青藤碱对肺癌细胞系NCI-H460增殖、凋亡的影响及其机制的研究
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摘要
前言
肺癌是严重威胁人类生命的疾病,最新统计资料显示,在世界范围内,肺癌所引起的死亡占所有肿瘤引起的死亡的首位。尽管目前多种治疗方法,包括分子靶向治疗,但肺癌治疗的效果始终不令人满意,其五年生存率低于15%。为了提高其5年生存率,更多的治疗方法正在被探索之中。目前中医药方法治疗肺癌受到了日益的关注
青藤碱(sinomenine, SIN)是抗风湿中药青风藤(Sinomeninum scutum Rehd. et Wils.)的单体提取物,其分子式为C19H23NO4,分子量329.38。青藤在中国应用于治疗风湿病已经有上千年历史。其目前在应用上主要以其盐酸盐形式为主即盐酸青藤碱为主。青藤碱具有抗炎、抗风湿、免疫抑制、镇痛、抗血管生成、抗心律失常等广泛的药理作用。新近研究报导其具有一定的抗肿瘤作用。
细胞凋亡,又称程序性死亡,包括一系列生物化学事件,引起细胞发生特征性形体变化和生物化学改变。一些药物通过诱导凋亡的方式抑制恶性肿瘤的增殖。细胞凋亡主要有三种通路：一种是以Fas和TNFR为代表死亡受体信号转导途径,凋亡发生过程中伴有caspase-8的活化；第二种是以线粒体为核心的凋亡途径,Cytochrome c从线粒体释放到胞浆与APaf-1继而与Caspase-9结合,引起Caspase-9自身剪切激活,启动凋亡过程中伴有caspase-9的活化；第三种是以内质网为核心的凋亡途径,凋亡过程中伴有caspase-12的活化。目前青藤碱对肺癌细胞是否有抑制作用的研究尚未见报导。
本实验研究青藤碱对非小细胞肺腺癌细胞系NCIH-460增殖和凋亡的影响,并研究其机制,为深入研究青藤碱抗肿瘤机制提供实验基础,应用于临床治疗肺癌提供理论依据。
实验材料与方法
一、青藤碱对肺癌细胞系NCI-H460增殖、凋亡的影响的研究
采用四甲基偶氮噻蓝(MTT)法检测青藤碱对NCI-H460细胞增殖的；采用流式细胞仪AnnexinV/PI双染法检测细胞凋亡；TdT酶介导的dUTP缺口末端标记(TUNNEL)方法观察细胞的形态变化；采用扫描电镜观察细胞超微结构的变化。
二、青藤碱诱导肺癌细胞系NCI-H460凋亡的机制的研究
采用罗丹明123(Rhodamine123)染色,通过流式细胞仪检测线粒体膜电位(△Ψm)；采用分光光度法检测caspase-3、caspase-8和caspase-9的活性的变化；采用Western blot方法检测SN对凋亡相关的蛋白如细胞色素C、Bcl-2、Bax的表达的影响。
三、MEK／ERK和PI3K／Akt信号通路参与青藤碱诱导NCI-H460细胞凋亡的研究
采用Western blot法检测SIN对NCI-H460细胞的ERK1／2、p-ERK1／2、Akt、p-Akt的表达的影响；采用流式细胞仪PI单染的方法检测信号通路阻滞剂与青藤碱联合对NCI-H460细胞凋亡的影响。
结果
一、青藤碱对肺癌细胞系NCI-H460增殖、凋亡的影响：
随着SIN浓度的加大和作用时间的延长,其对细胞的增殖有明显的抑制作用,呈现明显的时间-剂量效应关系。SIN 240μg／ml作用72 h,其抑制率达到最大的85.89%。流式细胞AnnexinV/PI检测细胞凋亡分析表明,SIN作用48 h,随浓度的增高,早期凋亡和晚期凋亡细胞的比例都逐渐增加,与对照组相比差异具有显著性(P<0.05)。SN 200μg／ml组可见凋亡细胞细胞核的棕黄色着色,并可见细胞内DNA呈片段化的改变,而对照组没有相应的变化。通过透射电镜观察,对照组NCI-H460的细胞形态规整,细胞核较大,线粒体丰富,膜相结构完整；青藤碱组凋亡细胞体积变小,胞质浓缩,胞浆内空泡增多,染色质固缩、边集或碎裂成不规则块状,线粒体肿胀。
二、青藤碱诱导肺癌细胞系NCI-H460凋亡的机制：
120gg／ml,200gg／ml SIN处理24 h后，结果表明caspase-3和caspase-9被活化，从而参与细胞凋亡的发生。120,200gg／ml SIN处理48 h后,处理的细胞内的△甲m峰值下降,分布在去极化区域的细胞增多,与对照组比较有显著差异(P<0.05),细胞内的△Ψm峰出现有意义的向左移的趋势。SIN(80,120,160,200μg／ml)对NCI-H460细胞作用48 h后,结果可见细胞浆内细胞色素C逐渐增多,而线粒体内细胞色素C逐渐减少,细胞色素C由胞浆释放到线粒体中。经过SIN(80,120,160,200μg／ml)处理NCI-H460细胞48 h后,Western blot方法检测结果显示,随SIN浓度的不断增加,Bax蛋白表达水平逐渐升高,而Bcl-2蛋白表达水平随浓度的增加逐渐降低,Bax/Bcl-2的比值逐渐升高。
三、MEK／ERK和PI3K／Akt信号通路参与青藤碱诱导NCI-H460细胞凋亡
随着SIN处理时间的逐渐延长, ERK1／2、p-ERK1／2、Akt、p-Akt表达逐渐上升。表明SIN处理可以诱导MEK／ERK以及P13K-AKT通路的活化。PD98059+SIN组、LY294002+SIN组与单纯SIN组相比,所诱导的凋亡明显升高,与之相比有明显的统计学差异(P<0.05)。结果表明MEK／ERK以及P13K／AKT通路抑制剂拮抗了其通路活化所发挥的抗凋亡作用,对青藤碱诱导的凋亡具有协同作用。
结论
1、青藤碱可以抑制肺癌细胞系NCI-H460的增殖,这种抑制作用具有时间和浓度依赖性。青藤碱抑制肺癌细胞系NCI-H460的增殖是通过诱导其凋亡来实现的。
2、青藤碱可能通过线粒体途径诱导肺癌细胞系NCI-H460的凋亡。青藤碱可能通过调控Bcl-2家族蛋白的表达,改变线粒体膜通透性,导致Cytochrome c从线粒体释放入胞浆,从而激活线粒体途径而诱发的细胞凋亡。
3、SIN处理后诱导NCI-H460细胞凋亡过程中,伴随着MEK/ERK以及P13K／Akt通路的活化。应用MEK/ERK以及P13K／Akt通路的抑制剂可以拮抗其抗凋亡作用,提高SIN所诱导的NCI-H460细胞的凋亡的效果。
Preface
Lung cancer is the leading cause of cancer mortality in the United States and worldwide in both men and women. Although multimodality therapies and some molecular targeted therapies have been applied, the clinical responses to chemotherapy in patients with lung cancer are still unsatisfactory. The 5-year overall survival in many countries generally is less than 15%. More effective chemo preventive and therapeutic approaches are apparently needed for those patients to improve 5-year survival rate.
Sinomenine (7,8-didehydro-4-hydroxyl-3,7-dimethoxy-17-meth ylmorphinan-6-one; SIN Figure 1) is a principal alkaloid isolated from the stem and root of Chinese medical plant Sinomenium acutum Rehd. et Wils (Family Menispermaceae), which has been successfully used for centuries in the treatment of various autoimmune diseases in Chinese folk medicine. Generally, SIN hydrochloride is the main chemical form for pharmaceutical purposes. Previous reports have demonstrated that SIN had a wide range of pharmacological actions, including anti-inflammatory, antirheumatic, analgesic, antiarrhythmic, anti-angiogenesis, anti-lipid-peroxidation and immunosuppressive effects.Furthermore, the antitumor effect of sinomenine has been reported in a few papers.
Apoptosis, or programmed cell death is defined as an active physiological process of cell self-destruction with specific morphological and biochemical changes. Some agents suppress the proliferation of malignant cells by inducing apoptosis. The mechanism of apoptosis mainly involves the mitochondrial, endoplasmic-reticulum-specific and cell death receptor signaling pathways. The key element in the mitochondrial pathway is the efflux of cytochrome C from mitochondria to the cytosol, where it subsequently forms a complex (apoptosome) with Apaf-1 and caspase-9, leading to activation of caspase-3. The cell death receptor pathway is characterized by the binding of cell death ligands and receptors, with subsequent activation of caspase-8 and caspase-3. The endoplasmic reticulum pathway is characterized by the activation of caspase-12.
In the present study we evaluated the effects of sinomenine on the growth of human lung cancer cell NCI-H460 and investigated its mechanism involved in the apoptosis induced by sinomenine. It will supply experimental basis for the futher invesitigation of anti-tumor mechanism of Sinomenine and will provide the theoretical basis for clinical treatment of lung cancer.
Materials and Methods
1 The research on the effects of sinomenine on the proliferation and apoptosis of lung cancer NC(?)H460 cells
The effect of SIN on NCI-H460 cell proliferation was determined by MTT assay. Annexin V-FITC and PI double staining was used to analyze apoptosis. Morphological changes of cells was observed by TUNNEL assay. Changes of Cell Ultrastructure stucture was observed by transmission electron microscopy.
2 The research on the mechanism of lung cancer cell line NCI-H460 apoptosis induced by Sinomenine
The mitochondrial membrane potential (△ψm) stained with Rhodamine123 were examined using flow cytometry. Caspase-3,-8 and-9 were detected by chromogenic substrate assay. The expression of apoptosis-related proteins,such as Cytochrome C、Bcl-2、Bax were evaluated by Western blot.
3 The research on the MEK/ERK和PI3K/Akt signal passway involved in the apoptosis induced by Sinomenine
The expression of ERK1/2、p-ERK1/2、Akt、p-Akt were evaluated by Western blot. The apoptosis induced by signal passway blockers combined with Sinomenine were detected by PI staining used flow cytometry.
Results
1 Effects of sinomenine on the proliferation and apoptosis of lung cancer NCI-H460 cells
SIN inhibited cell proliferation in NCI-H460 cell lines in a concentration-dependent and time-dependent manner. Maximal proliferation inhibition was observed at 72 h with 200μg/ml SIN, which inhibited proliferation in 85.89% of NCI-H460 cells. To further confirm the induction of apoptosis by SIN, we stained cells with Annexin V /PI, TUNEL assay and TEM were performed at the same time also. The proportion of Annexin V positive cells in SIN-treated cells were increased in a dose-dependent manner(*p<0.05 vs. the control group), which supports the finding that SIN-induced NCIH-460 cell death by apoptosis. As demonstrated by the results, treatment with SIN (120μg/ml and 200μg/ml) for 48 h significantly induced the apoptotic cell death with condensed nuclei and increase of TUNEL positive cells, suggesting that the DNA fragmentation was occurring in these cells. There were no corresponding changes in the control group. TEM analysis exhibited different morphological alterations inNCI-H460 cells after treatment with SIN (200μg/ml) for 48 h. In the control group, NCI-H460 cell remained regularity morphology with large nucleus, rich in mitochondria, membrane phase structural integrity. Cell volume shrinking, intracytoplasmic vacuoles increasing, Chromatin condensation and nuclear fragmentation mitochondrial swelling were observed in treated NCI-H460 cells.
2 The mechanism of lung cancer cell line NC(?)H460 apoptosis induced by Sinomenine
The activities of caspase-3, caspase-8 and caspase-9 were measured using caspase-3/-8/-9 activity Assay kit. Activation of caspase-3 and caspase-9 were significantly increased after SIN-treated 24 h, while no alteration of caspase-8 activity was observed in SIN treated cells. In order to further confirm the mitochondrial involvement in SIN-induced apoptotic cell death, we examined disruption in the mitochondrial membrane potential (△ψm) and release of cytochrome C from mitochondria into cytoplasm. As compared with controls, the proportion of depolarized cells in SIN-treated cells were increased in a dose-dependent manner, cells shifted towards left in case of SIN (120μg/ml and 200μg/ml) treatment for 48 h.The result indicated that SIN treatment induce significant disruption of△ψm. Western blot analysis revealed an increase in cytosolic cytochrome C after SIN treatment in NCI-H460 cells. Relative density of cytochrome C in mitochondrial fraction was decreased for SIN treated fraction as compared to control. These results suggest that cytochrome C release is involved in SIN-induced apoptosis. The Western blot analysis Western blot analysis showed that SIN treatment leads to decrease in Bcl-2 levels; increase in Bax levels as compared to control cells. The ratios of Bax/Bcl-2 increased as the concentration of SIN increased. These results indicated that SIN up-regulation Bax protein expression and down-regulation Bcl-2 protein expression.
3 MEK/ERK和PI3K/Akt signal passway involved in the apoptosis induced by Sinomenine
With gradual extension of time, the expression of ERK1/2、p-ERK1/2、Akt、p-Akt were increased after treated with Sinominen. The results indicated that Sinomenine could induce MEK/ERK and PI3K-AKT pathway activation. PD98059+SIN group, LY294002+SIN group compared with the pure Sinomenine group, the apoptosis significantly increased(*p<0.05 vs. the control group). The MEK/ERK and PI3K/AKT signal passway blockers performed anti-apoptotic effect by inhibited the activition of MEK/ERK and PI3K/AKT signal passway and enhanced the induction of apoptosis effect by Sinomenine.
Conclusion
1 Sinomenine could inhibted the proliferation of NCI-H460 cell lines in a concentration-dependent and time-dependent manner. Sinomenine inhibit lung cancer cell line NCI-H460 proliferation through the induction of apoptosis.
2 Sinomenine induced lung cancer cell line NCI-H460 apoptosis by mitochondrial pathway. Sinomenine possibly regulated the expression of Bcl-2 family protein and changed the mitochondrial membrane permeability. Cytochrome C released from mitochondria into the cytoplasm and activated the mitochondrial pathway and then induced apoptosis.
3 Sinomenine induced apoptosis of NCI-H460 cells followed by the activation of MEK/ERK and PI3K/AKT signal passway. Application of MEK/ERK and the PI3K/AKT pathway blockers could antagonize its anti-apoptotic effects and improve the SIN-induced apoptosis in NCI-H460 cells.
肺癌是严重威胁人类生命的疾病,最新统计资料显示,在世界范围内,肺癌所引起的死亡占所有肿瘤引起的死亡的首位。尽管目前多种治疗方法,包括分子靶向治疗,但肺癌治疗的效果始终不令人满意,其五年生存率低于15%。为了提高其5年生存率,更多的治疗方法正在被探索之中。目前中医药方法治疗肺癌受到了日益的关注
青藤碱(sinomenine, SIN)是抗风湿中药青风藤(Sinomeninum scutum Rehd. et Wils.)的单体提取物,其分子式为C19H23NO4,分子量329.38。青藤在中国应用于治疗风湿病已经有上千年历史。其目前在应用上主要以其盐酸盐形式为主即盐酸青藤碱为主。青藤碱具有抗炎、抗风湿、免疫抑制、镇痛、抗血管生成、抗心律失常等广泛的药理作用。新近研究报导其具有一定的抗肿瘤作用。
细胞凋亡,又称程序性死亡,包括一系列生物化学事件,引起细胞发生特征性形体变化和生物化学改变。一些药物通过诱导凋亡的方式抑制恶性肿瘤的增殖。细胞凋亡主要有三种通路：一种是以Fas和TNFR为代表死亡受体信号转导途径,凋亡发生过程中伴有caspase-8的活化；第二种是以线粒体为核心的凋亡途径,Cytochrome c从线粒体释放到胞浆与APaf-1继而与Caspase-9结合,引起Caspase-9自身剪切激活,启动凋亡过程中伴有caspase-9的活化；第三种是以内质网为核心的凋亡途径,凋亡过程中伴有caspase-12的活化。目前青藤碱对肺癌细胞是否有抑制作用的研究尚未见报导。
本实验研究青藤碱对非小细胞肺腺癌细胞系NCIH-460增殖和凋亡的影响,并研究其机制,为深入研究青藤碱抗肿瘤机制提供实验基础,应用于临床治疗肺癌提供理论依据。
实验材料与方法
一、青藤碱对肺癌细胞系NCI-H460增殖、凋亡的影响的研究
采用四甲基偶氮噻蓝(MTT)法检测青藤碱对NCI-H460细胞增殖的；采用流式细胞仪AnnexinV/PI双染法检测细胞凋亡；TdT酶介导的dUTP缺口末端标记(TUNNEL)方法观察细胞的形态变化；采用扫描电镜观察细胞超微结构的变化。
二、青藤碱诱导肺癌细胞系NCI-H460凋亡的机制的研究
采用罗丹明123(Rhodamine123)染色,通过流式细胞仪检测线粒体膜电位(△Ψm)；采用分光光度法检测caspase-3、caspase-8和caspase-9的活性的变化；采用Western blot方法检测SN对凋亡相关的蛋白如细胞色素C、Bcl-2、Bax的表达的影响。
三、MEK／ERK和PI3K／Akt信号通路参与青藤碱诱导NCI-H460细胞凋亡的研究
采用Western blot法检测SIN对NCI-H460细胞的ERK1／2、p-ERK1／2、Akt、p-Akt的表达的影响；采用流式细胞仪PI单染的方法检测信号通路阻滞剂与青藤碱联合对NCI-H460细胞凋亡的影响。
结果
一、青藤碱对肺癌细胞系NCI-H460增殖、凋亡的影响：
随着SIN浓度的加大和作用时间的延长,其对细胞的增殖有明显的抑制作用,呈现明显的时间-剂量效应关系。SIN 240μg／ml作用72 h,其抑制率达到最大的85.89%。流式细胞AnnexinV/PI检测细胞凋亡分析表明,SIN作用48 h,随浓度的增高,早期凋亡和晚期凋亡细胞的比例都逐渐增加,与对照组相比差异具有显著性(P<0.05)。SN 200μg／ml组可见凋亡细胞细胞核的棕黄色着色,并可见细胞内DNA呈片段化的改变,而对照组没有相应的变化。通过透射电镜观察,对照组NCI-H460的细胞形态规整,细胞核较大,线粒体丰富,膜相结构完整；青藤碱组凋亡细胞体积变小,胞质浓缩,胞浆内空泡增多,染色质固缩、边集或碎裂成不规则块状,线粒体肿胀。
二、青藤碱诱导肺癌细胞系NCI-H460凋亡的机制：
120gg／ml,200gg／ml SIN处理24 h后，结果表明caspase-3和caspase-9被活化，从而参与细胞凋亡的发生。120,200gg／ml SIN处理48 h后,处理的细胞内的△甲m峰值下降,分布在去极化区域的细胞增多,与对照组比较有显著差异(P<0.05),细胞内的△Ψm峰出现有意义的向左移的趋势。SIN(80,120,160,200μg／ml)对NCI-H460细胞作用48 h后,结果可见细胞浆内细胞色素C逐渐增多,而线粒体内细胞色素C逐渐减少,细胞色素C由胞浆释放到线粒体中。经过SIN(80,120,160,200μg／ml)处理NCI-H460细胞48 h后,Western blot方法检测结果显示,随SIN浓度的不断增加,Bax蛋白表达水平逐渐升高,而Bcl-2蛋白表达水平随浓度的增加逐渐降低,Bax/Bcl-2的比值逐渐升高。
三、MEK／ERK和PI3K／Akt信号通路参与青藤碱诱导NCI-H460细胞凋亡
随着SIN处理时间的逐渐延长, ERK1／2、p-ERK1／2、Akt、p-Akt表达逐渐上升。表明SIN处理可以诱导MEK／ERK以及P13K-AKT通路的活化。PD98059+SIN组、LY294002+SIN组与单纯SIN组相比,所诱导的凋亡明显升高,与之相比有明显的统计学差异(P<0.05)。结果表明MEK／ERK以及P13K／AKT通路抑制剂拮抗了其通路活化所发挥的抗凋亡作用,对青藤碱诱导的凋亡具有协同作用。
结论
1、青藤碱可以抑制肺癌细胞系NCI-H460的增殖,这种抑制作用具有时间和浓度依赖性。青藤碱抑制肺癌细胞系NCI-H460的增殖是通过诱导其凋亡来实现的。
2、青藤碱可能通过线粒体途径诱导肺癌细胞系NCI-H460的凋亡。青藤碱可能通过调控Bcl-2家族蛋白的表达,改变线粒体膜通透性,导致Cytochrome c从线粒体释放入胞浆,从而激活线粒体途径而诱发的细胞凋亡。
3、SIN处理后诱导NCI-H460细胞凋亡过程中,伴随着MEK/ERK以及P13K／Akt通路的活化。应用MEK/ERK以及P13K／Akt通路的抑制剂可以拮抗其抗凋亡作用,提高SIN所诱导的NCI-H460细胞的凋亡的效果。
Preface
Lung cancer is the leading cause of cancer mortality in the United States and worldwide in both men and women. Although multimodality therapies and some molecular targeted therapies have been applied, the clinical responses to chemotherapy in patients with lung cancer are still unsatisfactory. The 5-year overall survival in many countries generally is less than 15%. More effective chemo preventive and therapeutic approaches are apparently needed for those patients to improve 5-year survival rate.
Sinomenine (7,8-didehydro-4-hydroxyl-3,7-dimethoxy-17-meth ylmorphinan-6-one; SIN Figure 1) is a principal alkaloid isolated from the stem and root of Chinese medical plant Sinomenium acutum Rehd. et Wils (Family Menispermaceae), which has been successfully used for centuries in the treatment of various autoimmune diseases in Chinese folk medicine. Generally, SIN hydrochloride is the main chemical form for pharmaceutical purposes. Previous reports have demonstrated that SIN had a wide range of pharmacological actions, including anti-inflammatory, antirheumatic, analgesic, antiarrhythmic, anti-angiogenesis, anti-lipid-peroxidation and immunosuppressive effects.Furthermore, the antitumor effect of sinomenine has been reported in a few papers.
Apoptosis, or programmed cell death is defined as an active physiological process of cell self-destruction with specific morphological and biochemical changes. Some agents suppress the proliferation of malignant cells by inducing apoptosis. The mechanism of apoptosis mainly involves the mitochondrial, endoplasmic-reticulum-specific and cell death receptor signaling pathways. The key element in the mitochondrial pathway is the efflux of cytochrome C from mitochondria to the cytosol, where it subsequently forms a complex (apoptosome) with Apaf-1 and caspase-9, leading to activation of caspase-3. The cell death receptor pathway is characterized by the binding of cell death ligands and receptors, with subsequent activation of caspase-8 and caspase-3. The endoplasmic reticulum pathway is characterized by the activation of caspase-12.
In the present study we evaluated the effects of sinomenine on the growth of human lung cancer cell NCI-H460 and investigated its mechanism involved in the apoptosis induced by sinomenine. It will supply experimental basis for the futher invesitigation of anti-tumor mechanism of Sinomenine and will provide the theoretical basis for clinical treatment of lung cancer.
Materials and Methods
1 The research on the effects of sinomenine on the proliferation and apoptosis of lung cancer NC(?)H460 cells
The effect of SIN on NCI-H460 cell proliferation was determined by MTT assay. Annexin V-FITC and PI double staining was used to analyze apoptosis. Morphological changes of cells was observed by TUNNEL assay. Changes of Cell Ultrastructure stucture was observed by transmission electron microscopy.
2 The research on the mechanism of lung cancer cell line NCI-H460 apoptosis induced by Sinomenine
The mitochondrial membrane potential (△ψm) stained with Rhodamine123 were examined using flow cytometry. Caspase-3,-8 and-9 were detected by chromogenic substrate assay. The expression of apoptosis-related proteins,such as Cytochrome C、Bcl-2、Bax were evaluated by Western blot.
3 The research on the MEK/ERK和PI3K/Akt signal passway involved in the apoptosis induced by Sinomenine
The expression of ERK1/2、p-ERK1/2、Akt、p-Akt were evaluated by Western blot. The apoptosis induced by signal passway blockers combined with Sinomenine were detected by PI staining used flow cytometry.
Results
1 Effects of sinomenine on the proliferation and apoptosis of lung cancer NCI-H460 cells
SIN inhibited cell proliferation in NCI-H460 cell lines in a concentration-dependent and time-dependent manner. Maximal proliferation inhibition was observed at 72 h with 200μg/ml SIN, which inhibited proliferation in 85.89% of NCI-H460 cells. To further confirm the induction of apoptosis by SIN, we stained cells with Annexin V /PI, TUNEL assay and TEM were performed at the same time also. The proportion of Annexin V positive cells in SIN-treated cells were increased in a dose-dependent manner(*p<0.05 vs. the control group), which supports the finding that SIN-induced NCIH-460 cell death by apoptosis. As demonstrated by the results, treatment with SIN (120μg/ml and 200μg/ml) for 48 h significantly induced the apoptotic cell death with condensed nuclei and increase of TUNEL positive cells, suggesting that the DNA fragmentation was occurring in these cells. There were no corresponding changes in the control group. TEM analysis exhibited different morphological alterations inNCI-H460 cells after treatment with SIN (200μg/ml) for 48 h. In the control group, NCI-H460 cell remained regularity morphology with large nucleus, rich in mitochondria, membrane phase structural integrity. Cell volume shrinking, intracytoplasmic vacuoles increasing, Chromatin condensation and nuclear fragmentation mitochondrial swelling were observed in treated NCI-H460 cells.
2 The mechanism of lung cancer cell line NC(?)H460 apoptosis induced by Sinomenine
The activities of caspase-3, caspase-8 and caspase-9 were measured using caspase-3/-8/-9 activity Assay kit. Activation of caspase-3 and caspase-9 were significantly increased after SIN-treated 24 h, while no alteration of caspase-8 activity was observed in SIN treated cells. In order to further confirm the mitochondrial involvement in SIN-induced apoptotic cell death, we examined disruption in the mitochondrial membrane potential (△ψm) and release of cytochrome C from mitochondria into cytoplasm. As compared with controls, the proportion of depolarized cells in SIN-treated cells were increased in a dose-dependent manner, cells shifted towards left in case of SIN (120μg/ml and 200μg/ml) treatment for 48 h.The result indicated that SIN treatment induce significant disruption of△ψm. Western blot analysis revealed an increase in cytosolic cytochrome C after SIN treatment in NCI-H460 cells. Relative density of cytochrome C in mitochondrial fraction was decreased for SIN treated fraction as compared to control. These results suggest that cytochrome C release is involved in SIN-induced apoptosis. The Western blot analysis Western blot analysis showed that SIN treatment leads to decrease in Bcl-2 levels; increase in Bax levels as compared to control cells. The ratios of Bax/Bcl-2 increased as the concentration of SIN increased. These results indicated that SIN up-regulation Bax protein expression and down-regulation Bcl-2 protein expression.
3 MEK/ERK和PI3K/Akt signal passway involved in the apoptosis induced by Sinomenine
With gradual extension of time, the expression of ERK1/2、p-ERK1/2、Akt、p-Akt were increased after treated with Sinominen. The results indicated that Sinomenine could induce MEK/ERK and PI3K-AKT pathway activation. PD98059+SIN group, LY294002+SIN group compared with the pure Sinomenine group, the apoptosis significantly increased(*p<0.05 vs. the control group). The MEK/ERK and PI3K/AKT signal passway blockers performed anti-apoptotic effect by inhibited the activition of MEK/ERK and PI3K/AKT signal passway and enhanced the induction of apoptosis effect by Sinomenine.
Conclusion
1 Sinomenine could inhibted the proliferation of NCI-H460 cell lines in a concentration-dependent and time-dependent manner. Sinomenine inhibit lung cancer cell line NCI-H460 proliferation through the induction of apoptosis.
2 Sinomenine induced lung cancer cell line NCI-H460 apoptosis by mitochondrial pathway. Sinomenine possibly regulated the expression of Bcl-2 family protein and changed the mitochondrial membrane permeability. Cytochrome C released from mitochondria into the cytoplasm and activated the mitochondrial pathway and then induced apoptosis.
3 Sinomenine induced apoptosis of NCI-H460 cells followed by the activation of MEK/ERK and PI3K/AKT signal passway. Application of MEK/ERK and the PI3K/AKT pathway blockers could antagonize its anti-apoptotic effects and improve the SIN-induced apoptosis in NCI-H460 cells.
引文
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