Seleno-short-chain chitosan induces apoptosis in human breast cancer cells through mitochondrial apoptosis pathway in vitro

Abstract

Seleno-short-chain chitosan (SSCC) was a synthesized chitosan derivative with the molecular weight of 4826.986 Da. The study is aimed to investigate cytotoxicity of SSCC on human breast cancer MCF-7 and BT-20 cells and explore apoptosis-related mechanism in vitro. The MTT (3- [4,5-Dimethylthiazol-2-yl]-2, 5-diphenylterazolium bromide) assay showed that SSCC exhibited significantly cytotoxic effects on MCF-7 and BT-20 cells in a dose- and time-dependent manner, and the effective inhibitory concentration was 100 μg/ml and 200 μg/ml, respectively. Apoptosis assay of these two kinds of cells was determined by Hoechst 33,342/PI and Annexin V-FITC/PI double staining. The cell cycle assay showed that SSCC triggered S and G2/M phase cell cycle arrest in MCF-7 cells and S phase cell cycle arrest in BT-20 cells in a time-dependent manner. Further studies demonstrated that SSCC led to the generation of reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential (MMP) in these two kinds of cells. N- acetyl-L cysteine (NAC), as a radical scavenger, significantly inhibited the generation of ROS and decreased the apoptosis of MCF-7 and BT-20 cells. Moreover, the expression of mitochondrial apoptosis-related proteins was detected by western blot assay. SSCC up-regulated the expression of Bax, down-regulated the expression of Bcl-2, subsequently increased the release of cytochrome c from mitochondria to cytoplasm, and activated the cleavage of caspase-9 and -3, which finally induced apoptosis in MCF-7 and BT-20 cells in vitro. Consequently, these data indicated that SSCC could induce apoptosis of MCF-7and BT-20 cells in vitro by mitochondrial pathway.

Keywords: NAC; Seleno-short-chain chitosan; breast cancer BT-20 cells; breast cancer MCF-7 cells; mitochondrial apoptosis pathway.

Figure 1.

The cytotoxicity of SSCC on breast cancer MCF-7 and BT-20 cells and normal breast Hs 578Bst cells. (a) The chemical structure of seleno-short-chain chitosan (SSCC). (b), (c) and (d) Columns stand for inhibition rates of SSCC on normal breast cells, MCF-7 cells and BT-20 cells, after treatment with SSCC (25 – 600 μg/ml) for 8 – 24 h, separately. The inhibition rate of cells was determined by MTT method. *p ＜ 0.05 compared with control group was considered as statistically significant difference.

Figure 2.

Morphological changes of cells. (a) Morphological changes of MCF-7 cells were detected by inverted microscope (magnification, x20). Cells were exposed to 100 μg/ml SSCC for 8 – 24 h. (b) Morphological changes of BT-20 cells were observed by inverted microscope (magnification, x20), after incubation with 200 μg/ml SSCC for 8 – 24 h.

Figure 3.

SSCC induced apoptosis of breast cancer cells. (a) After incubation with appointed concentration of SSCC MCF-7 and BT-20 cells for 8 – 24 h, cells apoptosis was analyzed using Hoechst 33,342/PI double staining and observed under inverted fluorescence microscope (magnification, x 20). (b) Apoptosis rates of MCF-7 and BT-20 cells were detected by Annexin V-FITC/PI double staining. NAC, a kind of free radical scavenger, was used in this work. a and a’ Control group; b and b’ Treatment with 5 mM NAC for 1 h; c and c’ Treatment with SSCC for 8 h; d and d’ Treatment with SSCC for 16 h; e and e’ Treatment with SSCC for 24 h; f and f’ Treatment with 5 mM NAC for 1 h following treatment with SSCC for 24 h. (c) and (d) columns represent the proportion of two kinds of apoptotic cells after incubation with appointed concentration of SSCC for 8 – 24 h in the presence or absence of NAC. *p ＜ 0.05 compared with control group was considered as statistically significant difference.

Figure 4.

The effects of SSCC on cells cycle distribution. (a) MCF-7 cells (100 μg/ml) and BT-20 (200μg/ml) cells were treated with SSCC for 8 – 24 h and the number of cells in every phase was analyzed by flow cytometry. (b) and (c) Columns represent the percentages of corresponding cell cycle phase after treatment with SSCC for 8 – 24 h in MCF-7 cells and BT-20 cells, separately. *p ＜ 0.05 compared with control group was considered as statistically significant difference.

Figure 5.

ROS generation in MCF-7 and BT-20 cells was tested by DCFH-DA staining and analyzed using flow cytometry. NAC, a kind of free radical scavenger, inhibited SSCC-induced generation of ROS. (a) The fluorescence changes after incubation with SSCC for 8 – 24 h and the presence or absence of NAC in MCF-7 (100 μg/ml) and BT-20 cells (200 μg/ml). a and a’ Control group; b and b’ Treatment with 5 mM NAC for 1 h; c and c’ Treatment with SSCC for 8 h; d and d’ Treatment with SSCC for 16 h; e and e’ Treatment with SSCC for 24 h; f and f’ Treatment with 5 mM NAC for 1 h followed by treatment with SSCC for 24 h, separately. (b) and (c) Columns representing the levels of intracellular ROS after treatment with SSCC for 8 – 24 h and the presence or absence of NAC in MCF-7 cells and BT-20 cells, respectively. *p ＜ 0.05 compared with control group was considered as statistically significant difference.

Figure 6.

Mitochondrial membrane potential (MMP) disruption in MCF-7 and BT-20 cells was measured by Rhodamine 123 staining and analyzed using flow cytometry. (a) MCF-7 (100 μg/ml) and BT-20 cells (200 μg/ml) were treated with SSCC for 8 – 24 h. (b) and (c) Columns stand for the percentages of MMP disruption after treatment with specified concentration of SSCC for 8 – 24 h in MCF-7 and BT-20 cells, respectively. *p ＜ 0.05 compared with control group was considered as statistically significant difference.

Figure 7.

SSCC-induced mitochondrial apoptosis relevant-protein on MCF-7 cells and BT- 20 cells were measured by western blotting. Photos from A to I represent the ratio of Bax, Bcl-2, cytochrome c, pro-caspase-9, cleaved-caspase-9, pro-caspase-3, and cleaved-caspase-3 to β-actin after incubation with appointed concentration of SSCC for 8 – 24 h, respectively. *P＜ 0.05 compared with the control group was considered as significantly different.

Figure 7.

Continued.