Effect Of Rosmarinic Acid Derivative RAD-9 On Apoptosis Of Adriamycin-resistant Breast Cancer Cell Line MCF-7/adm And The Underlying Mechanisms

OBJECTIVE:（1）To study whether RAD-9 can reduce the resistance of MCF-7/ADM cells.（2）To investigate the mechanisms of apoptosis of MCF-7/ADM cells treated with RAD-9 in vitro.METHODS:（1）Identification of multidrug resistant cells of MCF-7/ADM:（1）MTT assay was used to evaluate the sensitivity of MCF-7/ADM and MCF-7cells to chemotherapeutic drugs,as well as calculation of IC₅₀0 and multiple drug resistance.（2）qRT-PCR was used to detect the mRNA expressions of MDR1 and MDR2 in MCF-7 and MCF-7/ADM cells.（3）Western blot was used to measure the expressions of P-pg,EGFR,NF-κB,Akt,p-Akt,ERK,p-ERK,p38 and p-p38 proteins in MCF-7 and MCF-7/ADM cells.（2）Effect of RAD-9 on apoptosis of MCF-7/ADM cells and its mechanisms:（1）MTT assay was taken to detect the survival of MCF-7/ADM cells treated with RAD-5,RAD-9,RAD-11 and ADM.（2）Colony assay was used to determine the cell colony formation rate on MCF-7/ADM cells treated with RAD-9.（3）The effect of RAD-9 on the morphology of MCF-7/ADM cells was observed with the optical microscope.（4）Hoechst 33258 staining was used to determine the effect of RAD-9 on morphology of MCF-7/ADM cells.（5）Flow cytometry was taken to detect the cell apoptosis of MCF-7/ADM cells treated with RAD-9.（6）qRT-PCR was used to determine the mRNA expressions of MDR1 and MDR2 in MCF-7/ADM cells treated with RAD-9.（7）Western blot was used to determine the protein expressions of P-pg、EGFR、Akt、p-Akt、ERK、p-ERK、p38 and p-p38 in MCF-7/ADM cells treated with RAD-9.RESULTS:（1）The result of MTT showed that ADM dramatically decreased the viability of MCF-7 cells（P<0.05）but not MCF-7/ADR cells;qRT-PCR showed that the mRNA expressions of MDR1 and MDR2 in MCF-7/ADM cells were significantly higher than that in MCF-7 cells;Western blot showed that the protein expressions of P-pg、EGFR、p-Akt and p-ERK in MCF-7/ADM cells were significantly higher than that in MCF-7 cells（P<0.05）.The protein expression of p-p38 in MCF-7/ADM cells was significantly higher than that in MCF-7 cells（P<0.05）.（2）Effect of RAD-9 on proliferation,colony formation and apoptosis of MCF-7 and MCF-7/ADM cells:（1）The result of MTT showed that RAD-9significantly diminished the proliferation of both MCF-7 and MCF-7/ADR cell lines in a dose-dependent manner compared with the control group（P<0.05）.However,the combination of non-cytotoxic RAD-9 and different concentrations of ADM could not enhance the anti-tumor effect of ADM.（2）Colony assay suggested that RAD-9 had an antiproliferative effect in MCF-7/ADM cells.（3）After being treated with RAD-9,the MCF-7/ADM cells became round and shrinked,then the connections between cells disappeared.Finally,cells started to separate from the surrounding ones and floate in the medium.（4）Hoechst33258 staining showed that the change of nucleus of MCF-7/ADM cells treated with RAD-9.（5）Western blot showed that the protein expression of Bcl-2 was significantly down-regulated after RAD-9 administration（P<0.05）,While the protein expressions of Bax and caspase-3 were significantly up-regulated after RAD-9 administrating（P<0.05）.These results suggested that RAD-9 can induce apoptosis of MCF-7/ADM cells.（3）Effect of RAD-9 on MDR1,MDR2 genes and P-pg protein:（1）qRT-PCR showed that the mRNA expressions of MDR1 and MDR2 were significantly dcreased after RAD-9 administrating（P<0.05）.（2）Western blot showed that the protein expression of P-pg was significantly reduced after RAD-9 administrating（P<0.05）.These results suggested that the mechanisms of MCF-7/ADM cell apoptosis induced by RAD-9 may be related to the down-regulation of MDR1,MDR2 and P-pg protein.RAD-9 can reduce the resistance of MCF-7/ADM cells.（4）Effect of RAD-9 on EGFR and its downstream PI3K/Akt,ERK/MAKP and p38/MAPK pathways:Western blot:（1）Both RAD-9 alone and combinated with EGFR inhibitor PD153035 can significantly down-regulate the expression of EGFR in MCF-7/ADM cells（P<0.05）.（2）RAD-9 can significantly reduce the expression of p-Akt（P<0.05）and it can reverse the activation of insulin to p-Akt（P<0.05）.（3）Both RAD-9 alone and combinated with ERK inhibitor SCH772984 can significantly down-regulate the expression of p-ERK in MCF-7/ADM cells（P<0.05）.（4）RAD-9 can activate the expression of p-p38 significantly（P<0.05）while it had no effect on p38（P>0.05）.These results suggested that RAD-9 induced apoptosis in MCF-7/ADM cells may be related to the inhibition of EGFR,PI3K/Akt and ERK/MAPK pathways and the activation of the p38/MAPK pathway.CONCLUSION:（1）RAD-9 can effectively inhibit the proliferation of MCF-7 and MCF-7/ADM cells and induce apoptosis of MCF-7/ADM cells.（2）RAD-9 can significantly down-regulate the expressions of MDR1 and MDR2 mRNA in MCF-7/ADM cells and down-regulate the expression of P-pg protein,which can reduce the resistance of MCF-7/ADM cells and induce apoptosis of MCF-7/ADM cells.（3）The mechanisms of apoptosis induced by RAD-9 in MCF-7/ADM cells may be related to the inhibition of EGFR signaling pathway and PI3K/Akt and ERK/MAPK signal pathways downstream of EGFR.