Restoration of klotho gene expression induces apoptosis and autophagy in gastric cancer cells: tumor suppressive role of klotho in gastric cancer

Cancer Cell International

Lianwen Yuan¹  Jinhui Chen²  Jiapeng Zhou¹  Dong-cai Liu¹  Guoshun Shu¹  Jianping Zhou¹  Biao Xie² 

[1]Departemt of Geriatric Surgery, Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, China

[2]Department of General Surgery, 8th Changsha Hospital, Changsha, Hunan, 410015, China

关键词: Autophagy;    Apoptosis;    Gastric cancer;    Klotho;   

Others  :  794310
DOI  :  10.1186/1475-2867-13-18

 received in 2013-01-20, accepted in 2013-02-13,  发布年份 2013

【 摘 要 】

Background

The loss of tumor suppressor gene expression is involved in the carcinogenesis of gastric cancer (GC). Klotho is a recently identified tumor suppressor gene that epigenetically inactivated in gastric cancer. However, the signaling pathways involved in the suppressive role of klotho have rarely been reported in gastric cancer. In this study, we investigated the involvement of klotho in gastric cancer cell proliferation, apoptosis, and autophagy as well as the associated signaling.

Methods

Methylation of klotho gene promoter in GC-7901, MNK-45 and AGS gastric cancer cells as well as GES-1 normal gastric epithelial cells was detected by bisulfate-based PCR. Restoration of klotho gene expression was established by applying a demethylating agent and delivering aklotho gene expression vector into GC-7901 cells. Cell viability was measured by CCK-8 assay. Cell apoptosis and cycling were analyzed by flow cytometry. Autophagy was measured by detecting LC3-I and LC3-II expression. Protein levels and phosphorylation were measured by Western blot assay.

Results

Methylation of klotho gene promoter and expression of the klotho gene were detected in GC cells. Restoration of klotho gene expression significantly inhibited cell proliferation, induced cell apoptosis, and increased LC3-I/LC3-II expression in GC cells. Restoration of klotho gene expression downregulated the phosphorylation levels of IGF-1 receptor, IRS-1, PI3K, Akt, and mTOR proteins. Both apoptosis and autophagy inhibitors blocked klotho-induced apoptosis and autophagy.

Conclusion

Klotho is a tumor suppressor in gastric cancer, which regulates IGF-1R phosphorylation and the subsequent activation of IRS-1/PI3K/Akt/mTOR signaling, tumor cell proliferation, apoptosis, and autophagy.

【 授权许可】

   
2013 Xie et al; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

【 参考文献 】

• [1]Kim K, Chun KH, Suh PG, Kim IH: Alterations in cell proliferation related gene expressions in gastric cancer. Crit Rev Eukaryot Gene Expr 2011, 21:237-254.
• [2]Jang BG, Kim WH: Molecular pathology of gastric carcinoma. Pathobiology 2011, 78:302-310.
• [3]Tamura G: Alterations of tumor suppressor and tumor-related genes in the development and progression of gastric cancer. World J Gastroenterol 2006, 12:192-198.
• [4]Wang L, Wang X, Wang X, Jie P, Lu H, Zhang S, Lin X, Lam EK, Cui Y, Yu J, Jin H: Klotho is silenced through promoter hypermethylation in gastric cancer. Am J Cancer Res 2011, 1:111-119.
• [5]Arking DE, Becker DM, Yanek LR, Fallin D, Judge DP, Moy TF, Becker LC, Dietz HC: KLOTHO allele status and the risk of early-onset occult coronary artery disease. Am J Hum Genet 2003, 72:1154-1161.
• [6]Chen CD, Podvin S, Gillespie E, Leeman SE, Abraham CR: Insulin stimulates the cleavage and release of the extracellular domain of Klotho by ADAM10 and ADAM17. Proc Natl Acad Sci USA 2007, 104:19796-19801.
• [7]Oshima T, Masuda M: Molecular targeted agents for gastric and gastroesophageal junction cancer. Surg Today 2012, 42:313-327.
• [8]Lin HM, Tseng HC, Wang CJ, Chyau CC, Liao KK, Peng PL, Chou FP: Induction of autophagy and apoptosis by the extract of Solanum nigrum Linn in HepG2 cells. J Agric Food Chem 2007, 55:3620-3628.
• [9]Ogata M, Hino S, Saito A, Morikawa K, Kondo S, Kanemoto S, Murakami T, Taniguchi M, Tanii I, Yoshinaga K, Shiosaka S, Hammarback JA, Urano F, Imaizumi K: Autophagy is activated for cell survival after endoplasmic reticulum stress. Mol Cell Biol 2006, 26:9220-9231.
• [10]Chaachouay H, Ohneseit P, Toulany M, Kehlbach R, Multhoff G, Rodemann HP: Autophagy contributes to resistance of tumor cells to ionizing radiation. Radiother Oncol 2011, 99:287-292.
• [11]Wang HB, Zhou CJ, Song SZ, Chen P, Xu WH, Liu B, Zhu KX, Yu WH, Wu HL, Wang HJ, Lin S, Guo JQ, Qin CY: Evaluation of Nrf2 and IGF-1 expression in benign, premalignant and malignant gastric lesions. Pathol Res Pract 2011, 207:169-173.
• [12]Chen B, Wang X, Zhao W, Wu J: Klotho inhibits growth and promotes apoptosis in human lung cancer cell line A549. J Exp Clin Cancer Res 2010, 29:99-106. BioMed Central Full Text
• [13]Pan J, Zhong J, Gan LH, Chen SJ, Jin HC, Wang X, Wang LJ: Klotho, an anti-senescence related gene, is frequently inactivated through promoter hypermethylation in colorectal cancer. Tumour Biol 2011, 32:729-735.
• [14]Lu L, Katsaros D, Wiley A, de la Longrais IA, Puopolo M, Yu H: Klotho expression in epithelial ovarian cancer and its association with insulin-like growth factors and disease progression. Cancer Invest 2008, 26:185-192.
• [15]Wolf I, Levanon-Cohen S, Bose S, Ligumsky H, Sredni B, Kanety H, Kuro-o M, Karlan B, Kaufman B, Koeffler HP, Rubinek T: Klotho: a tumor suppressor and a modulator of the IGF-1 and FGF pathways in human breast cancer. Oncogene 2008, 27:7094-70105.
• [16]Abramovitz L, Rubinek T, Ligumsky H, Bose S, Barshack I, Avivi C, Kaufman B, Wolf I: KL1 internal repeat mediates klotho tumor suppressor activities and inhibits bFGF and IGF-I signaling in pancreatic cancer. Clin Cancer Res 2011, 17:4254-4266.
• [17]Kurosu H, Yamamoto M, Clark JD, Pastor JV, Nandi A, Gurnani P, McGuinness OP, Chikuda H, Yamaguchi M, Kawaguchi H, Shimomura I, Takayama Y, Herz J, Kahn CR, Rosenblatt KP, Kuro-o M: Suppression of aging in mice by the hormone Klotho. Science 2005, 309:1829-1833.
• [18]Kooijman R: Regulation of apoptosis by insulin-like growth factor (IGF)-I. Cytokine Growth Factor Rev 2006, 17:305-323.
• [19]Yang S, Xiao X, Meng X, Leslie KK: A mechanism for synergy with combined mTOR and PI3 kinase inhibitors. PLoS One 2011, 6:e26343.
• [20]Cheng Y, Qiu F, Ye YC, Guo ZM, Tashiro S, Onodera S, Ikejima T: Autophagy inhibits reactive oxygen species-mediated apoptosis via activating p38-nuclear factor-kappa B survival pathways in oridonin-treated murine fibrosarcoma L929 cells. FEBS J 2009, 276:1291-1306.
• [21]Levine B, Yuan J: Autophagy in cell death: an innocent convict? J Clin Invest 2005, 115:2679-2688.
• [22]Lum JJ, Bauer DE, Kong M, Harris MH, Li C, Lindsten T, Thompson CB: Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Cell 2005, 120:237-248.
• [23]Arico S, Petiot A, Bauvy C, Dubbelhuis PF, Meijer AJ, Codogno P, Ogier-Denis E: The tumor suppressor PTEN positively regulates macroautophagy by inhibiting the phosphatidylinositol 3-kinase/protein kinase B pathway. J Biol Chem 2001, 276:35243-35246.
• [24]Degtyarev M, De Mazière A, Orr C, Lin J, Lee BB, Tien JY, Prior WW, van Dijk S, Wu H, Gray DC, Davis DP, Stern HM, Murray LJ, Hoeflich KP, Klumperman J, Friedman LS, Lin K: Akt inhibition promotes autophagy and sensitizes PTEN-null tumors to lysosomotropic agents. J Cell Biol 2008, 183:101-116.
• [25]Mammucari C, Milan G, Romanello V, Masiero E, Rudolf R, Del Piccolo P, Burden SJ, Di Lisi R, Sandri C, Zhao J, Goldberg AL, Schiaffino S, Sandri M: FoxO3 controls autophagy in skeletal muscle in vivo. Cell Metab 2007, 6:458-471.
• [26]Zhang X, Mi J, Wetsel WC, Davidson C, Xiong X, Chen Q, Ellinwood EH, Lee TH: PI3 kinase is involved in cocaine behavioral sensitization and its reversal with brain area specificity. Biochem Biophys Res Commun 2006, 340:1144-1150.