Alteronol Enhances the Anti-tumor Activity and Reduces the Toxicity of High-Dose Adriamycin in Breast Cancer

Abstract

The first-line chemotherapy drug adriamycin (ADM) is widely used for the treatment of breast cancer, but the acquired drug resistance and the normal tissue toxicity remain clinical challenges. Alteronol has been reported to exert wide-ranging anti-tumor activity. In this study, we firstly examined the synergistic anti-tumor effects and the underlying mechanisms of alteronol combined with ADM in breast cancer. We have found that the combination of alteronol and ADM significantly suppressed the expression levels of the cell cycle-related proteins (CDC2 and Cyclin B1) and induced cell cycle arrest at the G2/M phase, leading to cell proliferation inhibition in breast cancer 4T1 cells. Moreover, co-treatment of alteronol and ADM (i) remarkably activated p38 and JNK kinases, (ii) elevated ROS levels, (iii) triggered mitochondrial dysfunction, (iv) released cytochrome c into the cytoplasm, (v) upregulated apoptosis-related proteins, e.g., cleaved PARP, Bax, and cleaved caspase-3/9, and (vi) downregulated the expression of Bcl-2, followed by apoptosis. Furthermore, our in vivo studies showed that the low-dose combination of alteronol (2 mg/kg) and ADM (1 mg/kg) significantly inhibited tumor growth in tumor bearing mice, and the anti-tumor effect of the combination was the same as that of high-dose ADM (8 mg/kg). In addition, the low-dose combination group showed lower toxicities to major organs than the high-dose ADM group. Taken together, these data demonstrate that the low-dose combination of alteronol and ADM could notably improve the anti-tumor activity and have lower toxicities to major organs than those in high-dose ADM group.

Keywords: adriamycin; alteronol; apoptosis; breast cancer; cell cycle arrest; chemotherapy.

FIGURE 1

Combination of alteronol and ADM significantly inhibits cell viability in breast cancer 4T1 cells. (A) The anti-proliferative effect of alteronol on 4T1 cells was determined by MTT assay after 24 h or 48 h of treatment. (B) The anti-proliferative effect of ADM on 4T1 cells was determined by MTT assay after 24 h or 48 h of treatment. (C) After treatment with alteronol (5.68 μM), ADM (1.84 μM), or their combination for 24 or 48 h, respectively, the 4T1 cell viability was measured by MTT assay. ^∗P < 0.05, ^∗∗P < 0.01 vs. control group. ^#P < 0.05, ^##P < 0.01 vs. alteronol group; ^&P < 0.05, ^&&P < 0.01 vs. ADM group. All data are expressed as mean ± SD of three independent experiments.

FIGURE 2

The effect of the combination of alteronol and ADM on cell cycle distribution in 4T1 cells. (A) Cell cycle distribution of 4T1 cells was determined by flow cytometry after treatment with alteronol, ADM, or both. (B) Quantitative analysis of the cell cycle distribution in 4T1 cells after the indicated treatments. ^∗P < 0.05, ^∗∗P < 0.01 vs. the control group. ^#P < 0.05, ^##P < 0.01 vs. alteronol group. ^&P < 0.05, ^&&P < 0.01 vs. ADM group. All data are expressed as mean ± SD of three independent experiments.

FIGURE 3

The co-treatment with alteronol and ADM regulates the levels of cell cycle-related molecules in 4T1 cells. (A) Representative images of the protein levels of CDC2 and Cyclin B1 detected by western blot. (B) Western blot analysis of CDC2 and Cyclin B1 protein levels after treatment with alteronol, ADM, or both, normalized to β-actin. ^∗P < 0.05, ^∗∗P < 0.01 vs. the control group. ^#P < 0.05, ^##P < 0.01 vs. alteronol group. ^&P < 0.05, ^&&P < 0.01 vs. ADM group. Data are presented as mean ± SD of three independent experiments.

FIGURE 4

The effect of the combination of alteronol and ADM on apoptosis in 4T1 cells. (A) Morphological changes in 4T1 cells were examined by Hoechst 33258 staining after treatment with alteronol, ADM, or both. (B) Distribution of apoptosis rates in 4T1 cells as determined by Hoechst 33258 staining after the indicated treatments. (C) The apoptotic rates of 4T1 cells were detected by flow cytometry after the indicated treatments. (D) Quantitative analysis of apoptotic rates of 4T1 cells after the indicated treatments. ^∗P < 0.05, ^∗∗P < 0.01 vs. control group. ^#P < 0.05, ^##P < 0.01 vs. alteronol group. ^&P < 0.05, ^&&P < 0.01 vs. ADM group. All data are expressed as mean ± SD of three independent experiments.

FIGURE 5

The effect of the combination of alteronol and ADM on protein levels of apoptosis-related molecules in 4T1 cells. (A) The protein levels of Bax and Bcl-2 were measured by western blot. (B) Quantitative analysis of Bax and Bcl-2 protein levels in 4T1 cells after treatment with alteronol and/or ADM. (C) The protein levels of cleaved PARP, cleaved caspase-9, and cleaved caspase-3 were examined by western blot. (D) Quantitative analysis of cleaved PARP, cleaved caspase-9, and cleaved caspase-3 protein levels after the indicated treatments. ^∗P < 0.05, ^∗∗P < 0.01 vs. control group. ^#P < 0.05, ^##P < 0.01 vs. alteronol group. ^&P < 0.05, ^&&P < 0.01 vs. ADM group. All data are expressed as mean ± SD of three independent experiments.

FIGURE 6

Co-treatment with alteronol and ADM triggers ROS generation and MMP loss in 4T1 cells. (A) The ROS levels in 4T1 cells were evaluated by flow cytometry after treatment with alteronol and/or ADM. The ROS levels are indicated by DCF fluorescence. (B) Quantitative analysis of the fluorescence intensity of DCF after treatment with alteronol and/or ADM. (C) The representative images of MMP in 4T1 cells using JC-1 staining after the indicated treatments. (D) Quantitative analysis of the ratio of red fluorescence to green fluorescence after alteronol and/or ADM treatment. (E) The protein levels of cytoplasmic cytochrome c as analyzed by western blot. (F) Quantitative analysis of the protein levels of cytoplasmic cytochrome c. The relative protein levels of cytoplasmic cytochrome c were normalized to the value in the control group. ^∗P < 0.05, ^∗∗P < 0.01 vs. the control group. ^#P < 0.05, ^##P < 0.01 vs. alteronol group. ^&P < 0.05, ^&&P < 0.01 vs. ADM group. All data are presented as mean ± SD of three independent experiments.

FIGURE 7

The effect of the combination of alteronol and ADM on the protein levels of MAPK-related molecules in 4T1 cells. (A) The protein levels of MAPK-associated molecules as detected by western blot. (B) Western blot analysis of the protein levels of MAPK-associated molecules normalized by β-actin. ^∗P < 0.05, ^∗∗P < 0.01 vs. the control group. ^#P < 0.05, ^##P < 0.01 vs. alteronol group. ^&P < 0.05, ^&&P < 0.01 vs. ADM group. Data are presented as mean ± SD of three independent experiments.

FIGURE 8

The anti-tumor effect of the combination of alteronol and ADM in vivo. (A) The cell viability was measured by MTT assay after treatment with different doses of alteronol (5.68 and 8.52 μM) and/or ADM (1.84 and 14.72 μM) for 48 h. (B) Microscopic view of tumor tissue in mice. (C) The tumor weights isolated from breast cancer 4T1 cell bearing mice after the indicated treatments for 2 weeks. (D) Isolated tumor volumes from the tumor bearing mice. (E) Isolated spleen weight from breast cancer 4T1 cell bearing model. (F) Histological assessments of tissues using HE staining after treatment with alteronol, ADM, or both. ^∗P < 0.05, ^∗∗P < 0.01 vs. model group. ^#P < 0.05, ^##P < 0.01 vs. alteronol group (2 mg/kg). ^&P < 0.05, ^&&P < 0.01 vs. ADM group (1 mg/kg). ^$P < 0.05, ^$$P < 0.01 vs. the low-dose combination group.

FIGURE 9

Effect of the combination of alteronol and ADM on the heart/liver/kidney function markers in vivo. Effects of alteronol (2 mg/kg and 3 mg/kg), ADM (1 mg/kg and 8 mg/kg), or a combination on the levels of (A) LDH, (B) CK, (C) ALT, (D) AST, (E) BUN, and (F) Cr in mouse serum. ^∗P < 0.05, ^∗∗P < 0.01, vs. model group. ^#P < 0.05, ^##P < 0.01, vs. alteronol group (2 mg/kg); ^&P < 0.05, ^&&P < 0.01, vs. ADM group (1 mg/kg); ^$P < 0.05, ^$$P < 0.01, vs. the low-dose combination group.