| ObjectiveCancer seriously threatens to human health. In recent years, it has become a major cause of death worldwide. Chemotherapy is still one of the main means in the clinical treatment of cancer. However, due to its low rate of reaction, toxic-side effects, as well as multi-drug resistance, chemotherapy has failed to achieve good efficacy. Therefore, it is urgent to find effective and low-toxic drug candidates against cancer. Natural active ingredients extracted from medicinal plants are characterized by multi-target, high biological activity, low toxicity, and low resistance. They have become the international hot spots and focus on the study of anticancer drugs.Ethanol extract of Ilex hainanensis Merr. (IME) was extracted from the leaves of Ilex hainanensis Merr that belongs to the family Aquifoliaceae. Recently, we have demonstrated that IME possesses excellent lipid-modification and anti-inflammatory properties. And there is a close correlation between inflammation and cancer. Therefore, this study will further explore the anti-tumor activity of IME on the basis of the previous work, and elucidate its underlying molecular mechanism. The study would provide experimental support for the research that IME would be developed into a novel anti-tumor drug.MethodsPart one:The screening on anti-tumor activity of IME in vitroCulture 13 kinds of human tumor cell lines, BGC-803, MGC-803, SGC-7901, NCI-H460, A549, LETP-a-2, HepG2, SMMC-7721, MHCC, Hep3B, PC-3, and MDA-MB-231; 3 kinds of human tumor cell lines, B16-F10, H22, and Lewis. Using MTT and CCK-8 assay to detect the effect of IME on viability of tumor cells and calculate the 50% inhibitory concentration (IC50).Part two:Studies on anti-melanoma therapeutic effects of IME1. The effects of IME on growth of B16-F10 melanoma in tumor-bearing miceThe C57BL/6J mice were subcutaneously inoculated with B16-F10 cells at the right axillary flank. The tumor-bearing mice were randomized into model group, IME 25 mg/kg group, IME 50 mg/kg group, IME 100 mg/kg group, IME 200 mg/kg group and DTIC 70 mg/kg group. The mice in the IME treatment groups were intragastrically administered with IME 25,50,100 or 200 mg/kg, respectively. The mice in the model group were given equal volumes of 0.5% CMC-Na daily. The mice in the positive control group were intraperitoneally injected with DTIC at a dose of 70 mg/kg every 2 days. The drug administration was lasting for 14 days. The mice were individually observed for signs of acute toxicity and behavioral changes daily. The food consumption and body weight were recorded daily. The tumor volume was measured every 3 days with a caliper. On day 14, all the mice were sacrificed and tumor tissues were harvested, weighed and photographed. The inhibition rate of tumor growth was calculated. Moreover, main organs involving heart, liver, spleen, lung, and kidney were also sampled for organ index calculation and histological examination.2. The effects of IME on the survival time of tumor-bearing miceThe C57BL/6J mice were subcutaneously inoculated with B16-F10 cells at right axillary flank. The tumor-bearing mice were randomly grouped into model group, IME 50 mg/kg group, IME 100 mg/kg group, IME 200 mg/kg group and DTIC 70 mg/kg group. In the model group, the mice were gavaged daily with equal volumes of a 0.5% CMC aqueous solution. In the IME treatment groups, the mice were gavaged daily with IME at doses of 50,100, or 200 mg/kg/d, respectively. The mice in positive control group were intraperitoneally injected with DTIC at a dose of 70 mg/kg every other day. The drug administration was lasting for 14 days. All the groups kept being observed for 50 extra days. The median survival time and increase of survival time of the model or treatment groups were calculated.Part three:Studies on anti-melanoma mechanism of IMEThe B16-F10 cells were treated with IME at the indicated concentrations for 24,48 and 72 h, and the viability of B16-F10 cells was determined by MTT assay. Flow cytometry was employed to detect cell cycle and apoptosis. The gene expressions of NF-KB-p65, Bcl-2, Bcl-xL, and Bax in B16-F10 cells were detected by Real time-polymerase chain reaction (RT-PCR). The protein expressions of NF-κB-p65, P53, Bcl-2, Bcl-xL, Bax, Cytochrome C, CyclinD, CyclinE, CDK4, and CDK2 in B16-F10 cells were detected by western blot analyses. Caspases-3,-8, and-9 activities in B16-F10 cells were detected using the colorimetric method.ResultsPart one:The screening on anti-tumor activity of IME in vitroIME significantly inhibited the proliferation of 13 kinds of human tumor cell lines and 3 kinds of murine tumor cell lines in a dose-and time-dependent manner. The smallest IC50 values of IME on tumor cell lines are the murine B16-F10 melanoma cells and human HepG2 hepatoma cells, respectively.Part two:Studies on anti-melanoma therapeutic effects of IME1. IME suppressed growth of B16-F10 melanoma in tumor-bearing miceTo confirm the anti-melanoma effect of IME in vivo, a B16-F10 melanoma xenograft mouse model was established. Compared with the model group, IME significantly reduced the tumor volume and tumor weight of the tumor-bearing mice. The inhibition rate in the IME 25,50, 100,200 mg/kg groups was 38.41%,43.01%,49.25%, and 68.62%, respectively. The H&E and TUNEL staining of tumor tissues showed that IME induced intra-tumoral large areas of necrosis and apoptosis. During the experiment, the tumor-bearing mice from IME-treated groups did not appear obvious toxicity. In addition, no marked histopathological changes were observed in the organs from the IME treatment groups.2. IME prolonged the survival time of tumor-bearing mice.In the survival experiments of tumor-bearing mice, all the mice in the model group were died on day 33, whereas there were 70%,80%, and 90% of mice alive in the IME 50,100,200 mg/kg groups. At the end of the experiment, all the mice in the DTIC 70 mg/kg group had died, but one mouse in the IME 100 mg/kg group and two mice in the IME 200 mg/kg group were still alive. The increase rate of survival time in the IME 50,100,200 mg/kg groups was 45.10%, 45.10%, and 62.75%, respectively. These data demonstrated that IME remarkably prolonged the survival time of tumor-bearing mice.Part three:Studies on anti-melanoma mechanism of IMEIME significantly inhibited the proliferation of B16-F10 cells in a dose-and time-dependent manner. Flow cytometric analysis showed that IME induced G1/S cell cycle arrest and apoptosis. Western blot analysis showed that IME inhibited the expressions of G1/S cell cycle-related proteins including CyclinD, CyclinE, CDK4, and CDK2 in B16-F10 cells. RT-PCT assay showed that IME decreased the gene expressions of NF-κB-p65, Bcl-2, Bcl-xL, and increased the gene expressions of Bax in B16-F10 cells. Western blot analysis showed that IME inhibited translocation of NF-KB-p65 into the nucleus, decreased the protein expressions of Bcl-2 and Bcl-xL, increased protein expressions of P53 and Bax, and promoted the release of cytochrome c into the cytoplasm in B16-F10 cells. In addition, IME induced the activation of caspases-3,-8, and-9 in B16-F10 cells.Conclusions(1) In vitro, IME exerted the good anti-tumor activity, and the smallest IC50 values of IME on tumor cell lines were the murine B16-F10 melanoma cells and human HepG2 hepatoma cells, respectively.(2) IME effectively suppressed growth of B16-F10 melanoma in tumor-bearing mice, significantly prolonged the survival time of tumor-bearing mice, and had no obvious toxicity.(3) IME exerted the anti-melanoma effect by inducing G1/S cycle arrest and apoptosis in B16-F10 cells through regulation of G1/S cell cycle-related proteins, inhibition of translocation of NF-KB-p65 into the nucleus, up-regulation P53 protein experess, regulation of apoptosis-related proteins, and activation caspase cascade.(4) 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. |
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