14-噻吩基次亚甲基苦参碱抗鼻咽癌作用及机制的实验研究
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摘要
目的鼻咽癌(NPC)是严重威胁人民群众健康的常见恶性肿瘤之一,目前治疗鼻咽癌最有效的手段是以放疗为主并结合化疗的综合治疗,但这两种方式的副作用都较大,严重影响了治疗的进行。因此寻找新的抗肿瘤药物就成为当今研究的热点。近年来,研究发现苦参碱在抑制肿瘤生长、杀伤和诱导肿瘤细胞凋亡等方面表现出显著活性,并受到广泛关注。但由于苦参碱抑制肿瘤细胞生长的有效剂量较高,并不是一个高效治疗肿瘤的药物。因此,本课题组为了提高其抗癌效力,以苦参碱为先导化合物,通过化学合成方法对其结构进行改造,合成了20个结构新颖的苦参碱衍生物,并进行了抗鼻咽癌作用的筛选。从中初步筛出具有抗肿瘤活性成分——14-噻吩基次亚甲基苦参碱体(下文称改构体X),本实验室前期研究已发现改构体X具有强于苦参碱的体外抑制鼻咽癌细胞CNE2增殖的作用。为了进一步研究其抗肿瘤的作用和机制,本课题将系统地研究14-噻吩基次亚甲基苦参碱对人鼻咽癌细胞CNE1、 CNE2体内外的抗鼻咽癌作用,以其能找出确切有效的抗鼻咽癌的药物并为下一步获得自主知识产权治疗鼻咽癌的新药物提供实验依据。
研究内容：
第一部分14-噻吩基次亚甲基苦参碱体外抗鼻咽癌作用的实验研究
方法分别以14-噻吩基次亚甲基苦参碱和苦参碱对人鼻咽癌细胞株CNE1, CNE2进行干预,利用MTT法检测改构体X对细胞的生长抑制曲线,倒置显微镜观察细胞形态学变化,电镜下观察改构体X对细胞超微结构的影响,流式细胞仪检测细胞凋亡及细胞周期,Western blot检测相关凋亡蛋白表达等情况。
结果经MTT法发现在体外两种药物对CNE1及CNE2细胞均有抑制作用,其抑制作用呈剂量依赖性,且苦参碱经过结构改造后的化合物14-噻吩基次亚甲基苦参碱显示出强于其先导化合物苦参碱的抑制作用,其1-5天的IC50值分别低于对应天数的苦参碱的IC50。
形态学结果发现,14-噻吩基次亚甲基苦参碱作用鼻咽癌细胞后,光镜下可见细胞变圆变小；改构体X作用48h后电镜下观察可见两种鼻咽癌细胞均呈凋亡的形态改变,如细胞皱缩,核内异染色质明显增多,核周隙增大,凋亡小体形成等形态。
流式细胞仪检测结果显示,与对照组相比14-噻吩基次亚甲基苦参碱各剂量组、苦参碱IC50剂量组以及顺铂组对CNE1和CNE2细胞株均有促进细胞凋亡的作用,其P<0.05,有统计学意义。14-噻吩基次亚甲基苦参碱对CNE1的促凋亡作用从数据上显示强于对CNE2细胞的促凋亡作用。
细胞周期分析结果显示：对于细胞系CNE1,14-噻吩基次亚甲基苦参碱各剂量组和苦参碱处理组均可将CNE1细胞阻滞于G1期,随改构体X浓度的增加,其阻滞作用逐渐增强,其G1期的细胞数明显高于对照组G1期细胞(P<0.01)。顺铂则可使CNE1细胞阻滞于S期；对于细胞系CNE2,与对照组相比,可以看到改构体X各剂量组S期细胞比例均有所升高,其中,高剂量组差异具有显著性意义(P<0.05),苦参碱处理组和顺铂组则可将CNE2细胞阻滞于G1期并有显著差异(P<0.01)。
Western blot结果显示：药物作用CNE1细胞48h后,14-噻吩基次亚甲基苦参碱各剂量组细胞内Bax、p53蛋白含量随剂量的增大而升高,三种剂量处理下与阴性组相比均具统计学意义(P<0.05,P<0.01),并有一定的剂量依赖性,Caspase-3,-8和-9的含量也有不同程度升高,其中,中、高剂量组与阴性对照组相比具统计学意义(P<0.05,P<0.01)；苦参碱处理后Bax明显高于对照组(P<0.05),p53蛋白则未显示出统计学差异,Caspase-3、8、9的含量与对照组相比也显著增高(P<0.05)；顺铂组Bax、p53、Caspase-3,-8的含量与阴性对照组相比显著增高,均表现出统计学差异(P<0.05,P<0.01)；苦参碱、改构体X高剂量组以及顺铂组细胞Bcl-2含量显著低于对照组(P<0.05,P<0.01)。
对于CNE2细胞,药物作用48h后,14-噻吩基次亚甲基苦参碱中、高剂量组细胞内Bax和Caspase-9与阴性对照组相比显著升高(P<0.05,P<0.01),改构体X各剂量组Caspase-3蛋白均有不同程度的升高,且与对照组相比有显著性差异(P<0.05,P<0.01),抑制凋亡蛋白Bcl-2表达则显著降低(P<0.05,P<0.01)；苦参碱处理后Bax和Caspase-9表达有所增高,但不具统计学差异,Caspase-3的含量与对照组相比显著增高具有统计学意义(P<0.05),并能显著下调Bcl-2的表达；顺铂组各蛋白表达情况与苦参碱类似。
第二部分14-噻吩基次亚甲基苦参碱体内抗鼻咽癌的研究
方法将CNE1及CNE2细胞分别接种于裸鼠腋下建立人鼻咽癌裸鼠移植瘤模型,待肿瘤长至一定体积后设14-噻吩基次亚甲基苦参碱组、苦参碱、顺铂组及对照组进行治疗,每日测量裸鼠的体重、瘤体体积等,治疗15天后取出瘤体并测量瘤重。另结合光镜下观察瘤组织HE染色、透射电镜下观察瘤组织超微结构变化、原位组织化学法(TUNEL法)检测肿瘤组织细胞凋亡、免疫组织化学法检测肿瘤组织内Bcl-2, Bax、 p53、Caspase-3和-9的蛋白表达。
结果：随着14-噻吩基次亚甲基苦参碱给药剂量的增加可以发现瘤体积增长幅度越来越小、瘤重越来越轻。光镜下观察HE染色切片可见各给药组瘤细胞比例减少,大量炎症细胞浸润,肿瘤细胞被大量成纤维细胞包裹,电镜下可见给药组细胞细胞核核仁浓缩或碎裂,并见到类圆形的凋亡小体；在原位组织化学法(Tunnel法)检测中可见药物组凋亡细胞所占比例增加；在免疫组织化学法中可见给药后细胞内Bcl-2蛋白出现表达下调,Bax、p53、Caspase-3和-9蛋白的表达上调。
结论：14-噻吩基次亚甲基苦参碱在体内外均具有抑制人鼻咽癌CNE1和CNE2细胞增殖的作用。且产生抑制作用的剂量都低于其先导化合物苦参碱。其抑制细胞增殖的机制可能与细胞周期阻滞,上调促凋亡蛋白Bax、p53、Caspase-3、-8和-9等的表达,下调抑制凋亡蛋白Bcl-2的表达有关,并可能与Caspase依赖性的细胞凋亡途径有关。
Objective The nasopharyngeal carcinoma is one of the most common malignant tumors and threatens the people's health seriously. Now the main methods of curing with nasopharyngeal carcinoma include radiotherapy and chemotherapy, both of them have severe side effect and making the influence of further therapy. So to search new drug of anti-nasopharyngeal carcinoma is a top topic. Recently, some studies have shown that matrine can inhibit tumor growth, induce and kill apoptosis of tumor cell, and getting widely attention. But the dose of inhibit tumor growth of matrine is too high, and it is not a high efficient drug of anti-cancer. To make the higher effect of anti-cancer, we synthesized novel20matrine derivants that based on matrine as the lead compound by chemical derivant to transform its structure, and after carrying out the screening of anti-nasopharyngeal carcinoma, obtained dirivant X—14-thienyl methylene matrine as an optimal anti-tumor compound that have more higher effect of anti-nasopharyngeal carcinoma than matrine in prophase research. In order to further research the effects and its mechanism of14-thienyl methylene matrine on anti-nasopharyngeal carcinoma, NPC cell atrains, CNE1and CNE2will be used in these experimental researchs on its effects in vitro and in vivo so as to find a higher effective drug of anti-nasopharyngeal carcinoma.
Research contents
Part one
Studying on the inhibiting anti-nasopharyngeal carcinoma effect of14-thieny1methylene matrine in vitro
Methods:In order to research the effects and its mechanism of14-thienyl methylene matrine on anti-nasopharyngeal carcinoma, interfering NPC cell CNE1and CNE2with the14-thienyl methylene matrine and matrine. Use MTT to detect the grouth-inhibitory curves of cells. Observe the change of the cell shape by inverted microscope. Effect of mechanism of dirivant X of the Matrine on ultrastructures of CNE1cells was observed by transmission electron microscope. The cell apoptosis rates and cell cycle were analyzed by flow cytometry. The correlated apoptotic protein expression was examined by western blotting.
Results:Matrine and its dirivant X can inhibit the proliferation of CNE1and CNE2cells in dose dependent manners in vitro by MTT. Dirivant X which based on matrine for the lead compound through chemical synthesis to transform its structure, have more higher effect of CNE1and CNE2cells than matrine. The IC50(1-5days) of derivant X were lower than matrine's.
After incubation of CNE1and CNE2cells with derivant X of the Matrine for48h, cells alter to rounded and smaller. Characteristic morphology of apoptosis, such as shrinkage, heterochromatin significantly increased in nuclear, increased gap of periphery in nucleus uncertainly, and apoptotic bodies, etc were found under the transmission electron microscope.
Flow cytometry analysis revealed the apoptosis rate in different dose of derivant X, the dose of IC50of matrine and DDP group which showed the significant difference compared with the control group both in CNE1and CNE2cells(P<0.05). The apoptosis rate show that derivant X has stronger depressant effect on CNE1than on CNE2.
The detect of flow cytometry reveal, for CNE1cells, the cycle blockage happen at the stage of G1both in different dose of derivant X and matrine groups. The cell proportion of G1period is higher than the control group (p<0.01). The cycle blockage of the DDP group take place at the stage of S (P<0.01). Fellow the dose of derivant X increasing, the blockage effect of derivant X were increasing. For the CNE2, the cell proportion in different dose of derivant X of S period is higher than the control group. Compared with control group, high dose of derivant X can significant increase the cell proportion of S (p<0.01). The cycle blockage of the matrine and DDP group take place at the stage of G1(P<0.01).
After incubation of CNE1cells with derivant X of the Matrine for48h, Western blotting showed that the expression of Bax and p53protein were significantly increased by derivant X in dose dependent(P<0.05, P<0.01), at the same time, the expression of Caspase-3,-8and-9were increased in a dose dependent manner(P<0.05, P<0.01). Compared with the control group, the expression of Caspase-3,-8and-9were significantly increased in the middle and high dose of derivant X of the Matrine group (P<0.05, P<0.01); The expression of Bax, Caspase-3,-8,-9protein were significantly increased by matrine (P<0.05). In matrine group, the expression of p53was also increased but no significantly different. In DDP group, the expression of Bax, p53, Caspase-3,-8and-9were significantly increased compared with control group (P<0.05, P<0.01). The expression of Bcl-2were significantly decreased in matrine, derivant X and DDP group(P<0.05, P<0.01).
For the CNE2cell, the expression of Bax and Caspase-9protein were significantly increased by derivant X by derivant X of the Matrine in middle and high dose for48h(P<0.05, P<0.01). The expression of Caspase-3were increased in a dose dependent manner by matrine(P<0.05, P<0.01). At the same time, Bcl-2was significantly decreased by derivant X (P<0.05, P<0.01); The expression proteins of DDP group were similar with matrine gtoup. In matrine group, the expression of Bax and Caspase-9were also increased but no significantly difference. Caspase-3was significantly decreased and Bcl-2was significantly decreased by matrine (P<0.05).
Part one
Studying on the inhibiting anti-nasopharyngeal carcinoma effect of14-thienyl methylene matrine in vivo
Methods:The CNE1and CNE2cells were grafted in the null mice, when the tumor growed to a certain volume, the null divided into martrine group, derivant X group, DDP group and control group at random. The weight of the null mice and the volume of tumor were measured every day. The tumor was taken from mice and the weight of the tumor was measured after treatment of15days. The tumor issue was investigated by HE staining. Ultra microstructure changes of cells were observed by transmission electron microscope. TUNEL stain was used to detect the apoptosis of CNE1and CNE2cells. The expression of Bcl-2, Bax, p53, Caspase-3and Caspase-9were detected by immunohistochemistry.
Results:The volume and the weight of tumor were inhibited by14-thienyl methylene matrine. HE staining of tumor cell indicated that the number of cancer cell was reduced, the infiltration of a great amount of inflammatory cells were found, and the tumor cell was encapsulated by fiber tissue.Electron microscopy indicated the apoptosis of tumor cells with nuclei condensation and nuclei fragmentation, and apoptotic bodies were observed. The proportion of apoptotic cell of drug groups was higher than the control group. The expression of Bax、p53、Caspase-3and Caspase-9were increased and the expression of of the Bcl-2were decreased than control group.
Conclusion:14-thienyl methylene matrine can inhibit the growth of CNE1and CNE2cells both in vitro and in vivo. The effective dose is lower than matrine'. The possible mechanism is related with inducing apoptosis of cell, arresting the cell cycle, and increasing of the apoptotic protein Bax、 p53、Caspase-3、-8and-9expression level, decreasing of the anti-apoptotic protein Bcl-2protein expression level. This may indicate that the caspase-dependent apoptotic pathway were involved.
研究内容：
第一部分14-噻吩基次亚甲基苦参碱体外抗鼻咽癌作用的实验研究
方法分别以14-噻吩基次亚甲基苦参碱和苦参碱对人鼻咽癌细胞株CNE1, CNE2进行干预,利用MTT法检测改构体X对细胞的生长抑制曲线,倒置显微镜观察细胞形态学变化,电镜下观察改构体X对细胞超微结构的影响,流式细胞仪检测细胞凋亡及细胞周期,Western blot检测相关凋亡蛋白表达等情况。
结果经MTT法发现在体外两种药物对CNE1及CNE2细胞均有抑制作用,其抑制作用呈剂量依赖性,且苦参碱经过结构改造后的化合物14-噻吩基次亚甲基苦参碱显示出强于其先导化合物苦参碱的抑制作用,其1-5天的IC50值分别低于对应天数的苦参碱的IC50。
形态学结果发现,14-噻吩基次亚甲基苦参碱作用鼻咽癌细胞后,光镜下可见细胞变圆变小；改构体X作用48h后电镜下观察可见两种鼻咽癌细胞均呈凋亡的形态改变,如细胞皱缩,核内异染色质明显增多,核周隙增大,凋亡小体形成等形态。
流式细胞仪检测结果显示,与对照组相比14-噻吩基次亚甲基苦参碱各剂量组、苦参碱IC50剂量组以及顺铂组对CNE1和CNE2细胞株均有促进细胞凋亡的作用,其P<0.05,有统计学意义。14-噻吩基次亚甲基苦参碱对CNE1的促凋亡作用从数据上显示强于对CNE2细胞的促凋亡作用。
细胞周期分析结果显示：对于细胞系CNE1,14-噻吩基次亚甲基苦参碱各剂量组和苦参碱处理组均可将CNE1细胞阻滞于G1期,随改构体X浓度的增加,其阻滞作用逐渐增强,其G1期的细胞数明显高于对照组G1期细胞(P<0.01)。顺铂则可使CNE1细胞阻滞于S期；对于细胞系CNE2,与对照组相比,可以看到改构体X各剂量组S期细胞比例均有所升高,其中,高剂量组差异具有显著性意义(P<0.05),苦参碱处理组和顺铂组则可将CNE2细胞阻滞于G1期并有显著差异(P<0.01)。
Western blot结果显示：药物作用CNE1细胞48h后,14-噻吩基次亚甲基苦参碱各剂量组细胞内Bax、p53蛋白含量随剂量的增大而升高,三种剂量处理下与阴性组相比均具统计学意义(P<0.05,P<0.01),并有一定的剂量依赖性,Caspase-3,-8和-9的含量也有不同程度升高,其中,中、高剂量组与阴性对照组相比具统计学意义(P<0.05,P<0.01)；苦参碱处理后Bax明显高于对照组(P<0.05),p53蛋白则未显示出统计学差异,Caspase-3、8、9的含量与对照组相比也显著增高(P<0.05)；顺铂组Bax、p53、Caspase-3,-8的含量与阴性对照组相比显著增高,均表现出统计学差异(P<0.05,P<0.01)；苦参碱、改构体X高剂量组以及顺铂组细胞Bcl-2含量显著低于对照组(P<0.05,P<0.01)。
对于CNE2细胞,药物作用48h后,14-噻吩基次亚甲基苦参碱中、高剂量组细胞内Bax和Caspase-9与阴性对照组相比显著升高(P<0.05,P<0.01),改构体X各剂量组Caspase-3蛋白均有不同程度的升高,且与对照组相比有显著性差异(P<0.05,P<0.01),抑制凋亡蛋白Bcl-2表达则显著降低(P<0.05,P<0.01)；苦参碱处理后Bax和Caspase-9表达有所增高,但不具统计学差异,Caspase-3的含量与对照组相比显著增高具有统计学意义(P<0.05),并能显著下调Bcl-2的表达；顺铂组各蛋白表达情况与苦参碱类似。
第二部分14-噻吩基次亚甲基苦参碱体内抗鼻咽癌的研究
方法将CNE1及CNE2细胞分别接种于裸鼠腋下建立人鼻咽癌裸鼠移植瘤模型,待肿瘤长至一定体积后设14-噻吩基次亚甲基苦参碱组、苦参碱、顺铂组及对照组进行治疗,每日测量裸鼠的体重、瘤体体积等,治疗15天后取出瘤体并测量瘤重。另结合光镜下观察瘤组织HE染色、透射电镜下观察瘤组织超微结构变化、原位组织化学法(TUNEL法)检测肿瘤组织细胞凋亡、免疫组织化学法检测肿瘤组织内Bcl-2, Bax、 p53、Caspase-3和-9的蛋白表达。
结果：随着14-噻吩基次亚甲基苦参碱给药剂量的增加可以发现瘤体积增长幅度越来越小、瘤重越来越轻。光镜下观察HE染色切片可见各给药组瘤细胞比例减少,大量炎症细胞浸润,肿瘤细胞被大量成纤维细胞包裹,电镜下可见给药组细胞细胞核核仁浓缩或碎裂,并见到类圆形的凋亡小体；在原位组织化学法(Tunnel法)检测中可见药物组凋亡细胞所占比例增加；在免疫组织化学法中可见给药后细胞内Bcl-2蛋白出现表达下调,Bax、p53、Caspase-3和-9蛋白的表达上调。
结论：14-噻吩基次亚甲基苦参碱在体内外均具有抑制人鼻咽癌CNE1和CNE2细胞增殖的作用。且产生抑制作用的剂量都低于其先导化合物苦参碱。其抑制细胞增殖的机制可能与细胞周期阻滞,上调促凋亡蛋白Bax、p53、Caspase-3、-8和-9等的表达,下调抑制凋亡蛋白Bcl-2的表达有关,并可能与Caspase依赖性的细胞凋亡途径有关。
Objective The nasopharyngeal carcinoma is one of the most common malignant tumors and threatens the people's health seriously. Now the main methods of curing with nasopharyngeal carcinoma include radiotherapy and chemotherapy, both of them have severe side effect and making the influence of further therapy. So to search new drug of anti-nasopharyngeal carcinoma is a top topic. Recently, some studies have shown that matrine can inhibit tumor growth, induce and kill apoptosis of tumor cell, and getting widely attention. But the dose of inhibit tumor growth of matrine is too high, and it is not a high efficient drug of anti-cancer. To make the higher effect of anti-cancer, we synthesized novel20matrine derivants that based on matrine as the lead compound by chemical derivant to transform its structure, and after carrying out the screening of anti-nasopharyngeal carcinoma, obtained dirivant X—14-thienyl methylene matrine as an optimal anti-tumor compound that have more higher effect of anti-nasopharyngeal carcinoma than matrine in prophase research. In order to further research the effects and its mechanism of14-thienyl methylene matrine on anti-nasopharyngeal carcinoma, NPC cell atrains, CNE1and CNE2will be used in these experimental researchs on its effects in vitro and in vivo so as to find a higher effective drug of anti-nasopharyngeal carcinoma.
Research contents
Part one
Studying on the inhibiting anti-nasopharyngeal carcinoma effect of14-thieny1methylene matrine in vitro
Methods:In order to research the effects and its mechanism of14-thienyl methylene matrine on anti-nasopharyngeal carcinoma, interfering NPC cell CNE1and CNE2with the14-thienyl methylene matrine and matrine. Use MTT to detect the grouth-inhibitory curves of cells. Observe the change of the cell shape by inverted microscope. Effect of mechanism of dirivant X of the Matrine on ultrastructures of CNE1cells was observed by transmission electron microscope. The cell apoptosis rates and cell cycle were analyzed by flow cytometry. The correlated apoptotic protein expression was examined by western blotting.
Results:Matrine and its dirivant X can inhibit the proliferation of CNE1and CNE2cells in dose dependent manners in vitro by MTT. Dirivant X which based on matrine for the lead compound through chemical synthesis to transform its structure, have more higher effect of CNE1and CNE2cells than matrine. The IC50(1-5days) of derivant X were lower than matrine's.
After incubation of CNE1and CNE2cells with derivant X of the Matrine for48h, cells alter to rounded and smaller. Characteristic morphology of apoptosis, such as shrinkage, heterochromatin significantly increased in nuclear, increased gap of periphery in nucleus uncertainly, and apoptotic bodies, etc were found under the transmission electron microscope.
Flow cytometry analysis revealed the apoptosis rate in different dose of derivant X, the dose of IC50of matrine and DDP group which showed the significant difference compared with the control group both in CNE1and CNE2cells(P<0.05). The apoptosis rate show that derivant X has stronger depressant effect on CNE1than on CNE2.
The detect of flow cytometry reveal, for CNE1cells, the cycle blockage happen at the stage of G1both in different dose of derivant X and matrine groups. The cell proportion of G1period is higher than the control group (p<0.01). The cycle blockage of the DDP group take place at the stage of S (P<0.01). Fellow the dose of derivant X increasing, the blockage effect of derivant X were increasing. For the CNE2, the cell proportion in different dose of derivant X of S period is higher than the control group. Compared with control group, high dose of derivant X can significant increase the cell proportion of S (p<0.01). The cycle blockage of the matrine and DDP group take place at the stage of G1(P<0.01).
After incubation of CNE1cells with derivant X of the Matrine for48h, Western blotting showed that the expression of Bax and p53protein were significantly increased by derivant X in dose dependent(P<0.05, P<0.01), at the same time, the expression of Caspase-3,-8and-9were increased in a dose dependent manner(P<0.05, P<0.01). Compared with the control group, the expression of Caspase-3,-8and-9were significantly increased in the middle and high dose of derivant X of the Matrine group (P<0.05, P<0.01); The expression of Bax, Caspase-3,-8,-9protein were significantly increased by matrine (P<0.05). In matrine group, the expression of p53was also increased but no significantly different. In DDP group, the expression of Bax, p53, Caspase-3,-8and-9were significantly increased compared with control group (P<0.05, P<0.01). The expression of Bcl-2were significantly decreased in matrine, derivant X and DDP group(P<0.05, P<0.01).
For the CNE2cell, the expression of Bax and Caspase-9protein were significantly increased by derivant X by derivant X of the Matrine in middle and high dose for48h(P<0.05, P<0.01). The expression of Caspase-3were increased in a dose dependent manner by matrine(P<0.05, P<0.01). At the same time, Bcl-2was significantly decreased by derivant X (P<0.05, P<0.01); The expression proteins of DDP group were similar with matrine gtoup. In matrine group, the expression of Bax and Caspase-9were also increased but no significantly difference. Caspase-3was significantly decreased and Bcl-2was significantly decreased by matrine (P<0.05).
Part one
Studying on the inhibiting anti-nasopharyngeal carcinoma effect of14-thienyl methylene matrine in vivo
Methods:The CNE1and CNE2cells were grafted in the null mice, when the tumor growed to a certain volume, the null divided into martrine group, derivant X group, DDP group and control group at random. The weight of the null mice and the volume of tumor were measured every day. The tumor was taken from mice and the weight of the tumor was measured after treatment of15days. The tumor issue was investigated by HE staining. Ultra microstructure changes of cells were observed by transmission electron microscope. TUNEL stain was used to detect the apoptosis of CNE1and CNE2cells. The expression of Bcl-2, Bax, p53, Caspase-3and Caspase-9were detected by immunohistochemistry.
Results:The volume and the weight of tumor were inhibited by14-thienyl methylene matrine. HE staining of tumor cell indicated that the number of cancer cell was reduced, the infiltration of a great amount of inflammatory cells were found, and the tumor cell was encapsulated by fiber tissue.Electron microscopy indicated the apoptosis of tumor cells with nuclei condensation and nuclei fragmentation, and apoptotic bodies were observed. The proportion of apoptotic cell of drug groups was higher than the control group. The expression of Bax、p53、Caspase-3and Caspase-9were increased and the expression of of the Bcl-2were decreased than control group.
Conclusion:14-thienyl methylene matrine can inhibit the growth of CNE1and CNE2cells both in vitro and in vivo. The effective dose is lower than matrine'. The possible mechanism is related with inducing apoptosis of cell, arresting the cell cycle, and increasing of the apoptotic protein Bax、 p53、Caspase-3、-8and-9expression level, decreasing of the anti-apoptotic protein Bcl-2protein expression level. This may indicate that the caspase-dependent apoptotic pathway were involved.
引文
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