消癌解毒方通过自噬抑制人肝癌SMMC7721裸鼠移植瘤的研究
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摘要
目的:明确消癌解毒方(XAF)能否通过调控自噬抑制人肝癌移植瘤生长。方法:建立人肝癌SMMC7721裸鼠移植瘤模型,连续给药10d后,处死取瘤。对瘤体组织进行HE、TUNEL染色,并用Western Blot分析瘤体内LC3、p62、p-AKT、p-mTOR、Bax、Bcl-2、Cleaved Caspase-3蛋白表达水平。结果:XAF以剂量依赖方式抑制移植瘤生长(XAF低、中、高组IR分别为:19.80%,45.03%, 54.12%)。同时,硫酸羟氯喹(HCQ)降低了XAF的抑瘤作用(XAF+HCQ组IR:30.94%),而雷帕霉素(RAP)增强了XAF的抑瘤作用(XAF+RAP组IR:70.00%)。Western Blot结果显示,XAF能诱导LC3-Ⅱ蛋白表达,降低p62、p-AKT及p-mTOR蛋白表达。TUNEL和Western Blot结果均提示XAF能诱导移植瘤细胞凋亡,而且该凋亡诱导作用被HCQ减弱而被RAP增强。结论:XAF通过抑制AKT/mTOR信号通路激活的自噬,可能是其抑制移植瘤生长、促进肿瘤细胞凋亡的机制之一。
Objective: To determine Xiaoai Jiedu Formula inhibit the growth of human liver cancer transplanted tumor in nude mice by regulating autophagy. Methods: The human liver cancer SMMC7721 transplanted tumor models were established.After 10 d of treatment, mice were sacrificed and tumors were dissected. HE and TUNEL staining were used to detecte the tumors and apoptosis-related proteins in tumors by Western Blot. Results: Xiaoai Jiedu Formula could inhibit the growth of tumor dose-dependently(IR: 19.80%, 45.03%, 54.12% in low, medium and high Xiaoai Jiedu Formula groups). At the same time,hydroxychloroquine(HCQ) reduced the tumor inhibition effect of XAF(IR: 30.94% in XAF+HCQ group), while rapamycin(RAP)increased the tumor inhibition effect of XAF(IR: 70.00% in XAF+RAP group). Western Blot analysis showed that Xiaoai Jiedu Formula could increase the level of LC3-Ⅱ protein and decrease the levels of p62, p-AKT and p-mTOR proteins. The results of TUNEL and Western Blot assays indicated that Xiaoai Jiedu Formula could induce cell apoptosis of tumor tissues, which was weakened by HCQ and enhanced by RAP. Conclusion: The stimulated autophagy via inhibiting AKT/mTOR pathway by Xiaoai Jiedu Formula may be an important mechanism of Xiaoai Jiedu Formula inhibited tumor growth and induced apoptosis.
Objective: To determine Xiaoai Jiedu Formula inhibit the growth of human liver cancer transplanted tumor in nude mice by regulating autophagy. Methods: The human liver cancer SMMC7721 transplanted tumor models were established.After 10 d of treatment, mice were sacrificed and tumors were dissected. HE and TUNEL staining were used to detecte the tumors and apoptosis-related proteins in tumors by Western Blot. Results: Xiaoai Jiedu Formula could inhibit the growth of tumor dose-dependently(IR: 19.80%, 45.03%, 54.12% in low, medium and high Xiaoai Jiedu Formula groups). At the same time,hydroxychloroquine(HCQ) reduced the tumor inhibition effect of XAF(IR: 30.94% in XAF+HCQ group), while rapamycin(RAP)increased the tumor inhibition effect of XAF(IR: 70.00% in XAF+RAP group). Western Blot analysis showed that Xiaoai Jiedu Formula could increase the level of LC3-Ⅱ protein and decrease the levels of p62, p-AKT and p-mTOR proteins. The results of TUNEL and Western Blot assays indicated that Xiaoai Jiedu Formula could induce cell apoptosis of tumor tissues, which was weakened by HCQ and enhanced by RAP. Conclusion: The stimulated autophagy via inhibiting AKT/mTOR pathway by Xiaoai Jiedu Formula may be an important mechanism of Xiaoai Jiedu Formula inhibited tumor growth and induced apoptosis.
引文
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[2]程海波,吴勉华,周红光.周仲瑛从癌毒辨治恶性肿瘤的经验.北京中医药,2009,28(11):844-846
[3]Levine B, Yuan J.Autophagy in cell death:An innocent convict?J Clin Invest,2005,115(10):2679-2688
[4]Levine B.Cell biology:Autophagy and cancer.Nature,2007,446(7137):745-747
[5]White E,Mehnert J M,Chang C S.Autophagy,Metabolism,and Cancer.Clin Cancer Res,2015,21(22):5037-5046
[6]Chan K K,Wong O G,Wong E S,et al.Impact of iASPP on chemoresistance through PLK1 and autophagy in ovarian clear cell carcinoma.Int J Cancer,2018,143(6):1456-1469
[7]熊远珍.实验动物与人用药量的新换算.江西医学院学报,1997(4):41
[8]陈海彬,周红光,吴勉华,等.消癌解毒方对中晚期恶性肿瘤患者免疫功能的影响.南京医科大学学报(自然科学版),2009,29(9):1257-1259
[9]周红光,陈海彬,吴勉华,等.消癌解毒方配合化疗治疗中晚期恶性肿瘤临床疗效观察.中华中医药杂志,2010,25(7):1140-1143
[10]石海波.消癌解毒方联合化疗治疗晚期非小细胞肺癌(痰瘀郁毒型)的临床观察.南京:南京中医药大学,2018
[11]李黎.消癌解毒方对H22荷瘤小鼠和人肝癌SMMC-7721细胞的影响及机制研究.南京:南京中医药大学,2013
[12]李文婷,赵凤鸣,吴勉华,等.消癌解毒方含药血清对人肝癌SMMC-7721细胞端粒酶及凋亡相关基因m RNA表达的影响.中华中医药杂志,2016,31(4):1211-1214
[13]石文静,谭佳妮,程海波,等.消癌解毒方对结肠癌的抑制作用及机制探讨.中华中医药杂志,2018,33(1):69-72
[14]李文婷,周红光,吴勉华,等.消癌解毒方对移植性肝癌大鼠免疫功能影响研究.中华中医药学刊,2015,33(8):1880-1883
[15]Yang Z,Klionsky D J.An overview of the molecular mechanism of autophagy.Curr Top Microbiol Immunol,2009,335:1-32
[16]Chen S,Jiang Y Z,Huang L,et al.The residual tumor autophagy marker LC3B serves as a prognostic marker in local advanced breast cancer after neoadjuvant chemotherapy.Clin Cancer Res,2013,19(24):6853-6862
[17]Johansen T, Lamark T. Selective autophagy mediated by autophagic adapter proteins.Autophagy,2011,7(3):279-296
[18]Sooro M A,Zhang N,Zhang P.Targeting EGFR-mediated autophagy as a potential strategy for cancer therapy.Int J Cancer,2018,143(9):2116-2125
[19]Kondo Y,Kanzawa T,Sawaya R,et al.The role of autophagy in cancer development and response to therapy.Nat Rev Cancer,2005,5(9):726-734
[20]Nikoletopoulou V,Markaki M,Palikaras K,et al.Crosstalk between apoptosis,necrosis and autophagy.Biochim Biophys Acta,2013,1833(12):3448-3459
[21]Yang Z,Klionsky D J.Mammalian autophagy:Core molecular machinery andsignaling regulation.Curr Opin Cell Biol,2010,22(2):124-131