Study On Apoptosis Mechanism Induced By Pristimerin In Oral Carcinoma Cells

Oral cancer as a common human malignancy accounts for about 5%of human systemic malignancy,and now oral cancer treatment methods are mostly conventional treatment methods,such as surgical resection,radiotherapy and chemotherapy and so on.But these treatments have some limitations,which become important reasons for the failure in oral cancer treatment.Thus,novel low toxicity and efficiency therapeutics are urgently needed for the treatment of oral cancer.Pristimerin,a naturally occurring quinonemethide triterpenoid compound,is a traditional medicine derived from the Celastraceae and Hippocrateaceae families.A number of studies have shown pristimerin has the effect on apoptosis of some human tumor cell lines,such as human lung cancer,breast cancer,multiple myeloma,prostate cancer and cervical cancer and so on.It is found that the effect and potential mechanism of pristimerin on oral cancer cell has not been never reported.In this study,we investigated the apoptosis and the mechanism of oral cancer cells induced by pristimerin,and provided the theoretical basis for the clinical treatment of oral cancer.This study mainly includes the following contents:(1)The proliferation activity of oral cancer cal-27 cell and scc-25 cell was detected by MTS experiment after treatment with pristimerin.The results showed that the activity of proliferation of cal-27 cell and scc-25 cell was inhibited in a time and dose dependent manner by pristimerin.The half-effective inhibitory concentration of pristimerin in oral cancer cal27 cell and scc-25 cell was about 0.701 μM and 0.729μM,respectively,compared with cisplatin and 5-fluorouracil,the half-effective inhibitory concentration of pristimerin was the smallest,and the inhibitory effect on the proliferation of oral cancer cells was much better than that of cisplatin and 5Fluorouracil;(2)The effect of pristimerin on the long-term proliferation of oral cancer cal-27 cell and scc-25 cell was observed by colony formation experiment.The results showed that the pristimerin can inhibit the long-term proliferation of oral cancer cal-27 cell and scc-25 cell in both dose and time-dependent manner;(3)The effect of pristimerin on apoptosis and cell cycle distribution of cal-27 cell and scc-25 cell was detected by flow cytometry.The results showed that the pristimerin had the effect of inducing apoptosis in oral cancer cells cal-27 and scc-25,and which showed significant time and dose-dependent changes.Moreover,the pristimerin had induced G1 arrest in oral cancer cell cal-27 and scc-25;(4)The transcription level of gene p21,Cyclin D1 and Cyclin E was detected by Q-PCR after pristimerin treatment in oral cancer cell cal-27 and scc-25.The results showed that the transcription level of Cyclin D1 and Cyclin E was down-regulated and the transcription level of p21 was up-regulated as compared with the untreated group of cal-27 and scc-25 cell.The result suggested that the pristimerin down-regulated Cyclin D1 and Cyclin E expression and up-regulated p21 expression,which in turn resulted the cell cycle arrest in G1 phase in oral carcinoma cells;(5)The protein expression level of PARP,Pro-Caspase-3,p21,p-AKT and p-ERK1/2 were detected by Western Blot after pristimerin treatment in oral cancer cell cal-27 and scc-25.Results showed that the Cleaved-PARP protein level was up-regulated,while the Pro-Casepase-3 protein level was down-regulated,which both in a time and dose dependent manner.The changes in the level of these two molecules directly lead to cell apoptosis,which is consistent with the previous apoptosis changes by flow cytometry detection in cal-27 and scc-25 cells;The p21 protein level was up-regulated,which was consistent with previous p21 transcription level,indicating that cell cycle arrest in G1 phase was associated with p21;p-ERK 1/2、p-AKT expression level was down-regulated,indicating that pristimerin inhibited MAPK(ERK1/2)signaling pathway and PI3K/AKT signaling pathway in oral cancer cell cal-27 and scc-25,and thus inhibited cell proliferation and led to apoptosis of cal-27 and scc-25 cells;Conclusion:This study found that pristimerin inhibited the proliferation related pathways-MAPK(ERK1/2)pathway and PI3K/AKT pathway,and meanwhile down-regulated Cyclin D1 and Cyclin E transcription level and up-regulated p21 expression level in oral cancer cal-27 cell and scc-25 cell.The molecular regulation partern mediated by pristimerin mentioned above eventually converged cell cycle arrest in G1 phase,and finally inhibited cell proliferation and induced cell apoptosis in oral carcinoma cells.