| Objective: Breast cancer incidence and mortality increased year by year,women are facing serious health threats.With the medical research on breast cancer continues to deepen,and the continuous development of various disciplines,the current breast cancer has a standardized and systematic treatment,then formed comprehensive treatment which includes surgery,chemotherapy,radiotherapy,endocrine therapy and HER2 molecular targeted therapy.Gene P53 has been a hot topic since it was first reported in 1979.People gradually discover that wild-type P53 had lots of functions such as promoting cell apoptosis and maintenance of genome stability,and more than half of the malignant tumor had mutation of P53 gene.So in 2003,recombinant human P53 adenovirus injection(rAd-P53,also called Gendicine)was listed as the world's first gene therapy drug.It's mechanism is to import exogenous P53 into proliferative tumor cell and modified 5 Adenovirus is used as a carrier.Then it plays its function such as blocking growth,promoting apoptosis,promoting the role of chemotherapy and radiotherapy,inhibition of vascular growth and so on.It has been proven to have a good effect in head and neck squamous cell carcinoma,but the research of breast cancer is few.In this study,we investigated the effect of rAd-P53 combined with chemotherapeutic drugs on the breast cancer MCF-7 cell line to explore whether the two have a synergistic action of anti-proliferation and pro-apoptotic effect,and then studied the changes in its related gene expression to understand its mechanism of action.This study provides a preliminary theoretical basis for the combination of rAd-P53 and chemotherapeutic agents in the treatment of breast cancer.Methods: Firstly,we applied rAd-P53 combined with adriamycin(ADM)or cis-diamine dichloroplatinum(CDDP)to breast cancer MCF-7 cells for 96 hours.The absorbance of each well was measured by WST colorimetric method at OD450 nm,then we calculated whether there is a synergistic effect between the two by CalcuSyn.Based on the WST results,we calculated the combination index(CI)and the individual components(fractions affected,Fa),CI <1,CI = 1,CI> 1 were expressed as synergistic,additive and antagonistic actions.We selected a certain concentration which has synergism,then detected the change of cell cycle by flow cytometry.At last,quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the changes of related pro-apoptotic genes' expression after the use of rAd-P53 and chemotherapeutic drugs alone and in combination.Results:1 RAd-P53 combined with chemotherapeutic drugs ADM and CDDP have synergistic antiproliferative effect on breast cancer cell line MCF-7.The survival rate of the blank control group was setted to 100%,and the the concentration of rAd-P53 from high to low was 3×102U/cell,1×103U/cell,3×103U/cell,1×104U/cell,3×104U/cell,1×105U/cell and 3×105U/cell,the concentration of ADM and CDDP was setted as 0.003?M,0.01?M,0.03?M,0.1 ?M,0.3 ?M,1 ?M and0.03?M,0.1?M,0.3?M,1?M,3?M,10?M.After 96 hours of culturing,we detected cell suviral rate and growth inhibitory rate under different concentration by WST kit and drawed growth curve.CalcuSyn analysised that when rAd-P53 combined with chemotherapeutic drugs ADM,Fa between 0.2-0.6,CI <1,indicating that the two have a synergistic anti-proliferative effect on breast cancer cell line MCF-7.There was a similar effect when rAd-P53 combined with CDDP,when Fa was between 0.2-0.6,CI <1.It showed rAd-P53 and CDDP have a synergistic anti-proliferative effect on breast cancer cell line MCF-7.2 RAd-P53 combined with chemotherapeutic drugs ADM or CDDP induced changes of cell cycle on breast cancer MCF-7 cell line.Making the no treatment group as the control group,we applied rAd-P53(1×104U/cell),ADM(0.2?M),CDDP(2?M)on breast cancer MCF-7 for 96 hours.After harvesting and immobilizing cells,we used flow cytometry to analysis cell cycle.The levels of subG1 in the three monotherapy groups were higher than that in the control group(0.17% in the control group,1.37% in the ADM group,0.74% in the CDDP group and 0.80% in the rAd-P53 group),the difference was statistically significant(P <0.05),suggesting that in the case of monotherapy drugs,all three drugs have induced apoptosis of tumor cells.RAd-P53 combined with ADM or CDDP acted on breast cancer cell line MCF-7 cells for 96 hours,then we collected,immobilized cells,analysised by flow cytometry.The results showed that the levels of subG1 in every combined groups were higher than that in the monotherapy groups.(14.02% in the rAd-P53 combined with ADM group,10.31% in the rAd-P53 combined with CDDP group),compared with the control group and the monotherapy groups,the difference was statistically significant(P <0.05).3 The expression of P53,P21,P27,Bax and Noxa in MCF-7 cell line was regulated by rAd-P53 combined with chemotherapy.MCF-7 cells were divided into 6 groups: control group,rAd-P53 group(1×104U/cell),ADM group(0.2?M),CDDP group(2?M),rAd-P53 combined with ADM group,rAd-P53 combined with CDDP group.After the six groups worked on MCF-7 for 96 hours,we extracted RNA and reversed transcriptased it for cDNA,detecting the expression of P53,P21,P27,Bax and Noxa by qRT-PCR.The results showed that P53 expression levels in rAd-P53 group and each combination group were higher than that in control group,ADM group and CDDP group(P <0.05).Compared with control group,the expression level of P21 of each monotherapy group was higher(P<0.05).And compared with each monotherapy group,the expression level of P21 of rAd-P53 combined with ADM group and rAd-P53 combined with CDDP group was higher(P<0.05).The expression level of P27 of ADM combined with rAd-P53 group and CDDP combined with rAd-P53 group was significantly higher than that in each monotherapy group(P <0.05).The expression level of Bax of CDDP combined with rAd-P53 group and ADM combined with rAd-P53 group was significantly higher than that in the monotherapy group and control group(P<0.05).The expression level of Noxa of CDDP combined with rAd-P53 group and ADM combined with rAd-P53 group was significantly higher than that in each monotherapy group and control group(P <0.05).Conclusion: Chemotherapeutic drugs ADM or CDDP combined with rAd-P53 have synergistic antiproliferative effect on breast cancer cell line MCF-7.The mechanism of the action is that they increased the expression of subG1 in the cell cycle and up-regulated the expression level of P53,P21,P27,Bax and Noxa.And rAd-P53 can enhance the apoptosis induction of chemotherapeutic drug. |
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