复方大七气汤联合顺铂对卵巢癌SKOV3细胞的抑制作用
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摘要
目的探讨研究复方大七气汤(compound Daqiqi decoction,CDQD)联合顺铂对卵巢癌裸鼠皮下移植瘤抑制作用及其相关机制。方法采用人卵巢癌细胞株SKOV3构建60只裸鼠皮下移植瘤模型,随机分为6组,每组10只,分别为模型组(生理盐水),CDQD低剂量组(15.16 g·kg~(-1)),CDQD中剂量组(30.33 g·kg~(-1)),CDQD高剂量组(60.66 g·kg~(-1)),顺铂组(DDP,3mg·kg~(-1)),联合组(30.33 g·kg~(-1)CDQD+3 mg·kg~(-1)DDP),顺铂3 d·次~(-1),其余1 d·次~(-1),连续给药14 d,每3 d测瘤体积,治疗结束后剥取瘤体,称瘤重并计算抑瘤率,绘制肿瘤生长曲线。采用HE染色法观测各组肿瘤细胞形态,TUNEL法检测肿瘤组织细胞凋亡情况,实时荧光定量PCR和Western-blot检测肿瘤组织中miR-939、信号转导子与转录激活子3(STAT3)、血管内皮生长因子A(VEGFA)和血管内皮生长因子受体(EGFR)mRNA及蛋白的表达。结果与模型组相比,治疗组瘤体积、瘤重均减小(P<0.01),联合组瘤体积和瘤重均小于其余各组(P<0.01),联合组细胞凋亡率明显高于其余各组(P<0.01)。与模型组相比,治疗组中MiR-939、STAT3与VEGFA表达均下调,EGFR表达上升,其中联合组MiR-939、STAT3、VEGFA表达最低(P<0.05),EGFR表达最高(P<0.05)。结论 CDQD对卵巢癌裸鼠皮下移植瘤具有抑制作用,其机制可能与抑制MiR-939/STAT3通路激活,下调VEGFA、上调EGFR表达有关。CDQD对卵巢癌组织的抑制作用具有浓度依耐性,并且与DDP联合用药能增强DDP抗瘤作用,减轻DDP对荷瘤鼠的毒副作用。
OBJECTIVE To investigate the inhibitory effect of compound Daqiqi decoction( CDQD) combined with cisplatin on subcutaneously transplanted ovarian cancer in nude mice and its related mechanisms. METHODS The 60 subcutaneously transplanted model of nude mice was established with human ovarian cancer cell line SKOV3,and divided into 6 groups randomly,each group of 10 nude mice,which were model group that was treated with saline,CDQD low dose group with the CDQD dosage of 15. 16 g ·kg~(-1),CDQD medium dose group with the CDQD dosage of 30. 33 g·kg~(-1),CDQD high dose group with the CDQD dosage of 60. 66 g·kg~(-1),cisplatin group with the DDP dosage of 3 mg·kg~(-1) and combined group that was treated with the CDQD dosage of 30. 33 g·kg~(-1) and the DDP dosage of 3 mg·kg~(-1). Cisplatin was administered once every 3 d,and the remaining drugs were administered once a day. Then,the tumor-bearing mouse model was given the corresponding drugs for 14 consecutive days,and the tumor volume was measured every 3 d. After the end of treatment,the tumor was removed and weighed. The morphology of the tumor cells were observed by HE staining. The apoptosis of tumor cells was detected by TUNEL method. The mRNA and protein expression of miR939 and STAT3 and VEGFA and EGFR in tumor tissues were detected by real-time fluorescence quantitative PCR and Western-blot. RESULTS The tumor volume and the tumor weight of the treated groups were all decreased( P < 0. 01). Compared with the model group,the tumor volume and tumor weight of the combined group were less than those of the other groups( P < 0. 01),and the apoptosis rate of the combined group was significantly higher than other groups( P < 0. 01). The expression of miR939 and STAT3 and VEGFA were downregulated and the expression of EGFR were up-regulated in the treatment groups. Compared with the model group,the expression of MiR-939,STAT3 and VEGFA were down-regulated and the expression of EGFR was increased in the treatment group. MiR-939,STAT3,VEGFA expression in the combined group was the lowest( P < 0. 05),and the EGFR expression was highest( P < 0. 05).CONCLUSION Studies have shown that CDQD can inhibit ovarian cancer subcutaneously transplanted tumor in nude mice,and its mechanism may be related to inhibition of MiR-939/STAT3 pathway activation,down-regulation of VEGFA,and up-regulation of EGFR expression. The inhibitory effect of CDQD on ovarian cancer tissues has a concentration dependence. And the combination of CDQD and DDP can enhance the anti-tumor effect of DDP and reduce the side effects of DDP on tumor-bearing mice.
OBJECTIVE To investigate the inhibitory effect of compound Daqiqi decoction( CDQD) combined with cisplatin on subcutaneously transplanted ovarian cancer in nude mice and its related mechanisms. METHODS The 60 subcutaneously transplanted model of nude mice was established with human ovarian cancer cell line SKOV3,and divided into 6 groups randomly,each group of 10 nude mice,which were model group that was treated with saline,CDQD low dose group with the CDQD dosage of 15. 16 g ·kg~(-1),CDQD medium dose group with the CDQD dosage of 30. 33 g·kg~(-1),CDQD high dose group with the CDQD dosage of 60. 66 g·kg~(-1),cisplatin group with the DDP dosage of 3 mg·kg~(-1) and combined group that was treated with the CDQD dosage of 30. 33 g·kg~(-1) and the DDP dosage of 3 mg·kg~(-1). Cisplatin was administered once every 3 d,and the remaining drugs were administered once a day. Then,the tumor-bearing mouse model was given the corresponding drugs for 14 consecutive days,and the tumor volume was measured every 3 d. After the end of treatment,the tumor was removed and weighed. The morphology of the tumor cells were observed by HE staining. The apoptosis of tumor cells was detected by TUNEL method. The mRNA and protein expression of miR939 and STAT3 and VEGFA and EGFR in tumor tissues were detected by real-time fluorescence quantitative PCR and Western-blot. RESULTS The tumor volume and the tumor weight of the treated groups were all decreased( P < 0. 01). Compared with the model group,the tumor volume and tumor weight of the combined group were less than those of the other groups( P < 0. 01),and the apoptosis rate of the combined group was significantly higher than other groups( P < 0. 01). The expression of miR939 and STAT3 and VEGFA were downregulated and the expression of EGFR were up-regulated in the treatment groups. Compared with the model group,the expression of MiR-939,STAT3 and VEGFA were down-regulated and the expression of EGFR was increased in the treatment group. MiR-939,STAT3,VEGFA expression in the combined group was the lowest( P < 0. 05),and the EGFR expression was highest( P < 0. 05).CONCLUSION Studies have shown that CDQD can inhibit ovarian cancer subcutaneously transplanted tumor in nude mice,and its mechanism may be related to inhibition of MiR-939/STAT3 pathway activation,down-regulation of VEGFA,and up-regulation of EGFR expression. The inhibitory effect of CDQD on ovarian cancer tissues has a concentration dependence. And the combination of CDQD and DDP can enhance the anti-tumor effect of DDP and reduce the side effects of DDP on tumor-bearing mice.
引文
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[22]XIONG Y,QI L Y,ZHOU F,et al.MiR-939 promotes the proliferation of human ovarian cancer cells by repressing APC2 expression[J].Biomed Pharmacol,2015,71:64-69.
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[25]TANG M,JIANG L,LIN Y,et al.Platelet microparticle-mediated transfer of miR-939 to epithelial ovarian cancer cells promotes epithelial to mesenchymal transition[J].Oncot,2017,8(57):97464-97475.
[26]WRIGHT K L.Constitutive Stat3 activity up-regulates VEGF expression and tumor angiogenesis[J].Oncogene,2002,21(13):2000-2008.
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[2]FANG Y,CHEN R,ZHANG Q S,et al.Effect of compound Daqiqi Decoction on epithelial ovarian cancer in mice[J].Chin J Hosp Pharm(中国医院药学杂志),2013,33(14):1129-1134.
[3]CHEN Q.Chinese Medicine Pharmacological Research Methodology(中药药理研究方法学)[M].Beijing:People's Medical Publishing House,2011.
[4]DAI M,LUO R C,ZHENG D Y,et al.Effects of bevacizumab and cisplatin on human lung adenocarcinoma A549/DDP xenografts in nude mice[J].J Southern Med Univ(南京医科大学学报),2007,27(9):1402-1405.
[5]YANG Y B J,CHEN R,WANG S,et al.Effect of compound Daqiqi Decoction Combined with cisplatin on expression of Bcl-2/Bax in nude mice ovarian carcinoma subcutaneously transplanted[J].China J Chin Mater Med(中国中药杂志),2015,40(8):1575-1579.
[6]Marie France Poupon.Strong inhibition of ewing tumor xenograft growth by combination of human interferon-alpha or interferon-beta with ifosfamide[J].Oncogene,2002,21(50):7700-7709.
[7]GARRITY M M,BURGART L J,RIEHLE D L,et al.Identifying and quantifying apoptosis:navigating technical pitfalls[J].Mod Pathol Off J Unit States Canadian Acade Pathol Inc,2003,16(4):389.
[8]ZONDER J A,MOHRBACHER A F,SINGHAL S,et al.A phase 1,multicenter,open-label,dose escalation study of elotuzumab in patients with advanced multiple myeloma[J].Blood,2016,120(3):552-559.
[9]FOLKMAN J.Angiogenesis:an organizing principle for drug discovery?[J].Nature Reviews Drug Discovery,2007,6(4):273.
[10]XIONG Y,QI L Y,ZHOU F,et al.MiR-939 promotes the proliferation of human ovarian cancer cells by repressing APC2 expression[J].Biomed Pharmacol,2015,71:64-69.
[11]YU H,LEE H,HERRMANN A,et al.Revisiting STAT3 signalling in cancer:new and unexpected biological functions[J].Nat Rev Cancer,2014,14(11):736-746.
[12]TERME M,PERNOT S,MARCHETEAU E,et al.VEGFA-VEGFR pathway blockade inhibits tumor-induced regulatory T-cell proliferation in colorectal cancer[J].Cancer Res,2013,73(2):539-549.
[13]LI S C,ZHANG T,WEI X D.Anti-tumor effect of Sanjiao extract on H22 tumor-bearing mice[J].Heilongjiang Med J(黑龙江医药科学),2010,33(5):78-78.
[14]WANG Z,ZHANG J F,FU G F.Effects of Rhizoma Zedoariae and sparganum on apoptosis of human lung cancer cells[J].J Capit Univ Med Sci(首都医科大学学报),2001,22(4):304-305.
[15]LI X J,JIA Y J,ZHANG W Z,et al.Induction of apoptosis of SP and NON-SP cells in A549[J].Chin Tradit Herb Drugs(中草药),2013,44(18):2581-2584.
[16]WEI P Y,PU H Q,WEI X,et al.Effect of Scutellaria barbata extract on apoptosis of human lung cancer SPC-A-1 cells and its effect on expression of apoptosis-related genes[J].Chin Med Mater(中药材),2007,30(10):1270-1273.
[17]LV S,YANG P M,GAO G S.Advances in research on pharmacological effects and clinical application of Hedyotis diffusa[J].Chin J Inf Tradit Chin Med Pharm(中国中医药信息杂志),2016,23(3):122-125.
[18]ZHU H J,WANG K P,WANG R.Experimental study on antitumor effect of Bai Ying[J].Sci Tech Eng(科学技术与工程),2009,9(9):2304-2306.
[19]LEE Y,AHN C,HAN J,et al.The nuclear RNaseⅢdrosha initiates microRNA processing[J].Nature,2012,425(6956):415-419.
[20]ERIK V N,DIMOS G,JEAN H,et al.Inference of miRNA targets using evolutionary conservation and pathway analysis[J].Bmc Bioinf,2007,8(1):69.
[21]ZHAO M.miR-205 Targeting Down-regulation of PEG3 in endometrial adenocarcinoma cell lines[D].Jinan:Shandong University,2013.
[22]XIONG Y,QI L Y,ZHOU F,et al.MiR-939 promotes the proliferation of human ovarian cancer cells by repressing APC2 expression[J].Biomed Pharmacol,2015,71:64-69.
[23]ZHANG J X,XU Y,GAO Y,et al.Decreased expression of miR-939 contributes to chemoresistance and metastasis of gastric cancer via dysregulation of SLC34A2 and Raf/MEK/ERK pathway[J].Molec Cancer,2017,16(1):18.
[24]XIONG Y,QI L Y,ZHOU F,et al.MiR-939 promotes the proliferation of human ovarian cancer cells by repressing APC2 expression[J].Biomed Pharmacol,2015,71:64-69.
[25]TANG M,JIANG L,LIN Y,et al.Platelet microparticle-mediated transfer of miR-939 to epithelial ovarian cancer cells promotes epithelial to mesenchymal transition[J].Oncot,2017,8(57):97464-97475.
[26]WRIGHT K L.Constitutive Stat3 activity up-regulates VEGF expression and tumor angiogenesis[J].Oncogene,2002,21(13):2000-2008.
[27]DONG W J,GUO Y C,SONG H R.Effects of total saponins of Panax japonicus on the expression of vascular endothelial growth factor,angiopoietin-2 and receptor Tie-2 in synovium of collageninduced arthritis rats[J].Chin Pharm J(中国药学杂志),2013,48(2):101-105.