, USA; Department of Pharmacology & Chemical Biology, Pittsburgh Heart Lung Blood Vascular Institute, University of Pittsburgh Medical School, PA, USA
| | - Nidia Quillinan
- Department of Anesthesiology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA
| | - Paco S Herson
- Department of Anesthesiology, School of Medicine, University of Colorado Anschutz Medical Campus, Aurora, CO, USA
| | - Devika S Manickam
- Graduate School of Pharmaceutical Sciences and School of Pharmacy, Duquesne University, Pittsburgh, PA, USA.
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DNA Dyes-Highly Sensitive Reporters of Cell Quantification: Comparison with Other Cell Quantification Methods. Molecules 2021; 26:molecules26185515. [PMID: 34576986 PMCID: PMC8465179 DOI: 10.3390/molecules26185515] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2021] [Revised: 09/07/2021] [Accepted: 09/08/2021] [Indexed: 12/25/2022] Open
Abstract
Cell quantification is widely used both in basic and applied research. A typical example of its use is drug discovery research. Presently, plenty of methods for cell quantification are available. In this review, the basic techniques used for cell quantification, with a special emphasis on techniques based on fluorescent DNA dyes, are described. The main aim of this review is to guide readers through the possibilities of cell quantification with various methods and to show the strengths and weaknesses of these methods, especially with respect to their sensitivity, accuracy, and length. As these methods are frequently accompanied by an analysis of cell proliferation and cell viability, some of these approaches are also described.
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Evaluation of Fibroblast Viability Seeded on Acellular Human Amniotic Membrane. BIOMED RESEARCH INTERNATIONAL 2021; 2021:5597758. [PMID: 34124249 PMCID: PMC8169243 DOI: 10.1155/2021/5597758] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/16/2021] [Revised: 05/01/2021] [Accepted: 05/10/2021] [Indexed: 01/05/2023]
Abstract
Background Investigating the viability and proliferative rates of fibroblast cells on human amniotic membrane (HAM) as a scaffold will be an important subject for further research. The aim of this study was to assess the fibroblast viability seeded on acellular HAM, since foreskin neonatal allogenic fibroblasts seeded on HAM accelerate the wound healing process. Methods Fibroblasts were retrieved from the foreskin of a genetically healthy male infant, and we exploited AM of healthy term neonates to prepare the amniotic scaffold for fibroblast transfer. After cell culture, preparation of acellular HAM, and seeding of cells on HAM based on the protocol, different methods including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI), and propidium iodide (PI) staining were employed for assessment of fibroblast viability on HAM. Results Based on the results obtained from the DAPI and PI staining, the percentage of viable cells in the former staining was clearly higher than that of the dead cells in the latter one. The results of DAPI and PI staining were in accordance with the findings of MTT assay, confirming that fibroblasts were viable and even proliferate on HAM. Conclusion Our findings showed the viability of fibroblasts seeded on the acellular HAM using MTT assay, DAPI, and PI staining; however, this study had some limitations. It would be an interesting subject for future research to compare the viability and proliferation rate of fibroblasts seeded on both cellular and acellular HAM.
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Tabernilla A, dos Santos Rodrigues B, Pieters A, Caufriez A, Leroy K, Van Campenhout R, Cooreman A, Gomes AR, Arnesdotter E, Gijbels E, Vinken M. In Vitro Liver Toxicity Testing of Chemicals: A Pragmatic Approach. Int J Mol Sci 2021; 22:5038. [PMID: 34068678 PMCID: PMC8126138 DOI: 10.3390/ijms22095038] [Citation(s) in RCA: 22] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2021] [Revised: 05/04/2021] [Accepted: 05/05/2021] [Indexed: 02/07/2023] Open
Abstract
The liver is among the most frequently targeted organs by noxious chemicals of diverse nature. Liver toxicity testing using laboratory animals not only raises serious ethical questions, but is also rather poorly predictive of human safety towards chemicals. Increasing attention is, therefore, being paid to the development of non-animal and human-based testing schemes, which rely to a great extent on in vitro methodology. The present paper proposes a rationalized tiered in vitro testing strategy to detect liver toxicity triggered by chemicals, in which the first tier is focused on assessing general cytotoxicity, while the second tier is aimed at identifying liver-specific toxicity as such. A state-of-the-art overview is provided of the most commonly used in vitro assays that can be used in both tiers. Advantages and disadvantages of each assay as well as overall practical considerations are discussed.
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Affiliation(s)
| | | | | | | | | | | | | | | | | | | | - Mathieu Vinken
- Department of Pharmaceutical and Pharmacological Sciences, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium; (A.T.); (B.d.S.R.); (A.P.); (A.C.); (K.L.); (R.V.C.); (A.C.); (A.R.G.); (E.A.); (E.G.)
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27
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Seidel J, Leitzke S, Ahrens B, Sperrhacke M, Bhakdi S, Reiss K. Role of ADAM10 and ADAM17 in Regulating CD137 Function. Int J Mol Sci 2021; 22:2730. [PMID: 33800462 PMCID: PMC7962946 DOI: 10.3390/ijms22052730] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2021] [Revised: 03/01/2021] [Accepted: 03/05/2021] [Indexed: 12/20/2022] Open
Abstract
Human CD137 (4-1BB), a member of the TNF receptor family, and its ligand CD137L (4-1BBL), are expressed on immune cells and tumor cells. CD137/CD137L interaction mediates bidirectional cellular responses of potential relevance in inflammatory diseases, autoimmunity and oncology. A soluble form of CD137 exists, elevated levels of which have been reported in patients with rheumatoid arthritis and various malignancies. Soluble CD137 (sCD137) is considered to represent a splice variant of CD137. In this report, however, evidence is presented that A Disintegrin and Metalloproteinase (ADAM)10 and potentially also ADAM17 are centrally involved in its generation. Release of sCD137 by transfected cell lines and primary T cells was uniformly inhibitable by ADAM10 inhibition. The shedding function of ADAM10 can be blocked through inhibition of its interaction with surface exposed phosphatidylserine (PS), and this effectively inhibited sCD137 generation. The phospholipid scramblase Anoctamin-6 (ANO6) traffics PS to the outer membrane and thus modifies ADAM10 function. Overexpression of ANO6 increased stimulated shedding, and hyperactive ANO6 led to maximal constitutive shedding of CD137. sCD137 was functionally active and augmented T cell proliferation. Our findings shed new light on the regulation of CD137/CD137L immune responses with potential impact on immunotherapeutic approaches targeting CD137.
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Affiliation(s)
- Jana Seidel
- Department of Dermatology, University of Kiel, 24105 Kiel, Germany; (J.S.); (S.L.); (B.A.); (M.S.)
| | - Sinje Leitzke
- Department of Dermatology, University of Kiel, 24105 Kiel, Germany; (J.S.); (S.L.); (B.A.); (M.S.)
| | - Björn Ahrens
- Department of Dermatology, University of Kiel, 24105 Kiel, Germany; (J.S.); (S.L.); (B.A.); (M.S.)
| | - Maria Sperrhacke
- Department of Dermatology, University of Kiel, 24105 Kiel, Germany; (J.S.); (S.L.); (B.A.); (M.S.)
| | | | - Karina Reiss
- Department of Dermatology, University of Kiel, 24105 Kiel, Germany; (J.S.); (S.L.); (B.A.); (M.S.)
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Lee BH, Hasan MT, Lichthardt D, Gonzalez-Rodriguez R, Naumov AV. Manganese-nitrogen and gadolinium-nitrogen Co-doped graphene quantum dots as bimodal magnetic resonance and fluorescence imaging nanoprobes. NANOTECHNOLOGY 2021; 32:095103. [PMID: 33126228 DOI: 10.1088/1361-6528/abc642] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/21/2023]
Abstract
Graphene quantum dots (GQDs) are unique derivatives of graphene that show promise in multiple biomedical applications as biosensors, bioimaging agents, and drug/gene delivery vehicles. Their ease in functionalization, biocompatibility, and intrinsic fluorescence enable those modalities. However, GQDs lack deep tissue magnetic resonance imaging (MRI) capabilities desirable for diagnostics. Considering that the drawbacks of MRI contrast agent toxicity are still poorly addressed, we develop novel Mn2+ or Gd3+ doped nitrogen-containing graphene quantum dots (NGQDs) to equip the GQDs with MRI capabilities and at the same time render contrast agents biocompatible. Water-soluble biocompatible Mn-NGQDs and Gd-NGQDs synthesized via single-step microwave-assisted scalable hydrothermal reaction enable dual MRI and fluorescence modalities. These quasi-spherical 3.9-6.6 nm average-sized structures possess highly crystalline graphitic lattice structure with 0.24 and 0.53 atomic % for Mn2+ and Gd3+ doping. This structure ensures high in vitro biocompatibility of up to 1.3 mg ml-1 and 1.5 mg ml-1 for Mn-NGQDs and Gd-NGQDs, respectively, and effective internalization in HEK-293 cells traced by intrinsic NGQD fluorescence. As MRI contrast agents with considerably low Gd and Mn content, Mn-NGQDs exhibit substantial transverse/longitudinal relaxivity (r 2/r 1) ratios of 11.190, showing potential as dual-mode longitudinal or transverse relaxation time (T 1 or T 2) contrast agents, while Gd-NGQDs possess r 2/r 1 of 1.148 with high r 1 of 9.546 mM-1 s-1 compared to commercial contrast agents, suggesting their potential as T1 contrast agents. Compared to other nanoplatforms, these novel Mn2+ and Gd3+ doped NGQDs not only provide scalable biocompatible alternatives as T1/T2 and T1 contrast agents but also enable in vitro intrinsic fluorescence imaging.
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Affiliation(s)
- Bong Han Lee
- Department of Physics and Astronomy, Texas Christian University, TCU Box 298840, Fort Worth, Texas 76129, United States of America
| | - Md Tanvir Hasan
- Department of Physics and Astronomy, Texas Christian University, TCU Box 298840, Fort Worth, Texas 76129, United States of America
- Biosystems and Biomaterials Division, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, MD 20899, United States of America
| | - Denise Lichthardt
- Department of Physics and Astronomy, Texas Christian University, TCU Box 298840, Fort Worth, Texas 76129, United States of America
- Friedrich-Alexander University Erlangen-Nürnberg, Schlossplatz 4, 91054 Erlangen, Germany
| | - Roberto Gonzalez-Rodriguez
- Department of Physics and Astronomy, Texas Christian University, TCU Box 298840, Fort Worth, Texas 76129, United States of America
- Department of Physics, University of North Texas, 210 Avenue A, Denton, TX 76201, United States of America
| | - Anton V Naumov
- Department of Physics and Astronomy, Texas Christian University, TCU Box 298840, Fort Worth, Texas 76129, United States of America
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Zupin L, Barbi E, Sagredini R, Ottaviani G, Crovella S, Celsi F. In vitro effects of photobiomodulation therapy on 50B11 sensory neurons: evaluation of cell metabolism, oxidative stress, mitochondrial membrane potential (MMP), and capsaicin-induced calcium flow. JOURNAL OF BIOPHOTONICS 2021; 14:e202000347. [PMID: 33128434 DOI: 10.1002/jbio.202000347] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/01/2020] [Revised: 10/28/2020] [Accepted: 10/29/2020] [Indexed: 06/11/2023]
Abstract
The analgesic properties of photobiomodulation therapy (PBMT) have been raising increasing interest in the clinical community due to the positive effects observed on patients, nevertheless the mechanistic basis of its action on peripheral sensory neurons remains still elusive. In this study, the effect of near-infrared (NIR) PBMT at 800 and 970 nm of wavelength was investigated on the 50B11 immortalized nociceptive sensory neuronal cell line by evaluating capsaicin-induced calcium flow and different markers correlated to mitochondria, that is, ATP, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP). Calcium peak stimulated by capsaicin, the ligand of TRPV1 channel, was decreased in neurons pre-irradiated with the combination of the two wavelengths. Furthermore, delivering the 800 and 970 nm separately an increment of ATP, as well as MMP hyperpolarization were detected; notably, the 800 nm wavelength also increased ROS and O2- levels. Our findings, obtained on an in vitro model of nociception, show the positive effect of PBMT on two potential photo-targets of NIR light, namely the TRPV1 channel and the mitochondria.
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Affiliation(s)
- Luisa Zupin
- Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy
| | - Egidio Barbi
- Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy
- Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy
| | - Raffaella Sagredini
- Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy
| | - Giulia Ottaviani
- Department of Medicine, Surgery and Health Sciences, University of Trieste, Trieste, Italy
| | - Sergio Crovella
- Department of Biological and Environmental Sciences, College of Arts and Sciences, University of Qatar, Doha, Qatar
| | - Fulvio Celsi
- Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Italy
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Nitschke S, Petković S, Ahonen S, Minassian BA, Nitschke F. Sensitive quantification of α-glucans in mouse tissues, cell cultures, and human cerebrospinal fluid. J Biol Chem 2020; 295:14698-14709. [PMID: 32817315 DOI: 10.1074/jbc.ra120.015061] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2020] [Revised: 08/04/2020] [Indexed: 12/30/2022] Open
Abstract
The soluble α-polyglucan glycogen is a central metabolite enabling transient glucose storage to suit cellular energy needs. Glycogen storage diseases (GSDs) comprise over 15 entities caused by generalized or tissue-specific defects in enzymes of glycogen metabolism. In several, e.g. in Lafora disease caused by the absence of the glycogen phosphatase laforin or its interacting partner malin, degradation-resistant abnormally structured insoluble glycogen accumulates. Sensitive quantification methods for soluble and insoluble glycogen are critical to research, including therapeutic studies, in such diseases. This paper establishes methodological advancements relevant to glycogen metabolism investigations generally, and GSDs. Introducing a pre-extraction incubation method, we measure degradation-resistant glycogen in as little as 30 mg of skeletal muscle or a single hippocampus from Lafora disease mouse models. The digestion-resistant glycogen correlates with the disease-pathogenic insoluble glycogen and can readily be detected in very young mice where glycogen accumulation has just begun. Second, we establish a high-sensitivity glucose assay with detection of ATP depletion, enabling 1) quantification of α-glucans in cell culture using a medium-throughput assay suitable for assessment of candidate glycogen synthesis inhibitors, and 2) discovery of α-glucan material in healthy human cerebrospinal fluid, establishing a novel methodological platform for biomarker analyses in Lafora disease and other GSDs.
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Affiliation(s)
- Silvia Nitschke
- Departments of Pediatrics and Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA
| | - Sara Petković
- Program in Genetics and Genome Biology, The Hospital for Sick Children Research Institute, Toronto, Ontario, Canada
| | - Saija Ahonen
- Program in Genetics and Genome Biology, The Hospital for Sick Children Research Institute, Toronto, Ontario, Canada
| | - Berge A Minassian
- Departments of Pediatrics and Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA; Program in Genetics and Genome Biology, The Hospital for Sick Children Research Institute, Toronto, Ontario, Canada
| | - Felix Nitschke
- Departments of Pediatrics and Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
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Witt G, Keminer O, Leu J, Tandon R, Meiser I, Willing A, Winschel I, Abt JC, Brändl B, Sébastien I, Friese MA, Müller FJ, Neubauer JC, Claussen C, Zimmermann H, Gribbon P, Pless O. An automated and high-throughput-screening compatible pluripotent stem cell-based test platform for developmental and reproductive toxicity assessment of small molecule compounds. Cell Biol Toxicol 2020; 37:229-243. [PMID: 32564278 PMCID: PMC8012336 DOI: 10.1007/s10565-020-09538-0] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2020] [Accepted: 06/02/2020] [Indexed: 12/02/2022]
Abstract
The embryonic stem cell test (EST) represents the only validated and accepted in vitro system for the detection and classification of compounds according to their developmental and reproductive teratogenic potency. The widespread implementation of the EST, however, in particular for routine application in pharmaceutical development, has not been achieved so far. Several drawbacks still limit the high-throughput screening of potential drug candidates in this format: The long assay period, the use of non-homogeneous viability assays, the low throughput analysis of marker protein expression and the compatibility of the assay procedures to automation. We have therefore introduced several advancements into the EST workflow: A reduction of the assay period, an introduction of homogeneous viability assays, and a straightforward analysis of marker proteins by flow cytometry and high content imaging to assess the impact of small molecules on differentiation capacity. Most importantly, essential parts of the assay procedure have been adapted to lab automation in 96-well format, thus enabling the interrogation of several compounds in parallel. In addition, extensive investigations were performed to explore the predictive capacity of this next-generation EST, by testing a set of well-known embryotoxicants that encompasses the full range of chemical-inherent embryotoxic potencies possible. Due to these significant improvements, the augmented workflow provides a basis for a sensitive, more rapid, and reproducible high throughput screening compatible platform to predict in vivo developmental toxicity from in vitro data which paves the road towards application in an industrial setting.
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