The Study Of Survivin Gene Expression And Apoptosis In Hep-2 Cell Induced By Docetaxel

ObjectiveLaryngeal carcinoma is the most common malignant tumor of otolaryngology, and it occupies 11%-22% of the otolaryngology malignant tumor. The squamous cell carcinoma accounts for 95%-99% in all pathology types. With the development of technology, the early diagnosis technology constantly improveded. But there are still about 45.2% patients with advanced stage. The traditional treatment of laryngeal carcinoma is radical operation, radiotherapy and the five-year survival rate of laryngeal carcinoma is 30%-50%. With the development of medicine research since the 1980s,the treatment of laryngeal carcinoma, especially patients with advanced stage,has been suggested combination therapy which concluds surgery, radiotherapy, chemotherapy and immunotherapy. Recent years, massive researches have confirmed that the application of docetaxel to treat the head and neck tumors had already achieved a inspiring primary efficacy, and the efficiency of docetaxel monotherapy or combination could reach to 22%-75%. However, the tumor's chemotherapy resistance has been an important factor affecting further efficacy.At present, the resistance mechanism of chemotherapeutic drugs in tumor cells remains unknown. It's demonstrated that apoptosis is the main approach to induce tumor cells death by chemotherapeutic drugs recently. but the alteration of adjustment to apoptosis in tumor cells after drugs treatment may be the important factor in drug resisitance. The inhibitor of apoptosis protein (IAP) plays a key role in adjusting sensitivity of tumor cells to apoptosis. Survivin is one of the most discussed new members of IAP, which has the smallest molecular weight and the most powerful anti-apoptosis effect up to now. Its distribution is diferent from other members of IAP,restrictedly in tumor tissues and embryonic tissue. The expression and subcellular localization in different cell line are various. It inhibits cell apoptosis by means of inhibiting the activity of caspase-3 and caspase-7. The abnormal changes in survivin gene lead to disorder of apoptosis pathway and abnormal proliferation of cells,which has become a hot research in tumorigenesis, development, diagosis, treatment and antitumor drugs resistance.Human laryngeal cancer cell line Hep-2 cells were exposed to docetaxel in our experiment. Then we detected the alteration of survivin mRNA expression in different time point and drug concentration. Combinating the result of apoptosis of tumor cells, we analyzed the relationship between the survivin gene and drug-induced apoptosis of laryngeal cancer cells,so that to explore about the relationship between changes in survivin gene expression and drug resistance of laryngeal cancer cell.MethodsThe laryngeal cancer cell line Hep-2 cells were cultured in vitro. Meanwhile the experiment began when cells entered the logarithmic growth phase. Docetaxel was attenuated into 2000μg/ml,and it was diluted into seven concentration gradients. Then seven concentration gradients were added into the Hep-2 cells in period of logarithmic growth respectively. The MTT assay was used to detect the inhibition on the proliferation of Hep-2 cell in vitro by different drug concentrations. The inhibition rate was calculated, and then we obtained the IC₅₀ values. Two concentrations were selected, 2 IC₅₀(high concentration) and 1/2IC₅₀(low concentration), to treated cells. The alteration of survivin mRNA expression was evaluated by RT-PCR in laryngeal cancer cells, which had been cultured by docetaxel of high concentration for 24h or low concentration for12h,24h,48h. Meanwhile, the apoptosis ratio of cells was detected by FCM. Statistical analysis: SPSS statistical package program 13.0 were used to analysis. The quantitative data were tested by one-way ANOVA. Multiple comparisons between groups were compared with LSD analysis of variance. P<0.05 were deemed significant.Results1. The inhibitory effect of docetaxel in the largnx cancer cellsThe inhibition rate increased gradually with the increase of concentration of docetaxel in 24,48h. It was showed that the IC₅₀ values of docetaxel were 7.47μg/ml,95% confidence interval were [4.91-10.73] in 24h, and the IC₅₀ values of its were 4.32μg/ml,95% confidence interval were [2.54-6.39] in 48h.2. Morphological changes of Hep-2 cellsUnder the inverted microscope, we observed that the ability of adherence and refracting decreased, the cell proliferation was restricted under the drug effect.and we can observed the morphological changes in 4h, and floating in a large number of cells when the concentration reach to 80μg/ml in 72h. The cell morphology changed into round from Polygon or a long spindle.3. Apoptosis ratio detected by FCMIt was showed that with the increase of the concentration of drugs, the apoptosis ratio of cells increased also (P<0.05).In low concentration group (3.7ug/ml) apoptotic peak (sub G₁ peak) appeared in 24h,it has not obviously increase in following 48h group.We can aslo observed the G₂/M phase arrest after drugs. The peak of G₂/M phase arrest is in 12h at low concentration group. The arrest of 12h group is significantly higher than control,24,48h group (P<0.05) ,it is no obvious apoptotic peak appeared. The apoptosis peak appeared at cells were treated with docetaxel in 24h. Meanwhile,The high concentration group (15.0ug/ml) apoptotic peak (sub G₁ peak) appeared in 24h aslo,and the expression of high group was slightly higher than low's.4. Alteration of survivin mRNA's expression in Hep-2 cells treated with docetaxel.There was no significant variability of the expression of survivin mRNA beteween high and low concentration in Hep-2 cells treated with docetaxel , which were higher than the control group (P<0.05). However, the expression of survivin mRNA in Hep-2 cells increased in following treated time (P<0.05). The expression of survivin mRNA were negative control group of 1.40 times, 2.76 times, 3.68 times in survival cells after treated by drugs in 12h,24h,48h.Conclusion1. Docetaxel can significantly inhibit the proliferation of laryngeal cancer Hep-2 cells in a time-and dose-dependent manner.2. The apoptotic peak of Hep-2 cells exposed to docetaxel appears after the G2/M phase arrest,which suggests that G₂/M phase arrest induced by docetaxel is the imprtant of the apoptosis.3. The rising trend of the expression of survivin mRNA in Hep-2 cells exposed to docetaxel suggests that a high show expression of survivin in survival cells induced by docetaxel has the relationship with drug resistance of laryngeal cancer cells to docetaxel.