Role Of TWIST In The Paclitaxel-induced Apoptosis Of Human Laryngeal Carcinoma Hep-2 Cell Line

Laryngeal carcinoma is one of the most common tumors in the head and neck, which causes substantial annual morbidity and mortality. Over the past two decades, the survival rate of the patients has not considerably improved, despite multimodal therapeutic strategies, especially chemotherapy. Clinically used alone or in combination with other drugs, Paclitaxel (Taxol), has been shown effective inhibitory effect on certain types of human malignant tumors. Although in vitro studies have shown that paclitaxel-induced cell death occurs via the apoptotic pathway in several tumor cell lines.few studies have been conducted to examine how paclitaxel exerts its inhibitory effects on Hep-2 cells, the human laryngeal carcinoma cell line. Paclitaxel induced apoptosis in Hep-2 cell, which was related to the disturbance of cell cycle.However, the precise mechanism by which paclitaxel elicits apoptosis in Hep-2 cells is still unclear.TWIST, which belongs to basic helix-loop-helix family, is a highly conserved transcription factor. Previous research has confirmed that overexpression of TWIST is found in several kinds of human cancers, which not only promotes the immigration and invasion of cancers cells, but also decreases the sensitivity to chemotherapy. Overexpression of TWIST may a key for tumor metastasis and drug resistance, but the precise mechanism is still unclear.As yet, there are no studies on the role of TWIST in paclitaxel-exerted effect on Hep-2 cells.The present study was designed to investigate the possible role of TWIST activity in paclitaxel-induced apoptosis in Hep-2 cells.Objective To explore the relationship between the expression of transcription factor TWIST and apoptosis of Hep-2 cells induced by paclitaxel.Methods Morphological changes of Hep-2 cells were observed by reserved microscopy and acridine orange cytochemistry staining. Viability of Hep-2 cells treated with various concentrations of paclitaxel was detected by MTT assay. Apoptosis was examined by flow cytometry. The expressions of transcription factor TWIST at both mRNA and protein level in response to paclitaxel at 24 h,48 h and 72 h were then examined by reverse transcription-polymerase chain reaction(RT-PCR) and Western blotting, respectively.Results Typical morphological changes of apoptotic cells, i.e., cellular rounding-up, cytoplasmic contraction, chromatin condensation and, particularly, apoptotic body, the main morphological characteristic of apoptosis, were observed by reserved microscopy and acridine orange cytochemistry staining. The cell surviving rates significantly decreased in a concentration-and time-dependent manner as evidenced by MTT assay (P<0.05). Percent of apoptosis after 24 h,48 h or 72 h paclitaxel-treatment was 22.6%±5.30%,38.7%±7.90% and 52.4%±14.27%, respectively, whereas the percent of control was 9.85±5.83%. There existed a statistically significant difference between treatment and control (F=12.621,P<0.05). The expression of TWIST at both mRNA and protein levels for 24 h,48 h or 72 h in the paclitaxel-induced apoptosis of Hep-2 cells were decreased by 16.70%±5.83%, 46.85%±2.74%,76.87%±2.45%(F=10.407, P<0.05)and 16.44%±4.95%,33.56%±2.00%,69.62%±5.69%,(F=18.013,P<0.05) respectively.Conclusion TWIST, which was significantly decreased in expression in response to paclitaxel in Hep-2 cells, may play a pivotal role in paclitaxel-induced apoptosis of Hep-2 cells.