Patchoulenone Induce Apoptosis And Cell Cycle Arrest Of Human Gallbladder Carcinoma SGC-996 Cells And Its Mechanism

Background and objective:Gallbladder cancer(gallbladder carcinoma,GBC)is the most common biliary tract malignancy,represents the fifth place among gastrointestinal cancers.Gallbladder cancer has marked ethnic and geographical variations,although The Surveillance,Epidemiology,and End Results(SEER)program estimates that the incidence of GBC was at 2.5 cases per 1 × 10~5 people,the incidence of gallbladder carcinoma has an obvious increased trend,which can reach as high as 15.3 cases per1×10~5 people in developing countries.For the early gallbladder carcinoma progression is frequently rapid and silent,and has widely spread through direct infiltration,lymph node metastasis and blood supply transfer,it is hardly to make early diagnosis to gallbladder cancer.The majority of patients often has advanced gallbladder carcinoma when diagnosis,with poor curative effect and prognosis,and high mortality.The complete surgical tumor resection is the only curative treatment,GBC is not susceptible to the current traditional radiotherapy and chemotherapy.According to the statistics,The median survived time is only 6 months,and the 5-year survival rate is less than 5%.So founding new natural anti-tumor medicine for gallbladder cancer is a matter of great urgency for researchers.Patchoulenone is an important constituent of Pogostemon cablin,and possesses various bioactivitys like pesticidal activities,antibacterial,anti-inflammatory activity and immunoregulation activity.In current studies,Patchoulenone was shown anti-cancer activity in human prostatic cancer and colorectal cancer through induce autophagy and apoptosis in tumor cells,however the potential function of Patchoulenone in gallbladder cancer is not unclear.The present study was performed to investigate whether patchoulenone induced growth inhibition,apoptosis and cell cycle arrest in gallbladder cancer in vitro and in vivo,and the possible mechanisms,which could provide experimental evidence for the potential application of patchoulenone as a novel anti-gallbladder cancer agents.Method:1.Patchoulenone induce apoptosis and cell cycle arrest in gallbladder cancer SGC-996 cellsBy MTT assay and Colony formation assay the effect of patchoulenone on the proliferation of human gallbladder carcinoma SGC-996 cells.The effects of patchoulenone on the apoptosis of SGC-996 cells were investigated by annevin V/PI double-staining and Hoechst 33342 assay.The cell cycle of SGC-996 cells were investigated by flow cytometry assay.Then the in vivo efficacy of patchoulenone was detected via a SGC-996 xenograft model in athymic nude mice.2.The mechanism of apoptosis and cell cycle arrest induced by patchoulenoneThe activity of caspase-9 and caspase-3 was detected by caspases activity assay.The expression of cell cycle and apoptosis related proteins was analyzed by Western blotting.Result:1.After treatment with different concentrations of patchoulenone for different time,MTT assay confirm that patchoulenone showed a observably dose-and time-dependent inhibition effect on the proliferation of SGC-996 cells.By colony formation assay,the numbers and size of colons of SGC-996 cells were deeply suppressed.Flow cytometry assay showed that with the increase of patchoulenone concentration,the early and late apoptosis rate of SGC-996 cells had observably increased.Following typical apoptotic morphology changes.Meanwhile the proportion of cells in G0/G1 phase decreased significantly,while the proportion of cells in S phase increased remarkly compared with the control cells,indicating that patchoulenone induces S phase cell cycle arrest in gallbladder carcinoma SGC-996 cells.Intraperitoneal injection of patchoulenone(0,25,or 50 mg/kg)for 3 weeks significantly inhibited the growth of SGC-996 xenograft model in athymic nude mice.2.The activity of caspase-9 and caspase-3 increased significantly after patchoulenone treatment.Western blotting confirmed that patchoulenone treatment significantly up-regulated the expression levels of cleaved caspase-9,cleaved caspase-3,cleaved PARP-1 and the ratio of Bax to Bcl-2 in SGC-996 cells,Whereas down-regulated the expression levels of cyclin D1,cyclin A and cyclin B1,comparedwith the control group.Conclusion:Patchoulenone induce apoptosis and S phase cell cycle arrest of human gallbladder carcinoma SGC-996 cells.This effect may be related to regulating the expression levels of mitochondrial pathway-related proteins and cell cycle-related proteins by patchoulenone.