海芒果种子提取物对人肝癌HepG2细胞增殖、周期和凋亡的影响及其作用机制
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摘要
肝癌有原发性肝癌和继发性肝癌之分,原发性肝癌为我国常见恶性肿瘤之一,也是我国恶性肿瘤防治研究的重点。死亡率在恶性肿瘤中居第三位,仅次于胃癌和食管癌。手术切除是目前治疗早期肝癌唯一有效的方法,但大多数患者就诊时已达病程中晚期,手术疗效受限,因而寻找新的治疗方法尤为重要。随着对肿瘤发生机制的深入研究,探讨诱导肿瘤细胞凋亡和细胞周期阻滞的方法成为提高临床疗效的新思路。强心苷是一类对心肌有兴奋作用、具有强心生理活性的甾体化合物,能选择性地作用于心肌,加强心肌收缩力,临床上用于治疗充血性心力衰竭及某些心律失常。然而,这类化合物在预防和治疗肿瘤方面正扮演着越来越重要的角色。在过去五年内,许多研究已经表明强心苷对恶性细胞具有优先选择性杀伤作用,而不影响正常细胞的增殖。因此,强心苷有望成为一种靶向治疗肿瘤的新型抗肿瘤药物。海芒果全株有毒,其中果仁的毒性最大,其毒性在于海芒果毒素,其分子结构与异羟洋地黄毒甙(一种强心剂)非常相似,会阻断钙离子在心肌中的传输通道,造成中毒者迅速死亡。显然,了解海芒果毒素的毒理、药理及其影响因素对开发海芒果有重要意义。同时,海芒果在医药方面也有开发价值。海芒果中含有多种强心苷类化合物,均为甲型强心苷。海芒果植株还含具有抗皮肤癌、乳腺癌和肺癌活性的强心苷。Laphookhieo等从白花海芒果种子中成功分离出一系列具有抗癌活性的强心苷类化合物。β-D-glucosyl-(1-4)-α-L-thevetosides of 17β-digitoxigenin (GHSC-73)和2'-epi-2'-O-Acetylthevetin B(GHSC-74)是从植物海芒果种子中提取的两种具有抗肿瘤活性的天然产物,属强心苷。国内外文献未曾报道过这两种化合物的抗肿瘤活性。而HepG2细胞来源于人肝癌组织,与正常肝细胞在细胞增殖动力学上具有同源性,因此本论文选取人肝癌细胞株HepG2作为实验对象,来研究GHSC-73和GHSC-74对其增殖,周期和凋亡的影响及其作用机制。
第一部分β-D-glucosyl-(1-4)-α-L-thevetosides of 17β-digitoxigenin (GHSC-73)对人肝癌HepG2细胞增殖、周期和凋亡的影响及其作用机制
目的:研究β-D-glucosyl-(1-4)-α-L-thevetosides of 17β-digitoxigenin (GHSC-73)对人肝癌HepG2细胞增殖,周期和凋亡的影响及其作用机制。
方法:采用MTT法检测GHSC-73对人肝癌细胞株HepG2,人张氏肝细胞Chang liver和小鼠胚胎成纤维细胞株3T3-Swiss albino增殖的影响;采用流式细胞仪Propidium Iodide (PI)单标法检测GHSC-73对HepG2细胞周期进程的影响;Real-timePCR检测S期相关基因mRNA表达的变化;采用流式细胞仪AnnexinⅤ-FITC/PI双标法和PI单标的sub-G1(细胞亚二倍体凋亡峰)法检测细胞凋亡率;应用Hoechst33342和DAPI荧光染色法观察GHSC-73处理后细胞形态的变化;DNA琼脂糖凝胶电泳检测DNA梯状条带;western blotting检测凋亡诱导因子(apoptosis-inducing factor, AIF)和核转录因子-κB(nuclear factor-kapperB,NF-κB)亚基p65蛋白表达水平;采用流式细胞仪JC-1染色法检测线粒体膜电位(ΔΨm)水平;免疫荧光染色检测AIF和NF-κB亚基p65的核移位;采用流式细胞仪检测荧光探针DCFH-DA标记的细胞内活性氧(reactive oxygen species, ROS);利用caspase-3和caspase-8活性检测试剂盒检测细胞内caspase-3和caspase-8酶活性;采用流式细胞仪异硫氰酸荧光素(FITC)标记的anti-human Fas抗体和藻红蛋白(PE)标记的anti-human FasL抗体检测细胞内Fas和FasL的蛋白表达水平。
结果:GHSC-73可抑制HepG2细胞增殖,其作用具有时间和浓度依赖性,在作用HepG2细胞24h,48h,72h后,IC50分别为5.18±0.21,0.37±0.08,1.66±0.16μM,但对人张氏肝细胞Chang liver和小鼠胚胎成纤维细胞株3T3-Swiss albino显示出较低的毒性,80μM GHSC-73作用人张氏肝细胞Chang liver和小鼠胚胎成纤维细胞株3T3-Swiss albino 48h后的抑制率分别为31.5%和36.1%。流式细胞仪检测细胞周期进程和sub-G1的结果显示,与对照组相比,随着作用时间的延长,S期的细胞百分数逐渐增多(p<0.05),而G0/G1期的细胞百分数逐渐减少(p<0.05),G2/M期细胞百分数基本保持不变,表明GHSC-73阻滞细胞于S期,同时细胞亚二倍体凋亡峰sub-G1随着时间的延长而逐渐增多(p<0.05),说明GHSC-73具有诱导HepG2细胞凋亡的作用。Real-time PCR检测S期相关基因的结果显示:GHSC-73诱导HepG2细胞阻滞于S期可能与p21,GADD153,Cyclin D1基因上调和Cyclin A2,DHFR,TYMS基因下调有关。流式细胞仪AnnexinⅤ/PI双标检测凋亡率的结果也显示随着药物作用时间的延长,早期凋亡细胞(AnnexinⅤ+/PI-)占总细胞数的比例也不断增加(p<0.05),进一步证明GHSC-73具有诱导HepG2凋亡的作用。通过荧光染料Hoechst33342和DAPI检测细胞形态学改变的结果显示GHSC-73作用HepG2细胞48h后可见典型的凋亡形态学改变,表现为部分细胞胞质缩小,胞质浓缩,核染色体高度凝聚、边缘化,细胞核碎裂呈碎片状,可见典型的新月形凋亡小体,而正常对照组的细胞核完整。DNA琼脂糖凝胶电泳检测的结果显示药物处理组细胞DNA电泳可见典型的“梯状”条带。GHSC-73诱导HepG2细胞凋亡过程伴随以下一系列细胞内活动:(1)线粒体膜电位降低;(2)暂时增加细胞内活性氧水平;(3)凋亡诱导因子发生从线粒体到核的移位;(4)caspase-3和caspase-8随着药物作用时间延长,酶活性也逐渐提高;(5)抑制NF-κB亚基p65的核移位。然而细胞凋亡时Fas和FasL在蛋白水平上的表达与对照组细胞相比没有明显变化。除此之外,广谱caspase抑制剂z-VAD-fmk(40μM),caspase-3抑制剂Ac-DEVD-CHO(20μM)和抗氧化剂NAC(10mM)都不能抑制GHSC-73诱导的HepG2细胞凋亡,而且不影响凋亡诱导因子的核移位,表明GHSC-73诱导HepG2细胞凋亡是经不依赖caspase和活性氧的凋亡通路。GHSC-73与IκBа磷酸化抑制剂BAY 11-7082(5μM)共同作用于HepG2细胞48h后,能显著提高HepG2细胞的早期凋亡率,说明NF-κB在HepG2细胞中是被激活的,同时结合免疫荧光染色和western blotting的结果显示GHSC-73可通过抑制p65的核移位进而抑制NF-κB的活性。
结论:GHSC-73可选择性的控制人肝癌HepG2细胞增殖,但是不影响人张氏肝细胞Chang liver和小鼠胚胎成纤维细胞株3T3-Swiss albino的增殖。GHSC-73可通过S期阻滞和细胞凋亡来抑制HepG2细胞的增殖,并且通过Caspase非依赖的凋亡诱导因子介导的线粒体通路和抑制NF-κB活性来诱导HepG2细胞凋亡,但是凋亡过程并不依赖活性氧的产生和Fas/FasL的相互作用。GHSC-73具有诱导肿瘤细胞凋亡的作用,并能使细胞周期进程发生变化。
第二部分2'-epi-2'-O-Acetylthevetin B (GHSC-74)对人肝癌HepG2细胞增殖、周期和凋亡的影响及其作用机制
目的:研究2'-epi-2'-O-Acetylthevetin B (GHSC-74)对人肝癌HepG2细胞增殖、周期和凋亡的影响及其作用机制。
方法:采用MTT法检测GHSC-74对人肝癌细胞株HepG2,人张氏肝细胞Chang liver和小鼠胚胎成纤维细胞株3T3-Swiss albino增殖的影响;采用流式细胞仪Propidium Iodide (PI)单标法检测GHSC-74对HepG2细胞周期进程的影响;利用Giemsa染色检测细胞分裂指数(Mitotic Index, MI);采用流式细胞仪AnnexinⅤ-FITC/PI双标法和PI单标的sub-G1(细胞亚二倍体凋亡峰)法检测细胞凋亡率;应用Hoechst33342和DAPI荧光染色法观察GHSC-74处理后细胞形态的变化;DNA琼脂糖凝胶电泳检测DNA梯状条带;western blotting检测凋亡诱导因子(apoptosis-inducing factor, AIF),CDC2 ,Cyclin B1和Bcl-2蛋白表达水平;采用流式细胞仪JC-1染色法检测线粒体膜电位(ΔΨm)水平;免疫荧光染色检测AIF;采用流式细胞仪检测荧光探针DCFH-DA标记的细胞内活性氧(reactive oxygen species, ROS);采用流式细胞仪异硫氰酸荧光素(FITC)标记的anti-human Fas抗体和藻红蛋白(PE)标记的anti-human FasL抗体检测细胞内Fas和FasL的蛋白表达水平;采用流式细胞仪荧光探针Fluo-3 AM检测细胞内钙离子浓度。
结果:GHSC-74可抑制HepG2细胞增殖,其作用具有时间和浓度依赖性,在作用HepG2细胞24h,48h,72h后,IC50分别为3.66±0.28,0.66±0.12,0.41±0.01μM,但对人张氏肝细胞Chang liver和小鼠胚胎成纤维细胞株3T3-Swiss albino显示出较低的毒性,80μM GHSC-74作用人张氏肝细胞Chang liver和小鼠胚胎成纤维细胞株3T3-Swiss albino 48h后的抑制率分别为11.8%和34.7%。流式细胞仪检测细胞周期进程和sub-G1的结果显示,与对照组相比,随着作用时间的延长,S期和G2/M期的细胞百分数逐渐增多(p<0.05),而G0/G1期的细胞百分数逐渐减少(p<0.05),表明GHSC-74阻滞细胞于S和G2/M期,同时细胞亚二倍体凋亡峰sub-G1随着时间的延长而逐渐增多(p<0.05),说明GHSC-74具有诱导HepG2细胞凋亡的作用。流式细胞术并不能区分细胞处于细胞周期中的G2期还是M期,因此通过分裂指数(Mitotic Index, MI)的计算与流式细胞术对G2/M期细胞量的测定相结合的方法来观察GHSC-74是否对M期细胞有阻滞作用,结果显示HepG2细胞经GHSC-74处理后细胞周期是被阻滞于G2期,同时western blotting的结果显示细胞周期相关蛋白CDC2和Cyclin B1表达水平随着药物作用时间的延长而逐渐减少。流式细胞仪AnnexinⅤ/PI双标检测凋亡率的结果也显示随着药物作用时间的延长,早期凋亡细胞(AnnexinⅤ+/PI-)占总细胞数的比例也不断增加(p<0.05),进一步证明GHSC-74具有诱导HepG2凋亡的作用。通过荧光染料Hoechst33342和DAPI检测细胞形态学改变的结果显示GHSC-74作用HepG2细胞48h后可见典型的凋亡形态学改变,表现为部分细胞胞质缩小,胞质浓缩,核染色体高度凝聚、边缘化,细胞核碎裂呈碎片状,可见典型的新月形凋亡小体,而正常对照组的细胞核完整。DNA琼脂糖凝胶电泳检测的结果显示药物处理组细胞DNA电泳可见典型的“梯状”条带。GHSC-74诱导HepG2细胞凋亡过程伴随以下一系列细胞内活动:(1)线粒体膜电位降低;(2)细胞内活性氧水平持续增加;(3)凋亡诱导因子发生从线粒体到核的移位;(4)细胞内钙浓度持续升高(5)抑制NF-κB亚基p65的核移位(6)Bcl-2的蛋白表达水平降低。然而细胞凋亡时Fas和FasL在蛋白水平上的表达与对照组细胞相比没有明显变化。除此之外,抗氧化剂NAC(10mM)不能完全抑制GHSC-74诱导的HepG2细胞凋亡,而且不影响凋亡诱导因子的核移位,表明GHSC-74诱导HepG2细胞凋亡是经活性氧依赖和不依赖的凋亡通路。广谱caspase抑制剂z-VAD-fmk(80μM)不影响GHSC-74诱导的HepG2细胞凋亡诱导因子的核移位。细胞内钙敖合剂BAPTA-AM能部分抑制GHSC-74诱导的HepG2细胞凋亡和线粒体膜电位的降低,而细胞外钙敖合剂EGTA几乎不能抑制GHSC-74诱导的HepG2细胞凋亡,说明细胞内钙离子浓度的升高是通过胞内钙库中Ca2+的重新分布,而不是Ca2+的内流造成的,GHSC-74诱导的HepG2细胞凋亡与Ca2+介导的线粒体通路有关。
结论:GHSC-74可选择性的控制人肝癌HepG2细胞增殖,但是不影响人张氏肝细胞Chang liver和小鼠胚胎成纤维细胞株3T3-Swiss albino的增殖。GHSC-74可通过S和G2期阻滞和细胞凋亡来抑制HepG2细胞的增殖,并且通过Caspase非依赖的凋亡诱导因子核移位,Ca2+介导的线粒体通路和活性氧的产生来诱导HepG2细胞凋亡,但是凋亡过程并不依赖Fas/FasL的相互作用。GHSC-74具有诱导肿瘤细胞凋亡的作用,并能使细胞周期进程发生变化。
Hepatocellular carcinoma (HCC) is generally acknowledged as the sixth most prevalent cancer in the word and is currently the third most common cause of cancer death with a 5-year survival rate of 7%. Hepatic resection and liver transplantation are the two mainstays of curative treatment for HCC, but can only be applied to the early stage of HCC. The majority of patients with HCC are diagnosed at a late stage when curative treatment options are not applicable. Thus, developing new therapeutic and preventive strategies targeted at apoptosis inducing and cell cycle arrest could be effective in controlling the proliferation and invasiveness as well as in the prognosis advanced stages of HCC.β-D-glucosyl-(1-4)-α-L-thevetosides of 17β-digitoxigenin (GHSC-73) and 2'-epi-2'-O-Acetylthevetin B (GHSC-74) are isolated from the seeds of Cerbera manghas L., belonging to the class of steroid-like compounds designated as cardiac glycosides. Their continued efficacy in the treatment of congestive heart failure and dysrhythmia is well appreciated. However, there is little knowledge about the role of this category of compounds in the prevention and/or treatment of proliferative diseases such as cancer. New findings in recent five years have demonstrated that these compounds are involved in complex cell-signal transduction mechanisms, inducing selective control of human tumors rather than normal cellular proliferation, and as such represent a promising candidate for targeted cancer chemotherapy. In this study, we investigated in vitro effects of GHSC-73 and GHSC-74 on cell growth, cell cycle regulation, and apoptosis in HepG2 cells, and explored their cellular mechanisms.
SectionⅠ:β-D-glucosyl-(1-4)-а?-L-thevetosides of 17β-digitoxigenin extracted from seeds of Cerbera manghas L. induces cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells
β-D-glucosyl-(1-4)-а?-L-thevetosides of 17β-digitoxigenin (GHSC-73) is a cardiac glycoside isolated from the seeds of Cerbera manghas L. The aim of this study is to investigate in vitro effects of GHSC-73 on cell growth, cell cycle regulation, and apoptosis in HepG2 cells. It was found that GHSC-73 reduced viability of HepG2 cells in a time- and dose-dependent manner without decreasing the viability of Chang human liver cells and Swiss albino 3T3 fibroblasts. GHSC-73 induced S phase arrest of the cell cycle. S phase arrest was accompanied with down-regulation of Cyclin A2, DHFR and TYMS gene expression, and up-regulation of p21, GADD153 and Cyclin D1 gene expression. GHSC-73 efficiently stimulated apoptosis in HepG2 cells as evidenced by DNA fragmentation, flow cytometry of sub-G1 DNA content, DAPI staining, AnnexinⅤ/PI binding assay and Hoechst 33342 staining. This apoptotic process was accompanied by the loss of mitochondrial membrane potential (ΔΨm), translocation of apoptosis-inducing factor (AIF) from the mitochondrion to the nucleus, a transient increase in intracellular reactive oxygen species (ROS), activation of caspase-3 and caspase-8, and inactivation of nuclear factor-kappaB (NF-κB). However, exposure to GHSC-73 did not produce significant up-regulation of Fas and FasL in HepG2 cells as detected by flow cytometric analysis. In addition, a broad-spectrum caspase inhibitor (z-VAD-fmk), a caspase-3 inhibitor (Ac-DEVD-CHO) and an antioxidant N-acetyl-L-cysteine (NAC) tested in this experiment failed to rescue HepG2 cells from GHSC-73-induced cell death and did not affect translocation of AIF from the mitochondrion to the nucleus after GHSC-73 treatment, suggesting that caspase and ROS-independent pathway was involved in GHSC-73-induced apoptosis. GHSC-73 combined with a specific inhibitor of IκBаphosphorylation BAY 11-7082 significantly increased the apoptotic rate of HepG2 cells, and GHSC-73 inhibited p65 nuclear translocation as analyzed by immunofluorescence microscopy and Western blotting. In conclusion, our results provide the first evidence for a molecular mechanism of cytotoxicity of GHSC-73, showing that GHSC-73 inhibited growth of HepG2 cells by inducing S arrest of the cell cycle and by triggering apoptosis via caspase-independent AIF release from mitochondria, inactivation of NF-κB, independent of Fas/FasL interaction and ROS generation. We also demonstrated that GHSC-73 induced selective control of human tumors rather than normal cellular proliferation, thus GHSC-73 may be a potential candidate of anti-cancer drugs.
SectionⅡ: 2'-epi-2'-O-Acetylthevetin B extracted from seeds of Cerbera manghas L. induces cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells
2'-epi-2'-O-Acetylthevetin B (GHSC-74), a cardiac glycoside, is isolated from the seeds of Cerbera manghas L. The aim of this study is to investigate in vitro effects of GHSC-74 on cell growth, cell cycle regulation, and apoptosis in HepG2 cells. It was found that GHSC-74 reduced viability of HepG2 cells in a time- and dose-dependent manner without decreasing the viability of Chang human liver cells and Swiss albino 3T3 fibroblasts. Cell cycle flow cytometry demonstrated that HepG2 cells treated with GHSC-74 (4μM) resulted in S and G2 phase arrest in a time-dependent manner, as confirmed by mitotic index analysis. G2 phase arrest was accompanied with down-regulation of CDC2 and Cyclin B1 protein. GHSC-74 efficiently induced apoptosis in HepG2 cells as evidenced by AnnexinⅤ/PI binding assay, Hoechst 33342 staining, DNA fragmentation, DAPI staining, and flow cytometric detection of sub-G1 DNA content. This apoptotic process was accompanied by dissipation of mitochondrial membrane potential, sustained elevation of intracellular [Ca2+], down-regulation of Bcl-2, a significant increase in reactive oxygen species (ROS), and translocation of apoptosis-inducing factor (AIF) from the mitochondrion to the nucleus. Nevertheless, after GHSC-74 exposure, no significant Fas and FasL up-regulation was observed in HepG2 cells by flow cytometry. In addition, treatment with antioxidant N-acetyl-L-cysteine (NAC) largely prevented apoptosis but did not abrogate GHSC-74-induced nuclear translocation of AIF, indicating that oxidative stress was involved in GHSC-74-mediated cell death. Treatment with Pan-caspase inhibitor z-VAD-fmk did not abrogate GHSC-74-induced nuclear translocation of AIF, indicating that AIF released from the mitochondrion is caspase-independent. BAPTA-AM, an intracellular Ca2+ chelator, partly suppressed cell death and prevented mitochondrial membrane potential from losing in GHSC-74-treated HepG2 cells. In contrast, EGTA, an extracellular Ca2+ chelator, exhibited a weaker effect as compared to that of BAPTA-AM. In conclusion, we have demonstrated that GHSC-74 inhibited growth of HepG2 cells by inducing S and G2 arrest of the cell cycle and by triggering apoptosis via caspase-independent AIF released from the mitochondrion, Ca2+-mediated mitochondrial pathway and ROS generation, independent of Fas/FasL interaction. We also demonstrated that GHSC-74 induced selective control of human tumors rather than normal cellular proliferation, thus GHSC-74 may be a potential candidate of anti-cancer drugs.
第一部分β-D-glucosyl-(1-4)-α-L-thevetosides of 17β-digitoxigenin (GHSC-73)对人肝癌HepG2细胞增殖、周期和凋亡的影响及其作用机制
目的:研究β-D-glucosyl-(1-4)-α-L-thevetosides of 17β-digitoxigenin (GHSC-73)对人肝癌HepG2细胞增殖,周期和凋亡的影响及其作用机制。
方法:采用MTT法检测GHSC-73对人肝癌细胞株HepG2,人张氏肝细胞Chang liver和小鼠胚胎成纤维细胞株3T3-Swiss albino增殖的影响;采用流式细胞仪Propidium Iodide (PI)单标法检测GHSC-73对HepG2细胞周期进程的影响;Real-timePCR检测S期相关基因mRNA表达的变化;采用流式细胞仪AnnexinⅤ-FITC/PI双标法和PI单标的sub-G1(细胞亚二倍体凋亡峰)法检测细胞凋亡率;应用Hoechst33342和DAPI荧光染色法观察GHSC-73处理后细胞形态的变化;DNA琼脂糖凝胶电泳检测DNA梯状条带;western blotting检测凋亡诱导因子(apoptosis-inducing factor, AIF)和核转录因子-κB(nuclear factor-kapperB,NF-κB)亚基p65蛋白表达水平;采用流式细胞仪JC-1染色法检测线粒体膜电位(ΔΨm)水平;免疫荧光染色检测AIF和NF-κB亚基p65的核移位;采用流式细胞仪检测荧光探针DCFH-DA标记的细胞内活性氧(reactive oxygen species, ROS);利用caspase-3和caspase-8活性检测试剂盒检测细胞内caspase-3和caspase-8酶活性;采用流式细胞仪异硫氰酸荧光素(FITC)标记的anti-human Fas抗体和藻红蛋白(PE)标记的anti-human FasL抗体检测细胞内Fas和FasL的蛋白表达水平。
结果:GHSC-73可抑制HepG2细胞增殖,其作用具有时间和浓度依赖性,在作用HepG2细胞24h,48h,72h后,IC50分别为5.18±0.21,0.37±0.08,1.66±0.16μM,但对人张氏肝细胞Chang liver和小鼠胚胎成纤维细胞株3T3-Swiss albino显示出较低的毒性,80μM GHSC-73作用人张氏肝细胞Chang liver和小鼠胚胎成纤维细胞株3T3-Swiss albino 48h后的抑制率分别为31.5%和36.1%。流式细胞仪检测细胞周期进程和sub-G1的结果显示,与对照组相比,随着作用时间的延长,S期的细胞百分数逐渐增多(p<0.05),而G0/G1期的细胞百分数逐渐减少(p<0.05),G2/M期细胞百分数基本保持不变,表明GHSC-73阻滞细胞于S期,同时细胞亚二倍体凋亡峰sub-G1随着时间的延长而逐渐增多(p<0.05),说明GHSC-73具有诱导HepG2细胞凋亡的作用。Real-time PCR检测S期相关基因的结果显示:GHSC-73诱导HepG2细胞阻滞于S期可能与p21,GADD153,Cyclin D1基因上调和Cyclin A2,DHFR,TYMS基因下调有关。流式细胞仪AnnexinⅤ/PI双标检测凋亡率的结果也显示随着药物作用时间的延长,早期凋亡细胞(AnnexinⅤ+/PI-)占总细胞数的比例也不断增加(p<0.05),进一步证明GHSC-73具有诱导HepG2凋亡的作用。通过荧光染料Hoechst33342和DAPI检测细胞形态学改变的结果显示GHSC-73作用HepG2细胞48h后可见典型的凋亡形态学改变,表现为部分细胞胞质缩小,胞质浓缩,核染色体高度凝聚、边缘化,细胞核碎裂呈碎片状,可见典型的新月形凋亡小体,而正常对照组的细胞核完整。DNA琼脂糖凝胶电泳检测的结果显示药物处理组细胞DNA电泳可见典型的“梯状”条带。GHSC-73诱导HepG2细胞凋亡过程伴随以下一系列细胞内活动:(1)线粒体膜电位降低;(2)暂时增加细胞内活性氧水平;(3)凋亡诱导因子发生从线粒体到核的移位;(4)caspase-3和caspase-8随着药物作用时间延长,酶活性也逐渐提高;(5)抑制NF-κB亚基p65的核移位。然而细胞凋亡时Fas和FasL在蛋白水平上的表达与对照组细胞相比没有明显变化。除此之外,广谱caspase抑制剂z-VAD-fmk(40μM),caspase-3抑制剂Ac-DEVD-CHO(20μM)和抗氧化剂NAC(10mM)都不能抑制GHSC-73诱导的HepG2细胞凋亡,而且不影响凋亡诱导因子的核移位,表明GHSC-73诱导HepG2细胞凋亡是经不依赖caspase和活性氧的凋亡通路。GHSC-73与IκBа磷酸化抑制剂BAY 11-7082(5μM)共同作用于HepG2细胞48h后,能显著提高HepG2细胞的早期凋亡率,说明NF-κB在HepG2细胞中是被激活的,同时结合免疫荧光染色和western blotting的结果显示GHSC-73可通过抑制p65的核移位进而抑制NF-κB的活性。
结论:GHSC-73可选择性的控制人肝癌HepG2细胞增殖,但是不影响人张氏肝细胞Chang liver和小鼠胚胎成纤维细胞株3T3-Swiss albino的增殖。GHSC-73可通过S期阻滞和细胞凋亡来抑制HepG2细胞的增殖,并且通过Caspase非依赖的凋亡诱导因子介导的线粒体通路和抑制NF-κB活性来诱导HepG2细胞凋亡,但是凋亡过程并不依赖活性氧的产生和Fas/FasL的相互作用。GHSC-73具有诱导肿瘤细胞凋亡的作用,并能使细胞周期进程发生变化。
第二部分2'-epi-2'-O-Acetylthevetin B (GHSC-74)对人肝癌HepG2细胞增殖、周期和凋亡的影响及其作用机制
目的:研究2'-epi-2'-O-Acetylthevetin B (GHSC-74)对人肝癌HepG2细胞增殖、周期和凋亡的影响及其作用机制。
方法:采用MTT法检测GHSC-74对人肝癌细胞株HepG2,人张氏肝细胞Chang liver和小鼠胚胎成纤维细胞株3T3-Swiss albino增殖的影响;采用流式细胞仪Propidium Iodide (PI)单标法检测GHSC-74对HepG2细胞周期进程的影响;利用Giemsa染色检测细胞分裂指数(Mitotic Index, MI);采用流式细胞仪AnnexinⅤ-FITC/PI双标法和PI单标的sub-G1(细胞亚二倍体凋亡峰)法检测细胞凋亡率;应用Hoechst33342和DAPI荧光染色法观察GHSC-74处理后细胞形态的变化;DNA琼脂糖凝胶电泳检测DNA梯状条带;western blotting检测凋亡诱导因子(apoptosis-inducing factor, AIF),CDC2 ,Cyclin B1和Bcl-2蛋白表达水平;采用流式细胞仪JC-1染色法检测线粒体膜电位(ΔΨm)水平;免疫荧光染色检测AIF;采用流式细胞仪检测荧光探针DCFH-DA标记的细胞内活性氧(reactive oxygen species, ROS);采用流式细胞仪异硫氰酸荧光素(FITC)标记的anti-human Fas抗体和藻红蛋白(PE)标记的anti-human FasL抗体检测细胞内Fas和FasL的蛋白表达水平;采用流式细胞仪荧光探针Fluo-3 AM检测细胞内钙离子浓度。
结果:GHSC-74可抑制HepG2细胞增殖,其作用具有时间和浓度依赖性,在作用HepG2细胞24h,48h,72h后,IC50分别为3.66±0.28,0.66±0.12,0.41±0.01μM,但对人张氏肝细胞Chang liver和小鼠胚胎成纤维细胞株3T3-Swiss albino显示出较低的毒性,80μM GHSC-74作用人张氏肝细胞Chang liver和小鼠胚胎成纤维细胞株3T3-Swiss albino 48h后的抑制率分别为11.8%和34.7%。流式细胞仪检测细胞周期进程和sub-G1的结果显示,与对照组相比,随着作用时间的延长,S期和G2/M期的细胞百分数逐渐增多(p<0.05),而G0/G1期的细胞百分数逐渐减少(p<0.05),表明GHSC-74阻滞细胞于S和G2/M期,同时细胞亚二倍体凋亡峰sub-G1随着时间的延长而逐渐增多(p<0.05),说明GHSC-74具有诱导HepG2细胞凋亡的作用。流式细胞术并不能区分细胞处于细胞周期中的G2期还是M期,因此通过分裂指数(Mitotic Index, MI)的计算与流式细胞术对G2/M期细胞量的测定相结合的方法来观察GHSC-74是否对M期细胞有阻滞作用,结果显示HepG2细胞经GHSC-74处理后细胞周期是被阻滞于G2期,同时western blotting的结果显示细胞周期相关蛋白CDC2和Cyclin B1表达水平随着药物作用时间的延长而逐渐减少。流式细胞仪AnnexinⅤ/PI双标检测凋亡率的结果也显示随着药物作用时间的延长,早期凋亡细胞(AnnexinⅤ+/PI-)占总细胞数的比例也不断增加(p<0.05),进一步证明GHSC-74具有诱导HepG2凋亡的作用。通过荧光染料Hoechst33342和DAPI检测细胞形态学改变的结果显示GHSC-74作用HepG2细胞48h后可见典型的凋亡形态学改变,表现为部分细胞胞质缩小,胞质浓缩,核染色体高度凝聚、边缘化,细胞核碎裂呈碎片状,可见典型的新月形凋亡小体,而正常对照组的细胞核完整。DNA琼脂糖凝胶电泳检测的结果显示药物处理组细胞DNA电泳可见典型的“梯状”条带。GHSC-74诱导HepG2细胞凋亡过程伴随以下一系列细胞内活动:(1)线粒体膜电位降低;(2)细胞内活性氧水平持续增加;(3)凋亡诱导因子发生从线粒体到核的移位;(4)细胞内钙浓度持续升高(5)抑制NF-κB亚基p65的核移位(6)Bcl-2的蛋白表达水平降低。然而细胞凋亡时Fas和FasL在蛋白水平上的表达与对照组细胞相比没有明显变化。除此之外,抗氧化剂NAC(10mM)不能完全抑制GHSC-74诱导的HepG2细胞凋亡,而且不影响凋亡诱导因子的核移位,表明GHSC-74诱导HepG2细胞凋亡是经活性氧依赖和不依赖的凋亡通路。广谱caspase抑制剂z-VAD-fmk(80μM)不影响GHSC-74诱导的HepG2细胞凋亡诱导因子的核移位。细胞内钙敖合剂BAPTA-AM能部分抑制GHSC-74诱导的HepG2细胞凋亡和线粒体膜电位的降低,而细胞外钙敖合剂EGTA几乎不能抑制GHSC-74诱导的HepG2细胞凋亡,说明细胞内钙离子浓度的升高是通过胞内钙库中Ca2+的重新分布,而不是Ca2+的内流造成的,GHSC-74诱导的HepG2细胞凋亡与Ca2+介导的线粒体通路有关。
结论:GHSC-74可选择性的控制人肝癌HepG2细胞增殖,但是不影响人张氏肝细胞Chang liver和小鼠胚胎成纤维细胞株3T3-Swiss albino的增殖。GHSC-74可通过S和G2期阻滞和细胞凋亡来抑制HepG2细胞的增殖,并且通过Caspase非依赖的凋亡诱导因子核移位,Ca2+介导的线粒体通路和活性氧的产生来诱导HepG2细胞凋亡,但是凋亡过程并不依赖Fas/FasL的相互作用。GHSC-74具有诱导肿瘤细胞凋亡的作用,并能使细胞周期进程发生变化。
Hepatocellular carcinoma (HCC) is generally acknowledged as the sixth most prevalent cancer in the word and is currently the third most common cause of cancer death with a 5-year survival rate of 7%. Hepatic resection and liver transplantation are the two mainstays of curative treatment for HCC, but can only be applied to the early stage of HCC. The majority of patients with HCC are diagnosed at a late stage when curative treatment options are not applicable. Thus, developing new therapeutic and preventive strategies targeted at apoptosis inducing and cell cycle arrest could be effective in controlling the proliferation and invasiveness as well as in the prognosis advanced stages of HCC.β-D-glucosyl-(1-4)-α-L-thevetosides of 17β-digitoxigenin (GHSC-73) and 2'-epi-2'-O-Acetylthevetin B (GHSC-74) are isolated from the seeds of Cerbera manghas L., belonging to the class of steroid-like compounds designated as cardiac glycosides. Their continued efficacy in the treatment of congestive heart failure and dysrhythmia is well appreciated. However, there is little knowledge about the role of this category of compounds in the prevention and/or treatment of proliferative diseases such as cancer. New findings in recent five years have demonstrated that these compounds are involved in complex cell-signal transduction mechanisms, inducing selective control of human tumors rather than normal cellular proliferation, and as such represent a promising candidate for targeted cancer chemotherapy. In this study, we investigated in vitro effects of GHSC-73 and GHSC-74 on cell growth, cell cycle regulation, and apoptosis in HepG2 cells, and explored their cellular mechanisms.
SectionⅠ:β-D-glucosyl-(1-4)-а?-L-thevetosides of 17β-digitoxigenin extracted from seeds of Cerbera manghas L. induces cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells
β-D-glucosyl-(1-4)-а?-L-thevetosides of 17β-digitoxigenin (GHSC-73) is a cardiac glycoside isolated from the seeds of Cerbera manghas L. The aim of this study is to investigate in vitro effects of GHSC-73 on cell growth, cell cycle regulation, and apoptosis in HepG2 cells. It was found that GHSC-73 reduced viability of HepG2 cells in a time- and dose-dependent manner without decreasing the viability of Chang human liver cells and Swiss albino 3T3 fibroblasts. GHSC-73 induced S phase arrest of the cell cycle. S phase arrest was accompanied with down-regulation of Cyclin A2, DHFR and TYMS gene expression, and up-regulation of p21, GADD153 and Cyclin D1 gene expression. GHSC-73 efficiently stimulated apoptosis in HepG2 cells as evidenced by DNA fragmentation, flow cytometry of sub-G1 DNA content, DAPI staining, AnnexinⅤ/PI binding assay and Hoechst 33342 staining. This apoptotic process was accompanied by the loss of mitochondrial membrane potential (ΔΨm), translocation of apoptosis-inducing factor (AIF) from the mitochondrion to the nucleus, a transient increase in intracellular reactive oxygen species (ROS), activation of caspase-3 and caspase-8, and inactivation of nuclear factor-kappaB (NF-κB). However, exposure to GHSC-73 did not produce significant up-regulation of Fas and FasL in HepG2 cells as detected by flow cytometric analysis. In addition, a broad-spectrum caspase inhibitor (z-VAD-fmk), a caspase-3 inhibitor (Ac-DEVD-CHO) and an antioxidant N-acetyl-L-cysteine (NAC) tested in this experiment failed to rescue HepG2 cells from GHSC-73-induced cell death and did not affect translocation of AIF from the mitochondrion to the nucleus after GHSC-73 treatment, suggesting that caspase and ROS-independent pathway was involved in GHSC-73-induced apoptosis. GHSC-73 combined with a specific inhibitor of IκBаphosphorylation BAY 11-7082 significantly increased the apoptotic rate of HepG2 cells, and GHSC-73 inhibited p65 nuclear translocation as analyzed by immunofluorescence microscopy and Western blotting. In conclusion, our results provide the first evidence for a molecular mechanism of cytotoxicity of GHSC-73, showing that GHSC-73 inhibited growth of HepG2 cells by inducing S arrest of the cell cycle and by triggering apoptosis via caspase-independent AIF release from mitochondria, inactivation of NF-κB, independent of Fas/FasL interaction and ROS generation. We also demonstrated that GHSC-73 induced selective control of human tumors rather than normal cellular proliferation, thus GHSC-73 may be a potential candidate of anti-cancer drugs.
SectionⅡ: 2'-epi-2'-O-Acetylthevetin B extracted from seeds of Cerbera manghas L. induces cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells
2'-epi-2'-O-Acetylthevetin B (GHSC-74), a cardiac glycoside, is isolated from the seeds of Cerbera manghas L. The aim of this study is to investigate in vitro effects of GHSC-74 on cell growth, cell cycle regulation, and apoptosis in HepG2 cells. It was found that GHSC-74 reduced viability of HepG2 cells in a time- and dose-dependent manner without decreasing the viability of Chang human liver cells and Swiss albino 3T3 fibroblasts. Cell cycle flow cytometry demonstrated that HepG2 cells treated with GHSC-74 (4μM) resulted in S and G2 phase arrest in a time-dependent manner, as confirmed by mitotic index analysis. G2 phase arrest was accompanied with down-regulation of CDC2 and Cyclin B1 protein. GHSC-74 efficiently induced apoptosis in HepG2 cells as evidenced by AnnexinⅤ/PI binding assay, Hoechst 33342 staining, DNA fragmentation, DAPI staining, and flow cytometric detection of sub-G1 DNA content. This apoptotic process was accompanied by dissipation of mitochondrial membrane potential, sustained elevation of intracellular [Ca2+], down-regulation of Bcl-2, a significant increase in reactive oxygen species (ROS), and translocation of apoptosis-inducing factor (AIF) from the mitochondrion to the nucleus. Nevertheless, after GHSC-74 exposure, no significant Fas and FasL up-regulation was observed in HepG2 cells by flow cytometry. In addition, treatment with antioxidant N-acetyl-L-cysteine (NAC) largely prevented apoptosis but did not abrogate GHSC-74-induced nuclear translocation of AIF, indicating that oxidative stress was involved in GHSC-74-mediated cell death. Treatment with Pan-caspase inhibitor z-VAD-fmk did not abrogate GHSC-74-induced nuclear translocation of AIF, indicating that AIF released from the mitochondrion is caspase-independent. BAPTA-AM, an intracellular Ca2+ chelator, partly suppressed cell death and prevented mitochondrial membrane potential from losing in GHSC-74-treated HepG2 cells. In contrast, EGTA, an extracellular Ca2+ chelator, exhibited a weaker effect as compared to that of BAPTA-AM. In conclusion, we have demonstrated that GHSC-74 inhibited growth of HepG2 cells by inducing S and G2 arrest of the cell cycle and by triggering apoptosis via caspase-independent AIF released from the mitochondrion, Ca2+-mediated mitochondrial pathway and ROS generation, independent of Fas/FasL interaction. We also demonstrated that GHSC-74 induced selective control of human tumors rather than normal cellular proliferation, thus GHSC-74 may be a potential candidate of anti-cancer drugs.
引文
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