植物多酚类化合物联合阿霉素抑制MCF-7/ADM细胞的筛选及白藜芦醇逆转耐药性作用研究
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摘要
乳腺癌是一种严重影响妇女身心健康甚至威胁生命的常见病与多发病,而肿瘤的多药耐药(Multidrug Resistance,MDR)与化疗的毒副反应是临床上制约乳腺癌患者化疗的疗效从而影响生存与预后的两个严重问题。研究肿瘤MDR安全有效的逆转剂是肿瘤化疗中亟待解决的问题,亦是目前肿瘤研究的难点及热点领域。本研究从六种主要植物多酚类化合物中筛选出联合阿霉素抑制人乳腺癌多药耐药MCF-7/ADM细胞作用较强的化合物;研究该化合物联合阿霉素对MCF-7/ADM细胞产生的具体效应、增敏效果及增敏的可能原因;探讨增敏的可能机制;最后通过建立荷耐药移植瘤裸鼠模型来明确该化合物联合阿霉素治疗耐药肿瘤细胞移植瘤的体内效果。在筛选植物多酚类化合物中,我们率先尝试应用均匀设计的方法,大大缩短实验周期,得到理想的筛选结果,证实均匀设计是多因素、多水平的试验的良好处理方法。
研究目的
本研究通过体内外实验探讨植物多酚类化合物协同阿霉素抑制人乳腺癌多药耐药MCF-7/ADM细胞的作用,为寻找逆转肿瘤多药耐药性的新药物提供实验依据。
研究方法与结果
1.植物多酚类化合物联合阿霉素抑制人乳腺癌多药耐药MCF-7/ADM细胞作用的筛选
将六种植物多酚类化合物按配伍组设计,用MTT法初筛对MCF-7/ADM细胞具有抑制作用的植物多酚类化合物;再把初筛出的化合物和阿霉素经均匀设计,筛出联合阿霉素MCF-7/ADM细胞作用较强的化合物;最后用单细胞凝胶电泳(SCGE)检测筛出的化合物联合阿霉素对正常肝细胞的毒性作用。
结果显示:槲皮素、白藜芦醇和阿魏酸对MCF-7/ADM细胞的10%抑制浓度(IC_(10))分别是7.41μmol/L、10.70μmol/L、12.30μmol/L,儿茶素、大豆苷元、芝麻素在128μmol/L浓度下均无作用(P＞0.05);均匀设计的结果表明联合阿霉素(ADM)抑制MCF-7/ADM细胞的作用中,白藜芦醇(Res)较槲皮素和阿魏酸明显;SCGE试验证明12μmol/L白藜芦醇和10μmol/L阿霉素不论是单用还是联合应用均不会对正常肝细胞不会造成DNA的损伤。
2.白藜芦醇对人乳腺癌MCF-7/ADM细胞耐药性逆转的研究
采用MTT法测MCF-7/ADM细胞的耐药性及Res对MCF-7/S和MCF-7/ADM细胞的抑制作用;同时将ADM和Res联合作用于MCF-7/ADM细胞,通过计算联合作用后的抑制率IR%和半数抑制浓度IC_(50)值,得出Q值和逆转倍数,以判断Res的抑瘤增效及逆转作用;最后应用荧光分光光度法检测联合用药后MCF-7/ADM细胞内阿霉素浓度的变化。
结果表明:ADM对MCF-7/S细胞IC_(50)值为0.39μmol/L;对MCF-7/ADM细胞IC_(50)值为21.38μmol/L;MCF-7/ADM细胞的耐药倍数为54.82倍;Res对MCF-7/S细胞IC_(10)值为8.46μmol/L,Res对MCF-7/ADM细胞IC_(10)值为11.39μmol/L,Res在两种细胞之间无耐药性(1.35倍);从Res的抑瘤增效实验可知Res与ADM在体外均有相加或增强作用,绝大多数联合用药组的Q＞1.15,表明二者的作用主要表现为协同作用;同时Res具有提高MCF-7/ADM细胞对ADM的敏感性,其逆转倍数随着Res浓度的增高而增加(ADM联合4μmol/L Res时逆转倍数为1.950,联合8μmol/L Res时逆转倍数为2.178,联合12μmol/L Res时逆转倍数为2.355);荧光分光光度法检测结果提示:不论MCF-7/S细胞和MCF-7/ADM细胞,细胞内ADM浓度均随细胞外化疗药物ADM浓度的增加而升高,且耐药细胞MCF-7/ADM内ADM的浓度明显低于敏感细胞MCF-7/S内ADM的浓度(P＜0.01)。当加入Res后,与对照组相比,耐药细胞MCF-7/ADM内ADM的浓度明显升高(P＜0.05),且细胞内ADM浓度均随细胞外ADM浓度升高而升高。
3.白藜芦醇增加人乳腺癌MCF-7/ADM细胞内阿霉素浓度的机制探讨
通过RT-PCR方法检测Res对MCF-7/ADM细胞mdr-1mRNA转录的影响,Western-blot方法检测Res对MCF-7/ADM细胞mdr-1基因表达产物P-gp的影响,探讨白藜芦醇增加人乳腺癌MCF-7/ADM细胞内阿霉素蓄积性的机制。
结果提示联合治疗后MCF-7/ADM细胞mdr-1基因表达和mdr-1基因表达产物P-gp表达量均较空白组、阿霉素组和白藜芦醇组低(P＜0.05)。
4.白藜芦醇联合阿霉素对荷耐药移植瘤裸鼠的抑瘤作用研究
建立小鼠乳腺癌模型后,经腹腔注射不同药物后,测定小鼠体重、肿瘤体积和肿瘤重量,算出抑瘤率;并观察用药后的毒副作用;最后通过HE染色进行肿瘤组织形态学观察。
实验结果提示用药6W后,ADM+Res组移植瘤体积(1.203±0.338)cm~3明显小于其余各组,抑瘤率为50.58%,与单用ADM组(1.979±0.307)cm~3相比有差异(P＜0.05);肿瘤重量的结果与瘤体结果类似;未观察到用药后的毒副作用;经不同处理后,单独注射Res组小鼠体重较生理盐水组及ADM组有所增加(P＜0.05),而ADM+Res组治疗前后体重无差异(P＞0.05);HE结果显示与其他组相比,ADM+Res组肿瘤组织中出现大片坏死区域。
结论
1.Res联合ADM对人乳腺癌MCF-7/ADM细胞具有抑制作用,且对正常组织基本无毒副作用;
2.Res能逆转MCF-7/ADM细胞的多药耐药性,其原因与增加ADM药物浓度有关;
3.Res可提高ADM在MCF-7/ADM细胞内的蓄积的原因与下调MCF-7/ADM细胞mdr-1基因表达和减少mdr-1基因表达产物P-gp有关。
4.Res能在体内增强ADM对荷耐药移植瘤裸鼠的抑瘤作用,并且没有明显的毒副反应。
总之,Res在体内外均可协同ADM抑制人乳腺癌多药耐药MCF-7/ADM细胞的作用,本研究为Res成为逆转肿瘤多药耐药性的新药物提供科学依据,提示Res在抗乳腺癌多药耐药的作用上具有良好的研究价值。
Breast cancer is a kind of frequently-occurring disease which seriously damages the physical and mental health of feminality.Multidrug-resistance(MDR) and toxicity of chemoagents are the two major obstacles to the successful cancer chemotherapy.To investigate a safe and efficient MDR reversal reagent is an emergent problem to be solved,and it is one of the significant missions in the area of tumor therapy.Reversing multidrug resistance in cancer will provide the basis for future studies of overcoming drug resistance and ultimately improving chemotherapy and the outcome of cancer patients.
The study was to screen out the compound from six plant polyphenolses which could more efficient to inhibit human breast cancer cell line MCF-7/ADM combined with adriamycin(ADM,one of chemotherapeutic drug);and to research the compound reversed effect in cancer cells and possible mechanism of reversaling MDR on MCF-7/ADM cells in vitro;finally to evaluated the anticancer efficacy of Resveratrol(Res) combinated with ADM in vivo by tumor-bearing nude mice.
In this research we screened out the efficient compound by the uniform design which helped us to shorten experimental period and find out ideal result.The uniform design was a kind of good method to handle the test of several factors and several levels.
Objectives
Aims to investigate the multi-drug resistance reversal effects of plant polyphenolses on human breast cancer cell line MCF-7/ADM combined with ADM, to present a foundation of experiment for finding out a new drug to reverse MDR.
Methods and Results
1.Drug Screening from a series of Plant Polyphenolses to Resist Human Breast Cancer Cell Line MCF-7/ADM Combined with ADM
By randomized block design,to screen out some compounds which could efficient to inhibit human breast cancer cell line MCF-7/ADM combined with ADM from six plant polyphenolses such as Epigallocatechin gallate,Quercetin,Daidzein, Resveratrol,Fumalic acid and Sesamin;then find out the compound from latter by uniform design which was more efficient to inhibit the cancer cells,the safety of the drug was evaluated by SCGE assay on liver cells.
The results showed:Human breast cancer MCF-7/ADM cell line was cultured in vitro,the cytotoxicity of six ployphenolses was assessed against MCF-7/ADM cells by methyl thiazolyl tetrazolium(MTT) assay.Quercetin,Fumalic acid and Resveratrol alone inhibited MCF-7/ADM cells proliferation by randomized block design,10%inhibition concentration(IC_(10)) of Quercetin was 7.41μmol/L,of Resveratrol was 10.70μmol/L,and of Fumalic acid was 12.30μmol/L,however, other compounds such as Epigallocatechin gallate,Daidzein and Sesamin could not inhibite MCF-7/ADM cells(0～128μmol/L)(P＞0.05);Resveratrol(Res) was more efficient to inhibit the cancer cells than Quercetin and Fumalic acid by uniform design; SCGE test demonstrated that 12μmol/L Res& 10μmol/L ADM was safe to liver DNA.
2.Studies of reversal effects of Res on MDR cancer cells MCF-7/ADM
MTT assay was used to detect the inhibitory effect of Res and ADM on human breast cancer cell proliferation;to analyze the combination effect of Res with ADM on cancer cell MCF-7/ADM,according to inhibition ratio(IR) and 50%Inhibition Concentration(IC_(50)),calculated combined index Q and reversal index;Fluorescence Spectrophotometry was used to detect the concentration of ADM in MCF-7/S cells and MCF-7/ADM cells.
The result of MTT showed that Res or ADM was able to inhibit MCF-7/ADM cells and MCF-7 /S cells proliferation.On MCF-7/S cells,IC_(50) of ADM was 0.39μmol/L,IC_(10) of Res was 8.46μmol/L,On MCF-7/ADM cells,IC_(50) of ADM was 21.38μmol/L,IC_(10) of Res was 11.39μmol/L,the MDR cancer cell line MCF-7/ADM were 54.82 times more resistant to ADM in comparison with sensitive cell line MCF-7/S,Res inhibitive effect in MCF-7/ADM cells was as same as in MCF-7/S, The proliferation of MCF-7/ADM cells was inhibited by Res at a dose-dependent manner;Res combined with ADM synergistically inhibited MCF-7/ADM cells growth,the combined index Q＞1.15 mostly,the results demonstrated Res combined with ADM possessed the synergism effect;Res enhanced ADM to inhibit the MDR cell growth at a dose-dependent manner too,reversal index of ADM+ 4μmol/L Res was 1.950,of ADM+ 8μmol/L Res was 2.178,of ADM+ 12μmol/L Res was 2.355; by Fluorescence Spectrophotometry,we found that concentration of ADM in MCF-7/S cells and MCF-7/ADM cells gradually elevated with chemotherapeutic drug enhanced,concentration of ADM in MCF-7/ADM cells was lower than in MCF-7/S(P＜0.01),in comparison with the control,concentration of ADM was significantly increasing(P＜0.05) when Res combined with ADM.Res was able to elevate the concentration of ADM in MCF-7/ADM cells at a dose-dependent manner also.
3.Studies of the mechanism of Res functionally elevated the drug accumulation on MCF-7/ADM cells
Multidrug resistance gene 1(mdr-1) mRNA was measured quantitatively by reverse transcription-poly-merase chain reaction(RT-PCR).P-Glycoprotein(P-gp) which was a kind of protein expressed by mdr-1 was measured quantitatively by Western-blot.
The results demonstrated the mechanism of Res functionally elevated the drug accumulation on MCF-7/ADM cells by down-regulator expression of mdr-1 gene and its product P-gp.
4.Studies of the inhibit effect of Res combined with ADM on nude mice bearing implanted MCF-7/ADM tumor.
To observed the boby weigh,tumor volume,tumor weigh of tumor-bearing nude mice and side-effects of treatment after injected drug,then to calculate tumor inhibition ratio;the morphological changes of tumor tissues were assessed by HE staining.
Six weeks after drug intervention,we found,in ADM+Res group,the mean tumor volume of MCF-7/ADM cells implanted tumor was 1.203±0.338 cm~3 with statistical significance to compare with other groups(P＜0.05),decreased about 50.58%compared with the mean tumor volume of ADM alone group(1.979±0.307 cm~3);the result of the mean tumor weigh was alike to the mean tumor volume; compared with the control,Res could relieve weigh loss(P＜0.05),and the toxicity of treatment was not obvious;Microscopic examination showed that there was massive area of cytoclasis and apoptosis in the tumor tissue of the combination group comparing with other groups.
Conclusions
1.Res could enhance the inhibitory effect of ADM on human breast cancer cell line MCF-7/ADM cells and it was safety;
2.Res could reversal MDR on MCF-7/ADM cells,the reason was Res could elevated concentration of ADM in MCF-7/ADM cells;
3.The mechanism of elevated ADM accumulate was down-regulator the expression levels of mdr-1 gene and P-gp;
4.Res could inhibit tumor in vivo combined with ADM on nude mice bearing implanted MCF-7/ADM tumor,and it had not significant side-effects.
All together,our results indicate that Res could enhance the inhibitory effect of ADM in vitro and in vivo on MCF-7/ADM cells;to present a foundation of experiment what Res as a novel MDR reversal reagent.It is worthwhile to explore the therapeutic potential of Res in the therapy of breast cancer.
研究目的
本研究通过体内外实验探讨植物多酚类化合物协同阿霉素抑制人乳腺癌多药耐药MCF-7/ADM细胞的作用,为寻找逆转肿瘤多药耐药性的新药物提供实验依据。
研究方法与结果
1.植物多酚类化合物联合阿霉素抑制人乳腺癌多药耐药MCF-7/ADM细胞作用的筛选
将六种植物多酚类化合物按配伍组设计,用MTT法初筛对MCF-7/ADM细胞具有抑制作用的植物多酚类化合物;再把初筛出的化合物和阿霉素经均匀设计,筛出联合阿霉素MCF-7/ADM细胞作用较强的化合物;最后用单细胞凝胶电泳(SCGE)检测筛出的化合物联合阿霉素对正常肝细胞的毒性作用。
结果显示:槲皮素、白藜芦醇和阿魏酸对MCF-7/ADM细胞的10%抑制浓度(IC_(10))分别是7.41μmol/L、10.70μmol/L、12.30μmol/L,儿茶素、大豆苷元、芝麻素在128μmol/L浓度下均无作用(P＞0.05);均匀设计的结果表明联合阿霉素(ADM)抑制MCF-7/ADM细胞的作用中,白藜芦醇(Res)较槲皮素和阿魏酸明显;SCGE试验证明12μmol/L白藜芦醇和10μmol/L阿霉素不论是单用还是联合应用均不会对正常肝细胞不会造成DNA的损伤。
2.白藜芦醇对人乳腺癌MCF-7/ADM细胞耐药性逆转的研究
采用MTT法测MCF-7/ADM细胞的耐药性及Res对MCF-7/S和MCF-7/ADM细胞的抑制作用;同时将ADM和Res联合作用于MCF-7/ADM细胞,通过计算联合作用后的抑制率IR%和半数抑制浓度IC_(50)值,得出Q值和逆转倍数,以判断Res的抑瘤增效及逆转作用;最后应用荧光分光光度法检测联合用药后MCF-7/ADM细胞内阿霉素浓度的变化。
结果表明:ADM对MCF-7/S细胞IC_(50)值为0.39μmol/L;对MCF-7/ADM细胞IC_(50)值为21.38μmol/L;MCF-7/ADM细胞的耐药倍数为54.82倍;Res对MCF-7/S细胞IC_(10)值为8.46μmol/L,Res对MCF-7/ADM细胞IC_(10)值为11.39μmol/L,Res在两种细胞之间无耐药性(1.35倍);从Res的抑瘤增效实验可知Res与ADM在体外均有相加或增强作用,绝大多数联合用药组的Q＞1.15,表明二者的作用主要表现为协同作用;同时Res具有提高MCF-7/ADM细胞对ADM的敏感性,其逆转倍数随着Res浓度的增高而增加(ADM联合4μmol/L Res时逆转倍数为1.950,联合8μmol/L Res时逆转倍数为2.178,联合12μmol/L Res时逆转倍数为2.355);荧光分光光度法检测结果提示:不论MCF-7/S细胞和MCF-7/ADM细胞,细胞内ADM浓度均随细胞外化疗药物ADM浓度的增加而升高,且耐药细胞MCF-7/ADM内ADM的浓度明显低于敏感细胞MCF-7/S内ADM的浓度(P＜0.01)。当加入Res后,与对照组相比,耐药细胞MCF-7/ADM内ADM的浓度明显升高(P＜0.05),且细胞内ADM浓度均随细胞外ADM浓度升高而升高。
3.白藜芦醇增加人乳腺癌MCF-7/ADM细胞内阿霉素浓度的机制探讨
通过RT-PCR方法检测Res对MCF-7/ADM细胞mdr-1mRNA转录的影响,Western-blot方法检测Res对MCF-7/ADM细胞mdr-1基因表达产物P-gp的影响,探讨白藜芦醇增加人乳腺癌MCF-7/ADM细胞内阿霉素蓄积性的机制。
结果提示联合治疗后MCF-7/ADM细胞mdr-1基因表达和mdr-1基因表达产物P-gp表达量均较空白组、阿霉素组和白藜芦醇组低(P＜0.05)。
4.白藜芦醇联合阿霉素对荷耐药移植瘤裸鼠的抑瘤作用研究
建立小鼠乳腺癌模型后,经腹腔注射不同药物后,测定小鼠体重、肿瘤体积和肿瘤重量,算出抑瘤率;并观察用药后的毒副作用;最后通过HE染色进行肿瘤组织形态学观察。
实验结果提示用药6W后,ADM+Res组移植瘤体积(1.203±0.338)cm~3明显小于其余各组,抑瘤率为50.58%,与单用ADM组(1.979±0.307)cm~3相比有差异(P＜0.05);肿瘤重量的结果与瘤体结果类似;未观察到用药后的毒副作用;经不同处理后,单独注射Res组小鼠体重较生理盐水组及ADM组有所增加(P＜0.05),而ADM+Res组治疗前后体重无差异(P＞0.05);HE结果显示与其他组相比,ADM+Res组肿瘤组织中出现大片坏死区域。
结论
1.Res联合ADM对人乳腺癌MCF-7/ADM细胞具有抑制作用,且对正常组织基本无毒副作用;
2.Res能逆转MCF-7/ADM细胞的多药耐药性,其原因与增加ADM药物浓度有关;
3.Res可提高ADM在MCF-7/ADM细胞内的蓄积的原因与下调MCF-7/ADM细胞mdr-1基因表达和减少mdr-1基因表达产物P-gp有关。
4.Res能在体内增强ADM对荷耐药移植瘤裸鼠的抑瘤作用,并且没有明显的毒副反应。
总之,Res在体内外均可协同ADM抑制人乳腺癌多药耐药MCF-7/ADM细胞的作用,本研究为Res成为逆转肿瘤多药耐药性的新药物提供科学依据,提示Res在抗乳腺癌多药耐药的作用上具有良好的研究价值。
Breast cancer is a kind of frequently-occurring disease which seriously damages the physical and mental health of feminality.Multidrug-resistance(MDR) and toxicity of chemoagents are the two major obstacles to the successful cancer chemotherapy.To investigate a safe and efficient MDR reversal reagent is an emergent problem to be solved,and it is one of the significant missions in the area of tumor therapy.Reversing multidrug resistance in cancer will provide the basis for future studies of overcoming drug resistance and ultimately improving chemotherapy and the outcome of cancer patients.
The study was to screen out the compound from six plant polyphenolses which could more efficient to inhibit human breast cancer cell line MCF-7/ADM combined with adriamycin(ADM,one of chemotherapeutic drug);and to research the compound reversed effect in cancer cells and possible mechanism of reversaling MDR on MCF-7/ADM cells in vitro;finally to evaluated the anticancer efficacy of Resveratrol(Res) combinated with ADM in vivo by tumor-bearing nude mice.
In this research we screened out the efficient compound by the uniform design which helped us to shorten experimental period and find out ideal result.The uniform design was a kind of good method to handle the test of several factors and several levels.
Objectives
Aims to investigate the multi-drug resistance reversal effects of plant polyphenolses on human breast cancer cell line MCF-7/ADM combined with ADM, to present a foundation of experiment for finding out a new drug to reverse MDR.
Methods and Results
1.Drug Screening from a series of Plant Polyphenolses to Resist Human Breast Cancer Cell Line MCF-7/ADM Combined with ADM
By randomized block design,to screen out some compounds which could efficient to inhibit human breast cancer cell line MCF-7/ADM combined with ADM from six plant polyphenolses such as Epigallocatechin gallate,Quercetin,Daidzein, Resveratrol,Fumalic acid and Sesamin;then find out the compound from latter by uniform design which was more efficient to inhibit the cancer cells,the safety of the drug was evaluated by SCGE assay on liver cells.
The results showed:Human breast cancer MCF-7/ADM cell line was cultured in vitro,the cytotoxicity of six ployphenolses was assessed against MCF-7/ADM cells by methyl thiazolyl tetrazolium(MTT) assay.Quercetin,Fumalic acid and Resveratrol alone inhibited MCF-7/ADM cells proliferation by randomized block design,10%inhibition concentration(IC_(10)) of Quercetin was 7.41μmol/L,of Resveratrol was 10.70μmol/L,and of Fumalic acid was 12.30μmol/L,however, other compounds such as Epigallocatechin gallate,Daidzein and Sesamin could not inhibite MCF-7/ADM cells(0～128μmol/L)(P＞0.05);Resveratrol(Res) was more efficient to inhibit the cancer cells than Quercetin and Fumalic acid by uniform design; SCGE test demonstrated that 12μmol/L Res& 10μmol/L ADM was safe to liver DNA.
2.Studies of reversal effects of Res on MDR cancer cells MCF-7/ADM
MTT assay was used to detect the inhibitory effect of Res and ADM on human breast cancer cell proliferation;to analyze the combination effect of Res with ADM on cancer cell MCF-7/ADM,according to inhibition ratio(IR) and 50%Inhibition Concentration(IC_(50)),calculated combined index Q and reversal index;Fluorescence Spectrophotometry was used to detect the concentration of ADM in MCF-7/S cells and MCF-7/ADM cells.
The result of MTT showed that Res or ADM was able to inhibit MCF-7/ADM cells and MCF-7 /S cells proliferation.On MCF-7/S cells,IC_(50) of ADM was 0.39μmol/L,IC_(10) of Res was 8.46μmol/L,On MCF-7/ADM cells,IC_(50) of ADM was 21.38μmol/L,IC_(10) of Res was 11.39μmol/L,the MDR cancer cell line MCF-7/ADM were 54.82 times more resistant to ADM in comparison with sensitive cell line MCF-7/S,Res inhibitive effect in MCF-7/ADM cells was as same as in MCF-7/S, The proliferation of MCF-7/ADM cells was inhibited by Res at a dose-dependent manner;Res combined with ADM synergistically inhibited MCF-7/ADM cells growth,the combined index Q＞1.15 mostly,the results demonstrated Res combined with ADM possessed the synergism effect;Res enhanced ADM to inhibit the MDR cell growth at a dose-dependent manner too,reversal index of ADM+ 4μmol/L Res was 1.950,of ADM+ 8μmol/L Res was 2.178,of ADM+ 12μmol/L Res was 2.355; by Fluorescence Spectrophotometry,we found that concentration of ADM in MCF-7/S cells and MCF-7/ADM cells gradually elevated with chemotherapeutic drug enhanced,concentration of ADM in MCF-7/ADM cells was lower than in MCF-7/S(P＜0.01),in comparison with the control,concentration of ADM was significantly increasing(P＜0.05) when Res combined with ADM.Res was able to elevate the concentration of ADM in MCF-7/ADM cells at a dose-dependent manner also.
3.Studies of the mechanism of Res functionally elevated the drug accumulation on MCF-7/ADM cells
Multidrug resistance gene 1(mdr-1) mRNA was measured quantitatively by reverse transcription-poly-merase chain reaction(RT-PCR).P-Glycoprotein(P-gp) which was a kind of protein expressed by mdr-1 was measured quantitatively by Western-blot.
The results demonstrated the mechanism of Res functionally elevated the drug accumulation on MCF-7/ADM cells by down-regulator expression of mdr-1 gene and its product P-gp.
4.Studies of the inhibit effect of Res combined with ADM on nude mice bearing implanted MCF-7/ADM tumor.
To observed the boby weigh,tumor volume,tumor weigh of tumor-bearing nude mice and side-effects of treatment after injected drug,then to calculate tumor inhibition ratio;the morphological changes of tumor tissues were assessed by HE staining.
Six weeks after drug intervention,we found,in ADM+Res group,the mean tumor volume of MCF-7/ADM cells implanted tumor was 1.203±0.338 cm~3 with statistical significance to compare with other groups(P＜0.05),decreased about 50.58%compared with the mean tumor volume of ADM alone group(1.979±0.307 cm~3);the result of the mean tumor weigh was alike to the mean tumor volume; compared with the control,Res could relieve weigh loss(P＜0.05),and the toxicity of treatment was not obvious;Microscopic examination showed that there was massive area of cytoclasis and apoptosis in the tumor tissue of the combination group comparing with other groups.
Conclusions
1.Res could enhance the inhibitory effect of ADM on human breast cancer cell line MCF-7/ADM cells and it was safety;
2.Res could reversal MDR on MCF-7/ADM cells,the reason was Res could elevated concentration of ADM in MCF-7/ADM cells;
3.The mechanism of elevated ADM accumulate was down-regulator the expression levels of mdr-1 gene and P-gp;
4.Res could inhibit tumor in vivo combined with ADM on nude mice bearing implanted MCF-7/ADM tumor,and it had not significant side-effects.
All together,our results indicate that Res could enhance the inhibitory effect of ADM in vitro and in vivo on MCF-7/ADM cells;to present a foundation of experiment what Res as a novel MDR reversal reagent.It is worthwhile to explore the therapeutic potential of Res in the therapy of breast cancer.
引文
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