The Combined Effects Of ?-3 Polyunsaturated Fatty Acids And All-trans Retinoic Acid On Growth Inhibition Of Breast Cancer Cells

All-trans retinoic acid(ATRA),a natural active derivative of vitamin A,has been extensively investigated in clinical trials of leukemia and other carcinoma,including breast cancer.However,ATRA has toxic and side effects in high dose.An increasing number of studies have found ?-3 polyunsaturated fatty acids(?-3 PUFAs)can effectively reduce the risk of breast cancer.Whether ?-3 PUFAs could reduce the effective concentration of ATRA on breast cancer and play a combined effect on growth inhibition of breast cancer cells is unclear.In this paper,we analyzed the growth of three subtypes of human breast cancer cells(ER+ MCF7,HER2+ SK-BR-3 and triple-negative HCC1806)and HCC1806 breast xenografts by treating with combination of ?-3 PUFAs and ATRA.The following results:(1)The anti-proliferative effects of ?-3 PUFAs and ATRA on the growth of multiple human breast cancer cells were assessed by cell counting and CCK8 assay.80 ?M PUFAs or 20 ?M ATRA alone has the weak inhibitory effects on cell growth of three subtypes of human breast cancer cells.However,the number of breast cancer cells was significantly reduced when ?-3 PUFAs combined with ATRA,compared with the same concentration of single ?-3 PUFAs or ATRA,suggesting a synergistic effect of ?-3 PUFAs and ATRA on cell growth.(2)The synergetic anti-tumor effect could be attributed to inhibition of cell cycle progression and induction of apoptosis,which was detected by flow cytometry and immunoblotting.Compared with the control group and single drug groups,combination of ATRA and ?-3 PUFAs could induce an accumulation of cells in the G0/G1 phase,change the expression of p21 and p27,increase the expression of cleaved-PARP and decrease the expression of Bcl-2.These results suggest that the combination of ?-3 PUFAs and ATRA could synergistically inhibit cell growth through cell cycle arrest and inducing apoptosis.(3)To research the mechanism of the combination of ?-3 PUFAs and ATRA,we used the pan-caspase inhibitors to treat breast cancer cells.In our study,the apoptotic cell number was not changed by MG132 and CQ treatment,but it was significantly reduced by BOC-D-FMK and Z-VAD-FMK inhibitor treatment.Further study found,cleaved Caspase-3 proteins and down-regulation of total Caspase-6 and Caspase-7 proteins were observed in combination of ?-3 PUFAs and ATRA,suggesting caspase signals might be involved in cell death induced.(4)The growth inhibition effects of EPA combined with ATRA on breast tumor xenograft model in vivo was assessed detected by immunohistochemistry and immunoblotting.In our study,combination of ATRA and EPA could also inhibit the tumor growth,increase the expression of p21 and cleaved-PARP and decrease the expression of Ki-67 and Bcl-2.These results indicate that synergistic anti-tumor effects of ?-3 PUFAs and ATRA are also through proliferation inhibition and apoptosis induction.In conclusion,we reported that the combination of ?-3 polyunsaturated fatty acids and ATRA led to a synergistic inhibition of cell growth in three subtypes of human breast cancer cells by blocking cell cycle and inducing caspase-dependent apoptosis.In addition,the combined treatment of ?-3 PUFAs and ATRA could inhibit the growth of the human breast cancer cell HCC1806 xenografted tumor in nude mice.These results elucidate that the research on the effect of ?-3 PUFAs combined with ATRA could provide experimental reference for functional food auxiliary drug therapy of breast cancer patients.