Effects Of Ursolic Acid On Proliferation, Apoptosis Of KYSE150 Cells In Esophageal Carcinoma

Background and objectivesEsophageal cancer is one of the most prevalent and deadliest malignancies. Worldwide,esophageal cancer is the sixth leading cause of death from cancer,and even the most frequent malignant tumor in several regions of China. Although the research about its etiology and pathogenesis has made significant progress during the past 10 years,it still hasn't been clarified clearly enough, and the morbidity and mortality remain high in some areas. Esophageal cancer is one of the highest grade malignancy gastrointestinal tract tumors.There are about 300 thousands people died due to this disease every year. The treatment of esophageal cancer in the way of surgery and chemotherapy has been developing for decades. Although the chemotherapy treatment of esophageal cancer to improve prognosis is extremely important,the useful effective drugs are not enough. To find some effective and safe chemotherapy treatment of esophageal cancer drugs have become a hot focus.Ursolic acid (UA) is a versatile compound which exists widely in many kinds of traditional Chinese herbal medicines. At present, ursolic acid has been proved to possess various biological activities such as anti-tumor, anti-inflammatory, analgesic, liver protective, anti-hypertension, lowering blood glucose level, antioxidant, protecting cardiovascular and other pharmacological effects, it supposed to be a promising new medicine with high effect and low poisonous. Numerous studies have shown that ursolic acid can induce the apoptosis of endometrial cancer, colorectal cancer, prostate cancer, lung cancer, lymphoma and other malignant tumors, and also shown the effect of anti-tumor at some degree. But, at present the research about ursolic acid's effect to esophageal cancer is lacked. The experiments observing ursolic acid on esophageal cancer cell line KYSE150 in vitro study whether ursolic acid inhibit the proliferation of esophageal cancer cell line KYSE150. And the experiments exploring the effects of ursolic acid on apoptosis, cell cycle distribution of esophageal cancer KYSE150 cells, in the process of which the possible molecular mechanism. All these results are good basis for elucidating the underlying molecular mechanisms of ursolic acid's antitumor.Meterials and Methods1 Cytotoxicity was evaluated by the MTT assay after treated with UA. The human esophageal cancer cell line KYSE150 was adjusted to concentration of 1×105/mL after cultured conventionally to log period. KYSE150 cell was divided into 3 groups:①experimental group:At the different concentrations UA were administered to culture tumor cells with lOμmol/L,20μmol/L,30μmol/L,40μmol/L, 50μmol/L;②negative control group:RPMI-1640 medium was administered to culture tumor cells;③solvent control group:DMSO was administered to culture tumor cells with 0.1%.Every concentration of UA which was administered to KYSE150 cells, respectively, was set for 4 parallel holes. After treated with UA for 24h,48h,72h etc, the differences of cell proliferation inhibition rate between different groups were detected.2 Flow cytometry machine was used to detect the cell apoptosis and cell cycle distribution. The human esophageal cancer cell line KYSE150 was added RPMI-1640 medium to control group, and UA to experimental groups to make the concentration 40μmol/L, after it was cultured conventionally. The three bottles of KYSE150 cells from the control and experimental groups were collected respectively after the intervention for 24 hours,48 hours and 72 hours. The apoptosis of KYSE150 cells and cell cycle distribution were inspected. 3 The expression levels of bcl-2,bax and cyclinDl mRNA were detected by RT-PCR after the KYSE150 cells were treated with UA. The human esophageal cancer cell line KYSE150 was added RPMI-1640 medium to control group,and UA to experimental groups at the different concentrations with 10μmol/L,20μmol/L 30μmol/L,40μmol/L,50μmol/L,after which was cultured conventionally. The three bottles of KYSE150 cells from the control and experimental groups were collected respectively after the intervention for 48 hours. The expression levels of bcl-2, bax and cyclinD1 mRNA were detected by RT-PCR technology.4 The One-way ANOVA test and chi-square test in SPSS (13.0 version) were used for the comparison of groups and comparison of rates,α=0.05 level for the test, a values of P<0.05 were considered statistically significant.Results1 MTT showed UA had a significant antiproliferative effect on human esophageal cancer cell line KYSE150 in a dose-and time-dependent manner(P<0.05). The IC50 values of UA at 24h,48h and 72h.were 84.14μmol/L,36.72μmol/L and 25.06μmol/L respectively. KYSE150 cells that treated by ursolic acid in 50μmol/L dosage almost ceased proliferating.2 FCM:After treated by ursolic acid with 40μmol/L, the apoptosis rate of the KYSE150 cells heightened with the increasing of the time.It showed that the cell population reduced in S phase and cell cycle was arrested in G0/G1 phase.3 RT-PCR:After treated by ursolic acid for 48h, the expression level of bax mRNA increased while the expression levels of bcl-2, cyclinD1 decreased obviously with the increasing of the concentration.Conclusions1 The result of MTT experience shows that UA owns the inhibition effect to the proliferation of the human esophageal cancer cell line KYSE150, in a does and time-dependent manner.2 The result of flow cell shows that UA can affect KYSE150 cell cycle after it roles in KYSE150 cell, and stop KYSE150 at the period of G0/G1. The mechanism maybe is that UA lowered the expression of CyclinD1 mRNA who can adjust esophageal cancer cell cycle obviously. 3 Flow cytometry shows that UA can lead to apoptosis of esophageal cancer cell,one of mechanism may be that UA decreases the express level of Bcl-2 mRNA and increase that to Bax mRNA.