Screening From A Series Of Plant Polyphenolses To Resist Human Breast Cancer Cell Line MCF-7/ADM Combined With ADM And Study On The Reversal Effect Of Resveratrol On Multidrug-resistance

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.ObjectivesAims 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 ADMBy 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(IC₁₀) 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/ADMMTT 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(IC₅₀),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,IC₅₀ of ADM was 0.39μmol/L,IC₁₀ of Res was 8.46μmol/L,On MCF-7/ADM cells,IC₅₀ of ADM was 21.38μmol/L,IC₁₀ 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 cellsMultidrug 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 cm³ 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 cm³);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.Conclusions1.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.