Sp1 Protein Mediates The Molecular Mechanism Of Phloretin-induced Apoptosis Of Prostate Cancer Cells

Spl belongs to a specific protein Sp/Kruppel family of transcription factors,combined with genetic elements that are rich in GC,thus regulate gene transcription,cell apoptosis and cell cycle.Spl is over-expressed in a number of cancers,and Spl levels is related with the survival of patients.As a result,many studies have shown that in the treatment of cancer,Spl can be used as a target molecules of drug screening.Phloretin belongs to the chemical class of dihydrochalcones and phloretin is mainly present in apples,pears and other juicy fruits.Phloretin has many biological activities,such as anti-oxidation,anti-inflammatory,anti-tumor and protect blood vessels,and so on.A large number of research results showed that phloretin could inhibit a variety of cancer cell survival,proliferation and induced cell apoptosis.Therefore,we designed the experiment to explore that phloretin inhibited the molecular mechanisms of prostate cancer cells:First of all,MTT and CCK-8 experimental results showed that the different concentrations of phloretin(0,20,50 and 100μM)almost had no effect on prostate normal cells WPMY-1,but could significantly inhibit prostate cancer LNCaP,CWR22Rv1,PC-3 and DU 145 cell survival;Then,through western blot results,we found that the rate of prostate cancer cells apoptosis could been increased with increasing doses of phloretin,and the protein levels of c-Caspase 3,c-Caspase 8,c-Caspase 9 and c-PARP-1 also could been increased obviously,suggested that phloretin induced prostate cancer cells apoptosis;Another Flow cytometry(FCM)results showed that phloretin induced prostate cancer cell cycle arrest in G2/M.Secondly,according to the western blot results,we also found that phloretin inhibited PI3K-AKT and MEK-ERK1/2 signaling pathways,and then down-regulated the protein and mRNA levels of Spl and Sp3/4 and its downstream gene expression.In addition,we further explored and found the down-regulation of Spl and Sp3/4 proteins had nothing to do with ubiquitin proteasome degradation,instead of cutting nucleolin protein phosphorylation levels to reduce the 5’UTR of Sp1 mRNA so that down-regulated the translation of Sp1 protein levels.Finally,in vivo experimental results showed that the high and low concentrations of phloretin could inhibit tumor growth in vivo,and there was almost no effect on body weight of nude mice;We detected the protein levels of Spl,Sp3/4,Survivin and Ki-67 that significantly down-regulated,and the protein levels c-Caspase 3 and c-PARP-1 significantly up-regulated by western blot,showed that phloretin could inhibit the growth of xenograft tumor in vivo and induce tumor cells apoptosis.Above all,phloretin down-regulated the levels of Spl protein by transcription and translation levels,and then inhibited prostate cancer cells to survive,promoted prostate cancer cell cycle arrest and induced cells apoptosis.