Reseach Of Phloretin Induces Apoptosis Of Non-Small-Cell Lung Cancer A549 Cells And Pathways

Part one The effect of Phloretin on Non-Small-Cell Lung Cancer A549 CellsBackground:Lung cancer is a major cause of morbidity and mortality worldwide and the most common cause of cancer-related death.It is expected that the number of new diagnosed cases of lung cancer per year in China will be more than 1 million in 2025.Therefore,lung cancer is a great threat to human health.Non-small cell lung cancer(NSCLC)accounts for ~85% of all lung cancers.Although surgical and chemotherapeutic treatments have made great contributions in lung cancer,these methods may induce serious long-term adverse effects.Various natural herbal products have gained increasing attention due to their potential anticancer effects against NSCLC.Phloretin(Ph)(2’,4’,6’-trihydroxy-3-(4-hydroxyphenyl)-propiophenone)is a natural polyphenolic compound existing in apples,pears and other plants of the rosaceae family and has been found to have anti-inflammatory and immunosuppressive effects on both lymphoid-and myeloid-derived cell lines.Ph has also been shown to have antitumor activities by inducing apoptosis in human leukemia cells,bladder cancer and human colon cancer cells,and inhibiting the growth,invasiveness and migration of human liver cancer cells.However,little is known about its effects on human lung cancer cells.In the present study,we investigated the possible anticancer effects of Ph on A549 lung adenocarcinoma cells in vitro and in vivo,and discussed the underlying molecular mechanisms.Objective:The aim of the first part of the present study was to see whether Ph could induce apoptosis and reduce the migration ability of non-small cell lung cancer(NSCLC)A549cells.Methods:We use MTT assay to explore the effect of Ph on proliferation ability of A549 cells and use FITC-Annexin V apoptosis detection kit to explore the apoptosis condition of A549 cells after being treated with Ph.Furthermore we explore cell cycle by flowcytometry analysis.In addition we use Transwell migration assay to find the potential function of Ph on A549 tumor cell migration.Results:Through MTT assay we found that Ph inhibited the proliferation ability of A549 cells in a dose and time dependent manner.With the use of the FITC-Annexin V apoptosis detection kit,we found that Ph markedly induced cell apoptosis of NSCLC cell line A549.Cell cycle analysis by flow cytometry showed a dose-dependent increased accumulation of cell population in sub-G1 phase.The potential function of Ph on A549 tumor cell migration was characterized by Transwell migration assay.Conclusion:The results showed that Ph treatment slowed down the migration of A549 cells in a concentration-dependent manner,40 μM Ph markedly inhibited the migration of A549 cells.Part Two The Mechanism of Phloretin on A549 Cells and in Vivo StudyObjective:Explore the possible underlying mechanism of action in A549 cells and explore the in vivo effect of Ph.Methods:We use western blot analysis to explore whether caspase activation was involved in Ph-induced apoptosis.Mice bearing evident tumors were randomly divided into PBS control group,low-dose(10 mg/kg)Ph group,and high-dose(20 mg/kg)Ph group.Ph was dissolved in PBS for intraperitoneal(i.p.)administration to the mice every two days for three weeks.Female nude mice(BK Biotech)aged 5 weeks were used.A549 cells(5x106)were suspended in Matrigel(BD Biosciences)and injected subcutaneously(s.c.)into the mice.Tumor masses were isolated and tumor weight was measured.Results:We found that exposure of A549 cells to Ph(0,50,100 and 200 μM)for 24 h increased the number of cleaved fragments of caspase-3,caspase-9 and cleaved-PARP in a dose-dependent manner.Furthermore,Ph decreased Bcl-2,and increased the expressionlevel of P53 and Bax in a dose-dependent manner.We found that the phosphorylation of P38 MAPK,ERK1/2 and JNK1/2 was increased in a dose-dependent manner in parallel with Ph treatment.Inhibition of P38 MAPK and JNK1/2 by specific inhibitors significantly abolished the Ph-induced activation of the caspase-3 and-9.As anticipated,the tumor size was decreased significantly in both Ph groups,compared to that in the control group.The mean tumor mass in high-dose Ph group was 38% of that in the control group respectively,indicating that Ph had an inhibitory effect on lung carcinoma xenograft growth in mice.Conclusion:All these findings indicate that Ph is able to inhibit NSCLC A549 cell growth by inducing apoptosis through P38 MAPK and JNK1/2 pathways,and therefore may prove to be an adjuvant to the treatment of NSCLC.