氯化钴对人卵巢癌SKOV3细胞顺铂敏感性的影响
|
摘要
目的:观察氯化钴(CoCl_2)作用于人卵巢癌SKOV3细胞株后对SKOV3细胞顺铂敏感性的影响,并阐述其可能机制。方法:体外培养SKOV3细胞株,随机分为对照组、CoCl_2组、顺铂(DDP)组和CoCl_2联用DDP (联合)组,CoCl_2组细胞以200μmol·L~(-1) CoCl_2作用4h后换普通细胞培养基培养20h,DDP组细胞以含10 mg·L~(-1) DDP的细胞培养基培养24h,联合组细胞以200μmol·L~(-1) CoCl_2作用4h后更换含10mg·L~(-1) DDP的细胞培养基继续培养20h。MTT法检测各组细胞存活率,免疫荧光法检测各组细胞中低氧诱导因子1α(HIF-1α)和诱导型一氧化氮合酶(iNOS)阳性表达强度,Rhod 2-AM荧光探针检测各组细胞线粒体钙离子(Ca~(2+))水平,免疫蛋白印迹(Western blotting)法观察凋亡相关蛋白细胞色素C (cyto C)、半胱氨酸天冬氨酸蛋白酶3 (caspase 3)和活化半胱氨酸天冬氨酸蛋白酶3 (cleaved caspase 3)蛋白表达水平,Muse~凋亡试剂盒检测各组细胞凋亡率。结果:与对照组比较,CoCl_2组细胞存活率无明显变化(P>0.05),其余2组细胞存活率明显下降(P<0.05);且联合组高于DDP组(P<0.05)。与对照组比较,CoCl_2组和联合组细胞中HIF-1α阳性表达强度明显增强(P<0.05);DDP组和联合组细胞中iNOS阳性表达强度明显增强(P<0.05)。与对照组比较,DDP组和联合组细胞Ca~(2+)水平升高(P<0.05);且DDP组高于联合组(P<0.05)。与对照组比较,CoCl_2组细胞中cyto C、caspase 3和cleaved caspase 3蛋白表达水平无明显变化(P>0.05),DDP组细胞中cyto C、caspase 3和cleaved caspase 3蛋白表达水平明显升高(P<0.05);且联合组低于DDP组(P<0.05)。与对照组比较,DDP组细胞凋亡率明显升高(P<0.05);联合组细胞凋亡率较DDP组降低(P<0.05)。结论:CoCl_2可以通过抑制DDP诱导的人卵巢癌SKOV3细胞株iNOS表达增强来减少线粒体途径凋亡的发生,降低其顺铂敏感性。
Objective:To observe the effect of CoCl_2 on the cisplatin sensitivity of human ovarian cancer SKOV3 cells,and to clarify the possible mechanism.Methods:The SKOV3 cells were cultured in vitro and randomly divided into control group,CoCl_2 group,cisplatin(DDP)group and CoCl_2 combined with DDP(combination)group.The cells in CoCl_2 group were cultured in normal cell medium for 20 hafter cultured in 200μmol·L~(-1) CoCl_2 for 4 h,the cells in DDP group were cultured in normal cell medium containing 10 mg·L~(-1) DDP for 24 h,and the cells in combination group were cultured in 10 mg·L~(-1) DDP for 20 hafter cultured in 200μmol·L~(-1) CoCl_2 for 4 h.The survival rates of SKOV3 cells in various groups were detected by MTT method,and the positive expression intensities of hypoxia-inducible factor-1α(HIF-1α)and inducible nitric oxide synthase(iNOS)in the cells in various groups were detected by immunofluorescence method.Rhod 2-AM fluorescence probe was used to observe the levels of Ca~(2+)in mitochondria in the cells in various groups.Western blotting method was used to observe the expression levels of cytochrome C(cyto C),cysteinyl aspartase 3(caspase 3)and cleaved cysteinyl aspartase 3(cleaved caspase 3).Muse~ apoptosis assay kit was used to detect the apoptotic rates of cells in various groups.Results:Compared with control group,the survival rate of the cells in CoCl_2 group had no significant change(P>0.05),and the survival rates of the cells in DDP and combination groups were decreased(P<0.05);the survival rate in combination group was higher than that in DDP group(P<0.05).Compared with control group,the positive expression intensities of HIF-1αin CoCl_2 and combination groups were increased(P<0.05).Compared with control group,the positive expressions of iNOS in DDP and combination groups were increased(P<0.05).The Ca~(2+)levels in the cells in DDP group and combination groups were higher than that in control group(P<0.05)and the Ca~(2+)level in DDP group was higher than that in combination group(P<0.05).Compared with control group,the expression levels of cyto C,caspase 3 and cleaved caspase 3 proteins in the SKOV3 cells in CoCl_2 group had no significant changes(P>0.05),and the expression levels of cyto C,caspase 3 and cleaved caspase 3 in DDP group were increased significantly(P <0.05);compared with DDP group,they were lower than those in combination group(P<0.05).Compared with control group,the apoptotic rate of SKOV3 cells in DDP group was increased significantly(P<0.05);the apoptotic rate of SKOV3 cells in combination group was lower than that in DDP group(P<0.05).Conclusion:CoCl_2 can redece the mitochondrial apoptosis of human ovarian cancer SKOV3 cells by inhibiting the DDP-induced enhancement of iNOS expression and decrease the sensitivity of SKOV3 cells to cisplatin.
Objective:To observe the effect of CoCl_2 on the cisplatin sensitivity of human ovarian cancer SKOV3 cells,and to clarify the possible mechanism.Methods:The SKOV3 cells were cultured in vitro and randomly divided into control group,CoCl_2 group,cisplatin(DDP)group and CoCl_2 combined with DDP(combination)group.The cells in CoCl_2 group were cultured in normal cell medium for 20 hafter cultured in 200μmol·L~(-1) CoCl_2 for 4 h,the cells in DDP group were cultured in normal cell medium containing 10 mg·L~(-1) DDP for 24 h,and the cells in combination group were cultured in 10 mg·L~(-1) DDP for 20 hafter cultured in 200μmol·L~(-1) CoCl_2 for 4 h.The survival rates of SKOV3 cells in various groups were detected by MTT method,and the positive expression intensities of hypoxia-inducible factor-1α(HIF-1α)and inducible nitric oxide synthase(iNOS)in the cells in various groups were detected by immunofluorescence method.Rhod 2-AM fluorescence probe was used to observe the levels of Ca~(2+)in mitochondria in the cells in various groups.Western blotting method was used to observe the expression levels of cytochrome C(cyto C),cysteinyl aspartase 3(caspase 3)and cleaved cysteinyl aspartase 3(cleaved caspase 3).Muse~ apoptosis assay kit was used to detect the apoptotic rates of cells in various groups.Results:Compared with control group,the survival rate of the cells in CoCl_2 group had no significant change(P>0.05),and the survival rates of the cells in DDP and combination groups were decreased(P<0.05);the survival rate in combination group was higher than that in DDP group(P<0.05).Compared with control group,the positive expression intensities of HIF-1αin CoCl_2 and combination groups were increased(P<0.05).Compared with control group,the positive expressions of iNOS in DDP and combination groups were increased(P<0.05).The Ca~(2+)levels in the cells in DDP group and combination groups were higher than that in control group(P<0.05)and the Ca~(2+)level in DDP group was higher than that in combination group(P<0.05).Compared with control group,the expression levels of cyto C,caspase 3 and cleaved caspase 3 proteins in the SKOV3 cells in CoCl_2 group had no significant changes(P>0.05),and the expression levels of cyto C,caspase 3 and cleaved caspase 3 in DDP group were increased significantly(P <0.05);compared with DDP group,they were lower than those in combination group(P<0.05).Compared with control group,the apoptotic rate of SKOV3 cells in DDP group was increased significantly(P<0.05);the apoptotic rate of SKOV3 cells in combination group was lower than that in DDP group(P<0.05).Conclusion:CoCl_2 can redece the mitochondrial apoptosis of human ovarian cancer SKOV3 cells by inhibiting the DDP-induced enhancement of iNOS expression and decrease the sensitivity of SKOV3 cells to cisplatin.
引文
[1]LALRINPUII E,BHAGEERATHY P S,SEBASTIAN A,et al.Ovarian cancer in young women[J].Indian J Surg Oncol,2017,8(4):540-547.
[2]MUINAO T,DEKA BORUAH H P,PAL M.Diagnostic and prognostic biomarkers in ovarian cancer and the potential roles of cancer stem cells-An updated review[J].Exp Cell Res,2018,362(1):1-10.
[3]SHABANA A M,MONDAL U K,ALAM M R,et al.pH-Sensitive multiligand gold nanoplatform targeting carbonic anhydraseⅨenhances the delivery of doxorubicin to hypoxic tumor spheroids and overcomes the hypoxia-Induced chemoresistance[J].ACS Appl Mater Interfaces,2018,10(21):17792-17808.
[4]FU J,ZHANG J,GONG Y,et al.Regulation of HIF-1alpha by the proprotein convertases furin and PC7in human squamous carcinoma cells[J].Mol Carcinog,2015,54(9):698-706.
[5]COURTNAY R,NGO D C,MALIK N,et al.Cancer metabolism and the Warburg effect:the role of HIF-1and PI3K[J].Mol Biol Rep,2015,42(4):841-851.
[6]SONG X,YANG C,ZHANG H,et al.Hypoxia-inducible factor-1α(HIF-1α)expression on endothelial cells in juvenile nasopharyngeal angiofibroma:A review of 70cases and tissue microarray analysis[J].Ann Otol Rhinol Laryngol,2018,127(6):357-366.
[7]CHENG Y,FENG Y,XIA Z,,et al.ω-Alkynyl arachidonic acid promotes anti-inflammatory macrophage M2polarization against acute myocardial infarction via regulating the cross-talk between PKM2,HIF-1αand iNOS[J].Biochim Biophys Acta,2017,1862(12):1595-1605.
[8]BARBEN M,SAMARDZIJA M,GRIMM C.The role of hypoxia,hypoxia-inducible factor(HIF),and VEGF in retinal angiomatous proliferation[J].Adv Exp Med Biol,2018,1074:177-183.
[9]ZHANG X,LUO H.Effects of thalidomide on growth and VEGF-A expression in SW480colon cancer cells[J].Oncol Lett,2018,15(3):3313-3320.
[10]JIN P,KANG J,LEE M K,et al.Ferritin heavy chain controls the HIF-driven hypoxic response by activating the asparaginyl hydroxylase FIH[J].Biochem Biophys Res Commun,2018,499(3):475-481.
[11]CHEN P J,WENG J Y,HSU P H,et al.NPGPx modulates CPEB2-controlled HIF-1αRNA translation in response to oxidative stress[J].Nucleic Acids Res,2015,43(19):9393-3404.
[12]CHEN R,XU J,SHE Y,et al.Necrostatin-1 protects C2C12myotubes from CoCl2-induced hypoxia[J].Int J Mol Med,2018,41(5):2565-2572.
[13]PECORARO M,PINTO A,POPOLO A.Inhibition of Connexin 43translocation on mitochondria accelerates CoCl2-induced apoptotic response in a chemical model of hypoxia[J].Toxicol In Vitro,2018,47(3):120-128.
[14]ZHANG M,MA R,LI Q.Inhibitory action of CoCl2-induced MCF-7cell hypoxia model of breast cancer and its influence on vascular endothelial growth factor[J].J Biol Regul Homeost Agents,2015,29(3):671-676.
[15]WANG G L,SEMENZA G L.General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia[J].Proc Natl Acad Sci U S A,1993,90(9):4304-4308.
[16]HSU J T,LE P H,LIN C J,et al.Mechanism of salutary effects of melatonin-mediated liver protection after traumahemorrhage:p38 MAPK-dependent iNOS/HIF-1αpathway[J].Am J Physiol Gastrointest Liver Physiol,2017,312(5):427-433.
[17]PEA-MERCADO E,GARCIA-LORENZANA M,ARECHAGE-OCAMPO E,et al.Evaluation of HIF-1αand iNOS in ischemia/reperfusion gastric model:bioimpedance,histological and immunohistochemical analyses[J].Histol Histopathol,2018,33(8):815-823.
[18]RIEMANN A,REIME S THEWS O.Tumor acidosis and hypoxia differently modulate the inflammatory program:measurements in vitro and in vivo[J].Neoplasia,2017,19(12):1033-1042.
[19]CASILLAN A J,CHAO J,WOOD J G,et al.Acclimatization of the systemic microcirculation to alveolar hypoxia is mediated by an iNOS-dependent increase in nitric oxide availability[J].J Appl Physiol,2017,123(4):974-982.
[20]YU Y,XIE Q,LIU W,et al.Increased intracellular Ca2+decreases cisplatin resistance by regulating iNOS expression in human ovarian cancer cells[J].Biomed Pharmacother,2017,86(2):8-15.
[21]BERTERO E,MAACK C.Calcium signaling and reactive oxygen species in mitochondria[J].Circ Res,2018,122(10):1460-1478.
[2]MUINAO T,DEKA BORUAH H P,PAL M.Diagnostic and prognostic biomarkers in ovarian cancer and the potential roles of cancer stem cells-An updated review[J].Exp Cell Res,2018,362(1):1-10.
[3]SHABANA A M,MONDAL U K,ALAM M R,et al.pH-Sensitive multiligand gold nanoplatform targeting carbonic anhydraseⅨenhances the delivery of doxorubicin to hypoxic tumor spheroids and overcomes the hypoxia-Induced chemoresistance[J].ACS Appl Mater Interfaces,2018,10(21):17792-17808.
[4]FU J,ZHANG J,GONG Y,et al.Regulation of HIF-1alpha by the proprotein convertases furin and PC7in human squamous carcinoma cells[J].Mol Carcinog,2015,54(9):698-706.
[5]COURTNAY R,NGO D C,MALIK N,et al.Cancer metabolism and the Warburg effect:the role of HIF-1and PI3K[J].Mol Biol Rep,2015,42(4):841-851.
[6]SONG X,YANG C,ZHANG H,et al.Hypoxia-inducible factor-1α(HIF-1α)expression on endothelial cells in juvenile nasopharyngeal angiofibroma:A review of 70cases and tissue microarray analysis[J].Ann Otol Rhinol Laryngol,2018,127(6):357-366.
[7]CHENG Y,FENG Y,XIA Z,,et al.ω-Alkynyl arachidonic acid promotes anti-inflammatory macrophage M2polarization against acute myocardial infarction via regulating the cross-talk between PKM2,HIF-1αand iNOS[J].Biochim Biophys Acta,2017,1862(12):1595-1605.
[8]BARBEN M,SAMARDZIJA M,GRIMM C.The role of hypoxia,hypoxia-inducible factor(HIF),and VEGF in retinal angiomatous proliferation[J].Adv Exp Med Biol,2018,1074:177-183.
[9]ZHANG X,LUO H.Effects of thalidomide on growth and VEGF-A expression in SW480colon cancer cells[J].Oncol Lett,2018,15(3):3313-3320.
[10]JIN P,KANG J,LEE M K,et al.Ferritin heavy chain controls the HIF-driven hypoxic response by activating the asparaginyl hydroxylase FIH[J].Biochem Biophys Res Commun,2018,499(3):475-481.
[11]CHEN P J,WENG J Y,HSU P H,et al.NPGPx modulates CPEB2-controlled HIF-1αRNA translation in response to oxidative stress[J].Nucleic Acids Res,2015,43(19):9393-3404.
[12]CHEN R,XU J,SHE Y,et al.Necrostatin-1 protects C2C12myotubes from CoCl2-induced hypoxia[J].Int J Mol Med,2018,41(5):2565-2572.
[13]PECORARO M,PINTO A,POPOLO A.Inhibition of Connexin 43translocation on mitochondria accelerates CoCl2-induced apoptotic response in a chemical model of hypoxia[J].Toxicol In Vitro,2018,47(3):120-128.
[14]ZHANG M,MA R,LI Q.Inhibitory action of CoCl2-induced MCF-7cell hypoxia model of breast cancer and its influence on vascular endothelial growth factor[J].J Biol Regul Homeost Agents,2015,29(3):671-676.
[15]WANG G L,SEMENZA G L.General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia[J].Proc Natl Acad Sci U S A,1993,90(9):4304-4308.
[16]HSU J T,LE P H,LIN C J,et al.Mechanism of salutary effects of melatonin-mediated liver protection after traumahemorrhage:p38 MAPK-dependent iNOS/HIF-1αpathway[J].Am J Physiol Gastrointest Liver Physiol,2017,312(5):427-433.
[17]PEA-MERCADO E,GARCIA-LORENZANA M,ARECHAGE-OCAMPO E,et al.Evaluation of HIF-1αand iNOS in ischemia/reperfusion gastric model:bioimpedance,histological and immunohistochemical analyses[J].Histol Histopathol,2018,33(8):815-823.
[18]RIEMANN A,REIME S THEWS O.Tumor acidosis and hypoxia differently modulate the inflammatory program:measurements in vitro and in vivo[J].Neoplasia,2017,19(12):1033-1042.
[19]CASILLAN A J,CHAO J,WOOD J G,et al.Acclimatization of the systemic microcirculation to alveolar hypoxia is mediated by an iNOS-dependent increase in nitric oxide availability[J].J Appl Physiol,2017,123(4):974-982.
[20]YU Y,XIE Q,LIU W,et al.Increased intracellular Ca2+decreases cisplatin resistance by regulating iNOS expression in human ovarian cancer cells[J].Biomed Pharmacother,2017,86(2):8-15.
[21]BERTERO E,MAACK C.Calcium signaling and reactive oxygen species in mitochondria[J].Circ Res,2018,122(10):1460-1478.