Effects Of Umbelliferone On Mouse B16 Melanoma In Vivo

Malignant melanoma generates from nevus cells and melanocytes. It was a higher prevalence in white, while a low incidence in our country, but it caused mortality every year without enough understanding. Malignant melanoma was highly prone to distant metastasis, poor prognosis with chemotherapy, biological therapy insensitive. Therefore new drugs and the role of target were necessary to get to increase efficacy and improve the prognosis. Umbelliferone was a coumarin compounds, the active ingredient in many traditional Chinese medicine with antibacterial, anticoagulant, anti-mutagenic, and was confirmed it also had a certain anti-cancer effect in vitro. However, B16 mouse melanoma in vivo has not yet reported. The aim of this study was to observe the effects of umbelliferone on melanoma in mouse B16 melanoma model, and explore mechanism.Methods:1. C57 mince model bearing B16 melanoma were established.B16 cells were cultured in RPIM 1640 to logarithmic phase,then were inoculated to C57 subcutaneously in groin. After 14 days, the tumor was stripped and the cell suspension was made with concentration of 5x106 cells/ml, then the cell suspension were inoculated to C57 subcutaneously in groin. C57 mice including 42 female and 42 male, were randomly divided into 6 groups:Normal control group(The mice were not inoculated, and raised the daily routine); Negative control group (The day after inoculation,successive intragastricly physiological saline 14 days); Positive control group (The day after inoculation,100 mg/kg/d cyclophosphamide was injected intraperitoneally one day); low, middle, high dosage of umbelliferone (The day after inoculation,10 mg/kg,40 mg/kg,80 mg/kg of umbelliferone were respectively dissolved in 0.2ml physiological saline intragastricly every day for 14 days). The tumor volume was measured, and was weighed after 14 days administration, then inhibition rates was calculated.2. C57 mice including 40 female and 40 male were divided into 5 groups randomly,not setting normal control group. The melanoma model was established with the same method. The survivals of tumor-bearing mice were observed.3.18 indicators were detected on the three categories of blood cell analyzer respectively before inoculation, tumors in mice 10 d after inoculation and after administration.3. Tumor tissue samples and tumor-bearing mice with heart, liver, spleen, lung, kidney and thyroid were taken and examined by pyrogallol self-support method for the SOD activity, MDA-TBA assay for MDA content and polyacrylamide gel electrophoresis for LDH isoenzymes.4. HE staining analysis of the tumor tissue.5. Western-blot detection tumors in Bcl-2, Bax and Caspase-3 protein expression.Results:1. The mean tumor weight of low, middle, high dosage groups of umbelliferone (1.71±0.49g,1.32±0.61g,2.06±0.41g), was less than that of negative control group (2.13±0.51 g). The inhibition rates of tumor growth were 19.73%,37.78% and 2.96%. The mean survival time of low, middle, high dosage groups of umbelliferone (25.60±4.38 d, 27.60±3.95 d,24.20±3.40 d), and the middle group was obviously longer than that of negative control group (22.66±2.01 d) (P<0.05).2. The negative group, low, high dose group white blood cell, red blood cell decreased with tumor development. After administered umbelliferone, white blood cell of middle group significantly increased, and the negative group was statistically significant (P<0.05), while the smallest decline in red blood cell. Inoculated mice had a tumor on the rise of the platelet, but after treatment, the platelet of each group was down, and the smallest drop was in the high group.3. The SOD activity of tumor bearing mice had decreased compared to the control group, while the thyroid, spleen SOD activity of middle group higher than other groups, and kidney SOD activity was similar with normal mice. MDA content in tumor-bearing mice were higher than the normal control group.40 mg/kg dose of tumor tissue MDA content was lowest, while 10mg/kg dose of spleen, thyroid with the lowest MDA. The LDH activity in cardiac, lung, kidney and thyroid of 40 mg/kg dose group were close to normal control mice, and its LDH activity was lower in tumor tissue.4. HE staining showed that the tumor of 10 mg/kg,40 mg/kg and the 80 mg/kg dose group had different degrees of necrosis.5. Bcl-2 and P-actin ratio in positive control group,10 mg/kg, and 40 mg/kg dose group were lower than the negative control group, and the positive control group was the lowest,40 mg/kg dose group followed. In Caspase-3 andβ-actin ratio, negative control group was lowest, and positive control group was the highest,40 mg/kg dose group followed.The ratio of Bax andβ-actin in negative control group was the lowest, while positive control group and 40 mg/kg dosage group were relatively high.Conclusion:1. Umbelliferone mice can prolong the average survival time.2. Umbelliferone can increase the leucocyte, erythocytes number in tumor-bearing mice; improve the low immunity, anemia because of cancer.3. Umbelliferone can increase the antioxidant capacity of tumor-bearing mice, and recovery organ damage tumor caused.4. Umbelliferone may induce B16 melanoma cells apoptosis and necrosis, mediated by Bcl-2, Caspase-3 and Bax.