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Girotti AW, Bazak J, Korytowski W. Pro-Tumor Activity of Endogenous Nitric Oxide in Anti-Tumor Photodynamic Therapy: Recently Recognized Bystander Effects. Int J Mol Sci 2023; 24:11559. [PMID: 37511317 PMCID: PMC10380283 DOI: 10.3390/ijms241411559] [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: 06/21/2023] [Revised: 07/10/2023] [Accepted: 07/13/2023] [Indexed: 07/30/2023] Open
Abstract
Various studies have revealed that several cancer cell types can upregulate inducible nitric oxide synthase (iNOS) and iNOS-derived nitric oxide (NO) after moderate photodynamic treatment (PDT) sensitized by 5-aminolevulinic acid (ALA)-induced protoporphyrin-IX. As will be discussed, the NO signaled cell resistance to photokilling as well as greater growth and migratory aggressiveness of surviving cells. On this basis, it was predicted that diffusible NO from PDT-targeted cells in a tumor might enhance the growth, migration, and invasiveness of non- or poorly PDT-targeted bystander cells. This was tested using a novel approach in which ALA-PDT-targeted cancer cells on a culture dish were initially segregated from non-targeted bystander cells of the same type via impermeable silicone-rimmed rings. Several hours after LED irradiation, the rings were removed, and both cell populations were analyzed in the dark for various responses. After a moderate extent of targeted cell killing (~25%), bystander proliferation and migration were evaluated, and both were found to be significantly enhanced. Enhancement correlated with iNOS/NO upregulation in surviving PDT-targeted cancer cells in the following cell type order: PC3 > MDA-MB-231 > U87 > BLM. If occurring in an actual PDT-challenged tumor, such bystander effects might compromise treatment efficacy by stimulating tumor growth and/or metastatic dissemination. Mitigation of these and other negative NO effects using pharmacologic adjuvants that either inhibit iNOS transcription or enzymatic activity will be discussed.
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Affiliation(s)
- Albert W Girotti
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA
| | - Jerzy Bazak
- Department of Biophysics, Jagiellonian University, 31-007 Krakow, Poland
| | - Witold Korytowski
- Department of Biophysics, Jagiellonian University, 31-007 Krakow, Poland
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Bazak J, Korytowski W, Girotti AW. Hyper-Aggressiveness of Bystander Cells in an Anti-Tumor Photodynamic Therapy Model: Role of Nitric Oxide Produced by Targeted Cells. Crit Rev Oncog 2023; 28:15-25. [PMID: 37824384 DOI: 10.1615/critrevoncog.2022040016] [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: 10/14/2023]
Abstract
When selected tumor cells in a large in vitro population are exposed to ionizing radiation, they can send pro-survival signals to non-exposed counterparts (bystander cells). If there is no physical contact between irradiated and bystander cells, the latter respond to mediators from targeted cells that diffuse through the medium. One such mediator is known to be nitric oxide (NO). It was recently discovered that non-ionizing anti-tumor photodynamic therapy (PDT) can also elicit pro-survival/expansion bystander effects in a variety of human cancer cells. A novel silicone ring-based approach was used for distinguishing photodynamically-targeted cells from non-targeted bystanders. A key finding was that NO from upregulated iNOS in surviving targeted cells diffused to the bystanders and caused iNOS/NO upregulation there, which in turn stimulated cell proliferation and migration. The intensity of these responses depended on the extent of iNOS/NO induction in targeted cells of different cancer lines. Moreover, the responses could be replicated using NO from the chemical donor DETA/NO. This review will focus on these and related findings, their negative implications for clinical PDT, and how these might be averted by using pharmacologic inhibitors of iNOS activity or transcription.
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Affiliation(s)
- Jerzy Bazak
- Department of Biophysics, Jagiellonian University, Krakow, Poland
| | | | - Albert W Girotti
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226-3548, USA
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Niu J, Cheng M, Hong Z, Ling J, Di W, Gu L, Qiu L. The effect of 5-Aminolaevulinic Acid Photodynamic Therapy versus CO 2 laser in the Treatment of Cervical Low-grade Squamous Intraepithelial Lesions with High-Risk HPV Infection: A non-randomized, controlled pilot study. Photodiagnosis Photodyn Ther 2021; 36:102548. [PMID: 34562648 DOI: 10.1016/j.pdpdt.2021.102548] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2021] [Revised: 09/01/2021] [Accepted: 09/20/2021] [Indexed: 12/14/2022]
Abstract
BACKGROUND There are insufficient studies comparing the efficacy of 5-aminolaevulinic acid (ALA) photodynamic therapy (PDT) against CO2 laser therapy in the treatment of cervical low-grade squamous intraepithelial lesion (LSIL) with high-risk human papillomavirus (HR-HPV), especially for long-term efficacy. METHODS Patients with cervical LSIL and HR-HPV infection were divided into two treatment groups based on their own choice. All patients had a follow-up test including HPV testing, cytology and colposcopy at 4-6 months and 12 months after the treatment. RESULTS (1) Among 277 patients, 176 patients received 5-ALA PDT and 101 patients received CO2 laser therapy. (2) 4-6 months after treatment, there was no significant difference between two groups in the complete remission (CR) rates of cervical LSIL and the clearance rate of HR-HPV infection. (3) 12 months after treatment, compared with the CO2 laser group, the CR rates of cervical LSIL in the 5-ALA PDT group was significantly higher than the CO2 laser group. There was no statistical difference in the clearance rate of HR-HPV infection between the two groups. (4) 12 months after treatment, the recurrence rate of cervical lesions and the reinfection rate of HR-HPV infection in 5-ALA PDT group were significantly lower than those in CO2 laser group. CONCLUSION The effect of 5-ALA PDT is similar to CO2 laser at 4-6 months. The long-term efficacy of 5-ALA PDT appears better than CO2 laser. As a non-invasive treatment, 5-ALA PDT is a highly effective therapeutic procedure for cervical LSIL with HR-HPV infection.
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Affiliation(s)
- Jiaxin Niu
- Department of Obstetrics and Gynecology, Renji Hospital, Shanghai Jiaotong University School of Medicine; Shanghai Key Laboratory of Gynecologic Oncology, Renji Hospital, Shanghai Jiaotong University School of Medicine
| | - Mengxing Cheng
- Department of Obstetrics and Gynecology, Renji Hospital, Shanghai Jiaotong University School of Medicine; Shanghai Key Laboratory of Gynecologic Oncology, Renji Hospital, Shanghai Jiaotong University School of Medicine
| | - Zubei Hong
- Department of Obstetrics and Gynecology, Renji Hospital, Shanghai Jiaotong University School of Medicine; Shanghai Key Laboratory of Gynecologic Oncology, Renji Hospital, Shanghai Jiaotong University School of Medicine
| | - Jiayan Ling
- Department of Obstetrics and Gynecology, Renji Hospital, Shanghai Jiaotong University School of Medicine
| | - Wen Di
- Department of Obstetrics and Gynecology, Renji Hospital, Shanghai Jiaotong University School of Medicine; Shanghai Key Laboratory of Gynecologic Oncology, Renji Hospital, Shanghai Jiaotong University School of Medicine; State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine
| | - Liying Gu
- Department of Obstetrics and Gynecology, Renji Hospital, Shanghai Jiaotong University School of Medicine; Shanghai Key Laboratory of Gynecologic Oncology, Renji Hospital, Shanghai Jiaotong University School of Medicine.
| | - Lihua Qiu
- Department of Obstetrics and Gynecology, Renji Hospital, Shanghai Jiaotong University School of Medicine; Shanghai Key Laboratory of Gynecologic Oncology, Renji Hospital, Shanghai Jiaotong University School of Medicine; State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine.
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Martins WK, Belotto R, Silva MN, Grasso D, Suriani MD, Lavor TS, Itri R, Baptista MS, Tsubone TM. Autophagy Regulation and Photodynamic Therapy: Insights to Improve Outcomes of Cancer Treatment. Front Oncol 2021; 10:610472. [PMID: 33552982 PMCID: PMC7855851 DOI: 10.3389/fonc.2020.610472] [Citation(s) in RCA: 35] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2020] [Accepted: 12/03/2020] [Indexed: 12/24/2022] Open
Abstract
Cancer is considered an age-related disease that, over the next 10 years, will become the most prevalent health problem worldwide. Although cancer therapy has remarkably improved in the last few decades, novel treatment concepts are needed to defeat this disease. Photodynamic Therapy (PDT) signalize a pathway to treat and manage several types of cancer. Over the past three decades, new light sources and photosensitizers (PS) have been developed to be applied in PDT. Nevertheless, there is a lack of knowledge to explain the main biochemical routes needed to trigger regulated cell death mechanisms, affecting, considerably, the scope of the PDT. Although autophagy modulation is being raised as an interesting strategy to be used in cancer therapy, the main aspects referring to the autophagy role over cell succumbing PDT-photoinduced damage remain elusive. Several reports emphasize cytoprotective autophagy, as an ultimate attempt of cells to cope with the photo-induced stress and to survive. Moreover, other underlying molecular mechanisms that evoke PDT-resistance of tumor cells were considered. We reviewed the paradigm about the PDT-regulated cell death mechanisms that involve autophagic impairment or boosted activation. To comprise the autophagy-targeted PDT-protocols to treat cancer, it was underlined those that alleviate or intensify PDT-resistance of tumor cells. Thereby, this review provides insights into the mechanisms by which PDT can be used to modulate autophagy and emphasizes how this field represents a promising therapeutic strategy for cancer treatment.
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Affiliation(s)
- Waleska K Martins
- Laboratory of Cell and Membrane, Anhanguera University of São Paulo, São Paulo, Brazil
| | - Renata Belotto
- Perola Byington Hospital Gynecology - Lasertherapy Clinical Research Department, São Paulo, Brazil
| | - Maryana N Silva
- Laboratory of Cell and Membrane, Anhanguera University of São Paulo, São Paulo, Brazil
| | - Daniel Grasso
- CONICET, Instituto de Estudios de la Inmunidad Humoral (IDEHU), Universidad de Buenos Aires, Buenos Aires, Argentina
| | - Maynne D Suriani
- Institute of Chemistry, Federal University of Uberlândia, Uberlândia, Brazil
| | - Tayná S Lavor
- Institute of Chemistry, Federal University of Uberlândia, Uberlândia, Brazil
| | - Rosangela Itri
- Institute of Physics, University of São Paulo, São Paulo, Brazil
| | | | - Tayana M Tsubone
- Institute of Chemistry, Federal University of Uberlândia, Uberlândia, Brazil
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Girotti AW, Fahey JM, Korytowski W. Negative effects of tumor cell nitric oxide on anti-glioblastoma photodynamic therapy. JOURNAL OF CANCER METASTASIS AND TREATMENT 2020; 6:52. [PMID: 33564720 PMCID: PMC7869587 DOI: 10.20517/2394-4722.2020.107] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Glioblastomas are highly aggressive brain tumors that can persist after exposure to conventional chemotherapy or radiotherapy. Nitric oxide (NO) produced by inducible NO synthase (iNOS/NOS2) in these tumors is known to foster malignant cell proliferation, migration, and invasion as well as resistance to chemo- and radiotherapy. Minimally invasive photodynamic therapy (PDT) sensitized by 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) is a highly effective anti-glioblastoma modality, but it is also subject to NO-mediated resistance. Studies by the authors have revealed that glioblastoma U87 and U251 cells use endogenous iNOS/NO to not only resist photokilling after an ALA/light challenge, but also to promote proliferation and migration/invasion of surviving cells. Stress-upregulated iNOS/NO was found to play a major role in these negative responses to PDT-like treatment. Our studies have revealed a tight network of upstream signaling events leading to iNOS induction in photostressed cells and transition to a more aggressive phenotype. These events include activation or upregulation of pro-survival/ pro-expansion effector proteins such as NF-κB, phosphoinositide-3-kinase (PI3K), protein kinase-B (Akt), p300, Survivin, and Brd4. In addition to this upstream signaling and its regulation, pharmacologic approaches for directly suppressing iNOS at its activity vs. transcriptional level are discussed. One highly effective agent in the latter category is bromodomain and extra-terminal (BET) inhibitor, JQ1, which was found to minimize iNOS upregulation in photostressed U87 cells. By acting similarly at the clinical level, a BET inhibitor such as JQ1 should markedly improve the efficacy of anti-glioblastoma PDT.
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Affiliation(s)
- Albert W. Girotti
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, United States
| | - Jonathan M. Fahey
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, United States
| | - Witold Korytowski
- Department of Biophysics, Jagiellonian University, Krakow 30-387, Poland
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Girotti AW, Fahey JM, Korytowski W. Nitric oxide-elicited resistance to anti-glioblastoma photodynamic therapy. CANCER DRUG RESISTANCE (ALHAMBRA, CALIF.) 2020; 3:401-414. [PMID: 33073206 PMCID: PMC7558220 DOI: 10.20517/cdr.2020.25] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/20/2020] [Revised: 06/23/2020] [Accepted: 07/14/2020] [Indexed: 12/22/2022]
Abstract
Glioblastoma multiforme is a highly aggressive primary brain malignancy that resists most conventional chemoand radiotherapeutic interventions. Nitric oxide (NO), a short lived free radical molecule produced by inducible NO synthase (iNOS) in glioblastomas and other tumors, is known to play a key role in tumor persistence, progression, and chemo/radiotherapy resistance. Site-specific and minimally invasive photodynamic therapy (PDT), based on oxidative damage resulting from non-ionizing photoactivation of a sensitizing agent, is highly effective against glioblastoma, but resistance also exists in this case. Studies in the authors' laboratory have shown that much of the latter is mediated by iNOS/NO. For example, when glioblastoma U87 or U251 cells sensitized in mitochondria with 5-aminolevulinic acid -induced protoporphyrin IX were exposed to a moderate dose of visible light, the observed apoptosis was strongly enhanced by an iNOS activity inhibitor or NO scavenger, indicating that iNOS/NO had increased cell resistance to photokilling. Moreover, cells that survived the photochallenge proliferated, migrated, and invaded more aggressively than controls, and these responses were also driven predominantly by iNOS/NO. Photostress-upregulated iNOS rather than basal enzyme was found to be responsible for all the negative effects described. Recognition of NO-mediated hyper-resistance/hyper-aggression in PDT-stressed glioblastoma has stimulated interest in how these responses can be prevented or at least minimized by pharmacologic adjuvants such as inhibitors of iNOS activity or transcription. Recent developments along these lines and their clinical potential for improving anti-glioblastoma PDT are discussed.
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Affiliation(s)
- Albert W. Girotti
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA
| | - Jonathan M. Fahey
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA
| | - Witold Korytowski
- Department of Biophysics, Jagiellonian University, Krakow 31-008, Poland
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Girotti AW. Nitric Oxide-Mediated Resistance to Antitumor Photodynamic Therapy. Photochem Photobiol 2020; 96:500-505. [PMID: 31545517 PMCID: PMC7085955 DOI: 10.1111/php.13163] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2019] [Accepted: 09/05/2019] [Indexed: 12/11/2022]
Abstract
As an antitumor modality based on sensitizer photoexcitation by tumor-directed light, photodynamic therapy (PDT) has the advantage of being site-specific compared with conventional chemotherapy or radiotherapy. Like these other therapies, however, PDT is often limited by pre-existing or acquired resistance. One type of resistance, discovered in the author's laboratory, involves nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS) in tumor cells. Using human breast, prostate and brain cancer cell lines, we have shown that iNOS is dramatically upregulated after a moderate PDT challenge sensitized by 5-aminolevulinic acid-induced protoporphyrin IX. The elevated NO not only elicited a greater resistance to cell photokilling, but also an increase in the growth and migration/invasion rate of surviving cells. Greater iNOS/NO-based resistance was also demonstrated at the in vivo level using a breast tumor xenograft model. More recent studies have shown that NO from PDT-targeted cells can stimulate a progrowth/promigration response in non-targeted bystander cells. These novel effects of NO, their negative impact on PDT efficacy and possible mitigation thereof by anti-iNOS/NO pharmacologic agents will be discussed.
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Affiliation(s)
- Albert W. Girotti
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, 53226-3548
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Girotti AW, Fahey JM. Upregulation of pro-tumor nitric oxide by anti-tumor photodynamic therapy. Biochem Pharmacol 2019; 176:113750. [PMID: 31836386 DOI: 10.1016/j.bcp.2019.113750] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2019] [Accepted: 12/06/2019] [Indexed: 12/11/2022]
Abstract
Many malignant tumors use endogenous nitric oxide (NO) to promote survival, growth, and metastatic migration. This NO, which is typically generated by inducible nitric oxide synthase (iNOS), can also antagonize various anti-cancer therapies and its source is most often assumed to be constitutive or pre-existing iNOS. In this paper, we provide evidence (i) that many different cancer cells exhibit resistance to oxidative killing by photodynamic therapy (PDT), and (ii) that cells surviving the challenge grow, migrate and invade more aggressively, as do non-targeted bystander cells. Accompanying these effects are activation or upregulation of pro-survival/progression effector proteins such as NF-κB, Akt, and Survivin. Observed in the author's laboratory, these responses were not attributed to basal iNOS/NO in most cases, but rather to NO from enzyme that was strongly upregulated by photodynamic stress. Each of these effects and how they can be mitigated by inhibitors of iNOS activity or transcription, or by NO scavengers will be discussed. When approved for clinical use, such pharmacologic agents could improve PDT efficacy as well as reduce potentially negative side-effects of this therapy.
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Affiliation(s)
- Albert W Girotti
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, United States.
| | - Jonathan M Fahey
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, United States
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Fahey JM, Korytowski W, Girotti AW. Upstream signaling events leading to elevated production of pro-survival nitric oxide in photodynamically-challenged glioblastoma cells. Free Radic Biol Med 2019; 137:37-45. [PMID: 30991141 PMCID: PMC6526063 DOI: 10.1016/j.freeradbiomed.2019.04.013] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/12/2018] [Revised: 03/15/2019] [Accepted: 04/10/2019] [Indexed: 12/19/2022]
Abstract
Nitric oxide (NO) generated endogenously by inducible nitric oxide synthase (iNOS) promotes growth and migration/invasion of glioblastoma cells and also fosters resistance to chemotherapy and ionizing radiotherapy. Our recent studies revealed that glioblastoma cell iNOS/NO also opposes the cytotoxic effects of non-ionizing photodynamic therapy (PDT), and moreover stimulates growth/migration aggressiveness of surviving cells. These negative responses, which depended on PI3K/Akt/NF-κB activation, were strongly suppressed by blocking iNOS transcription with JQ1, a BET bromodomain inhibitor. In the present study, we sought to identify additional molecular events that precede iNOS transcriptional upregulation. Akt activation, iNOS induction, and viability loss in PDT-challenged glioblastoma U87 cells were all strongly inhibited by added l-histidine, consistent with primary involvement of photogenerated singlet oxygen (1O2). Transacetylase p300 not only underwent greater Akt-dependent activation after PDT, but greater interaction with NF-κB subunit p65, which in turn exhibited greater K310 acetylation. In addition, PDT promoted intramolecular disulfide formation and inactivation of tumor suppressor PTEN, thereby favoring Akt and p300 activation leading to iNOS upregulation. Importantly, deacetylase Sirt1 was down-regulated by PDT stress, consistent with the observed increase in p65-acK310 level, which fostered iNOS transcription. This study provides new mechanistic insights into how glioblastoma tumors can exploit iNOS/NO to not only resist PDT, but to attain a more aggressive survival phenotype.
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Affiliation(s)
- Jonathan M Fahey
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, 53226-3548, USA
| | | | - Albert W Girotti
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, 53226-3548, USA.
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Changes of microbial cell survival, metabolic activity, efflux capacity, and quorum sensing ability of Aggregatibacter actinomycetemcomitans due to antimicrobial photodynamic therapy-induced bystander effects. Photodiagnosis Photodyn Ther 2019; 26:287-294. [PMID: 31026616 DOI: 10.1016/j.pdpdt.2019.04.021] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/08/2018] [Revised: 04/22/2019] [Accepted: 04/22/2019] [Indexed: 12/27/2022]
Abstract
BACKGROUND The bystander effects, whereby naive (bystander) microbial cells near microbial cells directly exposed to certain treatment show responses that would not have happened in the absence of the directly targeted microbial cells, is recently documented in the field of microbiology. In this article, we discuss that substantial bystander responses are also observed after antimicrobial photodynamic therapy (aPDT) using curcumin (Cur). MATERIALS AND METHODS Bystander effects induced by whole bacterial cell suspension (WBCST), cell-free supernatants fluid (CFSFT), and bacterial cell pellet (BCPT) obtained from A. actinomycetemcomitans culture treated with Cur-aPDT on cell survival, quorum sensing (QS) ability, metabolic activity and efflux capacity of A. actinomycetemcomitans were determined using microbial viability assay, Escherichia coli-based bioassay, XTT reduction method, and ethidium bromide (EtBr) accumulation assay, respectively. RESULTS A. actinomycetemcomitans cell survival reduced by 82.7% (P = 0.001) and 76.2% (P = 0.01) after exposure to WBCST and CFSFT, respectively. The A. actinomycetemcomitans population increased by 5.5% (P = 0.7) after exposure to BCPT. Bacterial metabolic activity decreased by 42.6% (P = 0.02), 35.3% (P = 0.03), and 9.4% (P = 0.5) after exposure to WBCST, CFSFT, and BCPT, respectively. A. actinomycetemcomitans exposed to WBCST, CFSFT, and BCPT showed a reduction of 83.2% (P = 0.001), 77.2% (P = 0.01) and 21.9% (P = 0.09) in the QS mediator compared to the WBCSU, CFSFU, and BCPU of untreated A. actinomycetemcomitans, respectively. No significant change of the EtBr accumulation was observed in the three preparations of the Cur-aPDT-treated culture (i.e. WBCST, CFSFT, and BCPT) compared to their respective controls. CONCLUSIONS The results of the current study revealed that Cur-aPDT could significantly reduce microbial cell survival, cell metabolic activity, efflux capacity, and QS ability through the bystander effects. As a result, the bystander effects of Cur-aPDT along with the direct effect of Cur-aPDT can enhance the efficiency of aPDT as an adjunct therapeutic strategy for treatment of local infections.
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Fahey JM, Girotti AW. Nitric Oxide Antagonism to Anti-Glioblastoma Photodynamic Therapy: Mitigation by Inhibitors of Nitric Oxide Generation. Cancers (Basel) 2019; 11:E231. [PMID: 30781428 PMCID: PMC6406633 DOI: 10.3390/cancers11020231] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2018] [Revised: 01/25/2019] [Accepted: 02/09/2019] [Indexed: 12/14/2022] Open
Abstract
Many studies have shown that low flux nitric oxide (NO) produced by inducible NO synthase (iNOS/NOS2) in various tumors, including glioblastomas, can promote angiogenesis, cell proliferation, and migration/invasion. Minimally invasive, site-specific photodynamic therapy (PDT) is a highly promising anti-glioblastoma modality. Recent research in the authors' laboratory has revealed that iNOS-derived NO in glioblastoma cells elicits resistance to 5-aminolevulinic acid (ALA)-based PDT, and moreover endows PDT-surviving cells with greater proliferation and migration/invasion aggressiveness. In this contribution, we discuss iNOS/NO antagonism to glioblastoma PDT and how this can be overcome by judicious use of pharmacologic inhibitors of iNOS activity or transcription.
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Affiliation(s)
- Jonathan M Fahey
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
| | - Albert W Girotti
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
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Girotti AW. Upregulation of nitric oxide in tumor cells as a negative adaptation to photodynamic therapy. Lasers Surg Med 2018; 50:590-598. [PMID: 29504635 DOI: 10.1002/lsm.22807] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/01/2018] [Indexed: 12/14/2022]
Abstract
One of the advantages of PDT is that it can often circumvent tumor resistance to chemotherapeutic agents such as cisplatin and doxorubicin. However, pre-existing and acquired resistance to PDT has also been demonstrated. One type of resistance, which involves nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS/NOS2) in tumor cells, was discovered in the author's laboratory. When subjected to a 5-aminolevulinic acid (ALA)-based photodynamic challenge, several cancer lines, including breast, prostate, and glioma, underwent intrinsic apoptosis that could be substantially enhanced by iNOS enzymatic inhibitors or a NO scavenger, implying iNOS/NO-mediated resistance. In most cases, iNOS was significantly upregulated by the challenge and this appeared to be more important in the hyper-resistance than pre-existing enzyme. Of added importance was our observation that cells surviving ALA/light treatment typically exhibited a more aggressive phenotype, proliferating and migrating/invading more rapidly than controls in iNOS/NO-dependent fashion. Most of these in vitro PDT findings have recently been confirmed at the in vivo level, using a human breast tumor xenograft model. We have also shown that upregulated iNOS in PDT-targeted cells can elicit a pro-growth/migration response in non-targeted bystander cells, NO again playing a key role. Post-PDT resistance and potentially dangerous hyper-aggressiveness can be attenuated by inhibitors of iNOS enzymatic activity, some of which have seen pharmacologic use in non-cancer or PDT settings. These various aspects of PDT antagonism by tumor iNOS/NO and how they might be overcome will be discussed in this review. Lasers Surg. Med. 50:590-598, 2018.© 2018 Wiley Periodicals, Inc.
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Affiliation(s)
- Albert W Girotti
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin
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Bazak J, Fahey JM, Wawak K, Korytowski W, Girotti AW. Bystander effects of nitric oxide in anti-tumor photodynamic therapy. ACTA ACUST UNITED AC 2017; 4. [PMID: 29201944 DOI: 10.14800/ccm.1511] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Ionizing radiation of specifically targeted cells in a given population is known to elicit pro-death or pro-survival responses in non-targeted bystander cells, which often make no physical contact with the targeted ones. We have recently demonstrated a similar phenomenon for non-ionizing photodynamic therapy (PDT), showing that prostate cancer cells subjected to targeted photodynamic stress stimulated growth and migration of non-stressed, non-contacting bystander cells. Diffusible nitric oxide (NO) generated by stress-upregulated inducible nitric oxide synthase (iNOS) was shown to play a dominant role in these responses. Moreover, target-derived NO stimulated iNOS/NO induction in bystanders, suggesting a NO-mediated feed-forward field effect driven by targeted cells surviving the photodynamic challenge. In this research highlight, we will review these findings and discuss their potential negative implications on clinical PDT outcomes and how these might be mitigated through pharmacologic use of select iNOS inhibitors.
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Affiliation(s)
- Jerzy Bazak
- Department of Biophysics, Jagiellonian University, Krakow, 30-387, Poland
| | - Jonathan M Fahey
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, 53226, USA
| | - Katarzyna Wawak
- Department of Biophysics, Jagiellonian University, Krakow, 30-387, Poland
| | - Witold Korytowski
- Department of Biophysics, Jagiellonian University, Krakow, 30-387, Poland.,Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, 53226, USA
| | - Albert W Girotti
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, 53226, USA
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Fahey JM, Emmer JV, Korytowski W, Hogg N, Girotti AW. Antagonistic Effects of Endogenous Nitric Oxide in a Glioblastoma Photodynamic Therapy Model. Photochem Photobiol 2016; 92:842-853. [PMID: 27608331 PMCID: PMC5161550 DOI: 10.1111/php.12636] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2016] [Accepted: 08/05/2016] [Indexed: 12/21/2022]
Abstract
Gliomas are aggressive brain tumors that are resistant to conventional chemotherapy and radiotherapy. Much of this resistance is attributed to endogenous nitric oxide (NO). Recent studies revealed that 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) has advantages over conventional treatments for glioblastoma. In this study, we used an in vitro model to assess whether NO from glioblastoma cells can interfere with ALA-PDT. Human U87 and U251 cells expressed significant basal levels of neuronal NO synthase (nNOS) and its inducible counterpart (iNOS). After an ALA/light challenge, iNOS level increased three- to fourfold over 24 h, whereas nNOS remained unchanged. Elevated iNOS resulted in a large increase in intracellular NO. Extent of ALA/light-induced apoptosis increased substantially when an iNOS inhibitor or NO scavenger was present, implying that iNOS/NO was acting cytoprotectively. Moreover, cells surviving a photochallenge exhibited a striking increase in proliferation, migration and invasion rates, iNOS/NO again playing a dominant role. Also observed was a large iNOS/NO-dependent increase in matrix metalloproteinase-9 activity, decrease in tissue inhibitor of metalloproteinase-1 expression and increase in survivin and S100A4 expression, each effect being consistent with accelerated migration/invasion as a prelude to metastasis. Our findings suggest introduction of iNOS inhibitors as pharmacologic adjuvants for glioblastoma PDT.
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Affiliation(s)
- Jonathan M. Fahey
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA
| | - Joseph V. Emmer
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA
| | - Witold Korytowski
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA
- Department of Biophysics, Jagiellonian University, Krakow, Poland
| | - Neil Hogg
- Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI 53226, USA
| | - Albert W. Girotti
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA
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15
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Girotti AW. Modulation of the Anti-Tumor Efficacy of Photodynamic Therapy by Nitric Oxide. Cancers (Basel) 2016; 8:E96. [PMID: 27775600 PMCID: PMC5082386 DOI: 10.3390/cancers8100096] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2016] [Revised: 10/12/2016] [Accepted: 10/14/2016] [Indexed: 12/16/2022] Open
Abstract
Nitric oxide (NO) produced by nitric oxide synthase (NOS) enzymes is a free radical molecule involved in a wide variety of normophysiologic and pathophysiologic processes. Included in the latter category are cancer promotion, progression, and resistance to therapeutic intervention. Animal tumor photodynamic therapy (PDT) studies several years ago revealed that endogenous NO can reduce PDT efficacy and that NOS inhibitors can alleviate this. Until relatively recently, little else was known about this anti-PDT effect of NO, including: (a) the underlying mechanisms; (b) type(s) of NOS involved; and (c) whether active NO was generated in vascular cells, tumor cells, or both. In addressing these questions for various cancer cell lines exposed to PDT-like conditions, the author's group has made several novel findings, including: (i) exogenous NO can scavenge lipid-derived free radicals arising from photostress, thereby protecting cells from membrane-damaging chain peroxidation; (ii) cancer cells can upregulate inducible NOS (iNOS) after a PDT-like challenge and the resulting NO can signal for resistance to photokilling; (iii) photostress-surviving cells with elevated iNOS/NO proliferate and migrate/invade more aggressively; and (iv) NO produced by photostress-targeted cells can induce greater aggressiveness in non-targeted bystander cells. In this article, the author briefly discusses these various means by which NO can interfere with PDT and how this may be mitigated by use of NOS inhibitors as PDT adjuvants.
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Affiliation(s)
- Albert W Girotti
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
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16
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Ji YY, Ma YJ, Wang JW. Cytoprotective role of nitric oxide in HepG2 cell apoptosis induced by hypocrellin B photodynamic treatment. JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY 2016; 163:366-73. [DOI: 10.1016/j.jphotobiol.2016.09.006] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/30/2016] [Revised: 09/02/2016] [Accepted: 09/03/2016] [Indexed: 01/05/2023]
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Girotti AW. Role of Endogenous Nitric Oxide in Hyperaggressiveness of Tumor Cells that Survive a Photodynamic Therapy Challenge. Crit Rev Oncog 2016; 21:353-363. [PMID: 29431083 DOI: 10.1615/critrevoncog.2017020909] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Many malignant tumors exploit nitric oxide (NO) for a survival, growth, and migration/invasion advantage, and also to withstand the cytotoxic effects of chemo- and radiotherapies. Endogenous NO has also been shown to antagonize photodynamic therapy (PDT), a unique minimally invasive modality involving a photosensitizing (PS) agent, PS-exciting light in the visible- to near-infrared range, and molecular oxygen. The anti-PDT effects of NO were discovered about 20 years ago, but the underlying mechanisms are still not fully understood. More recent studies in the author's laboratory using breast, prostate, and brain cancer cell lines have shown that inducible NO synthase (iNOS/NOS2) is dramatically upregulated after a PDT challenge using 5-aminolevulinic acid (ALA-) -induced protoporphyrin IX as the PS. The parallel increase in NO resulted not only in a greater resistance to cell killing but also in a striking increase in the growth and migration/invasion rate of surviving cells. These in vitro findings and their recent recapitulation at the in vivo level are discussed in this article, along with how iNOS/NO's negative effects on PDT can be attenuated by the use of select iNOS inhibitors as PDT adjuvants.
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Affiliation(s)
- Albert W Girotti
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226-3548, USA
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18
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Juarez AV, Sosa LDV, De Paul AL, Costa AP, Farina M, Leal RB, Torres AI, Pons P. Riboflavin acetate induces apoptosis in squamous carcinoma cells after photodynamic therapy. JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY 2015; 153:445-54. [DOI: 10.1016/j.jphotobiol.2015.10.030] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/04/2015] [Revised: 10/26/2015] [Accepted: 10/31/2015] [Indexed: 12/24/2022]
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Chen JJ, Hong G, Gao LJ, Liu TJ, Cao WJ. In vitro and in vivo antitumor activity of a novel porphyrin-based photosensitizer for photodynamic therapy. J Cancer Res Clin Oncol 2015; 141:1553-61. [PMID: 25609073 DOI: 10.1007/s00432-015-1918-1] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2014] [Accepted: 01/12/2015] [Indexed: 01/09/2023]
Abstract
PURPOSE Photodynamic therapy (PDT) is a promising treatment in cancer therapy, based on the use of a photosensitizer activated by visible light in the presence of oxygen. Nowadays significant research efforts have been focused on finding a new photosensitizer. In the present paper, the antitumor effects of a novel porphyrin-based photosensitizer, {Carboxymethyl-[2-(carboxymethyl-{[4-(10,15,20-triphenylporphyrin-5-yl)-phenylcarbamoyl]-methyl}-amino)-ethyl]-amino}-acetic acid (ATPP-EDTA) on two types of human malignant tumor cells in vitro and a gastric cancer model in nude mice, were evaluated. METHODS The PDT efficacy with ATPP-EDTA in vitro was assessed by MTT assay. The intracellular accumulation was detected with fluorescence spectrometer, and the intracellular distribution was determined by laser scanning confocal microscopy. The mode of cell death was investigated by Hoechst 33342 staining and flow cytometer. BGC823-derived xenograft tumor model was established to explore the in vivo antitumor effects of ATPP-EDTA. RESULTS ATPP-EDTA exhibited intense phototoxicity on both cell lines in vitro in concentration- and light dose-dependent manners meanwhile imposing minimal dark cytotoxicity. The accumulation of ATPP-EDTA in two malignant cell lines was time-dependent and prior compared to normal cells. It was mainly localized at lysosomes, but induced cell death by apoptotic pathway. ATPP-EDTA significantly inhibited the growth of BGC823 tumors in nude mice (160 mW/cm(2), 100 J/cm(2)). CONCLUSIONS Present studies suggest that ATPP-EDTA is an effective photosensitizer for PDT to tumors. It distributed in lysosomes and caused cell apoptosis. ATPP-EDTA, as a novel photosensitizer, has a great potential for human gastric cancer treatment in PDT and deserves further investigations.
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Affiliation(s)
- Jing-Jing Chen
- Central Laboratory of Heping Hospital, Changzhi Medical College, Changzhi City, 046000, Shanxi Province, People's Republic of China
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Vannini F, Kashfi K, Nath N. The dual role of iNOS in cancer. Redox Biol 2015; 6:334-343. [PMID: 26335399 PMCID: PMC4565017 DOI: 10.1016/j.redox.2015.08.009] [Citation(s) in RCA: 385] [Impact Index Per Article: 38.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2015] [Revised: 08/07/2015] [Accepted: 08/10/2015] [Indexed: 01/02/2023] Open
Abstract
Nitric oxide (NO) is one of the 10 smallest molecules found in nature. It is a simple gaseous free radical whose predominant functions is that of a messenger through cGMP. In mammals, NO is synthesized by the enzyme nitric oxide synthase (NOS) of which there are three isoforms. Neuronal (nNOS, NOS1) and endothelial (eNOS, NOS3) are constitutive calcium-dependent forms of the enzyme that regulate neural and vascular function respectively. The third isoform (iNOS, NOS2), is calcium-independent and is inducible. In many tumors, iNOS expression is high, however, the role of iNOS during tumor development is very complex and quite perplexing, with both promoting and inhibiting actions having been described. This review will aim to summarize the dual actions of iNOS-derived NO showing that the microenvironment of the tumor is a contributing factor to these observations and ultimately to cellular outcomes.
NO is pro- and anti-tumorigenic. High concentrations of NO maybe anti-tumorigenic. iNOS produces high concentrations of NO and relates to tumor growth or its inhibition. iNOS is associated with cytotoxicity, apoptosis and bystander anti-tumor effects. Tumor- and stromal-iNOS, and the ‘cell situation’ contribute to anti or pro-tumor effects. Dual role of iNOS is influenced by the cell situation and is environment dependent.
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Affiliation(s)
- Federica Vannini
- Department of Physiology, Pharmacology and Neuroscience, Sophie Davis School of Biomedical Education, City University of New York Medical School, New York, NY 10031, United States
| | - Khosrow Kashfi
- Department of Physiology, Pharmacology and Neuroscience, Sophie Davis School of Biomedical Education, City University of New York Medical School, New York, NY 10031, United States
| | - Niharika Nath
- Department of Life Sciences, New York Institute of Technology, NY 10023, United States.
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Fahey JM, Girotti AW. Accelerated migration and invasion of prostate cancer cells after a photodynamic therapy-like challenge: Role of nitric oxide. Nitric Oxide 2015; 49:47-55. [PMID: 26068242 DOI: 10.1016/j.niox.2015.05.006] [Citation(s) in RCA: 64] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2015] [Revised: 05/14/2015] [Accepted: 05/26/2015] [Indexed: 11/25/2022]
Abstract
Employing an in vitro model for 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT), we recently reported that human prostate cancer PC3 cells rapidly and persistently overexpressed inducible nitric oxide synthase (iNOS) and nitric oxide (NO) after a moderate ALA/light challenge. The upregulated iNOS/NO was shown to play a key role in cell resistance to apoptotic photokilling and also in the dramatic growth spurt observed in surviving cells. In the present study, we found that PC3 cells surviving an ALA/light insult not only proliferated faster than non-stressed controls, but migrated and invaded faster as well, these effects being abrogated by an iNOS inhibitor or NO scavenger. Photostressed prostate DU145 cells exhibited similar behavior. Using in-gel zymography, we showed that PC3 extracellular matrix metalloproteinase-9 (MMP-9) was strongly activated 24 h after ALA/light treatment and that MMP-9 inhibitor TIMP-1 was downregulated, consistent with MMP-9 involvement in enhanced invasiveness. We also observed a photostress-induced upregulation of α6 and β1 integrins, implying their involvement as well. The MMP-9, TIMP-1, and integrin effects were strongly attenuated by iNOS inhibition, confirming NO's role in photostress-enhanced migration/invasion. This study reveals novel, potentially tumor-promoting, side-effects of prostate cancer PDT which may be averted through use of iNOS inhibitors as PDT adjuvants.
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Affiliation(s)
- Jonathan M Fahey
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226-3548, USA
| | - Albert W Girotti
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226-3548, USA.
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22
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Girotti AW. Tumor-generated nitric oxide as an antagonist of photodynamic therapy. Photochem Photobiol Sci 2015; 14:1425-32. [PMID: 25706541 DOI: 10.1039/c4pp00470a] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023]
Abstract
Nitric oxide (NO) is a multifunctional free radical molecule produced naturally by nitric oxide synthase (NOS) enzymes. Many tumors exploit NO for survival and growth signaling, and also to thwart the effects of therapeutic treatments, including PDT. The anti-PDT effects of NO were discovered using animal tumor models, but the mechanisms involved are still not fully understood. Recent in vitro studies on breast and prostate cancer cells have shown that inducible NOS (iNOS) along with NO is dramatically upregulated after an ALA-PDT-like challenge. Cells were more resistant to apoptosis after a photochallenge and survivors grew, migrated, and invaded more rapidly, iNOS/NO playing a key role in all these effects. This perspective briefly reviews what is currently known about NO's negative effects on PDT and some of the signaling mechanisms involved. It also provides insights into how these effects may be attenuated by pharmacologic use of iNOS inhibitors.
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Affiliation(s)
- Albert W Girotti
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
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23
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Basic and Clinical Aspects of Photodynamic Therapy. RESISTANCE TO TARGETED ANTI-CANCER THERAPEUTICS 2015. [DOI: 10.1007/978-3-319-12730-9_1] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
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Lamberti MJ, Vittar NBR, Rivarola VA. Breast cancer as photodynamic therapy target: Enhanced therapeutic efficiency by overview of tumor complexity. World J Clin Oncol 2014; 5:901-907. [PMID: 25493228 PMCID: PMC4259952 DOI: 10.5306/wjco.v5.i5.901] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/24/2013] [Revised: 04/04/2014] [Accepted: 07/15/2014] [Indexed: 02/06/2023] Open
Abstract
Photodynamic therapy is a minimally invasive and clinically approved procedure for eliminating selected malignant cells with specific light activation of a photosensitizer agent. Whereas interstitial and intra-operative approaches have been investigated for the ablation of a broad range of superficial or bulky solid tumors such as breast cancer, the majority of approved photodynamic therapy protocols are for the treatment of superficial lesions of skin and luminal organs. This review article will discuss recent progress in research focused mainly on assessing the efficacies of various photosensitizers used in photodynamic therapy, as well as the combinatory strategies of various therapeutic modalities for improving treatments of parenchymal and/or stromal tissues of breast cancer solid tumors. Cytotoxic agents are used in cancer treatments for their effect on rapidly proliferating cancer cells. However, such therapeutics often lack specificity, which can lead to toxicity and undesirable side effects. Many approaches are designed to target tumors. Selective therapies can be established by focusing on distinctive intracellular (receptors, apoptotic pathways, multidrug resistance system, nitric oxide-mediated stress) and environmental (glucose, pH) differences between tumor and healthy tissue. A rational design of effective combination regimens for breast cancer treatment involves a better understanding of the mechanisms and molecular interactions of cytotoxic agents that underlie drug resistance and sensitivity.
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25
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Pro-survival and pro-growth effects of stress-induced nitric oxide in a prostate cancer photodynamic therapy model. Cancer Lett 2013; 343:115-22. [PMID: 24080338 DOI: 10.1016/j.canlet.2013.09.025] [Citation(s) in RCA: 51] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2013] [Revised: 09/13/2013] [Accepted: 09/20/2013] [Indexed: 01/27/2023]
Abstract
We discovered recently that human breast cancer cells subjected to photodynamic therapy (PDT)-like oxidative stress localized in mitochondria rapidly upregulated nitric oxide synthase-2 (NOS2) and nitric oxide (NO), which increased resistance to apoptotic photokilling. In this study, we asked whether human prostate cancer PC-3 cells would exploit NOS2/NO similarly and, if so, how proliferation of surviving cells might be affected. Irradiation of photosensitized PC-3 cells resulted in a rapid (<1 h), robust (~12-fold), and prolonged (∼20 h) post-irradiation upregulation of NOS2. Caspase-3/7 activation and apoptosis were stimulated by NOS2 inhibitors and a NO scavenger, implying that induced NO was acting cytoprotectively. Cyclic GMP involvement was ruled out, whereas suppression of pro-apoptotic JNK and p38 MAPK activation was clearly implicated. Cells surviving photostress grew back ~2-times faster than controls. NOS2 inhibition prevented this and the large increase in cell cycle S-phase occupancy observed after irradiation. Thus, photostress upregulation of NOS/NO elicited both a pro-survival and pro-growth response, both of which could compromise clinical PDT efficacy unless suppressed, e.g. by pharmacological intervention with a NOS2 inhibitor.
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26
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Bhowmick R, Girotti AW. Cytoprotective signaling associated with nitric oxide upregulation in tumor cells subjected to photodynamic therapy-like oxidative stress. Free Radic Biol Med 2013; 57:39-48. [PMID: 23261943 PMCID: PMC3594367 DOI: 10.1016/j.freeradbiomed.2012.12.005] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/09/2012] [Revised: 12/04/2012] [Accepted: 12/07/2012] [Indexed: 12/12/2022]
Abstract
Photodynamic therapy (PDT) employs photoexcitation of a sensitizer to generate tumor-eradicating reactive oxygen species. We recently showed that irradiating breast cancer COH-BR1 cells after treating with 5-aminolevulinic acid (ALA, a pro-sensitizer) resulted in rapid upregulation of inducible nitric oxide (NO) synthase (iNOS). Apoptotic cell killing was strongly enhanced by an iNOS inhibitor (1400W), iNOS knockdown (kd), or a NO scavenger, suggesting that NO was acting cytoprotectively. Stress signaling associated with these effects was examined in this study. ALA/light-stressed COH-BR1 cells, and also breast adenocarcinoma MDA-MB-231 cells, mounted an iNOS/NO-dependent resistance to apoptosis that proved to be cGMP-independent. Immunocytochemistry and subcellular Western analysis of photostressed COH-BR1 cells revealed a cytosol-to-nucleus translocation of NF-κB which was negated by the NF-κB activation inhibitor Bay11. Bay11 also enhanced apoptosis and prevented iNOS induction, consistent with NF-κB involvement in the latter. JNK and p38 MAP kinase inhibitors suppressed apoptosis, implicating these kinases in death signaling. Post-irradiation extent and duration of JNK and p38 phosphorylation were dramatically elevated by 1400 W or iNOS-kd, suggesting that these activations were suppressed by NO. Regarding pro-survival stress signaling, rapid activation of Akt was unaffected by 1400 W, but prevented by Wortmannin, which also enhanced apoptosis. Thus, a link between upstream Akt activation and iNOS induction was apparent. Furthermore, p53 protein expression under photostress was elevated by iNOS-kd, whereas robust Survivin induction was abolished, consistent with p53 and Survivin being negatively and positively regulated by NO, respectively. Collectively, these findings enhance our understanding of cytoprotective signaling associated with photostress-induced NO and suggest iNOS inhibitor-based approaches for improving PDT efficacy.
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Affiliation(s)
- Reshma Bhowmick
- To whom correspondence may be addressed: Reshma Bhowmick, Ph.D. Department of Biochemistry Medical College of Wisconsin Milwaukee, WI, 53226 Tel: 414-955-8445
| | - Albert W. Girotti
- To whom correspondence may be addressed: Albert W. Girotti, Ph.D. Department of Biochemistry Medical College of Wisconsin Milwaukee, WI, 53226 Tel: 414-955-8432
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Rapozzi V, Della Pietra E, Zorzet S, Zacchigna M, Bonavida B, Xodo LE. Nitric oxide-mediated activity in anti-cancer photodynamic therapy. Nitric Oxide 2013; 30:26-35. [DOI: 10.1016/j.niox.2013.01.002] [Citation(s) in RCA: 67] [Impact Index Per Article: 5.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2012] [Revised: 01/08/2013] [Accepted: 01/17/2013] [Indexed: 02/05/2023]
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Kovaleva V, Berezhnaya E, Komandirov M, Rudkovskii M, Uzdensky A. Involvement of nitric oxide in photodynamic injury of neurons and glial cells. Nitric Oxide 2013; 29:46-52. [DOI: 10.1016/j.niox.2012.12.006] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2012] [Revised: 12/06/2012] [Accepted: 12/13/2012] [Indexed: 11/27/2022]
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29
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Liu L, Zhang Z, Xing D. Cell death via mitochondrial apoptotic pathway due to activation of Bax by lysosomal photodamage. Free Radic Biol Med 2011; 51:53-68. [PMID: 21530645 DOI: 10.1016/j.freeradbiomed.2011.03.042] [Citation(s) in RCA: 82] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/20/2010] [Revised: 03/29/2011] [Accepted: 03/31/2011] [Indexed: 01/02/2023]
Abstract
Lysosomal photosensitizers have been used in photodynamic therapy. The combination of such photosensitizers and light causes lysosomal photodamage, inducing cell death. Lysosomal disruption can lead to apoptosis but its signaling pathways remain to be elucidated. In this study, N-aspartyl chlorin e6 (NPe6), an effective photosensitizer that preferentially accumulates in lysosomes, was used to study the mechanism of apoptosis caused by lysosomal photodamage. Apoptosis in living human lung adenocarcinoma cells (ASTC-a-1) after NPe6-photodynamic treatment (NPe6-PDT) was studied using real-time single-cell analysis. Our results demonstrated that NPe6-PDT induced rapid generation of reactive oxygen species (ROS). The photodynamically produced ROS caused a rapid destruction of lysosomes, leading to release of cathepsins, and the ROS scavengers vitamin C and NAC prevent the effects. Then the following spatiotemporal sequence of cellular events was observed during cell apoptosis: Bcl-2-associated X protein (Bax) activation, cytochrome c release, and caspase-9/-3 activation. Importantly, the activation of Bax proved to be a crucial event in this apoptotic machinery, because suppressing the endogenous Bax using siRNA could significantly inhibit cytochrome c release and caspase-9/-3 activation and protect the cell from death. In conclusion, this study demonstrates that PDT with lysosomal photosensitizer induces Bax activation and subsequently initiates the mitochondrial apoptotic pathway.
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Affiliation(s)
- Lei Liu
- MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China
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Bhowmick R, Girotti AW. Rapid upregulation of cytoprotective nitric oxide in breast tumor cells subjected to a photodynamic therapy-like oxidative challenge. Photochem Photobiol 2011; 87:378-86. [PMID: 21143607 PMCID: PMC3048913 DOI: 10.1111/j.1751-1097.2010.00877.x] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
Many tumor cells produce nitric oxide (NO) as an antiapoptotic/progrowth molecule which also promotes antiogenesis and tumor expansion. This study was designed to examine possible antagonistic effects of endogenous NO on tumor eradication by photodynamic therapy (PDT). Using COH-BR1 breast cancer cells sensitized in mitochondria with 5-aminolevulinic acid (ALA)-generated protoporphyrin IX as a model for ALA-based PDT, we found that caspase-9 activation and apoptotic death following irradiation were strongly enhanced by 1400W, an inhibitor of inducible nitric oxide synthase (iNOS). RT-PCR and Western analyses revealed a substantial upregulation of both iNOS mRNA and protein, beginning ca 4 h after irradiation and persisting for at least 20 h. Accompanying this was a strong 1400W-inhibitable increase in intracellular NO, as detected with the NO probe, DAF-2-DA. Short hairpin RNA-based iNOS knockdown in COH-BR1 cells dramatically reduced NO production under photostress while enhancing caspase-9 activation and apoptosis. These findings suggest that cytoprotective iNOS/NO induction in PDT-treated tumor cells could reduce treatment efficacy, and point to pharmacologic intervention with iNOS inhibitors for counteracting this.
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Affiliation(s)
- Reshma Bhowmick
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA
| | - Albert W. Girotti
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA
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31
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Bhowmick R, Girotti AW. Cytoprotective induction of nitric oxide synthase in a cellular model of 5-aminolevulinic acid-based photodynamic therapy. Free Radic Biol Med 2010; 48:1296-301. [PMID: 20138143 PMCID: PMC2856718 DOI: 10.1016/j.freeradbiomed.2010.01.040] [Citation(s) in RCA: 62] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/09/2009] [Revised: 01/18/2010] [Accepted: 01/28/2010] [Indexed: 12/30/2022]
Abstract
Photodynamic therapy (PDT) employs a photosensitizing agent, molecular oxygen, and visible light to generate reactive species that kill tumor and tumor vasculature cells. Nitric oxide produced by these cells could be procarcinogenic by inhibiting apoptosis or promoting angiogenesis and tumor growth. The purpose of this study was to determine whether tumor cells upregulate NO as a cytoprotective measure during PDT. Breast tumor COH-BR1 cells sensitized in their mitochondria with 5-aminolevulinic acid (ALA)-derived protoporphyrin IX died apoptotically after irradiation, ALA- and light-only controls showing no effect. Western analysis revealed that inducible nitric oxide synthase (iNOS) was upregulated >3-fold within 4 h after ALA/light treatment, whereas other NOS isoforms were unaffected. Exposing cells to a NOS inhibitor (L-NAME or 1400W) during photochallenge enhanced caspase-3/7 activation and apoptotic killing up to 2- to 3-fold while substantially reducing chemiluminescence-assessed NO production, suggesting that this NO was cytoprotective. Consistently, the NO scavenger cPTIO enhanced ALA/light-induced caspase-3/7 activation and apoptotic kill by >2.5-fold. Of added significance, cells could be rescued from 1400W-exacerbated apoptosis by an exogenous NO donor, spermine-NONOate. This is the first reported evidence for increased tumor cell resistance due to iNOS upregulation in a PDT model. Our findings indicate that stress-elicited NO in PDT-treated tumors could compromise therapeutic efficacy and suggest NOS-based pharmacologic interventions for preventing this.
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Affiliation(s)
- Reshma Bhowmick
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, USA
| | - Albert W. Girotti
- Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, USA
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