Knockdown of clusterin sensitizes pancreatic cancer cells to gemcitabine chemotherapy by ERK1/2 inactivation

Journal of Experimental & Clinical Cancer Research

Yingsheng Jiang²  Shaochuan Sun²  Chunning Zheng²  Fenghua Liu¹  Yong Tang¹ 

[1] Pancreatic Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China;Department of general surgery, the affiliated hospital of Jinan central hospital, Shandong university, No105, Jiefang Road, District Lixia, Jinan, 250013, R. P. China

关键词: ERK1/2;    Clusterin;    Gene treatment;    Gemcitabine;    Chemoresistance;    Pancreatic cancer;   

Others  :  825989
DOI  :  10.1186/1756-9966-31-73

 received in 2012-07-18, accepted in 2012-08-07,  发布年份 2012

【 摘 要 】

Objectives

To study the hypothesis that gemcitabine treatment augments the chemoresistance to gemcitabine by clusterin (sCLU) upregulation. Clusterin inhibition could augment the chemosensitivity of human pancreatic cancer cells by inhibition of clusterin-dependent pERK1/2 activation.

Methods

Clusterin was silenced by serial concentration of OGX-011 transfection in pancreatic cancer MIAPaCa-2 and BxPC-3 cell lines, then treated with serial concentration of gemcitabine. After the cells were treated with OGX-011 for 8 h, the cells were then treated with 5 μM ERK inhibitor PD98059 for 18 h or transfected with a wt-pERK-expressing plasmid into these cells for 24 h, after which the cells were treated with 1.0 uM gemcitabine for 24–72 h. Cell proliferation was determined by MTT. Apoptosis was quantified by flow cytometry,.sCLU and pERK1/2 production was analyzed by western blot, and sCLU mRNA was analyzed by RT-PCR. Xenograft of established tumors was used to evaluate primary tumor growth and apoptosis after treatment with gemcitabine alone or in combination with OGX-011. Phosphorylated ERK1/2 and sCLU levels in tumor tissues were measured by TUNEL analysis.

Results

As detected by MTT and FACS assay, a combination of gemcitabine + OGX-011 reflected the chemotherapeutic sensitivity and increased the gemcitabine -induced apoptosis in MIAPaCa-2 and BxPC-3 cells. Western blotting and RT-PCR analysis revealed that the expression of clusterin was higher in gemcitabine -resistant MIAPaCa-2 cells, however, decreased significantly after pretreatment with OGX-011. Furthermore, the OGX-011 or combination of gemcitabine + OGX-011 decreased the gemcitabine -induced activation of pERK1/2. wt-pERK-re-expression decreased OGX-011+ gemcitabine -induced apoptosis. Finally, OGX-011 in combination with gemcitabine substantially decreased the in vivo tumor growth and promoted apoptosis. Taken together, clusterin confers gmcitabine resistance in pancreatic cancer cells.

Conclusions

Knockdown of clusterin by OGX-011 transfection sensitizes pancreatic cancer cells to gemcitabine by inhibition of gemcitabine -induced clusterin-pERK1/2 activation.

【 授权许可】

   
2012 Tang et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Jemal A, Siegel R, Ward E: Cancer statistics,2006. CA Cancer J Clin 2006, 56:106-130.
• [2]O’Reilly EM, Abou-Alfa GK: Cytotoxic therapy for advanced pancreatic adenocarcinoma. Semin Oncol 2007, 34:347-353.
• [3]Moore MJ, Goldstein D, Hamm J: Erlotinib plus gemcitabine compared with gemcitabine alone in patients with advanced pancreatic cancer: a phase III trial of the National Cancer Institute of Canada Clinical Trials Group. J Clin Oncol 2007, 25:1960-1966.
• [4]Gleave M, Chi KN: Knock-down of the cytoprotective gene, clusterin, to enhance hormone and chemosensitivity in prostate and other cancers. Ann N Y Acad Sci 2005, 1058:1-15.
• [5]Jones SE, Jomary C: Clusterin. Int J Biochem Cell Biol 2002, 34:427-431.
• [6]Springate CM, Jackson JK, Gleave ME, Burt HM: Efficacy of an intratumoral controlled release formulation of clusterin antisense oligonucleotide complexed with chitosan containing paclitaxel or docetaxel in prostate cancer xenograft models. Cancer Chemother Pharmacol. 2005, 56:239-247.
• [7]Zellweger T, Miyake H, July LV, Akbari M, Kiyama S, Gleave ME: Chemosensitization of human renal cell cancer using antisense oligonucleotides targeting the antiapoptotic gene clusterin. Neoplasia 2001, 3:360-367.
• [8]Redondo M, Tellez T, Roldan MJ: The role of clusterin (CLU) in malignant transformation and drug resistance in breast carcinomas. Adv Cancer Res 2009, 105:21-43.
• [9]Panico F, Rizzi F, Fabbri LM, Bettuzzi S, Luppi F: Clusterin (CLU) and lung cancer. Adv Cancer Res 2009, 105:63-76.
• [10]Bi J, Guo AL, Lai YR, Li B, Zhong JM, Wu HQ, Xie Z, He YL, Lv ZL, Lau SH, Wang Q, Huang XH, Zhang LJ, Wen JM, Guan XY: Overexpression of clusterin correlates with tumor progression, metastasis in gastric cancer: a study on tissue microarrays. Neoplasma 2010, 57:191-198.
• [11]Hazzaa SM, Elashry OM, Afifi IK: Clusterin as a diagnostic and prognostic marker for transitional cell carcinoma of the bladder. Pathol Oncol Res 2010, 16:101-109.
• [12]Lokamani I, Looi ML, Ali SA, Dali AZ, Jamal R: Clusterin as a potential marker in distinguishing cervical neoplasia. Anal Quant Cytol Histol 2011, 33:223-228.
• [13]Redondo M, Villar E, Torres-Muñoz J, Tellez T, Morell M, Petito CK: Overexpression of clusterin in human breast carcinoma. Am J Pathol 2000, 157:393-399.
• [14]Xie D, Lau SH, Sham JS, Wu QL, Fang Y, Liang LZ, Che LH, Zeng YX, Guan XY: Up-regulated expression of cytoplasmic clusterin in human ovarian carcinoma. Cancer 2005, 103:277-283.
• [15]Kang YK, Hong SW, Lee H, Kim WH: Overexpression of clusterin in human hepatocellular carcinoma. Hum Pathol 2004, 35:1340-1346.
• [16]Xie D, Sham JS, Zeng WF, Che LH, Zhang M, Wu HX, Lin HL, Wen JM, Lau SH, Hu L, Guan XY: Oncogenic role of clusterin overexpression in multistage colorectal tumorigenesis and progression. World J Gastroenterol 2005, 11:3285-3289.
• [17]Kurahashi T, Muramaki M, Yamanaka K, Hara I, Miyake H: Expression of the secreted form of clusterin protein in renal cell carcinoma as a predictor of disease extension. BJU Int 2005, 96:895-899.
• [18]Steinberg J, Oyasu R, Lang S, Sintich S, Rademaker A, Lee C, Kozlowski JM, Sensibar JA: Intracellular levels of SGP-2 (Clusterin) correlate with tumor grade in prostate cancer. Clin Cancer Res 1997, 3:1707-1711.
• [19]Bijian K, Mlynarek AM, Balys RL, Jie S, Xu Y, Hier MP, Black MJ, Di Falco MR, LaBoissiere S, Alaoui-Jamali MA: Serum proteomic approach for the identification of serum biomarkers contributed by oral squamous cell carcinoma and host tissue microenvironment. J Proteome Res. 2009, 8:2173-2185.
• [20]Li H, Liu S, Zhu X, Yang S, Xiang J, Chen H: Clusterin immunoexpression and its clinical significance in patients with non-small cell lung cancer. Lung 2010, 188:423-431.
• [21]Busam KJ, Kucukgol D, Eastlake-Wade S, Frosina D, Delgado R, Jungbluth AA: Clusterin expression in primary and metastatic melanoma. J Cutan Pathol 2006, 33:619-623.
• [22]Saffer H, Wahed A, Rassidakis GZ, Medeiros LJ: Clusterin expression in malignant lymphomas: a survey of 266 cases. Mod Pathol 2002, 15:1221-1226.
• [23]Zhong B, Sallman DA, Gilvary DL, Pernazza D, Sahakian E, Fritz D, Cheng JQ, Trougakos I, Wei S, Djeu JY: Induction of clusterin by AKT–role in cytoprotection against docetaxel in prostate tumor cells. Mol Cancer Ther 2010, 9:1831-1841.
• [24]Trougakos IP, Lourda M, Antonelou MH: Intracellular clusterin inhibits mitochondrial apoptosis by suppressing p53-activating stress signals and stabilizing the cytosolic Ku70-Bax protein complex. Clin Cancer Res 2009, 15:48-59.
• [25]Zhang H, Kim JK, Edwards CA, Xu Z, Taichman R, Wang CY: Clusterin inhibits apoptosis by interacting with activated Bax. Nat Cell Biol 2005, 7:909-915.
• [26]Ammar H, Closset JL: Clusterin activates survival through the phosphatidylinositol 3-kinase/Akt pathway. J Biol Chem 2008, 283:12851-12861.
• [27]Lee KB, Jeon JH, Choi I, Kwon OY, Yu K, You KH: Clusterin, a novel modulator of TGF-beta signaling, is involved in Smad2/3 stability. Biochem Biophys Res Commun 2008, 366:905-909.
• [28]Zoubeidi A, Ettinger S, Beraldi E: Clusterin facilitates COMMD1 and I-kB degradation to enhance NF-kB activity in prostate cancer cells. Mol Cancer ResIn press 2009
• [29]Chen Q, Wang Z, Zhang K, Liu X, Cao W, Zhang L, Zhang S, Yan B, Wang Y, Xia C: Clusterin confers gemcitabine resistance in pancreatic cancer. World J Surg Oncol. 2011, 24:9-59.
• [30]Lu Z, Xu S: ERK1/2 MAP kinases in cell survival and apoptosis. IUBMB Life. 2006, 58:621-31.
• [31]Boucher MJ, Morisset J, Vachon PH, Reed JC, Laine’ J, Rivard N: MEK/ERK signaling pathway regulates the expression of Bcl-2, Bcl-X(L), and Mcl-1 and promotes survival of human pancreatic cancer cells. J Cell Biochem 2000, 79:355-369.
• [32]Shin YJ, Kang SW, Jeong SY, Shim YJ, Kim YH, Kim BM, Kee SH, Park JJ, Park IS, Min BH: Clusterin enhances proliferation of primary astrocytes through extracellular signal-regulated kinase activation. NeuroReport 2006, 17:1871-5.
• [33]Shim YJ, Kang BH, Jeon HS, Park IS, Lee KU, Lee IK, Park GH, Lee KM, Schedin P, Min BH: Clusterin induces matrix metalloproteinase-9 expression via ERK1/2 and PI3K/Akt/NF-κB pathways in monocytes/macrophages. J Leukoc Biol 2011, 90:761-9.
• [34]Chou TY, Chen WC, Lee AC, Hung SM, Shih NY, Chen MY: Clusterin silencing in human lung adenocarcinoma cells induces a mesenchymal-to-epithelial transition through modulating the ERK/Slug pathway. Cell Signal 2009, 21:704-11.
• [35]Miyake H, Hara I, Gleave ME: Antisense oligodeoxynucleotide therapy targeting clusterin gene for prostate cancer: Vancouver experience from discovery to clinic. Int J Urol 2005, 12:785-94.
• [36]Sowery RD, Hadaschik BA, So AI: Clusterin knockdown using the antisense oligonucleotide OGX-011 re-sensitizes docetaxel-refractory prostate cancer PC-3 cells to chemotherapy. BJU Int 2008, 102:389-97.
• [37]Gleave M, Miyake H: Use of antisense oligonucleotides targeting the cytoprotective gene, clusterin, to enhance androgen- and chemo-sensitivity in prostate cancer. World J Urol 2005, 23:38-46.
• [38]Xue P, Thiruvengadam A, Tameyoshi Y, Levin PA, Vijaya R, Baoan J, Gabriel L-B, Vivas-Mejia PE, Sood AK, McConkey DJ, Logsdon CD: Nuclear Factor-KB p65/relA Silencing Induces Apoptosis and Increases Gemcitabine Effectiveness in a Subset of Pancreatic Cancer Cells. Clin Cancer Res 2008, 14:8143-8151.
• [39]Neoptolemos JP: Adjuvant treatment of pancreatic cancer. Eur J Cancer 2011, 47(Suppl 3):S378-80.
• [40]Katz MH, Fleming JB, Lee JE, Pisters PW: Current status of adjuvant therapy for pancreatic cancer. Oncologist 2010, 15:1205-1213.
• [41]Squadroni M, Fazio N: Chemotherapy in pancreatic adenocarcinoma. Eur Rev Med Pharmacol Sci 2010, 14:386-394.
• [42]Duxbury MS, Ito H, Zinner MJ, Ashley SW, Whang EE: Inhibition of SRC tyrosine kinase impairs inherent and acquired gemcitabine resistance in human pancreatic adenocarcinoma cells. Clin Cancer Res 2004, 10:2307-2318.
• [43]Gleave ME, Miyake H, Zellweger T, Chi K, July L, Nelson C, Rennie P: Use of antisense oligonucleotides targeting the antiapoptotic gene, clusterin/testosterone-repressed prostate message 2, to enhance androgen sensitivity and chemosensitivity in prostate cancer. Urology 2001, 58:39-49.
• [44]Miyake H, Hara I, Kamidono S, Gleave ME: Synergistic chemsensitization and inhibition of tumor growth and metastasis by the antisense oligodeoxynucleotide targeting clusterin gene in a human bladder cancer model. Clin Cancer Res 2001, 7:4245-4252.
• [45]Xue HY, Wong HL: Targeting megalin to enhance delivery of anti-clusterin small-interfering RNA nanomedicine to chemo-treated breast cancer. Eur J Pharm Biopharm 2012, 81(1):24-32.
• [46]Hassan MK, Watari H, Christenson L, Bettuzzi S, Sakuragi N: Intracellular clusterin negatively regulates ovarian chemoresistance: compromised expression sensitizes ovarian cancer cells to paclitaxel. Tumour Biol 2011, 32:1031-47.
• [47]Johnson GL, Lapadat R: Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science 2002, 298:1911-1912.
• [48]Guan J, Chen XP, Zhu H, Luo SF, Cao B, Ding L: Involvement of extracellular signal-regulated kinase/mitogen-activated protein kinase pathway in multidrug resistance induced by HBx in hepatoma cell line. World J Gastroenterol 2004, 10:3522-3527.
• [49]McCubrey JA, Steelman LS, Abrams SL, Lee JT, Chang F, Bertrand FE, Navolanic PM, Terrian DM, Franklin RA, D’Assoro AB: Roles of the RAF/MEK/ERK and PI3K/PTEN/AKT pathways in malignant transformation and drug resistance. Adv Enzyme Regul. 2006, 46:249-279.
• [50]Chen X, Xia S, Li R, Liu H, Huang Y, Qian X, Xiao X, Xu X, Lin X, Tian Y: Doxycycline enhances the Ras-MAPK signaling and proliferation of mouse thymic epithelial cells. J Cell Biochem 2009, 107:494-503.
• [51]Arti S, Hillegass JM, MacPherson MB, Beuschel SL, Vacek PM, Pass HI, Michele C, Testa JR, Mossman BT: Blocking of ERK1 and ERK2 sensitizes human mesothelioma cells to doxorubicin. Mol Cancer 2010, 9:314. BioMed Central Full Text