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. 2000 Nov;91(11):1161–1168. doi: 10.1111/j.1349-7006.2000.tb00900.x

Induction of Apoptosis and Cell Cycle Arrest in Mouse Colon 26 Cells by Benastatin A

Ikuko Kakizaki 1, Keizoh Ookawa 1, Takashi Ishikawa 2, Makoto Hayakari 1, Takaaki Aoyagi 3, Shigeki Tsuchida 1,✉
PMCID: PMC5926276  PMID: 11092982

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 μM 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 μM, 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 μM. In addition, β‐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.

Keywords: Glutathione transferase, Enzyme inhibitor, Apoptosis, Cell cycle, Colon cancer

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REFERENCES

  • 1. ) Jakoby , W. B.The glutathione S‐transferases: a group of multifunctional detoxification proteins . Adv. Enzymol. , 46 , 383 – 414 ( 1978. ). [DOI] [PubMed] [Google Scholar]
  • 2. ) Mannervik , B. , Ålin , P. , Guthenberg , C. , Jensson , H. , Tahir , M. K. , Warholm , M. and Jörnvall , H.Identification of three classes of cytosolic glutathione transferase common to several mammalian species: correlation between structural data and enzymatic properties . Proc. Natl. Acad. Sci. USA , 82 , 7202 – 7206 ( 1985. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. ) Satoh , K. , Kitahara , A. , Soma , Y. , Inaba , Y. , Hatayama , I. and Sato , K.Purification, induction, and distribution of placental glutathione transferase: a new marker enzyme for preneoplastic cells in the rat chemical hepatocarcinogenesis . Proc. Natl. Acad. Sci. USA , 82 , 3964 – 3968 ( 1985. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. ) Batist , G. , Tulpule , A. , Sinha , B. K. , Katki , A. G. , Myers , C. E. and Cowan , K. H.Overproduction of a novel anionic glutathione transferase in multidrug‐resistant human breast cancer cells . J. Biol. Chem. , 261 , 15544 – 15549 ( 1986. ). [PubMed] [Google Scholar]
  • 5. ) Tsuchida , S. and Sato , K.Glutathione transferases and cancer . Crit. Rev. Biochem. Mol. Biol. , 27 , 337 – 384 ( 1992. ). [DOI] [PubMed] [Google Scholar]
  • 6. ) Ban , N. , Takahashi , Y. , Takayama , T. , Kura , T. , Katahira , T. , Sakamaki , S. and Niitsu , Y.Transfection of glutathione S‐transferase (GST)‐π antisense complementary DNA increases the sensitivity of a colon cancer cell line to adriamycin, cisplatin, melpharan, and etoposide . Cancer Res. , 56 , 3577 – 3582 ( 1996. ). [PubMed] [Google Scholar]
  • 7. ) Tew , R. D. , Bomber , A. M. and Hoffman , S. J.Ethacrynic acid and piriprost as enhancers of cytotoxicity in drug‐resistant and sensitive cell lines . Cancer Res. , 48 , 3622 – 3625 ( 1988. ). [PubMed] [Google Scholar]
  • 8. ) Kerr , J. F. R. , Wyllie , A. H. and Currie , A. R.Apoptosis: a basic biological phenomenon with wide‐ranging implications in tissue kinetics . Br. J. Cancer , 26 , 239 – 257 ( 1972. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. ) Wyllie , A. H.Glucocorticoid‐induced thymocyte apoptosis is associated with endogenous endonuclease activation . Nature , 284 , 555 – 556 ( 1980. ). [DOI] [PubMed] [Google Scholar]
  • 10. ) Yonish‐Rouach , E. , Resnitzky , D. , Lotem , J. , Sachs , L. , Kimchi , A. and Oren , M.Wild‐type p53 induces apoptosis of myeloid leukaemic cells that is inhibited by interleukin‐6 . Nature , 352 , 345 – 347 ( 1991. ). [DOI] [PubMed] [Google Scholar]
  • 11. ) Nelson , W. G. and Kastan , M. B.DNA strand breaks: the DNA template alterations that trigger p53‐dependent DNA damage response pathways . Mol. Cell. Biol. , 14 , 1815 – 1823 ( 1994. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. ) Kastan , M. B. , Onyekwere , O. , Sidransky , D. , Vogelstein , B. and Craig , R. W.Participation of p53 protein in the cellular response to DNA damage . Cancer Res. , 51 , 6304 – 6311 ( 1991. ). [PubMed] [Google Scholar]
  • 13. ) Vaux , D. L. , Cory , S. and Adams , J. M.Bcl‐2 gene promotes haemopoietic cell survival and cooperates with c‐myc to immortalize pre‐B cells . Nature , 335 , 440 – 442 ( 1988. ). [DOI] [PubMed] [Google Scholar]
  • 14. ) Oltvai , Z. N. , Milliman , C. L. and Korsmeyer , S. J.Bcl‐2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death . Cell , 74 , 609 – 619 ( 1993. ). [DOI] [PubMed] [Google Scholar]
  • 15. ) Gupta , S. , Campbell , D. , Derjard , B. and Davis , R. J.Transcription factor ATF2 regulation by the JNK signal transduction pathway . Science , 267 , 389 – 393 ( 1995. ). [DOI] [PubMed] [Google Scholar]
  • 16. ) Xia , Z. , Dickens , M. , Raingeaud , J. , Davis , R. J. and Greenberg , M. E.Opposing effects of ERK and JNK‐p38 MAP kinases on apoptosis . Science , 270 , 1326 – 1331 ( 1995. ). [DOI] [PubMed] [Google Scholar]
  • 17. ) Kaufmann , S. H.Induction of endonucleolytic cleavage in human acute myelogenous leukemia cells by etoposide, camptothecin, and other cytotoxic anticancer drugs: a cautionary note . Cancer Res. , 49 , 5870 – 5878 ( 1989. ). [PubMed] [Google Scholar]
  • 18. ) Fisher , D. E.Apoptosis in cancer therapy: crossing the threshold . Cell , 78 , 539 – 542 ( 1994. ). [DOI] [PubMed] [Google Scholar]
  • 19. ) McCaughan , F. M. , Brown , A. L. and Harrison , D. J.The effect of inhibition of glutathione S‐transferase P on the growth of the Jurkat human T cell line . J. Pathol. , 172 , 357 – 362 ( 1994. ). [DOI] [PubMed] [Google Scholar]
  • 20. ) Asakura , T. , Ohkawa , K. , Takahashi , N. , Takabe , K. , Inoue , T. and Yokoyama , S.Glutathione‐doxorubicin conjugate expresses potent cytotoxicity by suppression of glutathione S‐transferase activity: comparison between doxorubicin‐sensitive and resistant rat hepatoma cells . Br. J. Cancer , 75 , 1333 – 1337 ( 1997. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. ) Aoyagi , T. , Aoyama , T. , Kojima , F. , Matsuda , N. , Maruyama , M. , Hamada , M. and Takeuchi , T.Benastatins A and B, new inhibitors of glutathione S‐transferase, produced by Streptomyces sp. MI 384‐DF12. I. Taxonomy, production, isolation, physico‐chemical properties and biological activities . J. Antibiot. (Tokyo) , 45 , 1385 – 1390 ( 1992. ). [DOI] [PubMed] [Google Scholar]
  • 22. ) Aoyama , T. , Naganawa , H. , Muraoka , Y. , Nakamura , H. , Aoyagi , T. , Takeuchi , T. and Iitaka , Y.Benastatins A and B, new inhibitors of glutathione S‐transferase, produced by Streptomyces sp. MI 384‐DF12. II. Structure determination of benastatins A and B . J. Antibiot. (Tokyo) , 45 , 1391 – 1396 ( 1992. ). [DOI] [PubMed] [Google Scholar]
  • 23. ) Murata , T. , Hatayama , I. , Kakizaki , I. , Satoh , K. , Sato , K. and Tsuchida , S.Lentinan enhances sensitivity of mouse colon 26 tumor to cis‐diamminedichloroplatinum(II) and decreases glutathione transferase expression . Jpn. J. Cancer Res. , 87 , 1171 – 1178 ( 1996. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24. ) Nicholson , D. W. , Ali , A. , Thornberry , N. A. , Vaillancourt , J. P. , Ding , C. K. , Gallant , M. , Gareau , Y. , Griffin , P. R. , Labelle , M. , Lazebnik , Y. A. , Munday , N. A. , Raju , S. M. , Smulson , M. E. , Yamin , T.‐T. , Yu , V. L. and Miller , D. K.Identification and inhibition of the ICE/CED‐3 protease necessary for mammalian apoptosis . Nature , 376 , 37 – 43 ( 1995. ). [DOI] [PubMed] [Google Scholar]
  • 25. ) Sakai , M. , Okuda , A. , Hatayama , I. , Sato , K. , Nishi , S. and Muramatsu , M.Structure and expression of the rat c‐jun messenger RNA: tissue distribution and increase during rat chemical hepatocarcinogenesis . Cancer Res. , 49 , 5633 – 5637 ( 1989. ). [PubMed] [Google Scholar]
  • 26. ) Tsujimoto , Y. and Croce , C. M.Analysis of the structure, transcripts, and protein products of bcl‐2, the gene involved in human follicular lymphoma . Proc. Natl. Acad. Sci. USA , 83 , 5214 – 5218 ( 1986. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. ) Imagawa , M. , Osada , S. , Koyama , Y. , Suzuki , T. , Hirom , P. C. , Diccianni , M. B. , Morimura , S. and Muramatsu , M.SFB that binds to a negative element in glutathione transferase P gene is similar or identical to trans‐activator LAP/IL‐6DBP . Biochem. Biophys. Res. Commun. , 179 , 293 – 300 ( 1991. ). [DOI] [PubMed] [Google Scholar]
  • 28. ) Ahmed , S. A. , Gogal , R. M. , Jr. and Walsh , J. E.A new rapid and simple non‐radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to 3H‐thymidine incorporation assay . J. Imunol. Methods , 170 , 211 – 224 ( 1994. ). [DOI] [PubMed] [Google Scholar]
  • 29. ) Stocker , F. W. , King , E. H. , Lucas , D. O. and Georgiade , N.A comparison of two different staining methods for evaluating corneal endothelial viability . Arch. Ophthalmol. , 76 , 833 – 835 ( 1966. ). [DOI] [PubMed] [Google Scholar]
  • 30. ) Sambrook , J. , Fritsch , E. F. and Maniatis , T. “ Molecular Cloning. A Laboratory Manual ,” 2nd Ed. ( 1989. ). Cold Spring Harbor Laboratory Press; , Cold Spring Harbor , NY . [Google Scholar]
  • 31. ) Habig , W. H. , Pabst , M. J. and Jakoby , W. B.Glutathione S‐transferase: the first enzymatic step in mercapturic acid formation . J. Biol. Chem. , 249 , 7130 – 7139 ( 1974. ). [PubMed] [Google Scholar]
  • 32. ) Bradford , M. M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein‐dye binding . Anal. Biochem. , 72 , 248 – 254 ( 1976. ). [DOI] [PubMed] [Google Scholar]
  • 33. ) Owens , C. W. and Belcher , R. V.A colorimetric micromethod micromethod for the determination of glutathione . Biochem. J. , 94 , 705 – 711 ( 1965. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34. ) Nakagawa , K. , Saijo , N. , Tsuchida , S. , Sakai , M. , Tsunokawa , Y. , Yokota , J. , Muramatsu , M. , Sato , K. , Terada , M. and Tew , K. D.Glutathione S‐transferase‐π as a determinant of drug resistance in transfectant cell lines . J. Biol. Chem. , 265 , 4296 – 4301 ( 1990. ). [PubMed] [Google Scholar]
  • 35. ) Laemmli , U. K.Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature , 227 , 680 – 685 ( 1970. ). [DOI] [PubMed] [Google Scholar]
  • 36. ) Hatayama , I. , Satoh , K. and Sato , K.Developmental and hormonal regulation of the major form of hepatic glutathione S‐transferase in male mice . Biochem. Biophys. Res. Commun. , 140 , 581 – 588 ( 1986. ). [DOI] [PubMed] [Google Scholar]
  • 37. ) Towbin , H. , Staehelin , T. and Gordon , J.Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications . Proc. Natl. Acad. Sci. USA , 76 , 4350 – 4354 ( 1979. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38. ) Hatayama , I. , Satoh , K. and Sato , K.A cDNA sequence coding a class pi glutathione S‐transferase of mouse . Nucleic Acids Res. , 18 , 4606 ( 1990. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39. ) Feinberg , A. P. and Vogelstein , B.A technique for radiolabeling DNA restriction endonuclease fragment to high specific activity . Anal. Biochem. , 137 , 266 – 267 ( 1984. ). [DOI] [PubMed] [Google Scholar]
  • 40. ) Zieve , G. W. , Turnbull , D. , Mullins , J. M. and McIntosh , J. R.Production of large numbers of mitotic mammalian cells by use of the reversible microtubule inhibitor nocodazole . Exp. Cell Res. , 126 , 397 – 405 ( 1980. ). [DOI] [PubMed] [Google Scholar]
  • 41. ) Martin , S. J. , Lennon , S. V. , Bonham , A. M. and Cotter , T. G.Induction of apoptosis (programmed cell death) in human leukemic HL‐60 cells by inhibition of RNA or protein synthesis . J. Immunol. , 145 , 1859 – 1867 ( 1990. ). [PubMed] [Google Scholar]
  • 42. ) Miura , M. , Zhu , H. , Rotello , R. , Hartwieg , E. A. and Yuan , J.Induction of apoptosis in fibroblasts by IL‐1β‐converting enzyme, a mammalian homolog of the C. elegans cell death gene ced‐3 . Cell , 75 , 653 – 660 ( 1993. ). [DOI] [PubMed] [Google Scholar]
  • 43. ) Lowe , S. W. , Schmitt , E. M. , Smith , S. W. , Osborne , B. A. and Jacks , T.p53 is required for radiation‐induced apoptosis in mouse thymocytes . Nature , 362 , 847 – 849 ( 1993. ). [DOI] [PubMed] [Google Scholar]
  • 44. ) Xiao , H. , Pearson , A. , Coulombe , B. , Truant , R. , Zhang , S. , Regier , J. L. , Triezenberg , S. J. , Reinberg , D. , Flores , O. , Ingles , C. J. and Greenblatt , J.Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53 . Mol. Cell. Biol. , 14 , 7013 – 7024 ( 1994. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45. ) Svejstrup , J. Q. , Vichi , P. and Egly , J.‐M.The multiple roles of transcription/repair factor TFIIH . Trends Biochem. Sci. , 21 , 346 – 350 ( 1996. ). [PubMed] [Google Scholar]
  • 46. ) Adler , V. , Yin , Z. , Fuchs , S. Y. , Benezra , M. , Rosario , L. , Tew , K. D. , Pincus , M. R. , Sardana , M. , Henderson , C. J. , Wolf , C. R. , Davis , R. J. and Ronai , Z.Regulation of JNK signaling by GSTp . EMBO J. , 18 , 1321 – 1334 ( 1999. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47. ) Nakano , H. , Hatayama , I. , Satoh , K. , Suzuki , S. , Sato , K. and Tsuchida , S.c‐Jun expression in single cells and preneoplastic foci induced by diethylnitrosamine in B6C3F1 mice: comparison with the expression of pi‐class glutathione S‐transferase . Carcinogenesis , 15 , 1853 – 1857 ( 1994. ). [DOI] [PubMed] [Google Scholar]

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