Alteration in TWIST expression: possible role in paclitaxel-induced apoptosis in human laryngeal carcinoma Hep-2 cell line

Abstract

Aim: To explore the relationship between alteration in the expression of TWIST, highly conserved transcription factor from the basic helix-loop-helix family, and apoptosis of Hep-2 cells induced by chemotherapeutic agent paclitaxel.

Methods: Morphological changes of Hep-2 cells were observed by acridine orange cytochemistry staining. Viability of Hep-2 cells treated with various concentrations of paclitaxel was examined by cell proliferation assay. Apoptosis was examined by flow cytometry. The mRNA and protein expression of TWIST in response to paclitaxel at 24 hours, 48 hours, and 72 hours was examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively.

Results: Typical morphological changes of apoptotic cells at 24 hours, 48 hours, or 72 hours after treatment wiyth paclitaxel (10x10(-9) mol/L) were observed. The cell survival rates significantly decreased in a concentration- and time-dependent manner (P=0.001). Paclitaxel-induced apoptosis increased with culture time (22.6+/-5.3% after 24 hours, 38.7+/-7.9% after 48 hours, and 52.4+/-14.3% after 72 hours; P=0.002). Both mRNA and protein expression of TWIST was markedly decreased at both mRNA levels and protein levels, at 24 hours, 48 hours, and 72 hours in the paclitaxel-induced apoptosis of Hep-2 cells (P<0.001).

Conclusion: TWIST, which has a significantly decreased expression in response to paclitaxel in Hep-2 cells, may play a pivotal role in paclitaxel-induced apoptosis of Hep-2 cells.

Figure 1

Effects of paclitaxel on cell viability of Hep-2 cells. The survival rates of Hep-2 cells decreased in concentration- and time-dependent manner. All experiments were performed in triplicate. Data are expressed as means ± standard deviation percent of the control. Significant differences vs control: asterisk – P = 0.001, closed triangle – P = 0.002, open triangle – P<0.001); open bars – 24 hours, closed bars – 48 hours, diagonally striped bars – 72 hours.

Figure 2

Morphological changes in Hep-2 cells treated with Paclitaxel (10 × 10⁻⁹mol/L) using acridine orange fluorescence microscopy ( × 200). (A) control; (B) 24 hours; (C) 48 hours; (D) 72 hours.

Figure 3

Flow cytometric analysis of apoptotic cells of Hep-2 cells induced by paclitaxel after staining with Annexin V(AN)-FITC/propidium iodide (PI). Histogram for AN/PI bivariate analysis. x-axis represents the density of PI, y-axis represents the density of Annexin V-FITC. D1, upper-left quadrant, contains AN+/PI-, early apoptotic cells; D2, upper-right quadrant, contains AN+/PI+, secondary necrotic cells; D3, lower-left quadrant, contains AN-/PI-, viable, non-apoptotic cells; D4, lower-right quadrant, contains AN-/PI+, damaged viable cells. (A) control; (B) 24 hours; (C) 48 hours; (D) 72 hours.

Figure 4

The alteration in expression of TWIST mRNA in Hep-2 cells at different times, as assessed by reverse transcription-polymerase chain reaction analysis.

Figure 5

The alteration in expression of TWIST protein in Hep-2 cells. (A) The alteration in expression levels of TWIST protein in different times treated with paclitaxel by Western blotting analysis. (B) The optical densities value of TWIST protein in each lane using National Institutes of Health software Image J system were demonstrated in different times treated with paclitaxel. Each data point in the figure represents the mean ± standard deviation of 3 separate experiments. All experiments were conducted 3 times. Asterisk represents statistically significant difference vs control (P < 0.001).