莱菔硫烷诱导人肝瘤HepG-2细胞G_2/M期阻滞及凋亡的机制研究
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摘要
据不完全统计,我国每年新增癌症病人160万。据WHO预测,到2020年世界癌症患者将增加到1470万人。因此,恶性肿瘤仍是本世纪严重危害人类健康的主要疾病之一。其中,肝癌是恶性程度很高的消化系肿瘤之一,在全世界范围内每年发病人数62.6万,发病率在全部恶性肿瘤中位居第六；每年死亡人数58.9万,致死率仅次于肺癌和胃癌位列全部癌症的第三位,且病死率呈逐年升高的趋势。目前,肝癌的治疗方法主要以手术、放疗和化疗为主,但这些方法大都毒副作用较大,很多患者最终不是死于癌症本身而是死于放化疗产生的毒副作用。因此,新型高效低毒抗癌药物的研制开发已经成为优化肿瘤治疗策略以及最终攻克癌症的希望所在。
莱菔硫烷(sulforaphane, SFN)是天然存在于十字花科植物中含有的一种异硫氰酸盐(isothiocyanates, ITCs)类物质,是十字花科植物中的硫代葡萄糖苷的酶解产物,在绿花椰菜中含量最高。大量研究表明,异硫氰酸盐可选择性地抑制Ⅰ相酶的活性,诱导Ⅱ相酶的活性,降低致癌物的活化,保护机体少受甚至不受损伤,显示出很强的化学预防作用。目前认为,SFN不仅在肿瘤发生的多个阶段发挥抑癌作用,而且它还是潜在的肿瘤治疗药物。SFN与肿瘤的关系研究正成为抗肿瘤药物研究领域的热点。
虽然关于肿瘤发病机制的学说很多,但总而言之,肿瘤是一类细胞周期紊乱和凋亡异常性疾病。以调控细胞周期和诱导细胞凋亡为靶点,寻找相关药物用于恶性肿瘤的治疗为抗肿瘤药物的研究提供了新的思路。本论文前期研究发现,绿花椰菜中的ITCs能够诱导人肝癌细胞系HepG-2细胞发生凋亡,进一步研究发现SFN是其中含量最高、活性最强的抗肝癌细胞活性成分。因此,本论文在前期工作的基础上,以人肝癌HepG-2细胞为对象,从细胞周期调控和诱导凋亡入手,系统研究SFN的抗肿瘤作用机制,为今后SFN作为抗肝癌药物的开发提供科学依据。
1莱菔硫烷抑制人肝癌HepG-2细胞增殖和诱导凋亡作用的研究
1.1莱菔硫烷对人肝癌细胞HepG-2增殖抑制作用研究
目的：研究SFN对人肝癌HepG-2细胞增殖的抑制作用。方法：采用MTT比色法和SRB法测定SFN对人肝癌HepG-2细胞增殖的抑制率,计算SFN对人肝癌HepG-2细胞的IC50和GI50；采用倒置显微镜观察细胞生长状态。结果：MTT法测定结果表明,不同浓度的SFN(5、10、20、40、80μmol/L)作用于HepG-2细胞24、48、72 h均可显著抑制细胞增殖(P<0.05或P<0.01),并呈一定的时间和剂量依赖性。经计算,SFN对HepG-2细胞24,48和72 h的IC50分别为32.03μmol/L±0.96μmol/L,20.90μmol/L±1.96μmol/L和13.87μmol/L±0.44μmol/L。SRB法测定结果表明,SFN作用于HepG-2细胞48h的IC50为21.80μmol/L,GI50为19.40μmol/L,与MTT结果相印证。倒置相差显微镜下观察到对照组细胞贴壁生长,细胞饱满,边缘清晰；而SFN作用后,贴壁细胞减少,细胞均逐渐变圆,体积变小,折光性减弱。结论：SFN对人肝癌HepG-2细胞的增殖具有较强的抑制作用。
1.2莱菔硫烷对人肝癌HepG-2细胞周期影响的研究
目的：研究SFN对人肝癌HepG-2细胞周期阻滞作用。方法：采用PI单染法,应用流式细胞仪分析SFN作用于人肝癌HepG-2细胞后细胞周期的分布变化。结果：10、20、40μmol/L的SFN作用HepG-2细胞48h后,各组G2/M期的细胞比例显著升高,且G2/M期细胞百分比随着药物浓度增加而增大,同时G1期细胞明显减少。SFN浓度达到40μmol/L时出现凋亡峰。结论：SFN可诱导人肝癌HepG-2细胞发生G2/M期阻滞。
1.3莱菔硫烷诱导人肝癌HepG-2细胞凋亡作用的研究目的：研究SFN对人肝癌HepG-2细胞凋亡诱导作用。方法：采用一步法TUNEL细胞凋亡检测试剂盒原位检测DNA片段化情况；采用Annexin V-FITC/PI双染,激光共聚焦显微镜观察细胞的早期凋亡情况；透射电镜观察肿瘤细胞超微结构变化；采用流式细胞仪(Annexin V-FITC/PI双染法)测定细胞凋亡率。结果：TUNEL法检测结果表明,SFN作用48h后,激光共聚焦显微镜下早期凋亡细胞核显绿色荧光。随着SFN剂量的增加凋亡细胞数量逐渐增多,而空白对照组细胞无明显着色。激光共聚焦显微镜下可见早期凋亡细胞的细胞膜被染成绿色,细胞核无明显着色。细胞膜被染成绿色,细胞核被染成红色是晚期凋亡细胞或坏死细胞。随着剂量的增加,镜下凋亡细胞数量也逐渐增加。电子显微镜下可见对照组细胞的细胞结构清晰,细胞器结构完整,核内染色质分布均匀,线粒体结构正常,细胞表面有微绒毛突起。而SFN作用后,细胞出现典型的凋亡形态,如微绒毛减少,细胞核内染色质固缩、凝集于核膜边界,形成新月状小体,核周间隙扩张,线粒体肿胀,胞浆空泡化,细胞形成凋亡小体。流式细胞仪检测结果表明,10、20、40μmol/L的SFN作用于HepG-2细胞48h后,早期凋亡细胞百分率分别达到27.42%±0.43%、46.53%±0.35%和58.92%±0.48%,与对照组1.6%±0.06%比较,差异有统计学意义(P<0.01)。结论：SFN可诱导人肝癌HepG-2细胞发生凋亡。
2莱菔硫烷诱导人肝癌HepG-2细胞G2/M期阻滞及凋亡的机制研究
2.1莱菔硫烷诱导人肝癌HepG-2细胞G2/M期阻滞的机制研究
目的：研究SFN诱导人肝癌HepG-2细胞G2/M期阻滞的分子作用机制。方法：采用Western Blot法检测SFN对人肝癌HepG-2细胞内CdKl,p-CdKl(Thr14), cyclinB1,Cdc25C,p21等G2/M期相关蛋白表达的影响。结果：10、20、40μmol/L的SFN作用HepG-2细胞48 h后,均可显著下调CdKl, cyclinB1, Cdc25C蛋白表达水平,与对照组比较差异具有统计学意义(P<0.01),同时显著上调p-CdKl(Thr14)和p21蛋白表达水平,与对照组比较差异具有统计学意义(P<0.05或P<0.01)。结论：SFN可通过降低CdK1-cyclinB1复合物的形成,同时通过下调Cdc25C表达、上调p21表达来抑制CdK1或CdKl-cyclinBl复合物的活性,诱导HepG-2细胞发生G2/M期阻滞。
2.2莱菔硫烷通过线粒体途径诱导人肝癌HepG-2细胞凋亡的机制研究
目的：研究SFN诱导人肝癌HepG-2细胞凋亡的分子作用机制。方法：采用酶活力检测试剂盒测定SFN对人肝癌HepG-2细胞Caspase-3,-8,-9活性的影响,采用激光共聚扫描焦显微镜测定细胞内线粒体膜电位(Δψm)和钙离子浓度,采用流式细胞仪测定人肝癌HepG-2细胞内ROS含量；采用Western blot法测定SFN对HepG-2细胞内Cyt-c蛋白表达的影响：采用Western blot法和RT-PCR法测定SFN对HepG-2细胞内Bcl-2、Bax表达的影响。结果：不同浓度的SFN作用于HepG-2细胞48h后,细胞线粒体膜电位Δψm随着SFN浓度的升高而逐渐降低,呈一定的剂量依赖关系；20、40μmol/L组FI值与对照组比较差异具有统计学意义(P<0.01)。细胞内钙离子浓度随着SFN浓度的升高而逐渐升高。10、20、40μmol/L SFN剂量组细胞内[Ca2+]i(FI)分别为45.68±10.51、88.62±16.09和176.33±25.14,与对照组比较差异具有统计学意义(P<0.01)。同时,细胞内活性氧水平显著升高,分别为28.74±4.59、52.90±6.61和90.32±5.95,与对照组比较差异具有统计学意义(P<0.01)。RT-PCR法和Western blot法检测结果表明,SFN能下调HepG-2细胞中Bcl-2 mRNA和蛋白的表达水平,同时上调Bax mRNA和蛋白表达水平,且Bcl-2/Bax和Bcl-2mRNA/Bax mRNA随剂量的增加而降低,与对照组比较差异具有统计学意义(P<0.05或P<0.01)。此外,SFN可上调细胞内Cyt-c蛋白表达量,并呈一定的剂量依赖性,与对照组相比差异具有统计学意义(P<0.05)。试剂盒检测结构表明,SFN可升高细胞内Caspase-3,-8,-9活性,并呈一定的剂量依赖性,各剂量组与对照组比较差异具有统计学意义(P<0.05或P<0.01)。结论：SFN可能是通过下调Bcl-2基因表达、上调Bax基因表达,进而诱导线粒体通透性转变孔道(PT孔道)的开放,促进线粒体内Cyt-c向胞浆的释放。释放的Cyt-c可能进一步与Apaf-1、procaspase-9结合形成凋亡体,活化的caspase-9进一步激活下游的caspase-3,最终完成SFN通过线粒体途径诱导HepG-2细胞的凋亡过程。
2.3莱菔硫烷通过MAPK信号转导途径诱导人肝癌HepG-2细胞凋亡机制研究
目的：阐明MAPK信号传导通路在SFN诱导人肝癌HepG-2细胞凋亡中的作用。方法：采用Western blot法研究SFN对人肝癌HepG-2细胞中MAPK信号通路相关蛋白P38、p-P38、ERK、p-ERK、JNK/SAPK和p-JNK/SAPK蛋白表达的影响。结果：10、20、40μmol/L的SFN作用人肝癌HepG-2细胞48h后,随着药物浓度的增加,p-P38和p-JNK蛋白表达量逐渐增加,而p-ERK的表达量逐渐降低,与对照组比较差异具有统计学意义(P<0.05或P<0.01)。同时,P38、ERK和JNK蛋白相对表达量与对照组相比并无显著性差异(P>0.05)。结论：MAPK信号转导途径在SFN诱导人肝癌HepG-2细胞周期阻滞和凋亡过程中发挥重要作用,SFN可通过启动MAPK信号通路诱导人肝癌HepG-2细胞G2/M期阻滞进而引起细胞凋亡。
结论
SFN对人肝癌HepG-2细胞具有显著地抗肿瘤作用,其可能的作用机制如下：①SFN可通过降低CdK1-cyclinB1复合物的形成,同时通过下调Cdc25C表达、上调p21表达来抑制CdK1或CdK1-cyclinB1复合物的活性,诱导HepG-2细胞发生G2/M期阻滞；②SFN可通过下调Bcl-2基因表达、上调Bax基因表达,进而诱导线粒体通透性转变孔道(PT孔道)的开放,促进线粒体内Cyt-c向胞浆的释放,进而激活caspase-9和下游的caspase-3,最终通过线粒体途径诱导HepG-2细胞发生凋亡；③SFN可通过激活p38MAPK信号转导通路,作用于Cdc25C磷酸酶而使其失活,导致其无法激活Cdk1而将细胞阻滞于G2/M期；同时,通过促进fas/fasL介导的细胞凋亡途径激活Caspase-8,启动Caspase级联反应而诱导HepG-2细胞凋亡,但这一推测仍有待研究证实；④SFN可通过下调p-ERK的表达抑制ERK通路的激活,进而抑制Bcl-2所致的抗凋亡作用,促进细胞凋亡；⑤SFN可通过促进JNK的磷酸化促进线粒体介导的细胞凋亡；同时,激活Fas/FasL介导的细胞凋亡途径而激活Caspase-8,最终激活Caspase-3诱导细胞凋亡；活化的JNK/SAPKs还可通过翻译后的磷酸化修饰稳定p21的活性,进而抑制Cdkl的活化,诱导细胞发生G2/M期阻滞。
本研究的创新点
1.首次研究发现启动G2/M期DNA损伤检查点调节机制是SFN诱导人肝癌HepG-2细胞周期阻滞的主要分子机制。
2.首次研究发现线粒体介导的凋亡通路是SFN诱导人肝癌HepG-2细胞凋亡的主要途径,下调Bcl-2基因表达、上调Bax基因表达是SFN启动内源性凋亡通路的关键。
3.首次研究发现MAPK信号通路是SFN诱导人肝癌HepG-2细胞G2/M期阻滞和凋亡的关键调节通路和重要作用靶点。
According to incomplete statistics, every year there occur about 1.6 million new cancer sufferers in our country:According to the predication of WHO, the number of cancer patients will increased to 14.7 million. Malignant tumor is one of the severe diseases harmful to human health. Liver cancer is a tumor of digestive system with high degree of malignancy, which annually causes more than 626,000 cases. The incidence of liver cancer ranks the 6th among whole malignant tumor. The death toll of liver cancer is 589,000 every year and the lethality occupies the first place next to lung carcinoma and gastric carcinoma. The mortality is higher gradually in recent years. Surgical procedures and postoperative chemotherapy and radiotherapy are the main treatments at present, but which have more side effects. Many of patients died with not liver cancer but the side effects of chemotherapy and radiotherapy. Therefore, the preparation of new-type antineoplastic agents of high efficiency and low toxicity holds the best hope for the success of tumor therapy.
Sulforaphane (SFN), one of naturally occurring isothiocyanates (ITCs) found in crucifer, is the enzymatic hydrolysate of Glucosinolates, which shows the highest amount in Brassica oleracea Linnaeus var. botrytis Linnaeus. A large number of studies show that ITCs can inhibit the activity of the phase I drug metabolizing enzyme, induce the activity of the phase II drug metabolizing enzyme and decrease activation of carcinogen, which can protect against injury. ITCs show a strong chemopreventive potential. At present, SFN not only restrains tumor during different stages of carcinogenesis, but also is a potential drug in the oncotherapy. The antitumor effect of SFN is a research focus in antineoplastic research field.
Though there are many theories on pathogenesis of cancer, in a word, cancer is a diseases associated to cell cycle and apoptosis deregulation. Researching the drug to regulate the cell cycle and induce apoptosis is a new way of tumor therapy. Our previous research proved that ITCs in Brassica oleracea Linnaeus var. botrytis Linnaeus could induce human hepatoma HepG-2 cells apoptosis. Further study found that SFN was the anti-hepatoma component and its content is the highest and the activity is the strongest. Therefore, based on previous research results, in this study, human hepatoma cell Line HepG-2 was chosen as research object. The objective of this article is to investigate systematically the antitumor mechanism of SFN starting from regulation of cell cycle and induction of apoptosis and to provide scientific basis for future the anti-hepatoma drug research and development.
1 Study on the effect of SFN on the proliferation inhibition and apoptosis induction in HepG-2 cells
1.1 Study on the effect of SFN on the proliferation inhibition in HepG-2 cells OBJECTIVE:To investigate the effect of SFN on the proliferation inhibition in HepG-2 cells. METHODS:MTT assay and SRB assay were used to determine the proliferation-inhibition ratio of SFN on HepG-2 cells and calculate IC50 and GI50; Inverted microscope was used to observe the growth situation of the cells. RESULTS:The results of MTT assay showed that SFN of different concentration (5,10,20,40 and 80μmol/L) could inhibit cell growth at 24h, 48h and 72h time points in a time and dose-dependent manner. IC50 of SFN of 24h,48h and 72h were 32.03±0.96,20.90±1.96和13.87±0.44μmol/L respectively. The results of MTT assay showed that IC50, GI50, TGI and LC50 were 21.80μmol/L,19.40μmol/L,26.68μmol/L and 61.87μmol/L, which agreed well with the results obtained by MTT assay. Under inverted microscope, the cells of control group grew adhering to the wall showing plump appearance and clear edge. After exposure to SFN, adherent cells decreased. HepG-2 cells became rounded and shrunken gradually with lower refraction. CONCLUSION:SFN can significantly inhibit the proliferation in SGC-7901 cells.
1.2 Study on the effect of SFN on cell cycle arrest in HepG-2 cells OBJECTIVE:To investigate cell cycle arrest effects of SFN in HepG-2 cells. METHODS: Cells were marked by PI and FCM was used to analysis the cell cycle distribution of HepG-2 cells after exposed to SFN. RESULTS:After SFN with different concentration (10,20,40μmol/L) treated on HepG-2 cells for 48 h, the proportion of G2/M phase cell increased significantly while the proportion of G1 phase cell decreased obviously. The percentage of cells in G2/M phase was increased with increasing concentrations of SFN. The subdiploid peak was founded in 40μmol/L dosage group of SFN. CONCLUSION:SFN can arrest HepG-2 cells on G2/M phase.
1.3 Study on the effect of SFN on the inducing apoptosis in HepG-2 cells OBJECTIVE:To investigate the apoptotic effect of SFN in HepG-2 cells. METHODS:In situ detection of DNA fragments by terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) was performed using the one-step TUNEL apotosis assay kit; Cells were doubly marked by Annexin V-FITC/PI, the typical morphological changes of early apoptosis in HepG-2 cells were observed with LCSM; TEM was used to observe the apoptotic morphology and the ultrastructure changes of HepG-2 cells; Cells were doubly marked by Annexin V-FITC/PI, the apoptosis rate of SGC-7901 cells was detected. RESULTS:The results of TUNEL assay showed that after SFN treated on HepG-2 cells for 48 h, under LCSM, the cell nuclear of the early apoptosis cells showed green fluorescent. The number of apoptosis cells was increased with increasing concentrations of SFN while the cells of control group were no significant coloring. Under LCSM, cells that stain positive for Annexin V-FITC (green) and negative for PI are undergoing apoptosis. Cells that stain positive for both Annexin V-FITC (green) and PI (red) are either in the end stage of apoptosis, or are undergoing necrosis. The number of early and late apoptotic cells increased in a dose-dependent manner. Under TEM, the morphological characteristics such as the clear cellularity, complete structure of organelles, the chromatin well distributing in the cell nuclear, the normal mitochondria and the microvilli on the cell membrane were commonly seen in these tumor cells of control group. After exposure to SFN, some tumor cells showed typical apoptotic morphology, including a decrease in microvilli, chromatin condensation, crescent margination of chromatin against the nuclear envelope, chromatin crescent formation, enlargement of perinuclear space, mitochondria swelling, cytoplasmic vacuolation and formation of apoptotic body. FCM analysis result showed that the apoptosis rates of HepG-2 cells treated with 10,20,40μmol/L of SFN for 48 h were 27.42%±0.43%,46.53%±0.35% and 58.92%±0.48%, which were significantly higher than that of the control (1.6%±0.06%) (P<0.01). CONCLUSION:SFN can induce apoptosis in SGC-7901 cells.
2 Study on the mechanism of G2/M phase arrest and apoptosis induced by SFN in HepG-2 cells
2.1 Study on the mechanism of G2/M phase arrest induced by SFN in HepG-2 cells
OBJECTIVE:To investigate the molecular mechanism of G2/M phase arrest induced by SFN in HepG-2 cells. METHODS:Western blot assay was used to investigate the G2/M phase correlative protein expression of CdKl, p-CdKl(Thrl4), cyclinBl, Cdc25C and p21. RESULTS:After treated with SFN at the dosage of 10,20,40μmol/L on HepG-2 cells for 48 h, the expression of CdKl, cyclinBl and Cdc25C protein were down-regulated, which were significantly lower than that of control group (P＜0.01). Meanwhile, the expression of p-CdKl(Thrl4) and p21 were significantly up-regulated (P＜0.05 or P＜0.01). CONCLUSION:SFN can induce G2/M phase arrest in HepG-2 cells through reducing complex formation of CdKl-cyclinBl and inhibiting the activity of CdKl or CdKl-cyclinBl by down-regulation of Cdc25C protein and up-regulation of p21 protein.
2.2 Study on the mechanism of apoptosis via mitochondria pathway induced by SFN in HepG-2 cells
OBJECTIVE:To investigate the molecular mechanism of cell apoptosis via mitochondria pathway induced by SFN in HepG-2 cells. METHODS:Caspase Assay Kit was used to detect the activity of caspase-3,-8,-9 in HepG-2 cells; LCSM was used to detect the mitochondrial transmembrane potential (△Ψm) and [Ca2+]i in HepG-2 cells; FCM was used to detect the cellular concentration of ROS in HepG-2 cells; Western blot assay was used to detect the expression of Cyt-c protein; RT-PCR and Western blot assay were used to detect the expression of Bcl-2 and Bax protein and mRNA. RESULTS:After treated with different concentration of SFN for 48 h,△Ψm decreased gradually in a dose dependent manner. At the dose of 10,20 and 40μmol/L of SFN, [Ca2+]I (FI) were 45.68±10.51,88.62±16.09 and 176.33±25.14 respectively, and the intracellular ROS level were 28.74±4.59,52.90±6.61 and 90.32±5.95, which were significantly higher than that in the control group (P＜0.01). The results of RT-PCR and Western blot assay showed that SFN could down-regulate the expression of Bcl-2 mRNA and protein, in the meantime, up-regulate the expression of Bax mRNA and protein. SFN could remarkably decrease the ratio of Bcl-2/Bax and Bcl-2 mRNA/Bax mRNA compared with control group (P＜0.05 or P＜0.01). Besides, SFN could significantly up-regulate the expression of Cyt-c in a dose dependent manner compared with control group (P＜0.05). Activity kit detection result showed that SFN could increase the activities of Caspase-3,-8,-9 in a dose dependent manner, which were remarkably higher than that of control group (P＜0.05 or P＜0.01). CONCLUSION:SFN can induce the opening of MPTP by down-regulating the Bcl-2 gene expression and up-regulating the Bax gene expression, and then, improve the release of Cyt-c from mitochondria to the cytoplasm. Cyt-c can further combine with Apaf-1 and procaspase-9 which formed an apoptosome. Then Caspase-9 activates Caspase-3 which may trigger the cytological events in the terminal stage of apoptosis induced by SFN.
2.3 Study on the mechanism of apoptosis via MAPK pathway induced by SFN in HepG-2 cells
OBJECTIVE:To investigate the molecular mechanism of cell apoptosis via MAPK pathway induced by SFN in HepG-2 cells. METHODS:Western blot assay was used to detect the MAPK correlative protein expression of P38, p-P38, ERK, p-ERK, JNK/SAPK and p-JNK/SAPK. RESULTS:After treated with SFN at the dosage of 10,20,40μmol/L on HepG-2 cells for 48 h, the expression of p-P38 and p-JNK protein were up-regulated, while p-ERK expression was down-regulated which showed significantly difference compared with control group (P<0.05 or P<0.01). Meanwhile, there were no significant changes on the expression of P38, ERK and JNK protein in HepG-2 cells (P>0.05). CONCLUSION:MAPK pathway plays an important role in the apoptosis induced by SFN in HepG-2 cells. SFN can induce G2/M phase arrest and cell apoptosis in HepG-2 cells through MAPK pathway.
CONCLUSION
SFN has remarkably antitumor effect on Human hepatoma HepG-2 cells, and its possible mechanism are as follows:①SFN can induce G2/M phase arrest in HepG-2 cells through reducing complex formation of CdKl-cyclinBl and inhibiting the activity of CdKl or CdKl-cyclinBl by down-regulation of Cdc25C protein and up-regulation of p21 protein.②SFN can induce the opening of MPTP by down-regulating the Bcl-2 gene expression and up-regulating the Bax gene expression, and then, improve the release of Cyt-c from mitochondria to the cytoplasm. Cyt-c can further activate caspase-9 and Caspase-3 which may trigger the cytological events in the terminal stage of apoptosis induced by SFN.③SFN can activate the p38MAPK signaling pathway by up-regulating the expression of p-P38, which can induce G2/M phase arrest by deactivating of Cdc25C and Cdkl. Meanwhile, the up-regulation of p-p38 can promote the activation of caspase-8 and then activate caspase cascade by activating the fas/fasL apoptosis pathway.④SFN can inhibit the activation of ERK pathway by down-regulating p-ERK protein, which can block the expression of Bcl-2 gene and inhibit the anti-apoptosis effect of Bcl-2. This may be one of the most important mechanism of apoptosis inducing effect of SFN.⑤SFN can promote cell apoptosis via mitochandial pathway by improving the phosphorylation of JNK. At the same time, the activation of JNK pathway can promote the activation of caspase-8 and then activate caspase cascade by activating the fas/fasL apoptosis pathway. It can also stabilize the activation of p21, which can inhibit the activation of Cdkl and induce G2/M phase arrest in HepG-2 cells.
INNOVATIVE POINTS OF CURRENT STUDY
1. For the first time, the activation of the of G2/M DNA damage checkpoint pathway is found to be the main molecular mechanism of SFN on the G2/M phase arrest in HepG-2 cells.
2. For the first time, mitochondria pathway is found to be the main approach of SFN on the induction of HepG-2 cell apoptosis, in which, down-regulation of bcl-2 gene expression and up-regulaiton of bax gene expression play a key role.
3. For the first time, MAPK signaling pathway is found to be the key regulation pathway and the most important effect target of SFN in the inducion of G2/M phase arrest and apoptosis of HepG-2 cells.
莱菔硫烷(sulforaphane, SFN)是天然存在于十字花科植物中含有的一种异硫氰酸盐(isothiocyanates, ITCs)类物质,是十字花科植物中的硫代葡萄糖苷的酶解产物,在绿花椰菜中含量最高。大量研究表明,异硫氰酸盐可选择性地抑制Ⅰ相酶的活性,诱导Ⅱ相酶的活性,降低致癌物的活化,保护机体少受甚至不受损伤,显示出很强的化学预防作用。目前认为,SFN不仅在肿瘤发生的多个阶段发挥抑癌作用,而且它还是潜在的肿瘤治疗药物。SFN与肿瘤的关系研究正成为抗肿瘤药物研究领域的热点。
虽然关于肿瘤发病机制的学说很多,但总而言之,肿瘤是一类细胞周期紊乱和凋亡异常性疾病。以调控细胞周期和诱导细胞凋亡为靶点,寻找相关药物用于恶性肿瘤的治疗为抗肿瘤药物的研究提供了新的思路。本论文前期研究发现,绿花椰菜中的ITCs能够诱导人肝癌细胞系HepG-2细胞发生凋亡,进一步研究发现SFN是其中含量最高、活性最强的抗肝癌细胞活性成分。因此,本论文在前期工作的基础上,以人肝癌HepG-2细胞为对象,从细胞周期调控和诱导凋亡入手,系统研究SFN的抗肿瘤作用机制,为今后SFN作为抗肝癌药物的开发提供科学依据。
1莱菔硫烷抑制人肝癌HepG-2细胞增殖和诱导凋亡作用的研究
1.1莱菔硫烷对人肝癌细胞HepG-2增殖抑制作用研究
目的：研究SFN对人肝癌HepG-2细胞增殖的抑制作用。方法：采用MTT比色法和SRB法测定SFN对人肝癌HepG-2细胞增殖的抑制率,计算SFN对人肝癌HepG-2细胞的IC50和GI50；采用倒置显微镜观察细胞生长状态。结果：MTT法测定结果表明,不同浓度的SFN(5、10、20、40、80μmol/L)作用于HepG-2细胞24、48、72 h均可显著抑制细胞增殖(P<0.05或P<0.01),并呈一定的时间和剂量依赖性。经计算,SFN对HepG-2细胞24,48和72 h的IC50分别为32.03μmol/L±0.96μmol/L,20.90μmol/L±1.96μmol/L和13.87μmol/L±0.44μmol/L。SRB法测定结果表明,SFN作用于HepG-2细胞48h的IC50为21.80μmol/L,GI50为19.40μmol/L,与MTT结果相印证。倒置相差显微镜下观察到对照组细胞贴壁生长,细胞饱满,边缘清晰；而SFN作用后,贴壁细胞减少,细胞均逐渐变圆,体积变小,折光性减弱。结论：SFN对人肝癌HepG-2细胞的增殖具有较强的抑制作用。
1.2莱菔硫烷对人肝癌HepG-2细胞周期影响的研究
目的：研究SFN对人肝癌HepG-2细胞周期阻滞作用。方法：采用PI单染法,应用流式细胞仪分析SFN作用于人肝癌HepG-2细胞后细胞周期的分布变化。结果：10、20、40μmol/L的SFN作用HepG-2细胞48h后,各组G2/M期的细胞比例显著升高,且G2/M期细胞百分比随着药物浓度增加而增大,同时G1期细胞明显减少。SFN浓度达到40μmol/L时出现凋亡峰。结论：SFN可诱导人肝癌HepG-2细胞发生G2/M期阻滞。
1.3莱菔硫烷诱导人肝癌HepG-2细胞凋亡作用的研究目的：研究SFN对人肝癌HepG-2细胞凋亡诱导作用。方法：采用一步法TUNEL细胞凋亡检测试剂盒原位检测DNA片段化情况；采用Annexin V-FITC/PI双染,激光共聚焦显微镜观察细胞的早期凋亡情况；透射电镜观察肿瘤细胞超微结构变化；采用流式细胞仪(Annexin V-FITC/PI双染法)测定细胞凋亡率。结果：TUNEL法检测结果表明,SFN作用48h后,激光共聚焦显微镜下早期凋亡细胞核显绿色荧光。随着SFN剂量的增加凋亡细胞数量逐渐增多,而空白对照组细胞无明显着色。激光共聚焦显微镜下可见早期凋亡细胞的细胞膜被染成绿色,细胞核无明显着色。细胞膜被染成绿色,细胞核被染成红色是晚期凋亡细胞或坏死细胞。随着剂量的增加,镜下凋亡细胞数量也逐渐增加。电子显微镜下可见对照组细胞的细胞结构清晰,细胞器结构完整,核内染色质分布均匀,线粒体结构正常,细胞表面有微绒毛突起。而SFN作用后,细胞出现典型的凋亡形态,如微绒毛减少,细胞核内染色质固缩、凝集于核膜边界,形成新月状小体,核周间隙扩张,线粒体肿胀,胞浆空泡化,细胞形成凋亡小体。流式细胞仪检测结果表明,10、20、40μmol/L的SFN作用于HepG-2细胞48h后,早期凋亡细胞百分率分别达到27.42%±0.43%、46.53%±0.35%和58.92%±0.48%,与对照组1.6%±0.06%比较,差异有统计学意义(P<0.01)。结论：SFN可诱导人肝癌HepG-2细胞发生凋亡。
2莱菔硫烷诱导人肝癌HepG-2细胞G2/M期阻滞及凋亡的机制研究
2.1莱菔硫烷诱导人肝癌HepG-2细胞G2/M期阻滞的机制研究
目的：研究SFN诱导人肝癌HepG-2细胞G2/M期阻滞的分子作用机制。方法：采用Western Blot法检测SFN对人肝癌HepG-2细胞内CdKl,p-CdKl(Thr14), cyclinB1,Cdc25C,p21等G2/M期相关蛋白表达的影响。结果：10、20、40μmol/L的SFN作用HepG-2细胞48 h后,均可显著下调CdKl, cyclinB1, Cdc25C蛋白表达水平,与对照组比较差异具有统计学意义(P<0.01),同时显著上调p-CdKl(Thr14)和p21蛋白表达水平,与对照组比较差异具有统计学意义(P<0.05或P<0.01)。结论：SFN可通过降低CdK1-cyclinB1复合物的形成,同时通过下调Cdc25C表达、上调p21表达来抑制CdK1或CdKl-cyclinBl复合物的活性,诱导HepG-2细胞发生G2/M期阻滞。
2.2莱菔硫烷通过线粒体途径诱导人肝癌HepG-2细胞凋亡的机制研究
目的：研究SFN诱导人肝癌HepG-2细胞凋亡的分子作用机制。方法：采用酶活力检测试剂盒测定SFN对人肝癌HepG-2细胞Caspase-3,-8,-9活性的影响,采用激光共聚扫描焦显微镜测定细胞内线粒体膜电位(Δψm)和钙离子浓度,采用流式细胞仪测定人肝癌HepG-2细胞内ROS含量；采用Western blot法测定SFN对HepG-2细胞内Cyt-c蛋白表达的影响：采用Western blot法和RT-PCR法测定SFN对HepG-2细胞内Bcl-2、Bax表达的影响。结果：不同浓度的SFN作用于HepG-2细胞48h后,细胞线粒体膜电位Δψm随着SFN浓度的升高而逐渐降低,呈一定的剂量依赖关系；20、40μmol/L组FI值与对照组比较差异具有统计学意义(P<0.01)。细胞内钙离子浓度随着SFN浓度的升高而逐渐升高。10、20、40μmol/L SFN剂量组细胞内[Ca2+]i(FI)分别为45.68±10.51、88.62±16.09和176.33±25.14,与对照组比较差异具有统计学意义(P<0.01)。同时,细胞内活性氧水平显著升高,分别为28.74±4.59、52.90±6.61和90.32±5.95,与对照组比较差异具有统计学意义(P<0.01)。RT-PCR法和Western blot法检测结果表明,SFN能下调HepG-2细胞中Bcl-2 mRNA和蛋白的表达水平,同时上调Bax mRNA和蛋白表达水平,且Bcl-2/Bax和Bcl-2mRNA/Bax mRNA随剂量的增加而降低,与对照组比较差异具有统计学意义(P<0.05或P<0.01)。此外,SFN可上调细胞内Cyt-c蛋白表达量,并呈一定的剂量依赖性,与对照组相比差异具有统计学意义(P<0.05)。试剂盒检测结构表明,SFN可升高细胞内Caspase-3,-8,-9活性,并呈一定的剂量依赖性,各剂量组与对照组比较差异具有统计学意义(P<0.05或P<0.01)。结论：SFN可能是通过下调Bcl-2基因表达、上调Bax基因表达,进而诱导线粒体通透性转变孔道(PT孔道)的开放,促进线粒体内Cyt-c向胞浆的释放。释放的Cyt-c可能进一步与Apaf-1、procaspase-9结合形成凋亡体,活化的caspase-9进一步激活下游的caspase-3,最终完成SFN通过线粒体途径诱导HepG-2细胞的凋亡过程。
2.3莱菔硫烷通过MAPK信号转导途径诱导人肝癌HepG-2细胞凋亡机制研究
目的：阐明MAPK信号传导通路在SFN诱导人肝癌HepG-2细胞凋亡中的作用。方法：采用Western blot法研究SFN对人肝癌HepG-2细胞中MAPK信号通路相关蛋白P38、p-P38、ERK、p-ERK、JNK/SAPK和p-JNK/SAPK蛋白表达的影响。结果：10、20、40μmol/L的SFN作用人肝癌HepG-2细胞48h后,随着药物浓度的增加,p-P38和p-JNK蛋白表达量逐渐增加,而p-ERK的表达量逐渐降低,与对照组比较差异具有统计学意义(P<0.05或P<0.01)。同时,P38、ERK和JNK蛋白相对表达量与对照组相比并无显著性差异(P>0.05)。结论：MAPK信号转导途径在SFN诱导人肝癌HepG-2细胞周期阻滞和凋亡过程中发挥重要作用,SFN可通过启动MAPK信号通路诱导人肝癌HepG-2细胞G2/M期阻滞进而引起细胞凋亡。
结论
SFN对人肝癌HepG-2细胞具有显著地抗肿瘤作用,其可能的作用机制如下：①SFN可通过降低CdK1-cyclinB1复合物的形成,同时通过下调Cdc25C表达、上调p21表达来抑制CdK1或CdK1-cyclinB1复合物的活性,诱导HepG-2细胞发生G2/M期阻滞；②SFN可通过下调Bcl-2基因表达、上调Bax基因表达,进而诱导线粒体通透性转变孔道(PT孔道)的开放,促进线粒体内Cyt-c向胞浆的释放,进而激活caspase-9和下游的caspase-3,最终通过线粒体途径诱导HepG-2细胞发生凋亡；③SFN可通过激活p38MAPK信号转导通路,作用于Cdc25C磷酸酶而使其失活,导致其无法激活Cdk1而将细胞阻滞于G2/M期；同时,通过促进fas/fasL介导的细胞凋亡途径激活Caspase-8,启动Caspase级联反应而诱导HepG-2细胞凋亡,但这一推测仍有待研究证实；④SFN可通过下调p-ERK的表达抑制ERK通路的激活,进而抑制Bcl-2所致的抗凋亡作用,促进细胞凋亡；⑤SFN可通过促进JNK的磷酸化促进线粒体介导的细胞凋亡；同时,激活Fas/FasL介导的细胞凋亡途径而激活Caspase-8,最终激活Caspase-3诱导细胞凋亡；活化的JNK/SAPKs还可通过翻译后的磷酸化修饰稳定p21的活性,进而抑制Cdkl的活化,诱导细胞发生G2/M期阻滞。
本研究的创新点
1.首次研究发现启动G2/M期DNA损伤检查点调节机制是SFN诱导人肝癌HepG-2细胞周期阻滞的主要分子机制。
2.首次研究发现线粒体介导的凋亡通路是SFN诱导人肝癌HepG-2细胞凋亡的主要途径,下调Bcl-2基因表达、上调Bax基因表达是SFN启动内源性凋亡通路的关键。
3.首次研究发现MAPK信号通路是SFN诱导人肝癌HepG-2细胞G2/M期阻滞和凋亡的关键调节通路和重要作用靶点。
According to incomplete statistics, every year there occur about 1.6 million new cancer sufferers in our country:According to the predication of WHO, the number of cancer patients will increased to 14.7 million. Malignant tumor is one of the severe diseases harmful to human health. Liver cancer is a tumor of digestive system with high degree of malignancy, which annually causes more than 626,000 cases. The incidence of liver cancer ranks the 6th among whole malignant tumor. The death toll of liver cancer is 589,000 every year and the lethality occupies the first place next to lung carcinoma and gastric carcinoma. The mortality is higher gradually in recent years. Surgical procedures and postoperative chemotherapy and radiotherapy are the main treatments at present, but which have more side effects. Many of patients died with not liver cancer but the side effects of chemotherapy and radiotherapy. Therefore, the preparation of new-type antineoplastic agents of high efficiency and low toxicity holds the best hope for the success of tumor therapy.
Sulforaphane (SFN), one of naturally occurring isothiocyanates (ITCs) found in crucifer, is the enzymatic hydrolysate of Glucosinolates, which shows the highest amount in Brassica oleracea Linnaeus var. botrytis Linnaeus. A large number of studies show that ITCs can inhibit the activity of the phase I drug metabolizing enzyme, induce the activity of the phase II drug metabolizing enzyme and decrease activation of carcinogen, which can protect against injury. ITCs show a strong chemopreventive potential. At present, SFN not only restrains tumor during different stages of carcinogenesis, but also is a potential drug in the oncotherapy. The antitumor effect of SFN is a research focus in antineoplastic research field.
Though there are many theories on pathogenesis of cancer, in a word, cancer is a diseases associated to cell cycle and apoptosis deregulation. Researching the drug to regulate the cell cycle and induce apoptosis is a new way of tumor therapy. Our previous research proved that ITCs in Brassica oleracea Linnaeus var. botrytis Linnaeus could induce human hepatoma HepG-2 cells apoptosis. Further study found that SFN was the anti-hepatoma component and its content is the highest and the activity is the strongest. Therefore, based on previous research results, in this study, human hepatoma cell Line HepG-2 was chosen as research object. The objective of this article is to investigate systematically the antitumor mechanism of SFN starting from regulation of cell cycle and induction of apoptosis and to provide scientific basis for future the anti-hepatoma drug research and development.
1 Study on the effect of SFN on the proliferation inhibition and apoptosis induction in HepG-2 cells
1.1 Study on the effect of SFN on the proliferation inhibition in HepG-2 cells OBJECTIVE:To investigate the effect of SFN on the proliferation inhibition in HepG-2 cells. METHODS:MTT assay and SRB assay were used to determine the proliferation-inhibition ratio of SFN on HepG-2 cells and calculate IC50 and GI50; Inverted microscope was used to observe the growth situation of the cells. RESULTS:The results of MTT assay showed that SFN of different concentration (5,10,20,40 and 80μmol/L) could inhibit cell growth at 24h, 48h and 72h time points in a time and dose-dependent manner. IC50 of SFN of 24h,48h and 72h were 32.03±0.96,20.90±1.96和13.87±0.44μmol/L respectively. The results of MTT assay showed that IC50, GI50, TGI and LC50 were 21.80μmol/L,19.40μmol/L,26.68μmol/L and 61.87μmol/L, which agreed well with the results obtained by MTT assay. Under inverted microscope, the cells of control group grew adhering to the wall showing plump appearance and clear edge. After exposure to SFN, adherent cells decreased. HepG-2 cells became rounded and shrunken gradually with lower refraction. CONCLUSION:SFN can significantly inhibit the proliferation in SGC-7901 cells.
1.2 Study on the effect of SFN on cell cycle arrest in HepG-2 cells OBJECTIVE:To investigate cell cycle arrest effects of SFN in HepG-2 cells. METHODS: Cells were marked by PI and FCM was used to analysis the cell cycle distribution of HepG-2 cells after exposed to SFN. RESULTS:After SFN with different concentration (10,20,40μmol/L) treated on HepG-2 cells for 48 h, the proportion of G2/M phase cell increased significantly while the proportion of G1 phase cell decreased obviously. The percentage of cells in G2/M phase was increased with increasing concentrations of SFN. The subdiploid peak was founded in 40μmol/L dosage group of SFN. CONCLUSION:SFN can arrest HepG-2 cells on G2/M phase.
1.3 Study on the effect of SFN on the inducing apoptosis in HepG-2 cells OBJECTIVE:To investigate the apoptotic effect of SFN in HepG-2 cells. METHODS:In situ detection of DNA fragments by terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) was performed using the one-step TUNEL apotosis assay kit; Cells were doubly marked by Annexin V-FITC/PI, the typical morphological changes of early apoptosis in HepG-2 cells were observed with LCSM; TEM was used to observe the apoptotic morphology and the ultrastructure changes of HepG-2 cells; Cells were doubly marked by Annexin V-FITC/PI, the apoptosis rate of SGC-7901 cells was detected. RESULTS:The results of TUNEL assay showed that after SFN treated on HepG-2 cells for 48 h, under LCSM, the cell nuclear of the early apoptosis cells showed green fluorescent. The number of apoptosis cells was increased with increasing concentrations of SFN while the cells of control group were no significant coloring. Under LCSM, cells that stain positive for Annexin V-FITC (green) and negative for PI are undergoing apoptosis. Cells that stain positive for both Annexin V-FITC (green) and PI (red) are either in the end stage of apoptosis, or are undergoing necrosis. The number of early and late apoptotic cells increased in a dose-dependent manner. Under TEM, the morphological characteristics such as the clear cellularity, complete structure of organelles, the chromatin well distributing in the cell nuclear, the normal mitochondria and the microvilli on the cell membrane were commonly seen in these tumor cells of control group. After exposure to SFN, some tumor cells showed typical apoptotic morphology, including a decrease in microvilli, chromatin condensation, crescent margination of chromatin against the nuclear envelope, chromatin crescent formation, enlargement of perinuclear space, mitochondria swelling, cytoplasmic vacuolation and formation of apoptotic body. FCM analysis result showed that the apoptosis rates of HepG-2 cells treated with 10,20,40μmol/L of SFN for 48 h were 27.42%±0.43%,46.53%±0.35% and 58.92%±0.48%, which were significantly higher than that of the control (1.6%±0.06%) (P<0.01). CONCLUSION:SFN can induce apoptosis in SGC-7901 cells.
2 Study on the mechanism of G2/M phase arrest and apoptosis induced by SFN in HepG-2 cells
2.1 Study on the mechanism of G2/M phase arrest induced by SFN in HepG-2 cells
OBJECTIVE:To investigate the molecular mechanism of G2/M phase arrest induced by SFN in HepG-2 cells. METHODS:Western blot assay was used to investigate the G2/M phase correlative protein expression of CdKl, p-CdKl(Thrl4), cyclinBl, Cdc25C and p21. RESULTS:After treated with SFN at the dosage of 10,20,40μmol/L on HepG-2 cells for 48 h, the expression of CdKl, cyclinBl and Cdc25C protein were down-regulated, which were significantly lower than that of control group (P＜0.01). Meanwhile, the expression of p-CdKl(Thrl4) and p21 were significantly up-regulated (P＜0.05 or P＜0.01). CONCLUSION:SFN can induce G2/M phase arrest in HepG-2 cells through reducing complex formation of CdKl-cyclinBl and inhibiting the activity of CdKl or CdKl-cyclinBl by down-regulation of Cdc25C protein and up-regulation of p21 protein.
2.2 Study on the mechanism of apoptosis via mitochondria pathway induced by SFN in HepG-2 cells
OBJECTIVE:To investigate the molecular mechanism of cell apoptosis via mitochondria pathway induced by SFN in HepG-2 cells. METHODS:Caspase Assay Kit was used to detect the activity of caspase-3,-8,-9 in HepG-2 cells; LCSM was used to detect the mitochondrial transmembrane potential (△Ψm) and [Ca2+]i in HepG-2 cells; FCM was used to detect the cellular concentration of ROS in HepG-2 cells; Western blot assay was used to detect the expression of Cyt-c protein; RT-PCR and Western blot assay were used to detect the expression of Bcl-2 and Bax protein and mRNA. RESULTS:After treated with different concentration of SFN for 48 h,△Ψm decreased gradually in a dose dependent manner. At the dose of 10,20 and 40μmol/L of SFN, [Ca2+]I (FI) were 45.68±10.51,88.62±16.09 and 176.33±25.14 respectively, and the intracellular ROS level were 28.74±4.59,52.90±6.61 and 90.32±5.95, which were significantly higher than that in the control group (P＜0.01). The results of RT-PCR and Western blot assay showed that SFN could down-regulate the expression of Bcl-2 mRNA and protein, in the meantime, up-regulate the expression of Bax mRNA and protein. SFN could remarkably decrease the ratio of Bcl-2/Bax and Bcl-2 mRNA/Bax mRNA compared with control group (P＜0.05 or P＜0.01). Besides, SFN could significantly up-regulate the expression of Cyt-c in a dose dependent manner compared with control group (P＜0.05). Activity kit detection result showed that SFN could increase the activities of Caspase-3,-8,-9 in a dose dependent manner, which were remarkably higher than that of control group (P＜0.05 or P＜0.01). CONCLUSION:SFN can induce the opening of MPTP by down-regulating the Bcl-2 gene expression and up-regulating the Bax gene expression, and then, improve the release of Cyt-c from mitochondria to the cytoplasm. Cyt-c can further combine with Apaf-1 and procaspase-9 which formed an apoptosome. Then Caspase-9 activates Caspase-3 which may trigger the cytological events in the terminal stage of apoptosis induced by SFN.
2.3 Study on the mechanism of apoptosis via MAPK pathway induced by SFN in HepG-2 cells
OBJECTIVE:To investigate the molecular mechanism of cell apoptosis via MAPK pathway induced by SFN in HepG-2 cells. METHODS:Western blot assay was used to detect the MAPK correlative protein expression of P38, p-P38, ERK, p-ERK, JNK/SAPK and p-JNK/SAPK. RESULTS:After treated with SFN at the dosage of 10,20,40μmol/L on HepG-2 cells for 48 h, the expression of p-P38 and p-JNK protein were up-regulated, while p-ERK expression was down-regulated which showed significantly difference compared with control group (P<0.05 or P<0.01). Meanwhile, there were no significant changes on the expression of P38, ERK and JNK protein in HepG-2 cells (P>0.05). CONCLUSION:MAPK pathway plays an important role in the apoptosis induced by SFN in HepG-2 cells. SFN can induce G2/M phase arrest and cell apoptosis in HepG-2 cells through MAPK pathway.
CONCLUSION
SFN has remarkably antitumor effect on Human hepatoma HepG-2 cells, and its possible mechanism are as follows:①SFN can induce G2/M phase arrest in HepG-2 cells through reducing complex formation of CdKl-cyclinBl and inhibiting the activity of CdKl or CdKl-cyclinBl by down-regulation of Cdc25C protein and up-regulation of p21 protein.②SFN can induce the opening of MPTP by down-regulating the Bcl-2 gene expression and up-regulating the Bax gene expression, and then, improve the release of Cyt-c from mitochondria to the cytoplasm. Cyt-c can further activate caspase-9 and Caspase-3 which may trigger the cytological events in the terminal stage of apoptosis induced by SFN.③SFN can activate the p38MAPK signaling pathway by up-regulating the expression of p-P38, which can induce G2/M phase arrest by deactivating of Cdc25C and Cdkl. Meanwhile, the up-regulation of p-p38 can promote the activation of caspase-8 and then activate caspase cascade by activating the fas/fasL apoptosis pathway.④SFN can inhibit the activation of ERK pathway by down-regulating p-ERK protein, which can block the expression of Bcl-2 gene and inhibit the anti-apoptosis effect of Bcl-2. This may be one of the most important mechanism of apoptosis inducing effect of SFN.⑤SFN can promote cell apoptosis via mitochandial pathway by improving the phosphorylation of JNK. At the same time, the activation of JNK pathway can promote the activation of caspase-8 and then activate caspase cascade by activating the fas/fasL apoptosis pathway. It can also stabilize the activation of p21, which can inhibit the activation of Cdkl and induce G2/M phase arrest in HepG-2 cells.
INNOVATIVE POINTS OF CURRENT STUDY
1. For the first time, the activation of the of G2/M DNA damage checkpoint pathway is found to be the main molecular mechanism of SFN on the G2/M phase arrest in HepG-2 cells.
2. For the first time, mitochondria pathway is found to be the main approach of SFN on the induction of HepG-2 cell apoptosis, in which, down-regulation of bcl-2 gene expression and up-regulaiton of bax gene expression play a key role.
3. For the first time, MAPK signaling pathway is found to be the key regulation pathway and the most important effect target of SFN in the inducion of G2/M phase arrest and apoptosis of HepG-2 cells.
引文
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