| ObjectiveCancer, as one of the most lethal diseases, is being turned into a public health issue and its incidence continues increase. Chemotherapy is the main treatment mean in clinical setting, but it exerts toxicity on both cancer cells and normal cells and thus elicits a series of side effects including immunosuppression, myelo suppress ion and kidney/liver damages. Side effects and multiple drug resistance limit the application of chemotherapy drugs. Traditional Chinese medicine is characterized by multiple components and multiple targets, and exerts anti-tumor activities on several vital processes of tumor growth. At the same time, it exerts anti-tumor activities with low toxicity, immunoenhancement and low resistance. Due to its beneficial effects above, traditional Chinese medicine and the phytochemicals contained have become focus of drug discovery.Ilexgenin A (IA) is extracted from the active fraction of Ilex hainanensis Merr.-"Sha 1v cha", which originates from leaves of Ilex hainanensis Merr. and possesses clearing heat and detoxicating as well as promoting blood circulation to open vessels. This study aims to evaluate the anti-tumor activity of IA and analyzes its potential toxicity to provide experiment data for further study.MethodsPart one:Firstly, we performed an MTT assay to determine cyto toxicity effects of IA on B16-F10 cells and calculated the suppression rate of IA (%) and the median inhibition concentration values (μM). Moreover, the cell cycle distribution of B16-F10 cells treated with IA was analyzed by flow cytometry to investigate possible changes in the cell cycle due to IA.Part two:B16-F10 murine melanoma cells were inoculated to the right armpits of C57BL/6J mice to establish tumor bearing mice model. The mice were randomly divided into model group, IA 15 mg/kg group, IA 30 mg/kg group and taxol 45 mg/kg group. The mice in the IA treatment groups were gavaged daily with IA (15 and 30 mg/kg) suspended in 0.5% sodium carboxymethyl (CMC-Na). The mice in the model group were gavaged daily with equal volumes of 0.5% CMC-Na. In the taxol 45 mg/kg group, the mice received intraperitoneal injection of 45 mg/kg taxol twice per week. The mice were monitored daily for general condition, and the body weights were measured every 2 days. After 10 days of treatment, all mice were sacrificed and tumor tissues and organs were harvested and weighted. The inhibition rate of tumor growth and organ index were calculated and tissue samples were fixed for histology.Part three:At the end of anti-tumor experiment, all mice were fasted prior to conducting the experiment (only food, not water, was withheld overnight) and sacrificed. The serum was collected and centrifuged. The concentrations of IL-6, TNF-a and IL-12/IL-23p40 were determined using mouse enzyme-linked immunosorbent assay. In addition, RAW 264.7 mouse macrophage cells were incubated with melanoma conditioned medium (MCM) (20% v/v) in the presence or absence of different concentrations of IA (5 to 40μM) for 6 h or 12 h. The culture supernatants were collected, and the level of IL-6 was measured by ELISA assay. Moreover, we employed MCM (100% v/v) inducing RAW 264.7 macrophages to differentiate into M2 tumor-associtaed macrophages. After treatment with IA for 24 h, total RNA was isolated and examined the mRNA levels of IL-6, VEGF and p65 by qRT-PCR.Part four:B16-F10 murine melanoma cells were inoculated to the right armpits of C57BL/6J mice to establish tumor bearing mice model. The mice were randomly divided into model group, IA 30 mg/kg group, IA 60 mg/kg group. The mice in the IA treatment groups were gavaged daily with IA (30 and 60 mg/kg) suspended in 0.5% CMC-Na. The mice in the model group were gavaged daily with equal volumes of 0.5% CMC-Na. The treatment lasted 30 days and went to withdrawal observation until the 100th day.ResultsPart one:IA inhibited the proliferation of B16-F10 cells in a dose-and time-dependent manner. The median inhibition concentration (ICso) values at 24 h and 48 h were 27.34 uM and 12.44 μM, respectively. In addition,20 μM IA treatment could induce growth arrest in the G1-S phase of the cell cycle in B16-F10 cells (p<0.05).Part two:The tumor weight of model group was 10.01 ± 3.14 g, whereas it was only 4.87 ± 3.71 g in IA 30 mg/kg group (51.13% decrease; p<0.01). Average tumor weight was 6.36±3.99 g (36.45%decrease;p<0.05) and 2.88±1.71 g (Inhibition rate:71.25%;p<0.001) in IA 15 mg/kg group and taxol 45 mg/kg group, respectively. Signs of illness, such as significant weight changes, signs of discomfort, or impaired movement, were not observed in the mice of the IA treatment groups during the whole treatment. Moreover, no marked histological changes and metastasis were observed in the organs from the IA treatment groups.Part three:Compared with the model group, the level of IL-6 was significant decreased (p<0.001) and the level of TNF-α was increased (p<0.05) in IA 30 mg/kg group. In addition, the IL-6 production in melanoma conditioned medium-stimulated RAW 264.7 mouse macrophage cells was significantly increased in the model group compared to the control group (p<0.001). IA significantly inhibited the IL-6 production of macrophage stimulated by MCM (20% ν/ν) (p<0.001). After co-cultured with MCM (100% ν/ν), the mRNA level of IL-6 was significantly decreased (p<0.01) and the mRNA level of Arg-1 and VEGF were increased (p<0.001). IA could increase the mRNA level of IL-6 in a dose-dependent manner, and this different was significant at 20 μM (P<0.05). At the same time, IA decreased the mRNA level of Arg-1 (p<0.01) and VEGF (p<0.001) in MCM (100% ν/ν) induced tumor-associated macrophages. Moreover, the mRNA level of p65 in model was significant icnreased compared with the control group (p<0.01), and IA exhibited an inhibitory effect on the mRNA level of p65 in a dose-dependnet manner (p<0.01).Part four:Thirty days after the onset of treatment,6 mice in the IA treatment groups were free of tumor, suggesting that Ilexgenin A significantly inhibited the growth of melanoma. Although two mice in the IA treatment groups experienced tumor recurrence after IA was removed for two weeks, four mice in IA 60 mg/kg group were alive with no sign of tumor at the end of experiment. Compared with 27 days of median survival time of model group, the median survival time of mice in IA 60 mg/kg group reached 79 days, indicating that IA significant prolonged the survival of tumor-bearing mice compared with the model group (p<0.001).Conclusions(1) IA inhibits the proliferation of B16-F10 murine melanoma cells in a dose- and time-dependent manner, and these effects are ascribed to cell cycle arrest at G0/G1.(2) IA has potent anti-melanoma activity with low toxicity. Moreover, IA may exert its anti-melanoma effects by regulating the production of tumor-associated cytokines in macrophages, especially the M2 tumor-associated macrophages in tumor microenvironment.(3) Ilexgenin A can improve the survival of tumor-bearing mice. |
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