水飞蓟宾对人宫颈癌和胃癌细胞凋亡作用及机制研究
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摘要
水飞蓟素（Silymarin）是从水飞蓟的种子中提取出来的一种黄酮木质素类混合物，它包括水飞蓟宾（Silibinin，或Sylibin）、异水飞蓟宾（isosylibinin）、水飞蓟亭（silicristin）、水飞蓟宁（silidianin）等成分。水飞蓟宾本身也是由等比例水飞蓟宾A和水飞蓟宾B构成的两个非对映体混合物，体外和动物研究认为其有很好的保护肝细胞免受毒素损伤的护肝作用，被称为“天然的保肝药”。在动物研究实验中没有明显的慢性毒副作用。近些年来的研究发现，水飞蓟宾还具有较强的抗肿瘤活性，其抗肿瘤作用和分子机制研究都取得了较大进展。体外研究表明，对于雄性激素依赖型和非依赖型前列腺癌、皮肤癌、膀胱癌、肺癌、结肠癌、乳腺癌、卵巢癌、肾癌、肝癌、宫颈癌和舌癌均具有抑制作用。
本研究的主要目的是探讨水飞蓟宾在宫颈癌及胃癌中的功能及其抑制肿瘤细胞增殖的机制。首先，我们以宫颈癌作为研究模型，流式细胞术的检测结果显示水飞蓟宾使宫颈癌HeLa细胞阻滞在G2/M期。用200μM的水飞蓟宾处理HeLa细胞24h后，G2/M期的细胞比例由对照组的5.64%增至22.67%。蛋白免疫印迹分析表明，水飞蓟宾能够以浓度依赖的方式下调CDK1（Cdc2）和CDK2蛋白水平；细胞周期蛋白Cyclin A和Cyclin B1没有明显变化；形态学分析表明，水飞蓟宾处理的细胞出现了染色质皱缩等细胞凋亡特征；流式细胞术的分析结果显示水飞蓟宾能够以浓度和时间依赖的方式诱导HeLa细胞凋亡，Caspase3,8,9被激活，PARP被切割； Bcl-2蛋白水平下降，Bax没有明显变化，细胞色素C从线粒体释放到胞浆中，Caspase9被激活，即细胞凋亡的线粒体通路被激活；同时， Fas、Fas L的蛋白水平明显上调， Caspase8被激活，即细胞凋亡的膜受体通路被激活。
迄今为止，水飞蓟宾在胃癌中的研究报道很少。因此，本文着重研究了水飞蓟宾在胃癌中的作用。我们应用胃癌细胞系SGC-7901, BGC-823, HGC-27作为研究模型，研究其诱导细胞凋亡和周期阻滞的分子机制。我们发现SGC-7901、BGC-823、HGC-27对水飞蓟宾有不同的敏感性。其中，HGC-27最为敏感，SGC-7901次之，BGC-823最不敏感。在SGC-7901细胞中，水飞蓟宾上调p53和p21的表达量，下调CDK1，进而减少CDK1-Cyclin B1复合体，使细胞阻滞在G2/M期。值得注意的是，水飞蓟宾能够诱导SGC-7901的凋亡，但并没有激活SGC-7901细胞的Caspase3,8,9的活性，是以一种caspase非依赖的方式诱导其凋亡。在BGC-823细胞中，水飞蓟宾的作用最不敏感，只能微弱下调CDK1(Cdc2)和微弱的激活Caspase3,9。因此，也只能引起较轻微的周期阻滞和细胞凋亡现象。然而，水飞蓟宾却显著的抑制HGC-27细胞的增殖；下调HGC-27细胞中CDK1、Cyclin B1的蛋白表达水平，引起G2/M期细胞周期阻滞；水飞蓟宾还激活了Caspase3, PARP被切割，导致细胞凋亡；在此过程中，Bcl-2明显减少，Bax增加，细胞色素C从线粒体释放到胞浆中，Caspase9被激活，即凋亡的线粒体途径被激活；并且，Caspase8也被激活，即凋亡的死亡受体通路也可能参与凋亡过程。
辅助化疗在晚期胃癌患者的治疗中起到了关键的作用。然而，大多数患者在起初对化疗药物是敏感的，但最后都对其化疗药物产生抗性而导致化疗失败。因此，具有低毒副作用、高抗癌活性的植物性治疗试剂可能与抗癌药物产生协同作用，是我们目前研究的最佳候选者。在此，我们评价了水飞蓟宾是否能与紫杉醇、阿霉素、喜树碱在胃癌细胞SGC-7901中产生协同作用。实验结果显示水飞蓟宾能够与紫杉醇产生很好的协同促进作用，诱导SGC-7901细胞G2/M期阻滞及凋亡。
Silymarin, an extract of the milk thistle seeds, is mixture of flavonolignansconsisting of among others of Silibinin, isosilibinin, silicristin, and silidianin.Silibinin itself is mixture of two diastereomers silibinin A and silybinin B inapproximately equimolar ratio. Both in vitro and animal research suggest thatsilibinin, as a ‘narural hepatoprotective drug’, has hepatoprotective (antihepatotoxic)properties that protect liver cells against toxins. Animal experiments showed thatSilibinin has no obivousely toxin side effect. Recent studies have showed strongerefficacy of Silibinin and more progress has been made towards antitumor role andmolecular mechanism. In vitro studies have also showed Silibinin processesinhibitory role against male hormone-dependent and hormone-nondependent cancerincluding prostate cancer, skin cancer, urinary bladder cancer, lung cancer, coloncancer, breast cancer, ovarian cancer, kindney cancer, hepatic cancer, cervical cancer,and tongure cancer.
The purpose of this study was to investigate the function and molecular mechanismsof Silibinin in cell growth in cervical cancer and gastric cancer. First, we had cervicalcancer (HeLa) as a model. Flow cytometry results showed Silibinin arrested HeLacells in G2/M phase. After24h treatment with200μM silibinin, percentages of G2/Mphase cells increased from5.64%(control) to22.67%. Using immunoblot analysis, wealso observed that Silibinin decreased the expression of CDK2and CDK1(Cdc2)levels in HeLa cell lines in a dose-dependent manner, which might be the possiblemolecular mechanism of Silibinin efficacy in growth inhibition in HeLa cells. In thestudies assessing Silibinin effect on cyclin levels, no changes were observed in CyclinA and Cyclin B1protein levels. Morphological analysis using DAPI staining revealedthe presence of nuclei with chromatin condensation and the formation of apoptoticbodies in cells cultured with Silibinin. In addition, flow cytometric analyses revealedenhanced apoptosis of HeLa cells treated with Silibinin in a concentration-dependentand time-dependent manner. Western-blot analysis revealed Silibinin significantly cleaved the PARP and reduced pro-caspase3/8/9in dose-dependent manner.Furthermore，our data also demonstrated that Silibinin significantly downregulated theratio of Bcl-2/Bax that was accompanied by a subsequent evident release ofcytochrome c from mitochondria to cytoplasm and activation of caspase-9. Meanwhile,the expression of Fas and Fas L were upregulated in a dose-dependent manner andcaspase8was activated. All these findings implicated that the Silibinin-drivenapoptosis was conducted in both the mitochondrial pathway and the deathreceptor-mediated pathway.
Silibinin, a flavonoid compound, has shown to be of chemopreventive potentialagainst many cancers. However, its efficacy against gastric cancer has not been wellelucidated. Here, we assessed the activity of Silibinin on apoptosis and cell-cyclearrest in human gastric cells culture system using SGC-7901, BGC-823, HGC-27asthe model. We observed that SGC-7901, BGC-823, HGC-27have different sensitivityto Silibinin. In SGC-7901cells, Silibinin showed a G2phase arrest upregulating theexpression p53, p21protein, downregulating the expression CDK1, and decreasing theactivity of CDK1-Cyclin B1complex. Silibinin induced G2phase arrest in thecell-cycle progression of SGC-7901cell line. Surprisingly, our data demonstrated thatSilibinin treatment did not show any increase in the activities of Caspase3,8,9. Thesefindings clearly demonstrated Silibinin treatment resulted in apoptotic death ofSGC-7901cells, which was independent of caspase activation. In BGC-823cells, theeffects of Silibinin are the most insensitive. Silibinin slightly downregulated CDK1and slightly activated Caspase3,9. However, Silibinin could significantlydownregulate the expression of CDK1and Cyclin B1in HGC-27cells. Furthermore,Silibinin could activite Caspase3,8,9, downregulate the Bcl-2, and upregulate theBax. These data demonstrated that Silibinin arrested G2/M phase and inducedapoptosis in both the mitochondrial pathway and the death receptor-mediated pathwayin HGC-27cells.
Palliative chemotherapy may play a very important role in the treatment of patientswith advanced gastric cancer. However, most patients who are sensitive to initial chemotherapy will eventually fail to react to further cytotoxic chemotherapeuticagents. Therefore, phytotherapeutic agents with high anticancer efficacy and leasttoxicity to normal tissues are suggested as possible candidates to be investigated fortheir synergistic efficacy in combination with anticancer drugs. Herein we assessedwhether Silibinin could synergize the therapeutic efficacy of Paclitaxel, or doxorubicin,or camptothecine in human gastric cancer cell line SGC-7901. The results showed thatSilibinin strongly synergized human gastric cancer SGC-7901cells toPaclitaxel-induced growth inbition, G2/M cell cycle arrest, and apoptosis. Together,we gained a deeper insight into synergetic efficacy of Silibinin in combination withPaclitaxel in human gastric cancer cells.
本研究的主要目的是探讨水飞蓟宾在宫颈癌及胃癌中的功能及其抑制肿瘤细胞增殖的机制。首先，我们以宫颈癌作为研究模型，流式细胞术的检测结果显示水飞蓟宾使宫颈癌HeLa细胞阻滞在G2/M期。用200μM的水飞蓟宾处理HeLa细胞24h后，G2/M期的细胞比例由对照组的5.64%增至22.67%。蛋白免疫印迹分析表明，水飞蓟宾能够以浓度依赖的方式下调CDK1（Cdc2）和CDK2蛋白水平；细胞周期蛋白Cyclin A和Cyclin B1没有明显变化；形态学分析表明，水飞蓟宾处理的细胞出现了染色质皱缩等细胞凋亡特征；流式细胞术的分析结果显示水飞蓟宾能够以浓度和时间依赖的方式诱导HeLa细胞凋亡，Caspase3,8,9被激活，PARP被切割； Bcl-2蛋白水平下降，Bax没有明显变化，细胞色素C从线粒体释放到胞浆中，Caspase9被激活，即细胞凋亡的线粒体通路被激活；同时， Fas、Fas L的蛋白水平明显上调， Caspase8被激活，即细胞凋亡的膜受体通路被激活。
迄今为止，水飞蓟宾在胃癌中的研究报道很少。因此，本文着重研究了水飞蓟宾在胃癌中的作用。我们应用胃癌细胞系SGC-7901, BGC-823, HGC-27作为研究模型，研究其诱导细胞凋亡和周期阻滞的分子机制。我们发现SGC-7901、BGC-823、HGC-27对水飞蓟宾有不同的敏感性。其中，HGC-27最为敏感，SGC-7901次之，BGC-823最不敏感。在SGC-7901细胞中，水飞蓟宾上调p53和p21的表达量，下调CDK1，进而减少CDK1-Cyclin B1复合体，使细胞阻滞在G2/M期。值得注意的是，水飞蓟宾能够诱导SGC-7901的凋亡，但并没有激活SGC-7901细胞的Caspase3,8,9的活性，是以一种caspase非依赖的方式诱导其凋亡。在BGC-823细胞中，水飞蓟宾的作用最不敏感，只能微弱下调CDK1(Cdc2)和微弱的激活Caspase3,9。因此，也只能引起较轻微的周期阻滞和细胞凋亡现象。然而，水飞蓟宾却显著的抑制HGC-27细胞的增殖；下调HGC-27细胞中CDK1、Cyclin B1的蛋白表达水平，引起G2/M期细胞周期阻滞；水飞蓟宾还激活了Caspase3, PARP被切割，导致细胞凋亡；在此过程中，Bcl-2明显减少，Bax增加，细胞色素C从线粒体释放到胞浆中，Caspase9被激活，即凋亡的线粒体途径被激活；并且，Caspase8也被激活，即凋亡的死亡受体通路也可能参与凋亡过程。
辅助化疗在晚期胃癌患者的治疗中起到了关键的作用。然而，大多数患者在起初对化疗药物是敏感的，但最后都对其化疗药物产生抗性而导致化疗失败。因此，具有低毒副作用、高抗癌活性的植物性治疗试剂可能与抗癌药物产生协同作用，是我们目前研究的最佳候选者。在此，我们评价了水飞蓟宾是否能与紫杉醇、阿霉素、喜树碱在胃癌细胞SGC-7901中产生协同作用。实验结果显示水飞蓟宾能够与紫杉醇产生很好的协同促进作用，诱导SGC-7901细胞G2/M期阻滞及凋亡。
Silymarin, an extract of the milk thistle seeds, is mixture of flavonolignansconsisting of among others of Silibinin, isosilibinin, silicristin, and silidianin.Silibinin itself is mixture of two diastereomers silibinin A and silybinin B inapproximately equimolar ratio. Both in vitro and animal research suggest thatsilibinin, as a ‘narural hepatoprotective drug’, has hepatoprotective (antihepatotoxic)properties that protect liver cells against toxins. Animal experiments showed thatSilibinin has no obivousely toxin side effect. Recent studies have showed strongerefficacy of Silibinin and more progress has been made towards antitumor role andmolecular mechanism. In vitro studies have also showed Silibinin processesinhibitory role against male hormone-dependent and hormone-nondependent cancerincluding prostate cancer, skin cancer, urinary bladder cancer, lung cancer, coloncancer, breast cancer, ovarian cancer, kindney cancer, hepatic cancer, cervical cancer,and tongure cancer.
The purpose of this study was to investigate the function and molecular mechanismsof Silibinin in cell growth in cervical cancer and gastric cancer. First, we had cervicalcancer (HeLa) as a model. Flow cytometry results showed Silibinin arrested HeLacells in G2/M phase. After24h treatment with200μM silibinin, percentages of G2/Mphase cells increased from5.64%(control) to22.67%. Using immunoblot analysis, wealso observed that Silibinin decreased the expression of CDK2and CDK1(Cdc2)levels in HeLa cell lines in a dose-dependent manner, which might be the possiblemolecular mechanism of Silibinin efficacy in growth inhibition in HeLa cells. In thestudies assessing Silibinin effect on cyclin levels, no changes were observed in CyclinA and Cyclin B1protein levels. Morphological analysis using DAPI staining revealedthe presence of nuclei with chromatin condensation and the formation of apoptoticbodies in cells cultured with Silibinin. In addition, flow cytometric analyses revealedenhanced apoptosis of HeLa cells treated with Silibinin in a concentration-dependentand time-dependent manner. Western-blot analysis revealed Silibinin significantly cleaved the PARP and reduced pro-caspase3/8/9in dose-dependent manner.Furthermore，our data also demonstrated that Silibinin significantly downregulated theratio of Bcl-2/Bax that was accompanied by a subsequent evident release ofcytochrome c from mitochondria to cytoplasm and activation of caspase-9. Meanwhile,the expression of Fas and Fas L were upregulated in a dose-dependent manner andcaspase8was activated. All these findings implicated that the Silibinin-drivenapoptosis was conducted in both the mitochondrial pathway and the deathreceptor-mediated pathway.
Silibinin, a flavonoid compound, has shown to be of chemopreventive potentialagainst many cancers. However, its efficacy against gastric cancer has not been wellelucidated. Here, we assessed the activity of Silibinin on apoptosis and cell-cyclearrest in human gastric cells culture system using SGC-7901, BGC-823, HGC-27asthe model. We observed that SGC-7901, BGC-823, HGC-27have different sensitivityto Silibinin. In SGC-7901cells, Silibinin showed a G2phase arrest upregulating theexpression p53, p21protein, downregulating the expression CDK1, and decreasing theactivity of CDK1-Cyclin B1complex. Silibinin induced G2phase arrest in thecell-cycle progression of SGC-7901cell line. Surprisingly, our data demonstrated thatSilibinin treatment did not show any increase in the activities of Caspase3,8,9. Thesefindings clearly demonstrated Silibinin treatment resulted in apoptotic death ofSGC-7901cells, which was independent of caspase activation. In BGC-823cells, theeffects of Silibinin are the most insensitive. Silibinin slightly downregulated CDK1and slightly activated Caspase3,9. However, Silibinin could significantlydownregulate the expression of CDK1and Cyclin B1in HGC-27cells. Furthermore,Silibinin could activite Caspase3,8,9, downregulate the Bcl-2, and upregulate theBax. These data demonstrated that Silibinin arrested G2/M phase and inducedapoptosis in both the mitochondrial pathway and the death receptor-mediated pathwayin HGC-27cells.
Palliative chemotherapy may play a very important role in the treatment of patientswith advanced gastric cancer. However, most patients who are sensitive to initial chemotherapy will eventually fail to react to further cytotoxic chemotherapeuticagents. Therefore, phytotherapeutic agents with high anticancer efficacy and leasttoxicity to normal tissues are suggested as possible candidates to be investigated fortheir synergistic efficacy in combination with anticancer drugs. Herein we assessedwhether Silibinin could synergize the therapeutic efficacy of Paclitaxel, or doxorubicin,or camptothecine in human gastric cancer cell line SGC-7901. The results showed thatSilibinin strongly synergized human gastric cancer SGC-7901cells toPaclitaxel-induced growth inbition, G2/M cell cycle arrest, and apoptosis. Together,we gained a deeper insight into synergetic efficacy of Silibinin in combination withPaclitaxel in human gastric cancer cells.
引文
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