Halofuginone and artemisinin synergistically arrest cancer cells at the G1/G0 phase by upregulating p21Cip1 and p27Kip1

Abstract

Combinational drug therapy is one of the most promising strategies in modern anticancer research. Traditional Chinese medicine (TCM) formulas represent a wealth of complex combinations proven successful over centuries of clinical application. One such formula used to treat a variety of diseases, including cancer, contains two herbs, whose main active components are Halofuginone (HF) and Artemisinin (ATS). Here we studied the anticancer synergism of HF and ATS in various cancer cell lines and in a xenograft nude mice model. We found that the HF-ATS combination arrested more cells at the G1/G0 phase than either one alone, with the concomitant increased levels of CDK2 inhibitors, p21Cip1 and p27Kip1. By knocking down p21Cip1 and p27Kip1 separately or simultaneously in HCT116 cells and MCF-7 cells, we found that p21Cip1 was required for HF induced G1/G0 arrest, whereas p21Cip1 and p27Kip1 were both required for ATS or HF-ATS combination-mediated cell cycle arrest. Moreover, HF-ATS combination synergistically inhibited tumor growth in xenograft nude mice, and this was associated with the increased levels of p21Cip1 and p27Kip1. Collectively, these data indicate that the upregulation of p21Cip1 and p27Kip1 contributes to the synergistic anticancer effect of the HF-ATS combination.

Keywords: artemisinin; cell cycle; cell proliferation; halofuginone; synergy.

Figure 1. HF-ATS combination synergistically inhibited the proliferation of cancer cells

A. HCT116 cells were treated with different dose of HF (5, 10, 20 nM), ATS (40, 80, 160 μM), or combinations of both. 24 h later, cell viabilities were measured by MTT assay. Combination indexes (CIs) were analyzed by the CompuSyn software. CI< 1 indicates synergism. B. Eight cancer cell lines were treated with increasing concentrations of HF-ATS combinations with the selected optimal fixed ratio. 24 h later, cell viabilities were measured by MTT assay. Combination indexes (CIs) were analyzed by using the CompuSyn software. CI < 1 indicates synergism.

Figure 2. HF–ATS combination markedly arrested cells at the G1/G0 phase

A. Flow cytometry analysis of HCT116 cells or MCF-7 cells treated with combination of HF (10 nM) and ATS (160 μM) for 12 h or 24 h, respectively, which cooperatively induced cell cycle arrest at the G1/G0 phase. *P < 0.05, **P < 0.01, compared with control group; aa P < 0.01, compared with HF; bb P < 0.01, compared with ATS. B. Quantitative RT-PCR analysis of p21^Cip1 and p27^Kip1 mRNA in HCT116 cells or MCF-7 cells treated with the combination of HF (10 nM) and ATS (160 μM) for 12 h or 24 h, respectively. *P < 0.05, **P < 0.01, compared with control group. C. Protein expression of p21^Cip1, p27^Kip1 and phospho-CDK2 in HCT116 cells or MCF-7 cells (left panel); quantitative analysis of protein expressions (right panel) treated with the combination of HF (10 nM) and ATS (160 μM) for 12 h or 24 h, respectively.

Figure 3. Knockdown of p21^Cip1, or p27^Kip1, or both in HCT116 cells and MCF-7 cells

A. Quantitative RT-PCR analysis of p21^Cip1 mRNA and p27^Kip1 mRNA in the knockdown HCT116 cells and MCF-7 cells. **P < 0.01, compared with control group. B. Protein expression levels of p21^Cip1 and p27^Kip1 (upper panel), and quantitative analysis of protein expressions (bottom panel) in knockdown HCT116 cells and MCF-7 cells.

Figure 4. Both p21^Cip1 and p27^Kip1 are required for HF-ATS combination-induced cell cycle arrest

A. Flow cytometry (left panel) and histogram (right panel) analyses of p21^Cip1 knockdown HCT116 cells or p21^Cip1 knockdown MCF-7 cells treated with the combination of HF (10 nM) and ATS (160 μM) for 12 h or 24 h, respectively. B. Flow cytometry (left panel) and histogram (right panel) analyses of p27^Kip1 knockdown HCT116 cells or p27^Kip1 knockdown MCF-7 cells treated with the combination of HF (10 nM) and ATS (160 μM) for 12 h or 24 h, respectively. C. Flow cytometry (left panel) and histogram (right panel) analyses of p21^Cip1-p27^Kip1 double knockdown HCT116 cells or p21^Cip1-p27^Kip1 double knockdown MCF-7 cells treated with the combination of HF (10 nM) and ATS (160 μM) for 12 h or 24 h, respectively. *P < 0.05, **P < 0.01, compared with control group; a P < 0.05, aa P < 0.01, compared with HF; b P < 0.05, bb P < 0.01, compared with ATS.

Figure 5. HF, ATS and HF-ATS combination differentially modulated p21^Cip1, p27^Kip1 and phospho-CDK2 in p21^Cip1 or/and p27^Kip1 knockdown cells

A. The expression of p21^Cip1, p27^Kip1, and phospho-CDK2 by combination of HF (10 nM) and ATS (160 μM) (left panel), and quantitative analysis of protein expressions (right panel) in p21^Cip1 knockdown HCT116 cells or p21^Cip1 knockdown MCF-7 cells. B. The expression of p21^Cip1, p27^Kip1 and phospho-CDK2 by combination of HF (10 nM) and ATS (160 μM) (left panel), and quantitative analysis of protein expressions (right panel) in p27^Kip1 knockdown HCT116 cells or p27^Kip1 knockdown MCF-7 cells. C. Expression of p21^Cip1, p27^Kip1 and phospho-CDK2 by combination of HF (10 nM) and ATS (160 μM) (left panel), and quantitative analysis of protein expressions (right panel) in p21^Cip1-p27^Kip1 double knockdown HCT116 cells or p21^Cip1-p27^Kip1 double knockdown MCF-7 cells.

Figure 6. HF–ATS combination exhibited synergistic anticancer activity in CRC xenograft nude mice

A. 6-week old nude mice were engrafted with HCT116 cells and randomly divided into 5 groups: vehicle group, HF group, ATS group, HF+ATS group, 5Fu group (n=5). Tumor volumes were measured and calculated by the length and width every day. B. The tumor weights of the 5 groups. *P < 0.05, **P < 0.01, compared with control group; a P < 0.05, aa P < 0.01, compared with HF; b P < 0.05, bb P < 0.01, compared with ATS. C. The xenograft tumors were dissected and measured. D. Expression levels of p21^Cip1, p27^Kip1 and phospho-CDK2 in xenograft tumors. E. H&E staining showed the decreased cell density and disrupted cell morphology in the tumor sections from combination of HF and ATS treated mice. Scale bar = 50 μm. F. Immunofluorescence staining for p21^Cip1, p27^Kip1 and phospho-CDK2 in xenograft tumors. Scale bar = 50 μm.