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Inhibition of Autophagy by 3-MA Enhances the Effect of 5-FU-Induced Apoptosis in Colon Cancer Cells

  • Laboratory and Translational Research
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Abstract

Background

5-fluorouracil-(5-FU)-based adjuvant chemotherapy is widely used for the treatment of colorectal cancer. However, 5-FU resistance in the course of treatment has become more common. Therefore, new therapeutic strategies and/or new adjuvant drugs still need to be explored.

Methods

Two colon-cancer-derived cell lines, colon26 and HT29, were used to investigate the effect of 5-FU, 3-methyladenine (3-MA, an autophagy inhibitor), or their combination on apoptotic cell death and autophagy. MTT assay, Hochest plus propidium iodide (PI) staining, and DNA fragmentation assay were used to observe apoptosis. Meanwhile, monodansylcadaverine (MDC) was used to detect autophagy. Finally, immunoblotting assay was used to explore the molecular change that occurred.

Results

We observed the apoptosis induced by 5-FU in colon cancer cells. Meanwhile, autophagy was also stimulated. The combination treatment of 3-MA and 5-FU significantly increased the apoptotic cell death. By isolating the subcellular fractions of mitochondria and cytosol, we observed that the release of cytochrome c was increased in combination-treated cells. Cytochrome c resulted in the activation of caspase-3, thus activating PARP. Moreover, the anti-apoptotic protein, Bcl-xL, was significantly downregulated by 3-MA.

Conclusions

Our results suggest that 5-FU-induced apoptosis in colon cancer cells can be enhanced by the inhibitor of autophagy, 3-MA. Autophagy might play a role as a self-defense mechanism in 5-FU-treated colon cancer cells, and its inhibition could be a promising strategy for the adjuvant chemotherapy of colon cancer.

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References

  1. Xu R, Zhou B, Fung PCW, Li X. Recent advances in the treatment of colon cancer. Histol Histopathol. 2006;21:867–72.

    PubMed  CAS  Google Scholar 

  2. Chau I, Cunningham D. Adjuvant therapy in colon cancer: current status and future directions. Cancer Treat Rev. 2002;28:223–36.

    Article  PubMed  CAS  Google Scholar 

  3. Chau I, Cunningham D. Adjuvant therapy in colon cancer-what, when and how? Ann Oncol. 2006;17:1347–59.

    Article  PubMed  CAS  Google Scholar 

  4. Huerta S, Goulet EJ, Livingston EH. Colon cancer and apoptosis. Am J Surg. 2006;191:517–26.

    Article  PubMed  CAS  Google Scholar 

  5. Nordman IC, Iyer S, Joshua AM, Clarke SJ. Advance in the adjuvant treatment of colorectal cancer. ANZ J Surg. 2006;76:373–80.

    Article  PubMed  Google Scholar 

  6. Wils J, O’Dwyer P, Labianca R. Adjuvant treatment of colorectal cancer at the turn of the century: European and US perspectives. Ann Oncol. 2001;12:13–22.

    Article  PubMed  CAS  Google Scholar 

  7. Giacchetti BS, Perpoint B, Zidani R, et al. Phase III multicenter randomized trial of oxaliplatin added to chronomodulated fluorouracil–leucovorin as first-line treatment of metastatic colorectal cancer. J Clin Oncol. 2000;18:136–47.

    PubMed  CAS  Google Scholar 

  8. Douillard JY, Cunningham D, Roth AD, et al. Irinotecan combined with fluorouracil compared with fluorouracil alone as first-line treatment for metastatic colorectal cancer: a multicentre randomised trial. Lancet. 2000;355:1041–7.

    Article  PubMed  CAS  Google Scholar 

  9. Longley DB, Harkin DP, Johnston PG. 5-Fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer. 2003;3:330–8.

    Article  PubMed  CAS  Google Scholar 

  10. Grem JL. 5-fluorouracil: forty-plus and still ticking. A review of its preclinical and clinical development. Invest New Drugs. 2000;18:299–313.

    Article  PubMed  CAS  Google Scholar 

  11. Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis. 2000;21:485–95.

    Article  PubMed  CAS  Google Scholar 

  12. Petiot A, Ogier-Denis E, Blommaart EFC, Meijer AJ, Codogno P. Distinct classes of phosphatidylinositol 3′ kinases are involved in signaling pathways that control macroautophagy in HT–29 cells. J Biol Chem. 2000;275:992–8.

    Article  PubMed  CAS  Google Scholar 

  13. Yan CH, Liang ZQ, Gu ZL, Yang YP, Reid P, Qin ZH. Contributions of autophagic and apoptotic mechanisms to CrTX induced death of K562 cells. Toxicon. 2006;47:521–30.

    Article  PubMed  CAS  Google Scholar 

  14. Cui Q, Tashiro S, Onodera S, Ikejima T. Augmentation of oridonin-induced apoptosis observed with reduced autophagy. J Pharmacol Sci. 2006;101:230–9.

    Article  PubMed  CAS  Google Scholar 

  15. Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science. 2000;290:1717–21.

    Article  PubMed  CAS  Google Scholar 

  16. Meijer AJ, Codogno P. Regulation and role of autophagy in mammalian cells. Int J Biochem Cell B. 2004;36:2445–62.

    Article  CAS  Google Scholar 

  17. Ng G, Huang JX. The significance of autophagy in cancer. Mol Carcinogen. 2005;43:183–7.

    Article  CAS  Google Scholar 

  18. Ogier-Denis E, Codogno P. Autophagy: a barrier or an adaptive response to cancer. BBA-Rev Cancer. 2003;1603:113–28.

    CAS  Google Scholar 

  19. Kondo Y, Kanzawa T, Sawaya R, Kondo S. The role of autophagy in cancer development and response to therapy. Nat Rev Cancer. 2005;5:726–34.

    Article  PubMed  CAS  Google Scholar 

  20. Hoorens A, Casteele MV, Klöppel G, Pipeleers D. Glucose promotes survival of rat pancreatic cells by activating synthesis of proteins which suppress a constitutive apoptotic program. J Clin Invest. 1996;98:1568–74.

    Article  PubMed  CAS  Google Scholar 

  21. Munafó DB, Colombo MI. A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation. J Cell Sci. 2001;114:3619–29.

    PubMed  Google Scholar 

  22. Abedin MJ, Wang D, McDonnell MA, Lehmann U, Kelekar A. Autophagy delays apoptotic cell death in breast cancer cells following DNA damage. Cell Death Differ. 2006;22: 1–11.

    Google Scholar 

  23. Biederbick A, Kern HF, Elsasser HP. Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles. Eur J Cell Biol. 1995;66:3–14.

    PubMed  CAS  Google Scholar 

  24. Shimizu S, Kanaseki T, Mizushima N, Mizuta T, Arakawa-Kobayashi S, Thompson CB, et al. Role of Bcl–2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes. Nat Cell Biol. 2004;6:1221–8.

    Article  PubMed  CAS  Google Scholar 

  25. Herman-Antosiewicz A, Johnson DE, Singh SV. Sulforaphane causes autophagy to inhibit release of cytochrome c and apoptosis in human prostate cancer cells. Cancer Res. 2006;66:5828–35.

    Article  PubMed  CAS  Google Scholar 

  26. Klionsky DJ, Levine B. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell. 2004;6:463–77.

    Article  PubMed  Google Scholar 

  27. Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H, et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature. 1999;402:672–6.

    Article  PubMed  CAS  Google Scholar 

  28. Qu XP, Yu J, Bhagat G, et al. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene. J Clin Invest. 2003;112:1809–20.

    PubMed  CAS  Google Scholar 

  29. Ding WX, Ni HM, Gao WT, et al. Differential effects of endoplasmic reticulum stress-induced autophagy on cell survival. J Biol Chem. 2007;282:4702–10.

    Article  PubMed  CAS  Google Scholar 

  30. Bauvy C, Gane P, Arico S, Codogon P, Ogier-Denis E. Autophagy delays Sulindac Sulfide-induced apoptosis in the human intestinal colon cancer cell line HT-29. Exp Cell Res. 2001;268:139–49.

    Article  PubMed  CAS  Google Scholar 

  31. Kanzawa T, Germano IM, Komata T, Kondo Y, Kondo S. Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells. Cell Death Differ. 2004;11:448–57.

    Article  PubMed  CAS  Google Scholar 

  32. Paglin S, Hollister T, Delohery T, et al. A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Res. 2001;61:439–44.

    PubMed  CAS  Google Scholar 

  33. Kanzawa T, Kondo Y, Ito H, Kondo S, Germano I. Induction of autophagic cell death in malignant glioma cells by arsenic trioxide. Cancer Res. 2003;63:2103–8.

    PubMed  CAS  Google Scholar 

  34. Gozuacik D, Kimchi A. Autophagy and cell death. Curr Top Dev Biol. 2007;78:217–45.

    Article  PubMed  CAS  Google Scholar 

  35. Bursch W, Ellinger A, Kienzl H, et al. Active cell death induced by the anti-estrogens tamoxifen and ICI164 384 in human mammary carcinoma cells (MCF–7) in culture: the role of autophagy. Carcinogenesis. 1996;17:1595–607.

    Article  PubMed  CAS  Google Scholar 

  36. Cao C, Subhawong T, Albert JM, et al. Inhibition of mammalian target of rapamycin or apoptotic pathway induces autophagy and radiosensitizes PTEN null prostate cancer cells. Cancer Res. 2006;66:10040–7.

    Article  PubMed  CAS  Google Scholar 

  37. Iwamaru A, Kondo Y, Iwado E, et al. Silencing mammalian target of rapamycin signaling by small interfering RNA enhances rapamycin-induced autophagy in malignant glioma cells. Oncogene. 2007;26:1840–51.

    Article  PubMed  CAS  Google Scholar 

  38. Zhu HB, Guo W, Zhang LD, et al. Bcl-xL small interfering RNA suppresses the proliferation of 5-fluorouracil-resistant human colon cancer cells. Mol Cancer Ther. 2005;4:451–6.

    Article  PubMed  CAS  Google Scholar 

  39. Tsujimoto Y. Cell death regulation by the Bcl–2 protein family in the mitochondria. J Cell Physiol. 2003;195:158–67.

    Article  PubMed  CAS  Google Scholar 

  40. Pattingre S, Tassa A, Qu XP, et al. Bcl-2 antiapoptotic proteins inhibit beclin 1-dependent autophagy. Cell. 2005;122:927–39.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by the 21st Century COE Program, Japan and the Japan Society for the Promotion of Science (JSPS) for A. Faried.

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Authors and Affiliations

  1. Department of General Surgical Science (Surgery I), Graduate School of Medicine, Gunma University, Maebashi, Gunma, 371-8511, Japan

    Jie Li MD, Ahmad Faried MD, PhD, Soichi Tsutsumi MD, PhD & Hiroyuki Kuwano MD, PhD

  2. Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Japan

    Ni Hou MD, PhD & Toshiyuki Takeuchi MD, PhD

Authors
  1. Jie Li MD
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  2. Ni Hou MD, PhD
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  3. Ahmad Faried MD, PhD
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  4. Soichi Tsutsumi MD, PhD
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  5. Toshiyuki Takeuchi MD, PhD
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  6. Hiroyuki Kuwano MD, PhD
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Correspondence to Jie Li MD.

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Li, J., Hou, N., Faried, A. et al. Inhibition of Autophagy by 3-MA Enhances the Effect of 5-FU-Induced Apoptosis in Colon Cancer Cells. Ann Surg Oncol 16, 761–771 (2009). https://doi.org/10.1245/s10434-008-0260-0

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  • DOI: https://doi.org/10.1245/s10434-008-0260-0

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