|
1
|
Materón EM, Shimizu FM, Figueiredo Dos Santos K, Nascimento GF, Geraldo VPN, Oliveira ON, Faria RC. Membrane model as key tool in the study of glutathione-s-transferase mediated anticancer drug resistance. Biomed Pharmacother 2021; 145:112426. [PMID: 34861633 DOI: 10.1016/j.biopha.2021.112426] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2021] [Revised: 11/05/2021] [Accepted: 11/12/2021] [Indexed: 11/02/2022] Open
Abstract
Glutathione-s-transferase is believed to be involved in the resistance to chemotherapeutic drugs, which depends on the interaction with the cell membranes. In this study, we employed Langmuir monolayers of a mixture of phospholipids and cholesterol (MIX) as models for tumor cell membranes and investigated their interaction with the anticancer drugs cisplatin (CDDP) and doxorubicin (DOX). We found that both DOX and CDDP expand and affect the elasticity of MIX monolayers, but these effects are hindered when glutathione-s-transferase (GST) and its cofactor glutathione (GSH) are incorporated. Changes are induced by DOX or CDDP on the polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) data for MIX/GST/GSH monolayers, thus denoting some degree of interaction that is not sufficient to alter the monolayer mechanical properties. Overall, the results presented here give support to the hypothesis of the inactivation of DOX and CDDP by GST and point to possible directions to detect and fight drug resistance.
Collapse
Affiliation(s)
- Elsa M Materón
- Chemistry Department, Federal University of São Carlos, CP 676, São Carlos 13565-905, São Paulo, Brazil; São Carlos Institute of Physics, University of São Paulo, P.O Box 369, 13560-970 São Carlos, SP, Brazil.
| | - Flavio M Shimizu
- São Carlos Institute of Physics, University of São Paulo, P.O Box 369, 13560-970 São Carlos, SP, Brazil; Department of Applied Physics, "Gleb Wataghin" Institute of Physics (IFGW), University of Campinas (UNICAMP), Campinas, SP 13083-859, Brazil.
| | | | - Gustavo F Nascimento
- São Carlos Institute of Physics, University of São Paulo, P.O Box 369, 13560-970 São Carlos, SP, Brazil
| | - Vananélia P N Geraldo
- São Carlos Institute of Physics, University of São Paulo, P.O Box 369, 13560-970 São Carlos, SP, Brazil
| | - Osvaldo N Oliveira
- São Carlos Institute of Physics, University of São Paulo, P.O Box 369, 13560-970 São Carlos, SP, Brazil.
| | - Ronaldo C Faria
- Chemistry Department, Federal University of São Carlos, CP 676, São Carlos 13565-905, São Paulo, Brazil.
| |
Collapse
|
|
2
|
Tatsunami R, Murao Y, Sato K. [Protective Effect of Epalrestat against Oxidative Stress-induced Cytotoxicity]. YAKUGAKU ZASSHI 2020; 140:1381-1388. [PMID: 33132274 DOI: 10.1248/yakushi.20-00167] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Epalrestat (EPS), approved in Japan, is currently the only aldose reductase inhibitor that is available for the treatment of diabetic neuropathy. Recently, we found that EPS at near-plasma concentration increases the intracellular levels of glutathione (GSH) in rat Schwann cells. GSH, the most abundant non-protein thiol antioxidant in cells, is important for protection against oxidative stress. Oxidative stress is associated with the development and progression of many pathological conditions, such as atherosclerosis, diabetes, and neurodegeneration. In this study, we tested the hypothesis that EPS enhances resistance to oxidative stress, by using rat Schwann cells. To determine whether EPS protects Schwann cells from oxidative stress, we performed experiments by using radical generators, drugs, and heavy metals as the source of oxidative stress. EPS reduced the cytotoxicity induced by 2,2-azobis-[2-(2-imidazolin-2-yl) propane] dihydrochloride, 6-hydroxydopamine, cisplatin, palmitate, cadmium chloride, and manganese (II) sulfate, indicating that EPS plays a role in protecting cells from oxidative stress. We suggest that EPS has the potential to prevent the development and progression of disorders caused by oxidative stress.
Collapse
Affiliation(s)
- Ryosuke Tatsunami
- Department of Public Health, Faculty of Pharmaceutical Sciences, Hokkaido University of Science
| | - Yu Murao
- Department of Public Health, Faculty of Pharmaceutical Sciences, Hokkaido University of Science
| | - Keisuke Sato
- Department of Public Health, Faculty of Pharmaceutical Sciences, Hokkaido University of Science
| |
Collapse
|
|
3
|
Amjadi I, Mohajeri M, Borisov A, Hosseini MS. Antiproliferative Effects of Free and Encapsulated Hypericum Perforatum L. Extract and Its Potential Interaction with Doxorubicin for Esophageal Squamous Cell Carcinoma. J Pharmacopuncture 2019; 22:102-108. [PMID: 31338250 PMCID: PMC6645344 DOI: 10.3831/kpi.2019.22.013] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2018] [Revised: 02/13/2019] [Accepted: 05/20/2019] [Indexed: 01/01/2023] Open
Abstract
Objectives Esophageal squamous cell carcinoma (ESCC) is considered as a deadly medical condition that affects a growing number of people worldwide. Targeted therapy of ESCC has been suggested recently and required extensive research. With cyclin D1 as a therapeutic target, the present study aimed at evaluating the anticancer effects of doxorubicin (Dox) or Hypericum perforatum L. (HP) extract encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles on the ESCC cell line KYSE30. Methods Nanoparticles were prepared using double emulsion method. Cytotoxicity assay was carried out to measure the anti-proliferation activity of Dox-loaded (Dox NPs) and HP-loaded nanoparticles (HP NPs) against both cancer and normal cell lines. The mRNA gene expression of cyclin D1 was evaluated to validate the cytotoxicity studies at molecular level. Results Free drugs and nanoparticles significantly inhibited KYSE30 cells by 55–73% and slightly affected normal cells up to 29%. The IC50 of Dox NPs and HP NPs was ~ 0.04–0.06 mg/mL and ~ 0.6–0.7 mg/mL, respectively. Significant decrease occurred in cyclin D1 expression by Dox NPs and HP NPs (P < 0.05). Exposure of KYSE-30 cells to combined treatments including both Dox and HP extract significantly increased the level of cyclin D1 expression as compared to those with individual treatments (P < 0.05). Conclusion Dox NPs and HP NPs can successfully and specifically target ESCC cells through downregulation of cyclin D1. The simultaneous use of Dox and HP extract should be avoided for the treatment of ESCC.
Collapse
Affiliation(s)
- Issa Amjadi
- Department of Biomedical Engineering, Wayne State University, Detroit, United States
| | - Mohammad Mohajeri
- Department of Medical Biotechnology, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Andrei Borisov
- Department of Biomedical Engineering, Wayne State University, Detroit, United States
| | - Motahare-Sadat Hosseini
- Biomaterials Group, Department of Biomedical Engineering, Amirkabir University of Technology, Tehran, Iran
| |
Collapse
|
|
4
|
Generation and characterization of a paclitaxel-resistant human gastric carcinoma cell line. Anticancer Drugs 2018; 29:491-502. [DOI: 10.1097/cad.0000000000000601] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
|
|
5
|
Merk D, Schubert-Zsilavecz M. The Linker Approach. METHODS AND PRINCIPLES IN MEDICINAL CHEMISTRY 2017. [DOI: 10.1002/9783527674381.ch8] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/23/2023]
Affiliation(s)
- Daniel Merk
- Goethe University Frankfurt; Institute of Pharmaceutical Chemistry; Max-von-Laue-Str. 9 60438 Frankfurt Germany
| | - Manfred Schubert-Zsilavecz
- Goethe University Frankfurt; Institute of Pharmaceutical Chemistry; Max-von-Laue-Str. 9 60438 Frankfurt Germany
| |
Collapse
|
|
6
|
Laborde E. Glutathione transferases as mediators of signaling pathways involved in cell proliferation and cell death. Cell Death Differ 2010; 17:1373-80. [DOI: 10.1038/cdd.2010.80] [Citation(s) in RCA: 265] [Impact Index Per Article: 17.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/01/2023] Open
|
|
7
|
Asakura T, Sasagawa A, Takeuchi H, Shibata SI, Marushima H, Mamori S, Ohkawa K. Conformational change in the active center region of GST P1-1, due to binding of a synthetic conjugate of DXR with GSH, enhanced JNK-mediated apoptosis. Apoptosis 2007; 12:1269-80. [PMID: 17431793 DOI: 10.1007/s10495-007-0053-0] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/23/2022]
Abstract
Treatment of cells with a synthetic conjugate of DXR with GSH via glutaraldehyde (GSH-DXR) caused cytochrome c to be released from the mitochondria to the cytosol following potent activation of caspase-3 and -9 by typical DNA fragmentation. This apoptosis was regulated by the JNK-signaling pathway. In the present experiment, binding of GSH-DXR to GST P1-1 allosterically led to the disappearance of its enzyme activity and activated the kinase activity of JNK without dissociation of the JNK-GST P1-1 complex. The recombinant GST P1-1 molecule with a mutation in the active center region (W38H and C47S) lost its GST activity when bound to JNK to the same degree as the wild-type, with the mutated GST P1-1 molecule failing to inhibit the activity of JNK. It has been reported that JNK-signaling is regulated by GST P1-1 via interaction with the C-terminus. We confirmed that GST P1-1 deletion mutant (Delta194-209) and a site-directed mutant (R201A) in the C-terminal region failed to bind and inhibit JNK. These results indicated that not only binding of the C-terminal region of GST P1-1 to the JNK molecule, but also the active center region of GST P1-1 play important roles in the regulation of JNK enzyme activity. The findings suggested that allosteric inhibition of GST P1-1 activity by the binding of GSH-DXR following conformational change may activate JNK and induce apoptosis via the mitochondrial pathway in the cells.
Collapse
Affiliation(s)
- Tadashi Asakura
- Department of Biochemistry (I), Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo, 105-8461, Japan.
| | | | | | | | | | | | | |
Collapse
|
|
8
|
Mamori S, Asakura T, Ohkawa K, Tajiri H. Survivin expression in early hepatocellular carcinoma and post-treatment with anti-cancer drug under hypoxic culture condition. World J Gastroenterol 2007; 13:5306-11. [PMID: 17879398 PMCID: PMC4171318 DOI: 10.3748/wjg.v13.i40.5306] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate the expression of survivin during the early stages of hepatocellular carcinoma (HCC).
METHODS: Immunohistochemical expression of survivin in liver tumor and non-tumor tissue specimens taken from 17 patients was compared. In addition, to determine the survivin expression in response to anti-cancer drugs in early stage HCC, the survivin expression was determined after the treatment of the HCC cells with anti-cancer drugs under hypoxic culture conditions.
RESULTS: Survivin proteins were expressed in 64.7% of cells in early HCC specimens. A correlation between the survivin expression rate in the peritumoral hepatocytes and the rate of expression in the HCC specimens (low-rate group vs high-rate group) was observed. The survivin protein concentration in HCC cells was increased by the combination of hypoxia and anti-cancer drugs.
CONCLUSION: This study suggests that survivin could be used as a therapeutic target in early HCC.
Collapse
Affiliation(s)
- Satoshi Mamori
- Division of Gastroenterology and Hepatology, Department of Internal Medicine, The Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Japan.
| | | | | | | |
Collapse
|
|
9
|
Xu Z, Schenk A, Hertweck C. Molecular analysis of the benastatin biosynthetic pathway and genetic engineering of altered fatty acid-polyketide hybrids. J Am Chem Soc 2007; 129:6022-30. [PMID: 17439117 DOI: 10.1021/ja069045b] [Citation(s) in RCA: 59] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
The entire gene locus encoding the biosynthesis of the potent glutathione-S-transferase inhibitors and apoptosis inducers benastatin A and B has been cloned and sequenced. The cluster identity was unequivocally proven by deletion of flanking regions and heterologous expression in S. albus and S. lividans. Inactivation and complementation experiments revealed that a KSIII component (BenQ) similar to FabH is crucial for providing and selecting the rare hexanoate PKS starter unit. In the absence of BenQ, several novel penta- and hexacyclic benastatin derivatives with antiproliferative activities are formed. In total, five new compounds were isolated and fully characterized, and the chemical analysis was confirmed by derivatization. The most intriguing observation is that the ben PKS can utilize typical straight and branched fatty acid synthase primers. If shorter straight-chain starters are utilized, the length of the polyketide backbone is increased, resulting in the formation of an extended, hexacyclic ring system reminiscent of proposed intermediates in the griseorhodin and fredericamycin pathways. Analysis and manipulation of the hybrid fatty acid polyketide pathway provides strong support for the hypothesis that the number of chain elongations is dependent on the total size of the polyketide chain that is accommodated in the PKS enzyme cavity. Our results also further substantiate the potential of metabolic engineering toward polyphenols with altered substituents and ring systems.
Collapse
Affiliation(s)
- Zhongli Xu
- Leibniz Institute for Natural Product Research and Infection Biology, HKI, Department of Biomolecular Chemistry, Beutenbergstrasse 11a, 07745 Jena, Germany
| | | | | |
Collapse
|
|
10
|
Jao SC, Chen J, Yang K, Li WS. Design of potent inhibitors for Schistosoma japonica glutathione S-transferase. Bioorg Med Chem 2005; 14:304-18. [PMID: 16275109 DOI: 10.1016/j.bmc.2005.07.077] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2005] [Revised: 07/22/2005] [Accepted: 07/22/2005] [Indexed: 11/18/2022]
Abstract
We implemented both structure-based drug design and the concept of polyvalency to discover a series of potent and unsymmetrical Schistosoma japonicum glutathione S-transferase (SjGST) inhibitors 10-12. This strategy achieved not only an excellent enhancement (10- to 490-fold) in the inhibitory potency, compared to the monofunctional analogues 1-5, but was also an effective modification by selecting a hydrophobic moiety with a flexible linker. The designed compounds with a low micromolar hit demonstrate special values in refining the new generation of SjGST inhibitors. The stoichiometry of the binding is one inhibitor molecule per SjGST monomer via isothermal titration calorimetric measurement.
Collapse
Affiliation(s)
- Shu-Chuan Jao
- Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan
| | | | | | | |
Collapse
|
|
11
|
Wu Z, Minhas GS, Wen D, Jiang H, Chen K, Zimniak P, Zheng J. Design, Synthesis, and Structure−Activity Relationships of Haloenol Lactones: Site-Directed and Isozyme-Selective GlutathioneS-Transferase Inhibitors. J Med Chem 2004; 47:3282-94. [PMID: 15163208 DOI: 10.1021/jm0499615] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
Overexpression of glutathione S-transferase (GST), particularly the GST-pi isozyme, has been proposed to be one of the biochemical mechanisms responsible for drug resistance in cancer chemotherapy, and inhibition of overexpressed GST has been suggested as an approach to combat GST-induced drug resistance. 3-Cinnamyl-5(E)-bromomethylidenetetrahydro-2-furanone (1a), a lead compound of site-directed GST-pi inactivator, has been shown to potentiate the cytotoxic effect of cisplatin on tumor cells. As an initial step to develop more potent and more selective haloenol lactone inactivators of GST-pi, we examined the relationship between the chemical structures of haloenol lactone derivatives and their GST inhibitory activity. A total of 16 haloenol lactone derivatives were synthesized to probe the effects of (1) halogen electronegativity, (2) electron density of aromatic rings, (3) molecular size and rigidity, (4) lipophilicity, and (5) aromaticity on the potency of GST-pi inactivation. The inhibitory potency of each compound was determined by time-dependent inhibition tests, and recombinant human GST-pi was used to determine their inhibitory activity. Our structure-activity relationship studies demonstrated that (1) reactivity of the halide leaving group plays a weak role in GST inactivation by the haloenol lactones, (2) aromatic electron density may have some influence on the potency of GST inactivation, (3) high rigidity likely disfavors enzyme inhibition, (4) lipophilicity is inversely proportional to enzyme inactivation, and (5) an unsaturated system may be important for enzyme inhibition. This work facilitated understanding of the interaction of GST-pi with haloenol lactone derivatives as site-directed and isozyme-selective inactivators, possibly potentiating cancer chemotherapy.
Collapse
Affiliation(s)
- Zhixing Wu
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 200031, China
| | | | | | | | | | | | | |
Collapse
|
|
12
|
Asakura T, Hashizume Y, Tashiro K, Searashi Y, Ohkawa K, Nishihira J, Sakai M, Shibasaki T. Suppression of GST-P by treatment with glutathione-doxorubicin conjugate induces potent apoptosis in rat hepatoma cells. Int J Cancer 2001; 94:171-7. [PMID: 11668494 DOI: 10.1002/ijc.1465] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
A conjugate of doxorubicin and glutathione via glutaraldehyde (GSH-DXR) inhibited glutathione S-transferase (GST) activity of rat hepatoma AH66 cells, and treatment of the cells with GSH-DXR induced caspase-3 activation and DNA fragmentation. After treatment of AH66 cells with 0.1 microM GSH-DXR, GST-P (placental type of rat GST isozymes) mRNA and its protein increased transiently and then decreased thereafter compared with the levels in nontreated cells. Caspase-3 activation and DNA fragmentation were induced following the suppression of GST-P expression by treatment with GSH-DXR. When the cells were treated with 100 microM ethacrynic acid (ECA), an inhibitor of GST, DNA fragmentation and caspase-3 activation were observed. In contrast, treatment of AH66 cells with a low concentration of ECA (1 microM) that showed little inhibition of GST activity induced slight, but significantly enhanced expression and activity of GST-P, and consequent prevention of DXR- and GSH-DXR-induced DNA fragmentation. Overexpression of GST-pi (placental type of human GST isozymes) by transfection of GST-pi sense cDNA into AH66 cells decreased sensitivities to DXR and GSH-DXR, and the suppression of GST-P by transfection of the antisense cDNA into the cells increased drug sensitivity. On the other hand, there was little change in drug sensitivity caused by overexpression of site-directedly mutated GST-P in which the active-site residue Tyr39 was replaced with His (W39H) or the substrate-binding site residue Cys48 was replaced with Ser (C48S) by transfection of those cDNAs into AH66 cells. These results suggested that the suppression of GST-P in AH66 cells treated with GSH-DXR must play an important role in the induction of apoptosis.
Collapse
Affiliation(s)
- T Asakura
- Department of Biochemistry (I), Jikei University School of Medicine, Tokyo, Japan.
| | | | | | | | | | | | | | | |
Collapse
|
|
13
|
|
|
14
|
Tashiro K, Asakura T, Fujiwara C, Ohkawa K, Ishibashi Y. Glutathione-S-transferase-pi expression regulates sensitivity to glutathione-doxorubicin conjugate. Anticancer Drugs 2001; 12:707-12. [PMID: 11604559 DOI: 10.1097/00001813-200109000-00010] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
We have reported that glutathione-doxorubicin conjugate (GSH-DXR) exhibited potent cytotoxicity against tumor cells and inhibited glutathione-S-transferase (GST) enzyme activity. In order to determine whether or not the expression of GST-pi lowered the cytotoxicity of GSH-DXR, cytocidal activity of the conjugate was examined using tumor cells in which the level of GST-pi expression was regulated by transfecting GST-pi cDNA in the correct or reverse direction and comparing with that of DXR. Enhancement of GST-pi expression by transfecting GST-pi sense cDNA into human hepatoblastoma HepG2 cells in which GST-pi expression was extremely low caused an increase in GST activity from 0.26 to 55.0 nmol/mg/min and a marked reduction in transfectant sensitivity to GSH-DXR to 1/120 (0.15-18 nM IC50) although the sensitivity to DXR was slightly decreased to 1/2.6 (380-990 nM IC50). By contrast, a high GST-pi-expressing human colon cancer cell line, HT29, showed a decrease in GST enzyme activity from 72.0 to 45.9 nmol/mg/min after transfecting GST-pi antisense cDNA and a marked improvement in transfectant sensitivity to GSH-DXR was observed (28-2.9 nM IC50) compared with the transfectant sensitivity to DXR (1020-320 nM IC50). Additionally, the expression of GST-pi in HepG2 cells caused a decrease in GSH-DXR-induced activation of caspase-3, which was an apoptotic marker, whereas the suppression of GST-pi in HT29 cells showed an increase in caspase-3 activation. These results suggested that the cytocidal efficacy of GSH-DXR, but not that of DXR, was controlled by the level of GST-pi expression in the cells.
Collapse
Affiliation(s)
- K Tashiro
- Department of Biochemistry (I), Jikei University School of Medicine, Tokyo 105-8461, Japan
| | | | | | | | | |
Collapse
|
|
15
|
Hashizume Y, Asakura T, Oikawa T, Yamauchi T, Soda K, Ohkawa K. Relationship between cytocidal activity and glutathione-S-transferase inhibition using doxorubicin coupled to stereoisomers of glutathione with different substrate specificity. Anticancer Drugs 2001; 12:549-54. [PMID: 11460002 DOI: 10.1097/00001813-200107000-00009] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
Abstract
To determine the cytotoxic mode of action of a glutathione (GSH)--doxorubicin (DXR) conjugate, which exhibited potent cytotoxicity against various multidrug-resistant as well as DXR-sensitive cell lines, the molecular interaction between covalent GSH--DXR conjugates and glutathione-S-transferase (GST), a possible molecular target of the conjugates, was investigated. The following four GSH molecules with stereoisomeric forms were prepared: L-Glu--L-Cys--Gly (LL-GSH), D-Glu--L-Cys--Gly (DL-GSH), L-Glu--D-Cys--Gly (LD-GSH) and D-Glu--D-Cys--Gly (DD-GSH). The enzymic activity of GST against each GSH stereoisomer was 88, 38, 8 and 4 nmol/mg/min, respectively, suggesting that the L-form cysteine residue in the molecule was an important substrate of GST. Addition of DXR conjugated with each isomer (10 microM) to a GSH-containing GST assay mixture inhibited the GST activity to 32% for LL-GSH--XR, 16% for DL-GSH-DXR and 61% for LD-GSH-DXR as compared with the solvent control. Moreover, IC50 values for these conjugates were 30, 20 and 250 nM, respectively. The cytocidal activity of each conjugate corresponded to the substrate specificity of GST activity for the GSH isomer. These conjugates bound to the GST molecule, and the binding ability was 0.746, 0.627 and 0.462 mol/mol of GST for LL-GSH--XR, DL-GSH-DXR and LD-GSH--XR, respectively. These findings suggested that GSH--DXR interacted with the substrate-binding site of the GST molecule and inhibition of GST activity exhibited potent cytotoxicity.
Collapse
Affiliation(s)
- Y Hashizume
- Departments of Biochemistry I, Jikei University School of Medicine, Tokyo 105-8461, Japan
| | | | | | | | | | | |
Collapse
|
|
16
|
Kakizaki I, Ookawa K, Ishikawa T, Hayakari M, Aoyagi T, Tsuchida S. Induction of apoptosis and cell cycle arrest in mouse colon 26 cells by benastatin A. Jpn J Cancer Res 2000; 91:1161-8. [PMID: 11092982 PMCID: PMC5926276 DOI: 10.1111/j.1349-7006.2000.tb00900.x] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022] Open
Abstract
Benastatin A, isolated from Streptomyces bacteria, is reported to inhibit mammalian glutathione transferases (GSTs). Since GST inhibitors such as ethacrynic acid are suggested to induce apoptosis in some cell lines, the effect of benastatin A on the survival of mouse colon 26 adenocarcinoma cells was compared with that of ethacrynic acid. When cells in stationary phase were treated with benastatin A, viable cells were found to be dose-dependently decreased after 3 days. In the case of ethacrynic acid, this became apparent within 24 h. Electrophoretic analysis revealed DNA fragmentation, indicating that cell loss was due to apoptosis in both cases. The dominant GST in colon 26 cells was identified as the class Pi-form (GST-II), and the activities in crude extracts as well as purified GST-II were almost completely inhibited by 50 microM ethacrynic acid. Immunoblot and northern blot analyses revealed increased GST-II protein and mRNA levels in cells treated with ethacrynic acid. Benastatin A did not significantly affect the activity in the crude extract even at 20 microM, a 10-fold higher concentration than that which almost completely inhibited the activity of purified GST-II. However, GST activity and GST-II protein were decreased in colon 26 cells treated with benastatin A for 5 days, no significant activity being detected in the range of 16 - 20 microM. In addition, beta-actin and bax mRNAs were also decreased in a dose-dependent manner. Furthermore, flow cytometric analysis of colon 26 cells revealed that benastatin A blocked the cell cycle at the G1/G0 phase. Thus, benastatin A also induces apoptosis of colon 26 cells, but this is unlikely to be due to inhibition of GST activity.
Collapse
Affiliation(s)
- I Kakizaki
- Second Department of Biochemistry, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan
| | | | | | | | | | | |
Collapse
|
|
17
|
Asakura T, Sawai T, Hashidume Y, Ohkawa Y, Yokoyama S, Ohkawa K. Caspase-3 activation during apoptosis caused by glutathione-doxorubicin conjugate. Br J Cancer 1999; 80:711-5. [PMID: 10360648 PMCID: PMC2362273 DOI: 10.1038/sj.bjc.6690414] [Citation(s) in RCA: 16] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
Glutathione-doxorubicin (GSH-DXR) effectively induced apoptosis in rat hepatoma cells (AH66) at a lower concentration than DXR. After 24 h of drug treatment, DNA fragmentation of the cells was observed at the concentration of 1.0 microM DXR or 0.01 microM GSH-DXR. Increase in caspase-3 activity and DNA fragmentation were observed within 12 h and 15 h after treatment with either drug. Intracellular caspase-3 activity was increased in a dose-dependent manner after treatment with DXR or GSH-DXR, and caspase-3 activity correlated well with the ability to induce DNA fragmentation. When the cells were treated with either DXR or GSH-DXR for only 6 h, apoptotic DNA degradation and caspase-3 activation occurred 24 h after treatment. DNA fragmentation caused by these drugs was prevented completely by simultaneous treatment with the caspase-3 inhibitor, acetyl-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO), at 10 microM. By contrast, DNA fragmentation was not prevented by the caspase-1 inhibitor, acetyl-Tyr-Val-Ala-Asp-aldehyde (YVAD-CHO), at the same concentration as DEVD-CHO, and caspase-1 was not activated at all by the treatment of AH66 cells with both DXR and GSH-DXR. These results demonstrate that DXR and GSH-DXR induce apoptotic DNA fragmentation via caspase-3 activation, but not via caspase-1 activation, and that GSH-DXR enhances the activation of caspase-3 approximately 100-fold more than DXR. Moreover, the findings suggested that an upstream apoptotic signal that can activate caspase-3 is induced within 6 h by treating AH66 cells with the drug.
Collapse
Affiliation(s)
- T Asakura
- Department of Biochemistry (I), Jikei University School of Medicine, Tokyo, Japan
| | | | | | | | | | | |
Collapse
|