Study Of Daphnetin On Its Antitumor And Immunoregulation Effects

Objective: To study the antitumor effect in vivo and in vitro of daphnetin and its immunity regulating effect in mice bearing tumor, and to provide the scientific basis of the development of medicinal daphnetin.Methods: 1. Daphne odora Var.Marginata extracts--- E002(daphnodorin I),E003(daphnetin)antitumor effects in vitro. MCF-7 and SMMC-7721 cells were treated respectively with E002 and E003 at different concentration, the growth inhibitory activity of E002,E003 were assessed using the MTT assay. 2. The antitumor effects of daphnetin and its immunity regulating effects in mice bearing-tumor in vivo.①Established S180-bearing mice model;②Animal grouping and drug use: Mice were randomly divided into six groups:a) Healthy control group;b)Daphnetin high group(4mg/kg);c)Daphnetin middle group(2mg/kg);d)Daphnetin low group(1mg/kg); e)Tumor-bearing control group;f)CY group (100mg/kg). Daphnetin treatment group were intraperitoneally injected different concentration daphnetin to each mouse with 0.2ml once a day, consecutively 10 days.Tumor-bearing control group and healthy control group were intraperitoneally injected NS to each mouse with 0.2ml once a day, consecutively 10 days. CY group were intraperitoneally injected 100mg/kg CY at 1,5,9 days to each mouse;③Detected the following indicators:a)The growth inhibition effect of Daphnetin on mice bearing tumor in vivo.Detected tumor weight and calculated inhibition rate after 10 days; b)RT-PCR was applied to detect IL-2 and IL-4 mRNA exression; c)MTT assay was used to detect killing activities of NK and LAK cells; d)The effect of lymphocyte proliferation was determined using MTT assay. e)RBC-C3RR was used to detect the immune function of RBC.Results: 1.①Both inhibition rate(%) were from 86.9±2.23 to 50.8±0.63, when SMMC-7721cells were treated by the E002,E003 with the concentration from 250 to 1.95(μg/ml).②Ihibition rate(%) were from 72.3±2.10 to 52.48±1.65, when MCF-7 cells were treated by the E002 with the concentration from 250 to 62.5(μg/ml).③ E002 on SMMC-7721 and MCF-7 cells IC50 value(μg/ml)were: 1.96±0.03 and 22.89±3.04. E003 on SMMC-7721 cells IC50 value(μg/ml)was 1.23±0.11. 2. The tumor weight of daphnetin treatment group was lower than tumor-bearing control group(P<0.05); Daphnetin treatment group inhibited obviously, the tumor growth inhibition rates were 80.28%(daphnetin high),71.18%(daphnetin middle) and 41.19%(daphnetin low), presented a dose-dependent tendency. 3. IL-2 mRNA expressions of daphnetin high and middle groups were significantly higher than tumor-bearing control group(P<0.05), and IL-4 mRNA expressions were opposite(P<0.05). IL-2 mRNA expressions of tumor-bearing control group was lower than healthy group(P<0.05); IL-4 mRNA expressions of tumor-bearing control group was higher than healthy control group(P<0.05), and tumor-bearing control group Showing a Th2-type response. IL-2 mRNA expressions of CY group and tumor-bearing control group were no significant difference( P﹥0.05). 4. NK,LAK cells killing rate of daphnetin treatment group were significantly higher than tumor-bearing control group and CY group(P<0.05); NK,LAK cells killing rate of daphnetin high group and healthy control group were no significant difference (P﹥0.05); NK,LAK cells killing rate of CY group was significantly lower than healthy control group and tumor-bearing control group(P<0.05).5. The lymphocyte proliferation stimulation index of daphnetin treatment group was higher than tumor-bearing control group and CY group(P<0.05); The lymphocyte proliferation stimulation index of CY group was lower than tumor-bearing control group and healthy control group(P<0.05).6. RBC-C3bRR of daphnetin high and middle groups were higher than tumor-bearing control group and CY group(P<0.05); RBC-C3bRR of daphnetin high group was no significant difference with healthy control group(P﹥0.05); RBC-C3bRR of CY group was lower than healthy control group(P<0.05), but was no significant difference with tumor-bearing control group(P<0.05).Conclusion: 1. In this study, we researched anti-tumor effects of E002(daphnodorin I)-----Daphne odora Var.Marginata extracts and anti-tumor effects in vivo of E003(daphnetin)-----Daphne odora Var.Marginata extracts in first time. 1. Daphnodorin I and daphnetin had varying degrees of inhibition effect on SMMC-7721 cell in the range of 0.12 ~ 250μg/ml, and showed dose-dependent; Daphnodorin I had inhibition effect on MCF-7 cell in the range of 0.12 ~ 250μg/ml, and showed dose-dependent. 2. Daphnetin can significantly enhance IL-2 mRNA expressions in the dose range of 2 ~4mg/kg?d, reduced IL-4 mRNA expressions at same time.To some extent, daphnetin improved the offset state of Th1/Th2 type which tumor-bearing lead. 3. Daphnetin had significant inbibition effect on murine transplanted S180 tumors in 1~4 mg/kg?d dose range ; Daphnetin can enhance the function of cell-mediated immune,non-specific immune and red-cell immune on murine transplanted S180 tumors.