丹皮酚诱导人肝癌细胞凋亡及其机制研究
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摘要
背景:肝细胞性肝癌(hepatocellular carcinoma, HCC)是我国常见的恶性肿瘤之一,其发病率为30.3/10万,占所有癌症的13.13%,居第3位,近二十年来,死亡率增加了41.7%,约占全世界HCC死亡人数的42%。近年来,由于丙型肝炎病毒感染增加,肝癌的发病率在发达国家也有上升的趋势。目前,手术切除仍是治疗HCC的最佳手段。但是80%的患者在诊断时已属晚期,失去手术机会,而对于那些手术患者,其两年内的复发率也高达50%。全身性化疗是治疗肝癌尤其是无手术指征的晚期肝癌的重要方法,但现有的化疗药物,不论是单药治疗还是多药联合治疗疗效均不理想。因此,探求新的有价值治疗手段和策略显得十分必要。从中药和其他天然产物中寻求高效、低毒的新型抗肝癌药物并深入探讨其抗肿瘤机制是当前国内外研究的重要课题。
丹皮酚(Paeonol, Pae)又称牡丹酚,是我省道地中药材毛莨科植物牡丹Paeonia Suffruicosa Andr根皮和萝摩科植物徐长卿Pycnostelma Paniculatum (Bunge) K. Schum干燥根或全草的主要有效成分,具有镇静催眠、解热镇痛、免疫调节及保护心脑血管等广泛的药理活性。我们在前期研究中发现,Pae对体内培养的多种肿瘤细胞的增殖有较强的抑制作用,体内应用有抗肿瘤作用。此外,我们也发现Pae能增强顺铂对人肝癌细胞HepG2、SMMC-7721和人食管癌细胞Eca-109、SEG-1的增殖抑制作用。本研究选用人肝癌细胞株HepG2、Bel-7402作为实验对象,通过观察Pae对人肝癌细胞株HepG2、Bel-7402的COX-2、Survivin、cIAP-1、XIAP表达的影响,探讨其可能的作用机制,以期为肝癌的临床治疗提供新的方法。
目的:探讨Pae诱导人肝癌细胞HepG2、Bel-7402凋亡的分子机制。
方法:采用MTT法测定Pae对HepG2、Bel-7402细胞的增殖抑制率;流式细胞仪检测细胞凋亡;原位细胞凋亡检测法观察细胞凋亡形态学变化;Western-blot方法及免疫细胞化学分析COX-2、cIAP-1、XIAP、Survivin变化。
结果:
1 Pae对人肝癌细胞株HepG2、Bel-7402增殖抑制作用
1.1 Pae对HepG2细胞的增殖抑制作用
采用MTT法,观察Pae对HepG2细胞的增殖抑制作用。结果显示(1)Pae7.81～62.5mg/L对HepG2细胞有增殖抑制作用,药物浓度越高,抑制作用越强。(2)系列浓度Pae作用于肝癌细胞HepG2 24h、48h及72h后观察细胞的生长情况,结果显示Pae作用时间越长,对HepG2细胞的抑制作用越强。
1.2 Pae对Bel-7402细胞的增殖抑制作用
采用MTT法,观察Pae对Bel-7402细胞的增殖抑制作用。结果显示(1)Pae7.81～62.5 mg/L对Bel-7402细胞有增殖抑制作用,药物浓度越高,抑制作用越强。(2)系列浓度Pae作用于肝癌细胞Bel-7402 24h、48h及72h后观察细胞的生长情况,结果显示Pae作用时间越长,对Bel-7402细胞的抑制作用越强。
2 Pae对人肝癌细胞株HepG2、Bel-7402凋亡的影响
2.1 Pae对人肝癌细胞株HepG2、Bel-7402凋亡的TUNEL分析
采用末端双脱氧核苷酸转移酶介导的dUTP缺口标记技术(terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, TUNEL)观察HepG2、Bel-7402细胞凋亡的形态学改变。典型的凋亡细胞形态特征包括核固缩、体积缩小、核染色质边集等。研究发现对照组细胞增殖旺盛,可见少许凋亡细胞;药物作用细胞48h后,细胞密度均明显减少,凋亡细胞数明显增加。
2.2 Pae对人肝癌细胞株HepG2、Bel-7402凋亡的FCM检测
应用流式细胞仪(Flow Cytometry, FCM)检测细胞凋亡率。在DNA图谱G0/G1期前出现了一个代表细胞凋亡的亚G1峰即凋亡峰。FCM结果显示,正常对照组可见低矮的凋亡峰,药物处理48h后,其凋亡峰增加。Pae7.81～62.5 mg/L作用HepG2细胞株时,凋亡率分别为:(2.3±1.8)%、(15.6±3.2)%、(27.6±3.9)%(33.2±2.7)%、(45.3±1.7)%;Pae7.81～62.5 mg/L作用Bel-7402细胞株时,凋亡率分别为:(1.8±1.2)%、(12.5±3.1)%、(25.1±4.2)%、(37.5±3.7)%、(50.3±4.5)%。
3 Pae对人肝癌细胞株HepG2、Bel-7402凋亡的可能分子机制
采用Western Blot和细胞免疫组化法检测COX-2、Survivin、cIAP-1、XIAP表达情况。结果表明,药物处理后HepG2、Bel-7402细胞COX-2、Survivin、cIAP-1、XIAP表达降低。
结论
本结果表明一定浓度的Pae能抑制HepG2、Bel-7402细胞株的增殖,并诱导细胞凋亡,其作用机制可能与下调COX-2、Survivin、cIAP-1、XIAP表达有关。
Background Hepatocellular carcinoma (HCC) is the third most common cancer in China where the incidence is 30.3/100 000. Over the past twenty years, the mortality was increased 41.7% which constituted 42% of all death number in the world. In recent years, the incidence of HCC is increasing in several developed countries, as a result of increased prevalence of HCV infection. The optimal treatment of HCC is hepatic resection. However, more than 80% of patients present with advanced or unresectable disease, and for those patients who do undergo resection, the recurrence rates can be as high as 50% at 2 years. Unfortunately, chemotherapy which is a significant way in the treatement of cancer has not play a key role for HCC patients.Systemic chemotherapy is an important means of hepatocellular carcinoma,especially no surgical indications of advanced hepatocellular carcinoma,Although various chemotherapeutic drugs have been used for the treatment of hepatocellular carcinoma, they are generally not effcetive either when used alone or in combination therapy with other drugs.There is a need for novel strategies to improve current therapy.It is imperative to develop more effective and low-toxic chemopreventive and chemotherapy agents for HCC.
Chinese herb medicine with the high performance and low venenosus characteristic has attentioned extensively in the prevention and cure of cancer. Paeonol (Pae), a major active component extracted from the herb Pycnostelma paniculatum (Bunge) K.S., and the root of the plant Paeonia Suffruticosa Andrew, possesses extensive pharmacological activities such as sedation, hypnosis, antipyresis, analgesic, antioxidation, antiinflammation, and immunoregulation. In our previous study, the antineoplastic activity of Pae has been demonstrated in vivo and in vitro. Furthermore, we also demonstrated that the Pae could enhanced the antiproliferative effect of cisplatin on human hepatocarcinoma cell lines HepG2, SMMC-7721 and human esophageal carcinoma cell lines Eca-109, SEG-1. The present study was designed to investigate the the possible mechanism of apoptosis in human hepatoma cell lines HepG2、Bel-7402 induced by Pae, in order to develop an effective therapy for HCC.
Objective to explore the effect of Pae on apoptosis in HepG2、Bel-7402cell lines and possible mechanism
Methods Pae on HepG2、Bel-7402 cell lines was measured by MTT assay.The apoptosis rate was deteceted by flow cytometry and the morphological change of apoptosis was observed by TUNEL. The expression of COX-2、cIAP-1、XIAP、Survivin were assayed by western-blot and immunohistochemical staining.
Results
1. Effect of Pae on the proliferation of human hepatoma cell lines HepG2、Bel-7402
1.1 Effect of Pae on the proliferation of human hepatoma cell lines HepG2
Investigated the effect of Pae on the proliferation of HepG2 by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-zolium bromide(MTT) assay. The results suggested that Pae at concentrations of 7.81-62.5mg/L, had the inhibitory effect on the proliferation of HepG2 cells which was dose- and time-dependence.
1.2 Effect of Pae on the proliferation of human hepatoma cell lines Bel-7402
Investigated the effect of Pae on the proliferation of Bel-7402 by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-zolium bromide(MTT) assay. The results suggested that Pae at concentrations of 7.81-62.5mg/L, had the inhibitory effect on the proliferation of Bel-7402 cells which was dose- and time-dependence.
2. The apoptotic effect of Pae on human hepatoma cell lines HepG2、Bel-7402
2.1 The apoptotic assay by TUNEL
Morphological evidence of apoptosis was assayed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL).The typically apoptotic changes include chromatin condensation and deformed and fragmented nuclei. The results suggested that the proliferation was prosperous in control group.However, the cell density were decreased significantly and the the number of apoptotic cells increased obviously in treated groups.
2.2 The apoptotic detection by FCM
A Flow Cytometry (FCM) assay was performed to analyze apoptotic rate. The sub-G1 peak, which appeared before the G0/G1 phase that represents apoptotic cell population, was observed clearly in the HepG2、Bel-7402 cell lines treated with Pae. The apoptotic peak was dramatically increased when the cells were exposed to Pae.The apoptotic rate of Pae at concentrations of 7.81-62.5mg/L on HepG2 is (2.3±1.8)%、(15.6±3.2)%、(27.6±3.9)%(33.2±2.7)%、(45.3±1.7)% respectively;The apoptotic rate of Pae at concentrations of 7.81-62.5mg/L on Bel-7402 is (1.8±1.2)%、(12.5±3.1)%、(25.1±4.2)%、(37.5±3.7)%、(50.3±4.5)%respectively.
3.Possible mechanism of Pae on the inducing apoptosis effect on human hepatoma cell lines HepG2、Bel-7402
The expression of COX-2 Survivin XIAP cIAP-1were assayed by Western-Blot and Immunocytochemical analysis. The results showed that COX-2、Survivin、XIAP、cIAP-1 were down-regulated after Pae treated.
Conclusion
Pae at some concentration had inhibitory effect and induce apoptosis on HepG2、Bel-7402 cell line, which may be associated with the decreased expression of COX-2、Survivin、XIAP、cIAP-1.
丹皮酚(Paeonol, Pae)又称牡丹酚,是我省道地中药材毛莨科植物牡丹Paeonia Suffruicosa Andr根皮和萝摩科植物徐长卿Pycnostelma Paniculatum (Bunge) K. Schum干燥根或全草的主要有效成分,具有镇静催眠、解热镇痛、免疫调节及保护心脑血管等广泛的药理活性。我们在前期研究中发现,Pae对体内培养的多种肿瘤细胞的增殖有较强的抑制作用,体内应用有抗肿瘤作用。此外,我们也发现Pae能增强顺铂对人肝癌细胞HepG2、SMMC-7721和人食管癌细胞Eca-109、SEG-1的增殖抑制作用。本研究选用人肝癌细胞株HepG2、Bel-7402作为实验对象,通过观察Pae对人肝癌细胞株HepG2、Bel-7402的COX-2、Survivin、cIAP-1、XIAP表达的影响,探讨其可能的作用机制,以期为肝癌的临床治疗提供新的方法。
目的:探讨Pae诱导人肝癌细胞HepG2、Bel-7402凋亡的分子机制。
方法:采用MTT法测定Pae对HepG2、Bel-7402细胞的增殖抑制率;流式细胞仪检测细胞凋亡;原位细胞凋亡检测法观察细胞凋亡形态学变化;Western-blot方法及免疫细胞化学分析COX-2、cIAP-1、XIAP、Survivin变化。
结果:
1 Pae对人肝癌细胞株HepG2、Bel-7402增殖抑制作用
1.1 Pae对HepG2细胞的增殖抑制作用
采用MTT法,观察Pae对HepG2细胞的增殖抑制作用。结果显示(1)Pae7.81～62.5mg/L对HepG2细胞有增殖抑制作用,药物浓度越高,抑制作用越强。(2)系列浓度Pae作用于肝癌细胞HepG2 24h、48h及72h后观察细胞的生长情况,结果显示Pae作用时间越长,对HepG2细胞的抑制作用越强。
1.2 Pae对Bel-7402细胞的增殖抑制作用
采用MTT法,观察Pae对Bel-7402细胞的增殖抑制作用。结果显示(1)Pae7.81～62.5 mg/L对Bel-7402细胞有增殖抑制作用,药物浓度越高,抑制作用越强。(2)系列浓度Pae作用于肝癌细胞Bel-7402 24h、48h及72h后观察细胞的生长情况,结果显示Pae作用时间越长,对Bel-7402细胞的抑制作用越强。
2 Pae对人肝癌细胞株HepG2、Bel-7402凋亡的影响
2.1 Pae对人肝癌细胞株HepG2、Bel-7402凋亡的TUNEL分析
采用末端双脱氧核苷酸转移酶介导的dUTP缺口标记技术(terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, TUNEL)观察HepG2、Bel-7402细胞凋亡的形态学改变。典型的凋亡细胞形态特征包括核固缩、体积缩小、核染色质边集等。研究发现对照组细胞增殖旺盛,可见少许凋亡细胞;药物作用细胞48h后,细胞密度均明显减少,凋亡细胞数明显增加。
2.2 Pae对人肝癌细胞株HepG2、Bel-7402凋亡的FCM检测
应用流式细胞仪(Flow Cytometry, FCM)检测细胞凋亡率。在DNA图谱G0/G1期前出现了一个代表细胞凋亡的亚G1峰即凋亡峰。FCM结果显示,正常对照组可见低矮的凋亡峰,药物处理48h后,其凋亡峰增加。Pae7.81～62.5 mg/L作用HepG2细胞株时,凋亡率分别为:(2.3±1.8)%、(15.6±3.2)%、(27.6±3.9)%(33.2±2.7)%、(45.3±1.7)%;Pae7.81～62.5 mg/L作用Bel-7402细胞株时,凋亡率分别为:(1.8±1.2)%、(12.5±3.1)%、(25.1±4.2)%、(37.5±3.7)%、(50.3±4.5)%。
3 Pae对人肝癌细胞株HepG2、Bel-7402凋亡的可能分子机制
采用Western Blot和细胞免疫组化法检测COX-2、Survivin、cIAP-1、XIAP表达情况。结果表明,药物处理后HepG2、Bel-7402细胞COX-2、Survivin、cIAP-1、XIAP表达降低。
结论
本结果表明一定浓度的Pae能抑制HepG2、Bel-7402细胞株的增殖,并诱导细胞凋亡,其作用机制可能与下调COX-2、Survivin、cIAP-1、XIAP表达有关。
Background Hepatocellular carcinoma (HCC) is the third most common cancer in China where the incidence is 30.3/100 000. Over the past twenty years, the mortality was increased 41.7% which constituted 42% of all death number in the world. In recent years, the incidence of HCC is increasing in several developed countries, as a result of increased prevalence of HCV infection. The optimal treatment of HCC is hepatic resection. However, more than 80% of patients present with advanced or unresectable disease, and for those patients who do undergo resection, the recurrence rates can be as high as 50% at 2 years. Unfortunately, chemotherapy which is a significant way in the treatement of cancer has not play a key role for HCC patients.Systemic chemotherapy is an important means of hepatocellular carcinoma,especially no surgical indications of advanced hepatocellular carcinoma,Although various chemotherapeutic drugs have been used for the treatment of hepatocellular carcinoma, they are generally not effcetive either when used alone or in combination therapy with other drugs.There is a need for novel strategies to improve current therapy.It is imperative to develop more effective and low-toxic chemopreventive and chemotherapy agents for HCC.
Chinese herb medicine with the high performance and low venenosus characteristic has attentioned extensively in the prevention and cure of cancer. Paeonol (Pae), a major active component extracted from the herb Pycnostelma paniculatum (Bunge) K.S., and the root of the plant Paeonia Suffruticosa Andrew, possesses extensive pharmacological activities such as sedation, hypnosis, antipyresis, analgesic, antioxidation, antiinflammation, and immunoregulation. In our previous study, the antineoplastic activity of Pae has been demonstrated in vivo and in vitro. Furthermore, we also demonstrated that the Pae could enhanced the antiproliferative effect of cisplatin on human hepatocarcinoma cell lines HepG2, SMMC-7721 and human esophageal carcinoma cell lines Eca-109, SEG-1. The present study was designed to investigate the the possible mechanism of apoptosis in human hepatoma cell lines HepG2、Bel-7402 induced by Pae, in order to develop an effective therapy for HCC.
Objective to explore the effect of Pae on apoptosis in HepG2、Bel-7402cell lines and possible mechanism
Methods Pae on HepG2、Bel-7402 cell lines was measured by MTT assay.The apoptosis rate was deteceted by flow cytometry and the morphological change of apoptosis was observed by TUNEL. The expression of COX-2、cIAP-1、XIAP、Survivin were assayed by western-blot and immunohistochemical staining.
Results
1. Effect of Pae on the proliferation of human hepatoma cell lines HepG2、Bel-7402
1.1 Effect of Pae on the proliferation of human hepatoma cell lines HepG2
Investigated the effect of Pae on the proliferation of HepG2 by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-zolium bromide(MTT) assay. The results suggested that Pae at concentrations of 7.81-62.5mg/L, had the inhibitory effect on the proliferation of HepG2 cells which was dose- and time-dependence.
1.2 Effect of Pae on the proliferation of human hepatoma cell lines Bel-7402
Investigated the effect of Pae on the proliferation of Bel-7402 by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetra-zolium bromide(MTT) assay. The results suggested that Pae at concentrations of 7.81-62.5mg/L, had the inhibitory effect on the proliferation of Bel-7402 cells which was dose- and time-dependence.
2. The apoptotic effect of Pae on human hepatoma cell lines HepG2、Bel-7402
2.1 The apoptotic assay by TUNEL
Morphological evidence of apoptosis was assayed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL).The typically apoptotic changes include chromatin condensation and deformed and fragmented nuclei. The results suggested that the proliferation was prosperous in control group.However, the cell density were decreased significantly and the the number of apoptotic cells increased obviously in treated groups.
2.2 The apoptotic detection by FCM
A Flow Cytometry (FCM) assay was performed to analyze apoptotic rate. The sub-G1 peak, which appeared before the G0/G1 phase that represents apoptotic cell population, was observed clearly in the HepG2、Bel-7402 cell lines treated with Pae. The apoptotic peak was dramatically increased when the cells were exposed to Pae.The apoptotic rate of Pae at concentrations of 7.81-62.5mg/L on HepG2 is (2.3±1.8)%、(15.6±3.2)%、(27.6±3.9)%(33.2±2.7)%、(45.3±1.7)% respectively;The apoptotic rate of Pae at concentrations of 7.81-62.5mg/L on Bel-7402 is (1.8±1.2)%、(12.5±3.1)%、(25.1±4.2)%、(37.5±3.7)%、(50.3±4.5)%respectively.
3.Possible mechanism of Pae on the inducing apoptosis effect on human hepatoma cell lines HepG2、Bel-7402
The expression of COX-2 Survivin XIAP cIAP-1were assayed by Western-Blot and Immunocytochemical analysis. The results showed that COX-2、Survivin、XIAP、cIAP-1 were down-regulated after Pae treated.
Conclusion
Pae at some concentration had inhibitory effect and induce apoptosis on HepG2、Bel-7402 cell line, which may be associated with the decreased expression of COX-2、Survivin、XIAP、cIAP-1.
引文
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