紫草素对JAR/MTX细胞毒性作用机制的蛋白质组学研究
摘要
恶性滋养细胞疾病的复发和耐药是当今妊娠滋养细胞肿瘤治疗的难题,迫切需要寻找新的化疗药物或是另辟新的治疗靶点。紫草素是我国传统中药,其对耐药的绒癌细胞作用少有报道,本研究首先以耐甲氨喋呤的绒毛膜癌JAR/MTX细胞为体外模型,采用细胞毒性实验、荧光染色法、流式细胞仪检测细胞凋亡、坏死及对细胞周期的影响,探讨紫草素对其增殖抑制和促进其凋亡、坏死及阻滞细胞周期的作用。然后采用蛋白质组学技术初步探讨紫草素对耐药绒癌细胞的毒性作用机制。
结果显示紫草素对JAR/MTX细胞具有明显的细胞毒性作用,其对绒癌耐药细胞生长的抑制率有明显的剂量和时间依赖性。Hoechst33342/PI双染显示细胞呈现亮蓝染色的核边集、核固缩、核碎裂等凋亡特征。流式细胞仪检测紫草素作用后,JAR/MTX细胞凋亡率及坏死率呈剂量及时间依赖性变化,随着药物浓度增加细胞周期G0/G1期比例升高,S期、G2/M期降低。这提示紫草素是通过抑制细胞增殖、促进细胞凋亡及坏死、阻滞细胞周期等多条途径发挥作用的。
另外,本研究首次应用蛋白质组学技术探讨紫草素对JAR/MTX细胞毒性作用机制,建立了紫草素处理前后JAR/MTX细胞的双向凝胶电泳图谱。Q-TOF质谱初步鉴定了9个差异蛋白点:明显上调的蛋白质包括ROA2、GNS、uMtCK、ANXA2、VDAC2、PRS8、ETFB,明显下调的蛋白质包括CWC15、ezrin。本研究首次发现紫草素与埃兹蛋白、VDAC2、ANXA2等蛋白的联系,初步揭示了紫草素复杂的作用机理,提示这些变化的蛋白质可能与紫草素的毒性作用有关,也可能是紫草素的作用靶点,也为揭示其分子机制提供了大量新的线索。
The malignant trophoblastic tumor become one of the curing solid tumor earlist due to its sensitivity to chemotherapy. But there are 20% patient with high risk and metastasis appear drug-resistant and relapse ,even to death. Now the therapy to the high risk , the drug-resistant and the relapse is Unsatisfactory. Their complete remission rate was only 30% -50% reported in the literature outside. This is directly related to the patient's fertility, and even the lives. Treatment of such patients is the tough problem of gestational trophoblastic tumor. The urgent need to find new chemotherapeutic drugs or open up new therapeutic targets. The naphthoquinone pigment, shikonin, and its derivatives are the active components isolated from the Chinese herbal therapeutic, Zicao. Multiple pharmacological actions have been attributed to shikonin. There are rare reports about shikonin to drug-resistant choriocarcinoma cells. The first part of this study use methotrexate-resistant choriocarcinoma JAR / MTX cells in vitro model to discuss Shikonin inhibited their proliferation and the promotion of its role in apoptosis and necrosis.
Part I Shikonin induced JAR / MTX cell apoptosis and necrosis
JAR/MTX cells were treated with different concentrations of shikonin(3μg/ml、1.5μg/ml、0.75μg/ml、0.375μg/ml、0.1875μg/ml)for 24h、48h and 72h, respectively. Cell morphological changes in JAR/MTX cells were examined by phase–contrast microscopy. The effect of shikonin on proliferation was detected by cycotoxicity test. The change of apoptosis rate and cell cycle disposition were analyzed by flow cyctometry assay, Cell apoptotic phenomenon was observed by fluorescence test.
We found that shikonin inhibited the proliferation of JAR/MTX cell line. The effect increased as the increase in concentration of the drug and the duration. The apoptosis rate and necrosis rate were increased in a dose-and time-dependent manner when the concentration of shikonin from 0.1875μg/ml to 0.75μg/ml for 24h,48h and 72h,respectively. Typical apoptotic nuclear morphological changes were observed such as condensed and fragmented nuclei. Shikonin significantly blocked the cell cycle progression in JAR/MTX cell line, decreased S, G2/M phase and increased G0 /G1 phase. This shows that shikonin play a role through a number of ways such as inhibiting cell proliferation, the promotion of apoptosis and necrosis, cell cycle arrest etc. This experiment for up to explore the toxic effects of shikonin mechanism at the protein level laid a solid foundation.
Part II Proteomics study of toxic mechanism of shikonin
In this paper, experiments have confirmed Shikonin induced JAR / MTX cell apoptosis and necrosis, but its mechanism is not clear. Many previous research limit to several genes, can not fully reveal its inherent mechanism. Proteomics research has the unique advantages in observing the overall changes in protein composition due to many genes. In this study, we apply two-dimensional gel electrophoresis and mass spectrometry technology to analysis the overall of dynamic changes of intracellular protein composition and activity of the law to reveal the possible mechanism of toxicity. The extracted protein of control group and drug group were separated with two-dimensional electrophoresis respectively and differentially expressed proteins were analyzed and identified by Hybrid Quadrupole Time of Flight Mass Spctrometer.
In this study, we first apply proteomics technology to research the cytotoxicity mechanism in JAR / MTX by Shikonin and set up the two-dimensional gel electrophoresis maps before and after the treatment Shikonin in JAR/ MTX cells, and then preliminary reveal the complex mechanism. The expressions of 26 proteins were significantly changed, of which 20 were up-regulated and 6 were down-regulated in shikonin-treated JAR/MTX cells compared with control.9 protein spots were identified by mass spectrometry. The up regulated protein include ROA2、GNS、uMtCK、ANXA2、VDAC2、PRS8、ETFB .T he down regulated protein include CWC15、ezrin,These changes in protein may be involved in the toxic effects of shikonin. Ezrin is a cytoskeleton -associated protein, the present study focused on inhibition of tumor cell apoptosis and promote tumor development, invasion and metastasis. Over-expression of ERM proteins is related to metastasis of many tumors. This study found for the first time ezrin reduced protein expression in the drug-resistant cells by Shikonin,which result is consistent with 2-DE and MS results. A decline in its expression not only mediated the JAR/MTX early morphological changes of apoptosis, but also inhibit their proliferation, induced apoptosis ,ezrin possible play an important role in the toxicity mechanism of Shikonin. VDAC2 located in the outer membrane of mitochondria is the composition of mitochondrial permeability transition pore, the closure can inhibit mitochondrial function, lead to changes in mitochondrial membrane permeability, which release cytochrome C, apoptosis and other factors eventually lead to apoptosis. A lot of evidence indicate that the VDAC is very important to the regulation of the mitochondria and cell function. We also found for the first time VDAC2 upregulation of drug group after Shikonin, which result is consistent with 2-DE and MS results .Through research and analysis about VDAC2, it indicate that shikonin-induced JAR / MTX cell apoptosis is likely through the mitochondrial pathway. The occurrence and development of tumor is closely related to ANXA2’S expression disorder and the information indicate that it may play an important role at the tumor invasion and metastasis . ANXA2 is different with the majority of tumor ,in the drug group in this experiment .It has a very significant increase ,maybe for the unique Shikonin mechanism. Heterogeneous ribonucleoprotein (hnRNP) are RNA-binding protein ,hnRNP play an important role in transcriptional regulation, pre-mRNA processing of a variety of biological processes. HnRNP A2/B1 study has shown that it is related to telomerase activity. In this experiment HnRNP A2/B1 is upregulate in drug group .Shikonin possible through the HnRNP A2/B1 impact the telomerase activity, which play a role in toxicity, the specific mechanism need to be further studied. uMtCK is the key enzyme in energy metabolism of energy conveying, muscle contraction and ATP regeneration. In this study uMtCK is upregulate in drug group due to Shikonin’s destructive effects. PRS8 is a sub-unit belong to the ubiquitin/ proteasome system. Ubiquitin / proteasome system in eukaryotic cells are the main channels of protein-selective degradation, while the proteasome is a great place of protein degradation. It has ATP-dependent, can selectively identify and remove the ubiquitined protein about 8O- 90% intracellular proteins. Thus, UPS is also involved in cell proliferation and differentiation, signal transduction, cell cycle, gene transcription regulation, apoptosis and many other kinds of biological functions. The study found that in drug group PRS8 significantly increase, suggesting its possible importance as a protein involved in shikonin cytotoxicity. Because a wide range of biological functions, its specific role is worthy of further study.
Change the above-mentioned proteins may be related to the toxic effects of shikonin, or it may be the role of Shikonin target. This is not only for the previous research results of others to provide a more powerful objective evidence, but also to reveal the molecular mechanism of a large number of new leads. This indicate Shikonin may become a new drug to drug-resistant choriocarcinoma. Those shikonin-related proteins’structure and function are worthy of further in-depth study, which may have provided for the treatment of drug-resistant choriocarcinoma new ideas.
结果显示紫草素对JAR/MTX细胞具有明显的细胞毒性作用,其对绒癌耐药细胞生长的抑制率有明显的剂量和时间依赖性。Hoechst33342/PI双染显示细胞呈现亮蓝染色的核边集、核固缩、核碎裂等凋亡特征。流式细胞仪检测紫草素作用后,JAR/MTX细胞凋亡率及坏死率呈剂量及时间依赖性变化,随着药物浓度增加细胞周期G0/G1期比例升高,S期、G2/M期降低。这提示紫草素是通过抑制细胞增殖、促进细胞凋亡及坏死、阻滞细胞周期等多条途径发挥作用的。
另外,本研究首次应用蛋白质组学技术探讨紫草素对JAR/MTX细胞毒性作用机制,建立了紫草素处理前后JAR/MTX细胞的双向凝胶电泳图谱。Q-TOF质谱初步鉴定了9个差异蛋白点:明显上调的蛋白质包括ROA2、GNS、uMtCK、ANXA2、VDAC2、PRS8、ETFB,明显下调的蛋白质包括CWC15、ezrin。本研究首次发现紫草素与埃兹蛋白、VDAC2、ANXA2等蛋白的联系,初步揭示了紫草素复杂的作用机理,提示这些变化的蛋白质可能与紫草素的毒性作用有关,也可能是紫草素的作用靶点,也为揭示其分子机制提供了大量新的线索。
The malignant trophoblastic tumor become one of the curing solid tumor earlist due to its sensitivity to chemotherapy. But there are 20% patient with high risk and metastasis appear drug-resistant and relapse ,even to death. Now the therapy to the high risk , the drug-resistant and the relapse is Unsatisfactory. Their complete remission rate was only 30% -50% reported in the literature outside. This is directly related to the patient's fertility, and even the lives. Treatment of such patients is the tough problem of gestational trophoblastic tumor. The urgent need to find new chemotherapeutic drugs or open up new therapeutic targets. The naphthoquinone pigment, shikonin, and its derivatives are the active components isolated from the Chinese herbal therapeutic, Zicao. Multiple pharmacological actions have been attributed to shikonin. There are rare reports about shikonin to drug-resistant choriocarcinoma cells. The first part of this study use methotrexate-resistant choriocarcinoma JAR / MTX cells in vitro model to discuss Shikonin inhibited their proliferation and the promotion of its role in apoptosis and necrosis.
Part I Shikonin induced JAR / MTX cell apoptosis and necrosis
JAR/MTX cells were treated with different concentrations of shikonin(3μg/ml、1.5μg/ml、0.75μg/ml、0.375μg/ml、0.1875μg/ml)for 24h、48h and 72h, respectively. Cell morphological changes in JAR/MTX cells were examined by phase–contrast microscopy. The effect of shikonin on proliferation was detected by cycotoxicity test. The change of apoptosis rate and cell cycle disposition were analyzed by flow cyctometry assay, Cell apoptotic phenomenon was observed by fluorescence test.
We found that shikonin inhibited the proliferation of JAR/MTX cell line. The effect increased as the increase in concentration of the drug and the duration. The apoptosis rate and necrosis rate were increased in a dose-and time-dependent manner when the concentration of shikonin from 0.1875μg/ml to 0.75μg/ml for 24h,48h and 72h,respectively. Typical apoptotic nuclear morphological changes were observed such as condensed and fragmented nuclei. Shikonin significantly blocked the cell cycle progression in JAR/MTX cell line, decreased S, G2/M phase and increased G0 /G1 phase. This shows that shikonin play a role through a number of ways such as inhibiting cell proliferation, the promotion of apoptosis and necrosis, cell cycle arrest etc. This experiment for up to explore the toxic effects of shikonin mechanism at the protein level laid a solid foundation.
Part II Proteomics study of toxic mechanism of shikonin
In this paper, experiments have confirmed Shikonin induced JAR / MTX cell apoptosis and necrosis, but its mechanism is not clear. Many previous research limit to several genes, can not fully reveal its inherent mechanism. Proteomics research has the unique advantages in observing the overall changes in protein composition due to many genes. In this study, we apply two-dimensional gel electrophoresis and mass spectrometry technology to analysis the overall of dynamic changes of intracellular protein composition and activity of the law to reveal the possible mechanism of toxicity. The extracted protein of control group and drug group were separated with two-dimensional electrophoresis respectively and differentially expressed proteins were analyzed and identified by Hybrid Quadrupole Time of Flight Mass Spctrometer.
In this study, we first apply proteomics technology to research the cytotoxicity mechanism in JAR / MTX by Shikonin and set up the two-dimensional gel electrophoresis maps before and after the treatment Shikonin in JAR/ MTX cells, and then preliminary reveal the complex mechanism. The expressions of 26 proteins were significantly changed, of which 20 were up-regulated and 6 were down-regulated in shikonin-treated JAR/MTX cells compared with control.9 protein spots were identified by mass spectrometry. The up regulated protein include ROA2、GNS、uMtCK、ANXA2、VDAC2、PRS8、ETFB .T he down regulated protein include CWC15、ezrin,These changes in protein may be involved in the toxic effects of shikonin. Ezrin is a cytoskeleton -associated protein, the present study focused on inhibition of tumor cell apoptosis and promote tumor development, invasion and metastasis. Over-expression of ERM proteins is related to metastasis of many tumors. This study found for the first time ezrin reduced protein expression in the drug-resistant cells by Shikonin,which result is consistent with 2-DE and MS results. A decline in its expression not only mediated the JAR/MTX early morphological changes of apoptosis, but also inhibit their proliferation, induced apoptosis ,ezrin possible play an important role in the toxicity mechanism of Shikonin. VDAC2 located in the outer membrane of mitochondria is the composition of mitochondrial permeability transition pore, the closure can inhibit mitochondrial function, lead to changes in mitochondrial membrane permeability, which release cytochrome C, apoptosis and other factors eventually lead to apoptosis. A lot of evidence indicate that the VDAC is very important to the regulation of the mitochondria and cell function. We also found for the first time VDAC2 upregulation of drug group after Shikonin, which result is consistent with 2-DE and MS results .Through research and analysis about VDAC2, it indicate that shikonin-induced JAR / MTX cell apoptosis is likely through the mitochondrial pathway. The occurrence and development of tumor is closely related to ANXA2’S expression disorder and the information indicate that it may play an important role at the tumor invasion and metastasis . ANXA2 is different with the majority of tumor ,in the drug group in this experiment .It has a very significant increase ,maybe for the unique Shikonin mechanism. Heterogeneous ribonucleoprotein (hnRNP) are RNA-binding protein ,hnRNP play an important role in transcriptional regulation, pre-mRNA processing of a variety of biological processes. HnRNP A2/B1 study has shown that it is related to telomerase activity. In this experiment HnRNP A2/B1 is upregulate in drug group .Shikonin possible through the HnRNP A2/B1 impact the telomerase activity, which play a role in toxicity, the specific mechanism need to be further studied. uMtCK is the key enzyme in energy metabolism of energy conveying, muscle contraction and ATP regeneration. In this study uMtCK is upregulate in drug group due to Shikonin’s destructive effects. PRS8 is a sub-unit belong to the ubiquitin/ proteasome system. Ubiquitin / proteasome system in eukaryotic cells are the main channels of protein-selective degradation, while the proteasome is a great place of protein degradation. It has ATP-dependent, can selectively identify and remove the ubiquitined protein about 8O- 90% intracellular proteins. Thus, UPS is also involved in cell proliferation and differentiation, signal transduction, cell cycle, gene transcription regulation, apoptosis and many other kinds of biological functions. The study found that in drug group PRS8 significantly increase, suggesting its possible importance as a protein involved in shikonin cytotoxicity. Because a wide range of biological functions, its specific role is worthy of further study.
Change the above-mentioned proteins may be related to the toxic effects of shikonin, or it may be the role of Shikonin target. This is not only for the previous research results of others to provide a more powerful objective evidence, but also to reveal the molecular mechanism of a large number of new leads. This indicate Shikonin may become a new drug to drug-resistant choriocarcinoma. Those shikonin-related proteins’structure and function are worthy of further in-depth study, which may have provided for the treatment of drug-resistant choriocarcinoma new ideas.
引文
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[94]杨义力.蛋白质泛素化系统[J].生命科学,2002,14:279-282
[95]何维,高晓明,曹雪涛,等.医学免疫学.北京:人民出版社,2005:232—234.
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