南蛇藤总萜对人乳腺癌MCF-7/ADM多药耐药的逆转作用以及相关机制的实验研究
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摘要
化学治疗在乳腺癌的综合治疗中占有重要地位,近年来,新的细胞毒性药物的出现使乳腺癌的治疗有了明显改观,尽管如此,肿瘤细胞对多种化疗产生多药耐药(multidrug resistance,MDR)仍是造成化疗失败的主要原因,MDR是指恶性肿瘤在接触一种化疗药物后,除了对这种药物产生耐药以外,还对其他结构及作用机制与之不同的化疗药物产生了交叉耐药的现象。
对于乳腺癌MDR的研究,目前已知有多种机制参与,包括①药物运输/摄取如P-糖蛋白(P-gp)、多药耐药相关蛋白(MRP)、肺耐药相关蛋白(LRP)等;②代谢解毒如谷胱甘肽(GSH)及其酶系统;③药物作用效靶如DNA拓扑异构酶Ⅱ(TopoⅡ);④凋亡途径改变等。其中认识最早并研究最深入的是P-gp/mdr1介导的MDR,而GSH及其酶系统对细胞药物解毒及抗氧化过程起重要作用,又可增加肿瘤细胞对抗肿瘤药物的代谢与运输能力,使细胞产生抗药性。
现已被证实具有逆转耐药活性的药物有钙通道阻滞剂、免疫调节剂、钙调蛋白拮抗剂等,因疗效欠佳、作用靶点单一、毒副作用大等难以应用于临床。中药因具有疗效确切、多靶点、毒副作用小等优点,具有很大的研究价值,所以从中药中寻找安全有效的耐药逆转剂是当今研究的热点。南蛇藤(Celastrus orbiculatus)为卫矛科南蛇藤(celastrus)属植物,分布广泛,其藤茎、根、叶、果均可入药。我们课题组研究证实南蛇藤具有较强的抗肿瘤活性,诱导细胞凋亡是其杀伤肿瘤细胞的途径之一。
目的:
本课题应用高效液相色谱(HPLC)法、生物化学法、免疫组化法等探讨南蛇藤总萜是否具有逆转人乳腺癌MCF-7/ADM多药耐药的作用,并对其可能的逆转机制进行研究,以期为临床应用提供理论和实验依据。
方法:
1.MTT法
以MTT法测定南蛇藤总萜对MCF-7(敏感细胞)以及MCF-7/ADM(耐药细胞)的直接细胞毒作用,以无细胞毒性的南蛇藤总萜作为逆转剂,用MTT法体外观察其对MCF-7/ADM的逆转作用,测定阿霉素(ADM)对两种细胞的毒性作用并计算出耐药倍数。
2.高效液相色谱(HPLC)法
用HPLC法测定南蛇藤总萜作用于MCF-7以及耐药细胞MCF-7/ADM后,细胞内ADM浓度变化。
3.流式细胞仪(FCM)
用流式细胞仪测定南蛇藤总萜作用于MCF-7以及耐药细胞MCF-7/ADM后,细胞的凋亡率;通过间接免疫荧光标记法测定南蛇藤总萜作用于MCF-7以及耐药细胞MCF-7/ADM后,细胞内P-gp的表达变化。
4.生物化学法
用生物化学法测定南蛇藤总萜作用于MCF-7/ADM前后,细胞内谷胱甘肽(GSH)含量的变化。
5.细胞免疫组化法
采用细胞免疫组化S-P染色法检测MCF-7以及耐药细胞MCF-7/ADM中P-gp的表达率,并检测南蛇藤总萜作用后,细胞内P-gp的表达变化。
结果:
1.南蛇藤总萜在10μg/ml浓度时对MCF-7以及耐药细胞MCF-7/ADM没有毒性,无细胞毒性的南蛇藤总萜(5μg/ml、10μg/ml)可以增强ADM对MCF-7/ADM的细胞毒作用,ADM对MCF-7的半数抑制率(IC_(50))是1.09μg/ml,对MCF-7/ADM的IC_(50)是62.62μg/ml,耐药倍数是57.45。
2.南蛇藤总萜(5μg/ml、10μg/ml)能够增加人乳腺癌耐药细胞MCF-7/ADM内的ADM的聚积浓度。
3.不同浓度南蛇藤总萜作用后,人乳腺癌敏感细胞MCF-7以及耐药细胞MCF-7/ADM的凋亡细胞比例升高。利用流式细胞免疫荧光技术检测人乳腺癌耐药细胞株MCF-7/ADM中P-gp的表达,其表达随着南蛇藤总萜作用浓度的增加而减少。
4.南蛇藤总萜(10μg/ml)能使MCF-7/ADM细胞内GSH含量下降,考虑是其逆转耐药的机制之一。
5.南蛇藤总萜(5μg/ml、10μg/ml)作用于人乳腺癌耐药细胞MCF-7/ADM后,细胞内P-gp的表达有所下降。
结论:
南蛇藤总萜具有一定体外抗乳腺癌作用,能够下调MCF-7/ADM细胞中GSH以及P-gp的表达量,可以增加MCF-7/ADM的凋亡率,可以增加MCF-7/ADM内的ADM浓度,从而部分逆转人乳腺癌耐药细胞MCF-7/ADM的多药耐药,可能成为一种有效安全的多药耐药逆转剂。
Chemotherapy play an important role in synthetic therapy of breast cancer.Resently new anticancer drugs has.greatly promoted the treating effect of many carcinoma including solid tumor.Multidrug resistance(MDR) remains to be a capital problem in chemotherapy.Once the tumor develop resistant to a certain anticancer drug,it will resist to drugs that are structurally and chemically unrelated.
Many mechanisms are now know contribute to MDR,including changes①in drug transport such as Permeability-glycoprotein(P-gp)、Multidrug Resistance Associated Protein(MRP)、Lung Resistance-related Protein(LRP);②in metabolic detoxificationg such as Glutation(GSH);③in drug target,such as topoisoeraseⅡ(TopoⅡ)、in DNA repair;④in apoptosis etc.Among these,the mechanism of P-gp/mdrl mediated was recognized earliestly and was researched most extensively.GSH and its enzyme systems not only play important roles in cellular drug detoxification and in the course of resisting oxidation,but also enhance the metabolism and transportation potency of anticancer drugs in cancer cells,the cancer cells then become drug resistance cell.
Now the active drugs which can reverse multidrug-resistance are as follow:calcium channel blocker,immunomodulator,calmodulin inhibitor and so on.Because the curative effect is not good,target is single and toxic action is obvious,thy are not frequently applied in clinic.Chinese traditional drag possesses the good quality of high performance,multitarget and little toxic action,so it has the value of being investigated.So searching safe and active reversal drug for multidrug resistance from Chinese traditional drug is a hot spot now.Celastrusorbiculatus is the plant of celastrus,distribute generally,its rattan,root,leafage,fruit are all put in drug.The modern chemical pharmacy has confirmed that some component of Celastrus possess doughty antineoplasmic activity,inducing cell apoptosis is one of the pathway that kill and wound the tumor cell.
1.Purpose:
The purposes of this research was to study on reversal effect and possible mechanism of MDR in MCF-7 /ADM cell line by the total terpenes of Celastrus orbiculatus with methods of high performance liquid chroma-tography(HPLC)、biochemical analyses,immunohisto chemical technique and so on to provide the theoretically and experimental bases for clinical use.
2.Method:
2.1 MTT method
Apply MTT deoxidized method to measure direct cell toxicant effect on MCF-7(sensitive strain) and MCF-7/ADM(multidrug-resistant strain) by the total terpenes of Celastrus orbiculatus.To observe the reverse effect of the total terpenes of Celastrus orbiculatus which has screened for nontoxicant concentration to MCF-7/ ADM by MTT.To measure toxicant effect of ADM to both strains for resistant index and resistance fold by MTT;
2.2 Hing performance liquid chromatography(HPLC)
Apply Hing performance liquid chromatography to detect cellular ADM concentration in MCF-7、MCF-7/ADM which were treated with the total terpenes of Celastrus orbiculatus.
2.3 Flow cytometry
Apply Flow cytometry to detect apoptosis cell population in MCF-7、MCF-7/ADM which were treated with the total terpenes of Celastrus orbiculatus.Apply Flow cytometry to survey P-gp proteinum transmutation in MCF-7/ADM which were treated with the total terpenes of Celastrus orbiculatus through indirect fluorescent labeling methond.
2.4 Biochemical analyse
Apply Biochemical analyse with DTNB to detect celluar GSH concentration of MCF-7、MCF-7/ADM which were treated with total terpenes of Celastrus orbiculatus.
2.5 Cell immunohistochemistry
Apply Cell immunohistochemistry S-P staining method to meaure P-gp proteinum masculine expressing rate in MCF-7/ADM which were treated with total terpenes of Celastrus orbiculatus.
3.Result
3.1 There is no obvious cell toxicity of the total terpenes of Celastrus orbiculatus to MCF-7 and MCF-7/ADM cell when the concentration of the total terpenes of Celastrus orbiculatus is 10μg/ml below.The total terpenes of Celastrus orbiculatus(5μg/ml、10μg/ml) can increased cell toxic effect of ADM to MCF-7/ADM.Inhibitive concentration 50 of ADM to MCF-7 and MCF-7/ADM are1.09μg/ml and 62.62μg/ml respectively,resistance fold of MCF-7/ADM is57.45.
3.2 The total terpenes of Celastrus orbiculatus(5μg/ml、10μg/ml)can increased MCF-7/ADM's accumulated concentration of ADM.
3.3 MCF-7 and MCF-7/ADM are effected by different densities of the total terpenes of Celastrus orbiculatus,the rate of apoptisis cell step up obviously,and to offer dose dependent.Utilizing Flow cytometry immunofluorescence technic detect P-gp gene product which decreased along with the increase of dosage of the total terpenes of Celastrus orbiculatus.
3.4 The total terpenes of Celastrus orbiculatus(10μg/ml) can decrease the celluar GSH concentration in MCF-7/ADM cell line,decreasing of celluar GSH concentration was one of the reversal mechanisms of MDR in MCF-7/ADM cell line by the total terpenes of Celastrus orbiculatus.
3.5 After treated with the total terpenes of Celastrus orbiculatus(5μg/ml、10μg/ml), P-gp proteinum expression all have different degree decrease in MCF-7/ADM cell line.
4.Conclusion
The total terpenes of Celastrus orbiculatus has certain breast tumout-resistant effect,it can increase MCF-7/ADM'S accumulated concentration of ADM,it can reduce MCF-7/ADM'S P-gp and GSH expression,it can increase the apoptosis cell population of MCF-7/ADM,the total terpenes of Celastrus orbiculatus can partly revese multidrug resistance of MCF-7/ADM in vitro.So Celastrus orbiculatus may be a kind of safe and efficient reversal agent for clinic.
对于乳腺癌MDR的研究,目前已知有多种机制参与,包括①药物运输/摄取如P-糖蛋白(P-gp)、多药耐药相关蛋白(MRP)、肺耐药相关蛋白(LRP)等;②代谢解毒如谷胱甘肽(GSH)及其酶系统;③药物作用效靶如DNA拓扑异构酶Ⅱ(TopoⅡ);④凋亡途径改变等。其中认识最早并研究最深入的是P-gp/mdr1介导的MDR,而GSH及其酶系统对细胞药物解毒及抗氧化过程起重要作用,又可增加肿瘤细胞对抗肿瘤药物的代谢与运输能力,使细胞产生抗药性。
现已被证实具有逆转耐药活性的药物有钙通道阻滞剂、免疫调节剂、钙调蛋白拮抗剂等,因疗效欠佳、作用靶点单一、毒副作用大等难以应用于临床。中药因具有疗效确切、多靶点、毒副作用小等优点,具有很大的研究价值,所以从中药中寻找安全有效的耐药逆转剂是当今研究的热点。南蛇藤(Celastrus orbiculatus)为卫矛科南蛇藤(celastrus)属植物,分布广泛,其藤茎、根、叶、果均可入药。我们课题组研究证实南蛇藤具有较强的抗肿瘤活性,诱导细胞凋亡是其杀伤肿瘤细胞的途径之一。
目的:
本课题应用高效液相色谱(HPLC)法、生物化学法、免疫组化法等探讨南蛇藤总萜是否具有逆转人乳腺癌MCF-7/ADM多药耐药的作用,并对其可能的逆转机制进行研究,以期为临床应用提供理论和实验依据。
方法:
1.MTT法
以MTT法测定南蛇藤总萜对MCF-7(敏感细胞)以及MCF-7/ADM(耐药细胞)的直接细胞毒作用,以无细胞毒性的南蛇藤总萜作为逆转剂,用MTT法体外观察其对MCF-7/ADM的逆转作用,测定阿霉素(ADM)对两种细胞的毒性作用并计算出耐药倍数。
2.高效液相色谱(HPLC)法
用HPLC法测定南蛇藤总萜作用于MCF-7以及耐药细胞MCF-7/ADM后,细胞内ADM浓度变化。
3.流式细胞仪(FCM)
用流式细胞仪测定南蛇藤总萜作用于MCF-7以及耐药细胞MCF-7/ADM后,细胞的凋亡率;通过间接免疫荧光标记法测定南蛇藤总萜作用于MCF-7以及耐药细胞MCF-7/ADM后,细胞内P-gp的表达变化。
4.生物化学法
用生物化学法测定南蛇藤总萜作用于MCF-7/ADM前后,细胞内谷胱甘肽(GSH)含量的变化。
5.细胞免疫组化法
采用细胞免疫组化S-P染色法检测MCF-7以及耐药细胞MCF-7/ADM中P-gp的表达率,并检测南蛇藤总萜作用后,细胞内P-gp的表达变化。
结果:
1.南蛇藤总萜在10μg/ml浓度时对MCF-7以及耐药细胞MCF-7/ADM没有毒性,无细胞毒性的南蛇藤总萜(5μg/ml、10μg/ml)可以增强ADM对MCF-7/ADM的细胞毒作用,ADM对MCF-7的半数抑制率(IC_(50))是1.09μg/ml,对MCF-7/ADM的IC_(50)是62.62μg/ml,耐药倍数是57.45。
2.南蛇藤总萜(5μg/ml、10μg/ml)能够增加人乳腺癌耐药细胞MCF-7/ADM内的ADM的聚积浓度。
3.不同浓度南蛇藤总萜作用后,人乳腺癌敏感细胞MCF-7以及耐药细胞MCF-7/ADM的凋亡细胞比例升高。利用流式细胞免疫荧光技术检测人乳腺癌耐药细胞株MCF-7/ADM中P-gp的表达,其表达随着南蛇藤总萜作用浓度的增加而减少。
4.南蛇藤总萜(10μg/ml)能使MCF-7/ADM细胞内GSH含量下降,考虑是其逆转耐药的机制之一。
5.南蛇藤总萜(5μg/ml、10μg/ml)作用于人乳腺癌耐药细胞MCF-7/ADM后,细胞内P-gp的表达有所下降。
结论:
南蛇藤总萜具有一定体外抗乳腺癌作用,能够下调MCF-7/ADM细胞中GSH以及P-gp的表达量,可以增加MCF-7/ADM的凋亡率,可以增加MCF-7/ADM内的ADM浓度,从而部分逆转人乳腺癌耐药细胞MCF-7/ADM的多药耐药,可能成为一种有效安全的多药耐药逆转剂。
Chemotherapy play an important role in synthetic therapy of breast cancer.Resently new anticancer drugs has.greatly promoted the treating effect of many carcinoma including solid tumor.Multidrug resistance(MDR) remains to be a capital problem in chemotherapy.Once the tumor develop resistant to a certain anticancer drug,it will resist to drugs that are structurally and chemically unrelated.
Many mechanisms are now know contribute to MDR,including changes①in drug transport such as Permeability-glycoprotein(P-gp)、Multidrug Resistance Associated Protein(MRP)、Lung Resistance-related Protein(LRP);②in metabolic detoxificationg such as Glutation(GSH);③in drug target,such as topoisoeraseⅡ(TopoⅡ)、in DNA repair;④in apoptosis etc.Among these,the mechanism of P-gp/mdrl mediated was recognized earliestly and was researched most extensively.GSH and its enzyme systems not only play important roles in cellular drug detoxification and in the course of resisting oxidation,but also enhance the metabolism and transportation potency of anticancer drugs in cancer cells,the cancer cells then become drug resistance cell.
Now the active drugs which can reverse multidrug-resistance are as follow:calcium channel blocker,immunomodulator,calmodulin inhibitor and so on.Because the curative effect is not good,target is single and toxic action is obvious,thy are not frequently applied in clinic.Chinese traditional drag possesses the good quality of high performance,multitarget and little toxic action,so it has the value of being investigated.So searching safe and active reversal drug for multidrug resistance from Chinese traditional drug is a hot spot now.Celastrusorbiculatus is the plant of celastrus,distribute generally,its rattan,root,leafage,fruit are all put in drug.The modern chemical pharmacy has confirmed that some component of Celastrus possess doughty antineoplasmic activity,inducing cell apoptosis is one of the pathway that kill and wound the tumor cell.
1.Purpose:
The purposes of this research was to study on reversal effect and possible mechanism of MDR in MCF-7 /ADM cell line by the total terpenes of Celastrus orbiculatus with methods of high performance liquid chroma-tography(HPLC)、biochemical analyses,immunohisto chemical technique and so on to provide the theoretically and experimental bases for clinical use.
2.Method:
2.1 MTT method
Apply MTT deoxidized method to measure direct cell toxicant effect on MCF-7(sensitive strain) and MCF-7/ADM(multidrug-resistant strain) by the total terpenes of Celastrus orbiculatus.To observe the reverse effect of the total terpenes of Celastrus orbiculatus which has screened for nontoxicant concentration to MCF-7/ ADM by MTT.To measure toxicant effect of ADM to both strains for resistant index and resistance fold by MTT;
2.2 Hing performance liquid chromatography(HPLC)
Apply Hing performance liquid chromatography to detect cellular ADM concentration in MCF-7、MCF-7/ADM which were treated with the total terpenes of Celastrus orbiculatus.
2.3 Flow cytometry
Apply Flow cytometry to detect apoptosis cell population in MCF-7、MCF-7/ADM which were treated with the total terpenes of Celastrus orbiculatus.Apply Flow cytometry to survey P-gp proteinum transmutation in MCF-7/ADM which were treated with the total terpenes of Celastrus orbiculatus through indirect fluorescent labeling methond.
2.4 Biochemical analyse
Apply Biochemical analyse with DTNB to detect celluar GSH concentration of MCF-7、MCF-7/ADM which were treated with total terpenes of Celastrus orbiculatus.
2.5 Cell immunohistochemistry
Apply Cell immunohistochemistry S-P staining method to meaure P-gp proteinum masculine expressing rate in MCF-7/ADM which were treated with total terpenes of Celastrus orbiculatus.
3.Result
3.1 There is no obvious cell toxicity of the total terpenes of Celastrus orbiculatus to MCF-7 and MCF-7/ADM cell when the concentration of the total terpenes of Celastrus orbiculatus is 10μg/ml below.The total terpenes of Celastrus orbiculatus(5μg/ml、10μg/ml) can increased cell toxic effect of ADM to MCF-7/ADM.Inhibitive concentration 50 of ADM to MCF-7 and MCF-7/ADM are1.09μg/ml and 62.62μg/ml respectively,resistance fold of MCF-7/ADM is57.45.
3.2 The total terpenes of Celastrus orbiculatus(5μg/ml、10μg/ml)can increased MCF-7/ADM's accumulated concentration of ADM.
3.3 MCF-7 and MCF-7/ADM are effected by different densities of the total terpenes of Celastrus orbiculatus,the rate of apoptisis cell step up obviously,and to offer dose dependent.Utilizing Flow cytometry immunofluorescence technic detect P-gp gene product which decreased along with the increase of dosage of the total terpenes of Celastrus orbiculatus.
3.4 The total terpenes of Celastrus orbiculatus(10μg/ml) can decrease the celluar GSH concentration in MCF-7/ADM cell line,decreasing of celluar GSH concentration was one of the reversal mechanisms of MDR in MCF-7/ADM cell line by the total terpenes of Celastrus orbiculatus.
3.5 After treated with the total terpenes of Celastrus orbiculatus(5μg/ml、10μg/ml), P-gp proteinum expression all have different degree decrease in MCF-7/ADM cell line.
4.Conclusion
The total terpenes of Celastrus orbiculatus has certain breast tumout-resistant effect,it can increase MCF-7/ADM'S accumulated concentration of ADM,it can reduce MCF-7/ADM'S P-gp and GSH expression,it can increase the apoptosis cell population of MCF-7/ADM,the total terpenes of Celastrus orbiculatus can partly revese multidrug resistance of MCF-7/ADM in vitro.So Celastrus orbiculatus may be a kind of safe and efficient reversal agent for clinic.
引文
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5.白雪英,靳英.槲皮素对人乳腺癌MCF-7/ADR细胞P-gp表达的影响[J].实用医技杂志,2003,10(12):1348-1349.
6.靳英,周庚寅,张延国,等。植物多酚化合物逆转肿瘤多药耐药的筛选[J].山东大学学报(医学版),2002,40(6):497-499.
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8.唐小卿,曹建国,廖端芳,等.甲基莲心碱对人乳腺癌细胞阿霉素多药耐药的逆转[J].癌症,2001,20(8):831-833.
9.Cao J G,Tang X Q,zhou H,et al.Reveral of Multidrug Resistance by neferine in adriamycin resistant human breast cancer cell line MCF-7/ADM.The.Chinese-German[J].ClinOncol,2004.3(2):93.
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