Ethanol Extract of Ilex Hainanensis Merr. Exhibits Anti-Melanoma Activity by Induction of G1/S Cell-Cycle Arrest and Apoptosis

Objective： To evaluate anti-melanoma effect of ethanol extract of Ilex hainanensis Merr. （IME） and elucidate its underlying mechanism. Methods： Thirty-six tumor-bearing mice were randomized into 6 groups （n=6） as follows： model group, IME 25, 50, 100, and 200 mg/kg groups and dacarbazine （DTIC） 70 mg/kg group. The mice in the IME treatment groups were intragastrically administered with IME 25, 50, 100 or 200 mg/kg per day, respectively. The mice in the DTIC group were intraperitoneally injected with DTIC 70 mg/kg every 2 days. The drug administration was lasting for 14 days. The cell viability was evaluated by 3-（4,5-dime-thylthylthiazol-2-yl）-2, 5-diphenyl-tetrazolium bromide （MTT） assay. Flow cytometry was employed to detect cell cycle and apoptosis. The gene and protein expressions of nuclear factor κB-p65 （NF-κB-p65）, Bcl-2, B-cell lymphoma-extra large （Bcl-xL） and Bax were detected by quantitative real-time polymerase chain reaction and Western blot analyses. Caspases-3, -8, and -9 activities were detected using the colorimetric method. In addition, a B16-F10 melanoma xenograft mouse model was used to evaluate the anti-cancer activity of IME in vivo. Furthermore, a survival experiment of tumor-bearing mice was also performed to evaluate the possible toxicity of IME. Results： IME significantly inhibited the proliferation of B16-F10 cells （P〈0.01）. Flow cytometric analysis showed that IME induced G1/S cell cycle arrest and apoptosis （both P〈0.01）. IME inhibited activation of NF-κB, decreased the gene and protein expressions of Bcl-2, Bcl-xL, and increased the gene and protein expressions of Bax （all P〈0.01）. In addition, IME induced the activation of Caspases-3, -8, and -9 in B16-F10 cells. The study in vivo showed that IME significantly reduced tumor volume （P〈0.01）, and the inhibitory rate came up to 68.62%. IME also induced large areas of necrosis and intra-tumoral apoptosis that correlated with a reduction in tumor volume. Survival experiment showed that treatment with IME for 14 days significantly prolonged survival time and 20% of mice in the IME 200 mg/kg group were still alive until the 50th day. Notably, IME showed no apparent side-effects during the treatment period. Conclusion： IME exhibited significant anti-melanoma activity in vitro and in vivo, suggesting that IME might be a promising effective candidate with lower toxic for malignant melanoma therapy.