The role of Six1 signaling in paclitaxel-dependent apoptosis in MCF-7 cell line

Abstract

The resistance of cancer cells to chemotherapeutic agents represents the main problem in cancer treatment. Despite intensive research, mechanisms of resistance have not yet been fully elucidated. Six1 signaling has an important role in the expansion of progenitor cell populations during early embryogenesis. Six1 gene overexpression has been strongly associated with aggressiveness, invasiveness, and poor prognosis of different cancers. In this study, we investigated the role of Six1 signaling in resistance of MCF-7 breast cancer cells to taxanes. We first established in vitro paclitaxel-resistant MCF-7 breast cancer cells. Morphological modifications in paclitaxel-resistant cells were examined via light microscopic images and fluorescence-activated cell sorting analysis. Applying quantitative real-time polymerase chain reaction, we measured Six1, B-cell lymphoma/leukemia(BCL-2), BAX, and P53 mRNA expression levels in both non-resistant and resistant cells. Resistant cells were developed from the parent MCF-7 cells by applying increasing concentrations of paclitaxel up to 64 nM. The inhibitory concentration 50% value in resistant cells increased from 3.5 ± 0.03 to 511 ± 10.22 nM (p = 0.015). In paclitaxel-resistant cells, there was a significant increase in Six1 and BCL-2 mRNA levels (p = 0.0007) with a marked decrease in pro-apoptotic Bax mRNA expression level (p = 0.03); however, there was no significant change in P53 expression (p = 0.025). Our results suggest that identifying cancer patients with high Six1 expression and then inhibition of Six1 signaling can improve the efficiency of chemotherapeutic agents in the induction of apoptosis.

FIGURE 1

Determination of inhibitory concentration 50% of paclitaxel against MCF-7 cell. 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide results of parent MCF-7 cell line (a), MCF-7/Pac 64 nM (b).

FIGURE 2

Effect of 64 nM concentration of paclitaxel on morphology of cells. Apoptotic cells were characterized by condense nuclei and non-apoptotic dead cells: Diaminophenylindole images of cells treated with 64 nM concentration of paclitaxel in MCF-7 (a) and MCF-7/Pac 64 nM (b).

FIGURE 3

The effects of different paclitaxel concentrations (nM) on the rates of apoptosis of MCF-7/Pac 64 nM cells. The values were shown in percentage (%) from the most appropriate individual test. Flow cytometric measurement was performed by staining with annexin-V and the corresponding scatter plots were depicted for different treated concentrations of paclitaxel as control, 0 µM (a), 64 (b), 200 (c), 500 (d), and 700 (e). Apoptosis, the percentage of necrosis and viability of these effects were also underlined in each scatter plot.

FIGURE 4

Gene expression patterns of Six1 and P53 gene in MCF-7 and MCF-7/Pac 64 nM cells. Ribosomal β-actin was used as housekeeping gene. Effects of paclitaxel on pro/anti-apoptotic genes: (a) Level of mRNA of Six1 was increased significantly in MCF-7/Pac 64 nM (p = 0.0007) compared with MCF-7 cell line, (b) Level of p53 did not change significantly in paclitaxel-resistant cell line compare with parent MCF-7 cell line MCF-7/Pac 64 nM.