内质网相关降解通路小分子抑制剂与传统抗癌药物联合运用对肿瘤细胞的抑制作用
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摘要
目的探究内质网相关降解通路(endoplasmic reticulum-associated degradation,ERAD)小分子抑制剂EeyarestatinⅠ(EerⅠ)与传统抗癌药物联合运用对肿瘤细胞的抑制作用。方法不同剂量的EerⅠ处理结肠癌SW480和胰腺癌PANC-1细胞,MTS法检测细胞增殖,流式细胞仪检测细胞凋亡率;EerⅠ单独或与抗癌药物顺铂(cisplatin,CDDP)和硼替佐米(bortezomib,BTZ)联用处理SW480和PANC-1细胞,Western blot法检测细胞中Poly ADP-ribose polymerase(PARP)及其剪切体和糖调节蛋白(GRP78/Bi P)的蛋白表达。结果 EerⅠ抑制SW480和PANC-1细胞增殖,促进细胞凋亡(P<0.01),且呈剂量-效应关系;联合用药的实验结果显示EerⅠ提高了SW480和PANC-1细胞对CDDP和BTZ的敏感度(P<0.01);Western blot法检测结果表明,联合用药促进了PARP在SW480和PANC-1细胞中的剪切,同时BTZ处理可提高Bi P蛋白的表达,提示激活内质网应激(endoplasmic reticulum stress,ERS)反应。结论 ERAD通路抑制剂EerⅠ可明显增强传统抗癌药物CDDP和BTZ的抗癌活性,这为抗癌新药研发及肿瘤耐药性逆转剂的研究提供了新的线索。
Objective To characterise the inhibitory effect of Eer Ⅰ,a small molecule targeting the key protein of the ERAD pathway,p97,alone or in combination with the traditional anticancer drugs on tumor cells. Methods The effect of Eer Ⅰ singularly and combined with cisplatin( CDDP) or bortezomib( BTZ) on tumor cell proliferation and apoptosis was measured through MTS assay and flow cytometry. Western blot was used to detect the cleavge of PARP and GRP78 in SW480 and PANC- 1 cells after treatments. Results Eer Ⅰ significantly inhibited cell proliferation and induced apoptosis of SW480 and PANC- 1 in a dose- dependent manner compared with the control group( P < 0. 01). MTS assay and flow cytometry assay showed that the combination treatment remarkably enhanced the cytotoxic effects of CDDP and BTZ( P < 0. 01). Western blot results demonstrated the combination treatment further enhanced the PARP cleavage,which was a marker of cell apoptosis. Interestingly,the expression of Bi P,a marker for the unfolded protein response,was elevated after treatment of BTZ. Conclusion Eer Ⅰ combined with other chemotherapeutic drugs significantly inhibits the growth of SW480 and PANC- 1,thus establishes p97 as a novel target in the treatment of colon and pancreatic cancer.
Objective To characterise the inhibitory effect of Eer Ⅰ,a small molecule targeting the key protein of the ERAD pathway,p97,alone or in combination with the traditional anticancer drugs on tumor cells. Methods The effect of Eer Ⅰ singularly and combined with cisplatin( CDDP) or bortezomib( BTZ) on tumor cell proliferation and apoptosis was measured through MTS assay and flow cytometry. Western blot was used to detect the cleavge of PARP and GRP78 in SW480 and PANC- 1 cells after treatments. Results Eer Ⅰ significantly inhibited cell proliferation and induced apoptosis of SW480 and PANC- 1 in a dose- dependent manner compared with the control group( P < 0. 01). MTS assay and flow cytometry assay showed that the combination treatment remarkably enhanced the cytotoxic effects of CDDP and BTZ( P < 0. 01). Western blot results demonstrated the combination treatment further enhanced the PARP cleavage,which was a marker of cell apoptosis. Interestingly,the expression of Bi P,a marker for the unfolded protein response,was elevated after treatment of BTZ. Conclusion Eer Ⅰ combined with other chemotherapeutic drugs significantly inhibits the growth of SW480 and PANC- 1,thus establishes p97 as a novel target in the treatment of colon and pancreatic cancer.
引文
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11 Wang Q,Mora-Jensen H,Weniger MA,et al.ERAD inhibitors integrate ER stress with an epigenetic mechanism to activate BH3-only protein NOXA in cancer cells[J].Proc Natl Acad Sci U S A,2009,106(7):2200-2205
12 Mandic A,Hansson J,Linder S,et al.Cisplatin induces endoplasmic reticulum stress and nucleus-independent apoptotic signaling[J].J Biol Chem,2003,278(11):9100-9106
13 Kretowski R,Borzym-Kluczyk M,Cechowska-Pasko M.Efficient induction of apoptosis by proteasome inhibitor:bortezomib in the human breast cancer cell line MDA-MB-231[J].Mol Cell Biochem,2014,389(1-2):177-185
14 Xu S,Peng G,Wang Y,et al.The AAA-ATPase p97 is essential for outer mitochondrial membrane protein turnover[J].Mol Biol Cell,2011,22(3):291-300
15 Rumpf S,Lee SB,Jan LY,et al.Neuronal remodeling and apoptosis require VCP-dependent degradation of the apoptosis inhibitor DIAP1[J].Development,2011,138(6):1153-1160
16 Raman M,Havens CG,Walter JC,et al.A genome-wide screen identifies p97 as an essential regulator of DNA damage-dependent CDT1 destruction[J].Mol Cell,2011,44(1):72-84
17 Meyer H,Bug M,Bremer S.Emerging functions of the VCP/p97AAA-ATPase in the ubiquitin system[J].Nat Cell Biol,2012,14(2):117-123
18 Magnaghi P,D'Alessio R,Valsasina B,et al.Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death[J].Nat Chem Biol,2013,9(9):548-556
19 Brem GJ,Mylonas I,Bruning A.Eeyarestatin causes cervical cancer cell sensitization to bortezomib treatment by augmenting ER stress and CHOP expression[J].Gynecol Oncol,2013,128(2):383-390
20 Auner HW,Moody AM,Ward TH,et al.Combined inhibition of p97and the proteasome causes lethal disruption of the secretory apparatus in multiple myeloma cells[J].PLo S One,2013,8(9):e74415
2 Healy SJ,Gorman AM,Mousavi-Shafaei P,et al.Targeting the endoplasmic reticulum-stress response as an anticancer strategy[J].Eur J Pharmacol,2009,625(1-3):234-246
3 Tsai YC,Weissman AM.The Unfolded Protein Response,Degradation from Endoplasmic Reticulum and Cancer[J].Genes Cancer,2010,1(7):764-778
4 Liu Y,Ye Y.Proteostasis regulation at the endoplasmic reticulum:a new perturbation site for targeted cancer therapy[J].Cell Res,2011,21(6):867-883
5 Luo B,Lee AS.The critical roles of endoplasmic reticulum chaperones and unfolded protein response in tumorigenesis and anticancer therapies[J].Oncogene,2013,32(7):805-818
6 Chapman E,Fry AN,Kang M.The complexities of p97 function in health and disease[J].Mol Biosyst,2011,7(3):700-710
7 Valle CW,Min T,Bodas M,et al.Critical role of VCP/p97 in the pathogenesis and progression of non-small cell lung carcinoma[J].PLo S One,2011,6(12):e29073
8 Tsujimoto Y,Tomita Y,Hoshida Y,et al.Elevated expression of valosin-containing protein(p97)is associated with poor prognosis of prostate cancer[J].Clin Cancer Res,2004,10(9):3007-3012
9 Yamamoto S,Tomita Y,Hoshida Y,et al.Increased expression of valosin-containing protein(p97)is associated with lymph node metastasis and prognosis of pancreatic ductal adenocarcinoma[J].Ann Surg Oncol,2004,11(2):165-172
10 Yi P,Higa A,Taouji S,et al.Sorafenib-mediated targeting of the AAA(+)ATPase p97/VCP leads to disruption of the secretory pathway,endoplasmic reticulum stress,and hepatocellular cancer cell death[J].Mol Cancer Ther,2012,11(12):2610-2620
11 Wang Q,Mora-Jensen H,Weniger MA,et al.ERAD inhibitors integrate ER stress with an epigenetic mechanism to activate BH3-only protein NOXA in cancer cells[J].Proc Natl Acad Sci U S A,2009,106(7):2200-2205
12 Mandic A,Hansson J,Linder S,et al.Cisplatin induces endoplasmic reticulum stress and nucleus-independent apoptotic signaling[J].J Biol Chem,2003,278(11):9100-9106
13 Kretowski R,Borzym-Kluczyk M,Cechowska-Pasko M.Efficient induction of apoptosis by proteasome inhibitor:bortezomib in the human breast cancer cell line MDA-MB-231[J].Mol Cell Biochem,2014,389(1-2):177-185
14 Xu S,Peng G,Wang Y,et al.The AAA-ATPase p97 is essential for outer mitochondrial membrane protein turnover[J].Mol Biol Cell,2011,22(3):291-300
15 Rumpf S,Lee SB,Jan LY,et al.Neuronal remodeling and apoptosis require VCP-dependent degradation of the apoptosis inhibitor DIAP1[J].Development,2011,138(6):1153-1160
16 Raman M,Havens CG,Walter JC,et al.A genome-wide screen identifies p97 as an essential regulator of DNA damage-dependent CDT1 destruction[J].Mol Cell,2011,44(1):72-84
17 Meyer H,Bug M,Bremer S.Emerging functions of the VCP/p97AAA-ATPase in the ubiquitin system[J].Nat Cell Biol,2012,14(2):117-123
18 Magnaghi P,D'Alessio R,Valsasina B,et al.Covalent and allosteric inhibitors of the ATPase VCP/p97 induce cancer cell death[J].Nat Chem Biol,2013,9(9):548-556
19 Brem GJ,Mylonas I,Bruning A.Eeyarestatin causes cervical cancer cell sensitization to bortezomib treatment by augmenting ER stress and CHOP expression[J].Gynecol Oncol,2013,128(2):383-390
20 Auner HW,Moody AM,Ward TH,et al.Combined inhibition of p97and the proteasome causes lethal disruption of the secretory apparatus in multiple myeloma cells[J].PLo S One,2013,8(9):e74415