Molecular mechanism and cytotoxicity of allicin and all-trans retinoic acid against CD44(+) versus CD117(+) melanoma cells

Background: All-trans retinoic acid (ATRA) is a differentiating agent that inhibits cancer cell growth during the cell cycle. However, despite its potent antitumor properties, some melanoma cells are resistant to ATRA therapy. Purpose: Here, we hypothesized that allicin can sensitize malignant melanoma cells to ATRA treatment. To clarify this mechanism, we determined the sensitivity to ATRA, allicin and allicin/ATRA in CD44(+) and CD117(+) melanoma cell subpopulations. Methods: The CD44(+) and CD117(+) cells were sorted from A375 melanoma cell line using the magnetic-activated cell sorting (MACS). The potential anticancer effects of ATRA, allicin and allicin/ATRA were examined using cell proliferation MTT assay. In addition, flow cytometry was used to detect cell cycle arrest. The efficacy of the treatments in controlling cancer cell proliferation was assessed by quantitative realtime polymerase chain reaction (RT-PCR). Results: Here, we demonstrated that CD44(+) melanoma cells were more resistant to allicin and ATRA than CD117(+) cells. Importantly, we observed that allicin sensitized melanoma cell to ATRA-induced cell death. The combination treatment with allicin and ATRA significantly reduced the IC50 value obtained for ATRA alone in CD44(+) melanoma cells. In CD44(+) cells, the IC50 value of ATRA was 37.43 +/- 0.54, while the IC50 value of allicin/ATRA treatment was 17.53 +/- 0.2 mu M. Allicin treatment resulted in significant increases in the percentage of cells at the G2/M and G0/G1 phases in the CD44(+) and CD117(+) cells, respectively. The combination treatment caused the inhibition of CD44(+) and CD117(+) melanoma cells at the S phases compared to ATRA alone. Allicin, ATRA, and allicin/ATRA increased the expression of cyclin D1 mRNA in both CD44(+) and CD117(+) cells. Allicin combination with ATRA increased the mRNA level of RAR beta in CD117(+) cells. Furthermore, allicin alone caused a remarkable reduction of MMP-9 mRNA expression in both CD44(+) and CD117(+) cells. In contrast, ATRA and the combination treatment significantly increased MMP-9 gene expression in CD44(+) cells. Conclusion: Overall, our results indicate that allicin reinforces the ATRA-mediated inhibitory effects on CD44(+) and CD117(+) melanoma cells and may provide a new approach for the treatment of malignant melanoma.