The Effect Of Total Oil In Stahlianthus Involucratus On Apoptosis And Cell Cycle Arrest In Nasopharyngeal Carcinoma Cell Line CNE-2

Objective: To research the cell apoptotic and cell cycle distribution at different time in CNE-2 induced by total oil in Stahlianthus involucratus, investigate the changes of cell cycle related gene expression from cell cycle regulation perspective, and evaluate its possible tumor suppressor mechanism.Methods:1. The essential oil from the dry rhizoma of Stahlianthus involucratus was extracted by steam distillation,which was carried out by a conventional met hod for five times. Collect the upperoil and calculate the oil yield;2. CNE-2 cells were cultured in vitro, which were induced by different concentrations of total oil in Stahlianthus involucratus(20, 40, 60, 80, 160 and 240μg·m L-1). The inhibition of CNE-2 cells proliferation and half inhibition concentration(IC50) were measured by 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay at the time of 24, 48, 72 and 96 h;3. The morphological changes of CNE-2 cells, which were induced by different concentrations of drug, were observed with an inverted microscope. The hological changes of apoptosis of CNE-2 cells were determined by Hoechst 333258 fluorescent staining at the time of 24 h;4. The effect of total oil in Stahlianthus involucratus on cell cycle distribution and apoptosis induction of CNE-2 cells was evaluated with flow cytometry(FCM);5. Use the method of SYBR Green fluorescent dye Quantitative Real-time PCR to screen the changes of 47 cell cycle related genes expression profile in CNE-2 cells induced by total oil in Stahlianthus involucratus;6. Use the method of western blot to validation the expression leve of cell cycle arrest and the cell apoptosis related genes, which were screen by Quantitative Real-time PCR. Results:1.After the dry rhizoma of Stahlianthus involucratus was extracted by stea m distillation for 4 hours, the separation of the upper oil, dissolved in anhydrousdiethyl ether, and anhydrous sodium sulfate dehydrate, collect orange essential oil, its oil yield was 3.78%;2. The total oil in Stahlianthus involucratus can significantly inhibited the proliferation on the CNE-2 cells in a dose-and time-dependent manner in certain range(20, 40, 60, 80, 120, 160 and 240μg·m L-1), the corresponding IC50 was 71.12, 60.03, 53.17 and 34.75μg·m L-1;3. Under the inverted microscope, it shows that the cells reduced in size, the cells decreased in number and the cellular outlines gradually blur and granular, cytoplasm uneven distribution, exfoliated cells and small fragments of cells gradually increased with the increasing of drug concentration. Under a fluorescent microscope, it shows a strong blue fluorescence, which was the nucleolus of apoptosis cell. It could be seen apoptotic bodies; when the drug concentration reaches 100μg·m L-1, under the microscope, it could rarely find a complete cell nucleus, the chromatin gradually been splited into pieces;4. After treated by different concentrations of total oil in Stahlianthus involucratus for 24, 48, 72 and 96 h, the CNE-2 cells were detected by flow cytometry(FCM), the Annexin V FITC/PI staining shows that total oil in Stahlianthus involucratus could induce apoptosis and the effect was enhanced as the dosage increased. The PI staining shows that showing that total oil in Stahlianthus involucratus leads CNE-2 cells to obstructing of G1 stage;5. After treated by total oil in Stahlianthus involucratus for 24 h, the related genes expression profile of cell cycle regulation genes were detected by Quantitative Real-time PCR, which show that the expression of TP53, CDKN1A(p21), CDKN1B(p27), CDKN3, caspase-3, and GADD45 A m RNA increased when compared with control group, when the drug concentration reach 100μg·m L-1, it has significant difference(P<0.05). The expression level of E2F1, ATM, Skp2, CCND1, CDK6, CDC34, CDC25 A, CCNE1, CUL1, ABL1, CDC6, MCM3, MCM4, WEE1, Bcl-2, MDM2, CCNA2, CCNB1, CCNB2, CCND2, RAD17 and Hus1 m RNA in different degrees of decline;6. The CNE-2 cells were treated by different concentrations of total oil in Stahlianthus involucratus(0, 40, 80, 100 and 120μg·m L-1) for 24 h, the cycle regulation related geneswere detected which were screen by PCR. Western blot results showed that, compared with the control group, the protein expression of p53, p21, PPARγ and caspase-3 increased in drug-treated group, and the protein expression of cyclin D1 and Bcl-2 down-regulation; when drug concentration reach 80μg·m L-1, it has significant difference(P<0.01).Conclusions: The growth of CNE-2 cells could significantly inhibited by total oil in Stahlianthus involucratus in a dose-and a time-dependent manners, and play anti-tumor effect by inducing cell cycle arrest and apoptosis, and its mechanism may related with p53-dependent pathway and ATM-Chk2-Cdc25 pathway which could interfere with DNA damage repair and other signals pathway. Therefore, total oil in Stahlianthus involucratus is a potential drug for the resistance in nasopharyngeal carcinoma.