Astragalus Polysaccharide RAP Selectively Attenuates Paclitaxel-Induced Cytotoxicity Toward RAW 264.7 Cells by Reversing Cell Cycle Arrest and Apoptosis

Abstract

Purpose: The purpose of this study was to determine if an Astragalus polysaccharide (RAP) can protect immune cells from the toxic side effects of paclitaxel (Taxol), a powerful anti-tumor drug whose equally powerful side effects limit its clinical use. Methods: We hypothesized that RAP can reduce the toxic effects induced by Taxol. To test this hypothesis, we conducted a series of studies in vivo and in vitro. First, we confirmed RAP's effects in vivo utilizing BALB/c mice inoculated with 4T1 mouse breast cancer cells as the tumor model. Mice were treated with RAP and/or Taxol, and the differences in the life spans were recorded. Second, a co-culture cell model was used to study the protective effect of RAP on cells vis-a-vis Taxol. The cell cycle and apoptosis of RAW 264.7 cells that were treated with RAP with/without Taxol were checked by flow cytometry and Hoechst staining. Proteins involved in the cell cycle and apoptosis were also tested by Western blot to reveal the probable mechanism. Results: RAP prolonged the life span of tumor-bearing mice treated with Taxol. The in vitro experiments showed that Taxol suppressed the proliferation of RAW 264.7 cells while RAP protected the RAW 264.7 cells from Taxol-induced suppression. The protection is selective because RAP had no effect on 4T1 cells. Furthermore, Taxol clearly led to cell cycle arrest mainly at the G2/M phase and generated cytotoxicity against RAW 264.7 cells, while RAP blocked cell cycle arrest and protected cells from apoptosis. Taxol up-regulated the protein levels of P-H₂A, PARP, Chk1, p53, and p21 and down-regulated Bcl-Xl and Mcl-1, and RAP reversed the expression of all these proteins. Conclusion: These results suggested that RAP can protect immune cells from Taxol-induced toxicity, by changing the cell cycle and apoptosis.

Keywords: Astragalus polysaccharide; apoptosis; cell cycle; cytotoxicity; protective effect.

FIGURE 1

Long time survival of 4T1 breast tumor-bearing mice treated with RAP with/without Taxol.

FIGURE 2

Effects of RAP with/without Taxol on 4T1 and RAW 264.7 cells. (A) 4T1 and RAW 264.7 cells co-culture model; (B) 4T1 and RAW 264.7 cells were not stimulated by RAP directly; (C) Apoptosis of 4T1 cells in co-culture system induced by RAP (50 μg/mL); (D) Proliferation of 4T1 and (E) RAW 264.7 cells in co-culture model treated by RAP with/ without Taxol. Data are presented as mean ± SD. Compared with the control group. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001.

FIGURE 3

G2/M cell cycle arrest and related proteins of RAW 264.7 cells treated by RAP (50 μg/mL) with/without Taxol (10 μM). (A) Diagram of cell cycle analysis in RAW 264.7 cells by flow cytometry; (B) Statistical analysis of G0-G1, S, and G2/M populations in RAW 264.7 cells; (C) Western blotting analysis of the level of phosphorylation of Chk1 (MW: 56 kDa), p53 (MW: 53 kDa), and p21 (MW: 21 kDa) protein expression; (D) Quantitative analysis of Chk 1, p53, and p21 (^∗p < 0.05 compared with Taxol alone group).

FIGURE 4

Apoptosis of RAW 264.7 cells treated by RAP (50 μg/mL) with/without Taxol (10 μM). (A) Apoptotic effect of RAP with/without Taxol on RAW 264.7 cells detected by flow cytometry; (B) Statistical analysis of apoptotic effect of RAP with/without Taxol detected by flow cytometry; (C) Apoptosis that assessed by Hoechst 33342 staining: red arrow showed some of the typical “apoptotic bodies.” (D) Statistical analysis of apoptotic effect of RAP with/without Taxol detected by Hoechst staining; (E) Western blotting analysis of Phospho-Histone H2A. X (MW: 15 kDa), PARP (full length, MW: 116 kDa), Bcl-Xl (MW: 30 kDa) and Mcl-1 (MW: 35 kDa); (F) Quantitative analysis of Phospho-Histone H2A. X, PARP, Bcl-Xl, and Mcl-1 (^∗p < 0.05 compared with Taxol alone group). Data are presented as mean ± SD. Compared with the control group. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.