雷公藤内酯醇对肝癌细胞株HepG2的作用和作用机制
摘要
研究背景
雷公藤内酯醇(Triptlide, TPL)是从卫矛科雷公藤属植物中提取出来的具有多种生物活性的小分子化合物,是雷公藤提取物中主要的活性成分,主要用于自身免疫性疾病及炎性疾病的治疗。最近研究发现TPL对白血病、卵巢癌、肾癌等多种肿瘤均有很强的抑制作用,具有广谱的抗肿瘤作用,其抗肿瘤作用主要与caspase通路诱导细胞凋亡有关。肝癌是常见恶性肿瘤,其发病率居常见肿瘤第五位,死亡率仅次于胃癌和食管癌,而在我国仅次于肺癌,是恶性肿瘤的第二致死原因。目前有关TPL对肝癌的作用研究较少,其作用及作用机制不清。
研究目的
研究TPL对肝癌细胞株HepG2增殖抑制和凋亡诱导作用及HepG2由caspase-3、8、9与PARP(聚ADP核糖聚合酶)蛋白的表达变化情况,探讨TPL抗肿瘤作用及作用机制,为其临床应用提供实验依据。
研究方法
以加培养基细胞为空白对照组,按预计量加入TPL,使TPL终浓度为10、20、40、80nmol/L的HepG2细胞为实验组。分别收集培养24、48和72小时HepG2细胞,MTT法检测TPL对细胞增殖的影响；收集培养24小时HepG2细胞,hoechst荧光染色观察凋亡细胞形态变化,AnnexinV/PI流式细胞仪标记法检测TPL对HepG2细胞凋亡作用的影响,western blot检测caspase-3、8、9与PARP的表达情况。
研究结果
经MTT检测,10、20、40、80nmol/L浓度的TPL作用24小时HepG2细胞的抑制率分别为15.2、22.5、34.9、40.7%；48小时抑制率分别为21.5、34.8、44.1、44.8%；72小时抑制率分别为31.6、47.2、51.6、56.0%；抑制作用呈时间和剂量的依赖性。TPL对HepG2的IC50为38nmol/L。10、20、40、80nmol/L浓度的TPL作用24小时的HepG2细胞,hoechst荧光染色显微镜下均出现明显的凋亡细胞；AnnexinV/PI流式细胞仪检测其凋亡率分别为8.89%、9.81%、14.69%、22.04%,对照组抑制率为7.35%,其凋亡率呈浓度依赖性；western blot结果显示,其caspase-3、9、PARP蛋白活化显影,且均较对照组显影增强,显影强度随TPL浓度的增加而增强,未见caspase-8活化显影。
研究结论
TPL对HepG2细胞有增殖抑制作用,抑制作用呈时间和剂量依赖性；
TPL可以诱导HepG2发生凋亡,凋亡率随浓度的增高而增高；
TPL诱导细胞凋亡的机制与caspase-3、9活化有关即与线粒体通路相关,与caspase-8即死亡受体通路无关。
Background
Triptolide is a diterpenoid isolated from a traditional Chinese medicinal plant Tripterygium wilfordii Hook F, which has been widely used as traditional Chinese medicine to treat inflammatory and autoimmune diseases such as rheumatoid arthritis、systemic lupus and psoriatic arthritis.TPL is also the primary element of Tripterygium wilfordii Hook F,which has potent activity in not only anti-inflammation and immune modulation, but also antiproliferative and proapoptotic activity in many different types of cancer cells. The mechanism of TPL may be related to caspase pathway. Hepatocellular carcinoma is the fifth most common malignance worldwide and the second leading cause of cancer-related death in China. Only a few reports about the effect and mechanism of TPL on hepatocellular carcinoma were published.The mechanisms underlying triptolide-induced apoptosis in hepatocellular carcinoma are not clarified.
Purposes
To investigate the effect of the proliferation and apoptosis, and the expression of actived caspase-3.8.9 and PARP of Hep G2 cells treated by TPL, and explore the effect and mechanism of TPL on Hep G2 cells.
Methods
TPL was added into HepG2 cells with the working concentration of 10、20、40、80nmol/L as the TPL treated groups, and the culture medium was added as the control groups. HepG2 cells were harvested which had cultured for 24、48 and 72 houres and the inhibitory rates in the HepG2 cells were assessed by MTT assay. In the HepG2 cells which had cultured for 24 hours, the morphological characters were observed by hoechst staining, the apoptosis rates were determined by AnnexinV/PI flow cytometry, and the expression of actived caspase-3.8.9 and PARP proteins was examined by western blot.
Results
The inhibitory rates of HepG2 had cultured for 24 hours with 10.20.40.80nmol/L concentration respectively were 15.2、22.5、34.9、40.7%, for 48 hours were 21.5、34.8、44.1、44.8%, for 72 hours were 31.6、47.2、51.6、56.0%. The inhibitory rates were in a dose-and time-dependent.IC50 of TPL on HepG2 was 38nmol/L. In the HepG2 cells treated by TPL had cultured for 24 hours, typical apoptotic cells were observed via hoechst staining. The apoptosis rates of the HepG2 cells treated by TPL with 10、20、40、80nmol/L concentration were 8.89%、9.81%、14.69%、22.04%respectively via FITC-Annexin V/PI flow cytometry. The expression of actived caspase-3、9 and PARP proteins in the cells that treated with TPL were higher compared to the control groups, and the expression increased with the concentration of TPL.
Conclusions
Our results suggest that TPL could inhibit the proliferation and induce the apoptosis of HepG2. The apoptosis mechanism of TPL on HepG2 cells was related with the expression of actived caspase-3.9 and PARP proteins and the mitochondrial pathway, not related with caspase-8 and the death receptor pathway.
雷公藤内酯醇(Triptlide, TPL)是从卫矛科雷公藤属植物中提取出来的具有多种生物活性的小分子化合物,是雷公藤提取物中主要的活性成分,主要用于自身免疫性疾病及炎性疾病的治疗。最近研究发现TPL对白血病、卵巢癌、肾癌等多种肿瘤均有很强的抑制作用,具有广谱的抗肿瘤作用,其抗肿瘤作用主要与caspase通路诱导细胞凋亡有关。肝癌是常见恶性肿瘤,其发病率居常见肿瘤第五位,死亡率仅次于胃癌和食管癌,而在我国仅次于肺癌,是恶性肿瘤的第二致死原因。目前有关TPL对肝癌的作用研究较少,其作用及作用机制不清。
研究目的
研究TPL对肝癌细胞株HepG2增殖抑制和凋亡诱导作用及HepG2由caspase-3、8、9与PARP(聚ADP核糖聚合酶)蛋白的表达变化情况,探讨TPL抗肿瘤作用及作用机制,为其临床应用提供实验依据。
研究方法
以加培养基细胞为空白对照组,按预计量加入TPL,使TPL终浓度为10、20、40、80nmol/L的HepG2细胞为实验组。分别收集培养24、48和72小时HepG2细胞,MTT法检测TPL对细胞增殖的影响；收集培养24小时HepG2细胞,hoechst荧光染色观察凋亡细胞形态变化,AnnexinV/PI流式细胞仪标记法检测TPL对HepG2细胞凋亡作用的影响,western blot检测caspase-3、8、9与PARP的表达情况。
研究结果
经MTT检测,10、20、40、80nmol/L浓度的TPL作用24小时HepG2细胞的抑制率分别为15.2、22.5、34.9、40.7%；48小时抑制率分别为21.5、34.8、44.1、44.8%；72小时抑制率分别为31.6、47.2、51.6、56.0%；抑制作用呈时间和剂量的依赖性。TPL对HepG2的IC50为38nmol/L。10、20、40、80nmol/L浓度的TPL作用24小时的HepG2细胞,hoechst荧光染色显微镜下均出现明显的凋亡细胞；AnnexinV/PI流式细胞仪检测其凋亡率分别为8.89%、9.81%、14.69%、22.04%,对照组抑制率为7.35%,其凋亡率呈浓度依赖性；western blot结果显示,其caspase-3、9、PARP蛋白活化显影,且均较对照组显影增强,显影强度随TPL浓度的增加而增强,未见caspase-8活化显影。
研究结论
TPL对HepG2细胞有增殖抑制作用,抑制作用呈时间和剂量依赖性；
TPL可以诱导HepG2发生凋亡,凋亡率随浓度的增高而增高；
TPL诱导细胞凋亡的机制与caspase-3、9活化有关即与线粒体通路相关,与caspase-8即死亡受体通路无关。
Background
Triptolide is a diterpenoid isolated from a traditional Chinese medicinal plant Tripterygium wilfordii Hook F, which has been widely used as traditional Chinese medicine to treat inflammatory and autoimmune diseases such as rheumatoid arthritis、systemic lupus and psoriatic arthritis.TPL is also the primary element of Tripterygium wilfordii Hook F,which has potent activity in not only anti-inflammation and immune modulation, but also antiproliferative and proapoptotic activity in many different types of cancer cells. The mechanism of TPL may be related to caspase pathway. Hepatocellular carcinoma is the fifth most common malignance worldwide and the second leading cause of cancer-related death in China. Only a few reports about the effect and mechanism of TPL on hepatocellular carcinoma were published.The mechanisms underlying triptolide-induced apoptosis in hepatocellular carcinoma are not clarified.
Purposes
To investigate the effect of the proliferation and apoptosis, and the expression of actived caspase-3.8.9 and PARP of Hep G2 cells treated by TPL, and explore the effect and mechanism of TPL on Hep G2 cells.
Methods
TPL was added into HepG2 cells with the working concentration of 10、20、40、80nmol/L as the TPL treated groups, and the culture medium was added as the control groups. HepG2 cells were harvested which had cultured for 24、48 and 72 houres and the inhibitory rates in the HepG2 cells were assessed by MTT assay. In the HepG2 cells which had cultured for 24 hours, the morphological characters were observed by hoechst staining, the apoptosis rates were determined by AnnexinV/PI flow cytometry, and the expression of actived caspase-3.8.9 and PARP proteins was examined by western blot.
Results
The inhibitory rates of HepG2 had cultured for 24 hours with 10.20.40.80nmol/L concentration respectively were 15.2、22.5、34.9、40.7%, for 48 hours were 21.5、34.8、44.1、44.8%, for 72 hours were 31.6、47.2、51.6、56.0%. The inhibitory rates were in a dose-and time-dependent.IC50 of TPL on HepG2 was 38nmol/L. In the HepG2 cells treated by TPL had cultured for 24 hours, typical apoptotic cells were observed via hoechst staining. The apoptosis rates of the HepG2 cells treated by TPL with 10、20、40、80nmol/L concentration were 8.89%、9.81%、14.69%、22.04%respectively via FITC-Annexin V/PI flow cytometry. The expression of actived caspase-3、9 and PARP proteins in the cells that treated with TPL were higher compared to the control groups, and the expression increased with the concentration of TPL.
Conclusions
Our results suggest that TPL could inhibit the proliferation and induce the apoptosis of HepG2. The apoptosis mechanism of TPL on HepG2 cells was related with the expression of actived caspase-3.9 and PARP proteins and the mitochondrial pathway, not related with caspase-8 and the death receptor pathway.
引文
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[1]张鑫,陈元仲,雷公藤内酯醇抗肿瘤作用研究进展。中国医学研究与临床,2008,6(4)： 41-44
[2]许建华,李常春,黄自强,雷公藤内酯对L1210等癌细胞的体外杀伤作用。福建医学院学报,1993,27(3)：197-200
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[5]张天虹,高箐,李玉茹等,雷公藤内酯醇对人喉癌Hep-2细胞增殖的影响。哈尔滨医科大学学报,2008,42(2)：118-121
[6]Choi YJ, Kim TG, Kim YH, et al. Immunosuppressant PG490(triplolide) induces apoptosis through the activation of caspase-3 and down-regulation of XIAP in U937 cells[J], Biochem Pharmacol,2003,66(2):273-280
[7]王恒邦,许建华,温彩霞等,雷公藤内酯醇体内外抗肿瘤作用[J]。福建医科大学学报,2007,41(4)：304—307
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[1]张鑫,陈元仲,雷公藤内酯醇抗肿瘤作用研究进展。中国医学研究与临床,2008,6(4)： 41-44
[2]许建华,李常春,黄自强,雷公藤内酯对L1210等癌细胞的体外杀伤作用。福建医学院学报,1993,27(3)：197-200
[3]黄秀旺,许建华,陈元仲,雷公藤内酯醇在大鼠体内的组织分布。中国临床药理学与治疗学,2008,13(7)：764—767
[4]刘明星,董静,杨亚江等,雷公藤内酯醇的研究进展[J]。中国中药杂志,2005,30(3)：170—174
[5]张天虹,高箐,李玉茹等,雷公藤内酯醇对人喉癌Hep-2细胞增殖的影响。哈尔滨医科大学学报,2008,42(2)：118-121
[6]Choi YJ, Kim TG, Kim YH, et al. Immunosuppressant PG490(triplolide) induces apoptosis through the activation of caspase-3 and down-regulation of XIAP in U937 cells[J], Biochem Pharmacol,2003,66(2):273-280
[7]王恒邦,许建华,温彩霞等,雷公藤内酯醇体内外抗肿瘤作用[J]。福建医科大学学报,2007,41(4)：304—307
[8]崔国惠,陈卫华,张纯等, 淋巴瘤细胞系Raji细胞血管内皮细胞生长因子表达及基质胶中ECV304细胞血管形成与雷公藤内酯醇的干预.中国组织工程研究与临床康复,2008,12(11)：2039-2042
[9]Yang S, Chen J. Guo Z. et al. Triptolide inhibits the growth and metastasis of solid tumors[J]. Mol Cancer Ther,2003,2(1):65-72
[1 0]刘芳,崔国惠,王妍等.雷公藤内酯醇对K562细胞增殖周期的影响及对比研究[j].中国中西医结合杂志,2004,24(6)：255—256
[11]HUANG Huiwei, QIU Yihua. Inhibitory effect of triptolide on proliferation of PC12 cells[J]. Chinese Journal of Applied Physiology,2005,21(4):466-468
[12]Wang X, Matta R, Shen Q et al. Mechanism of triptolide-induced apoptosis:Effect on caspase activation and Bid cleavage and essentiality of the hydroxyl group of triptolide J Mol Med,2006,84(5):405-415.
[13]Wan CK, Wang C, Cheung HY, et al. Triptolide induces Bc 1-2 cleavage and Mitochindria dependent apoptosis in p53-deficien HL-60cells.Cancer Lett,2006, 241(1):31-41
[14]Dseagher S, Martinou JC. Mitochondria as the central control point of apoptosis[J]. Trends Cell Biol,2000,10(9):369-377
[15]Ashkenazi A, Dixit VM. Death receptors:signaling and modulation[J]. Science, 1998,281(5381):1305-1308
[16]欧阳曙明,杨业金,程静等,雷公藤内酯醇诱导非小细胞肺癌细胞凋亡及其机制初探。中国药师,2007,10(7)：629-631
[17]顾江红,董陆,宁维朔,朱晓玲,雷公藤内酯醇抑制卵巢癌细胞增殖并诱导凋亡作用初探。中医药学刊,2006,24(11)：2062-2064
[18]GX Zhou, XLDing,JF Huang et al,Apoptosis of human pancreatic cancer cells induced by Triptolide. World J Gastroenterol,2008,14(10):1504-1509
[19]Satchll MA, Zhang X, Kochanek PM, et al. A dual role for poly ADP-ribosylation in spa-tial memory acquisition after traumatic brain injury in mice involving NAD +depletion and ribosylation of 14-33gamma[J]. J Neurocbem,2003,85(3): 697-708
[20]高俊,陈洪雷,caspase-3、PARP蛋白在肺癌组织中的表达及临床意义。Journal of Mathematical Medicine,2006,19(1):34-36
[21]沈悦娣,邵雪婷,倪有娣等,虫草多糖增强雷公藤内酯醇诱导的HL-60细胞凋亡作用及其机制。浙江大学学报,2009,38(2),158-162
[22]胡可斌,刘志红,刘栋等,雷公藤内酯醇对内皮细胞血管内皮细胞生长因子活性的影响。肾脏病与透析肾移植杂志,2000,9(3)：229-233