| ObjectiveThe present study analyzed the anti-tumor effect of Icaritin in vitro and in vivo; and also to explore how does the effect work by Icaritin alone or Icaritin combined with the commonly used anticancer drugs.Methods1. To explore the effect of Icaritin anti-tumor at cellular level:Using MTT assay method for endometrial carcinoma of Ishikawa/Hec-lb cells and cervical cancer Siha/Hela cells under four concentrations:1μmol/L,5μmol/L,10μmol/L,20μmol/L; After24h,48h,72h treatment hours, testing the inhibitory rate of the four cell lines under different concentrations.2. To explore the effect of Icaritin anti-tumor at molecular level:Using RT-PCR method for endometrial carcinoma of Ishikawa/Hec-lb cells and cervical cancer Siha/Hela cells lines with different drug treatment:Icaritin alone, cisplatin alone, tamoxifen alone, Icaritin combined with cisplatin, Icaritin combined with tamoxifen and a blank control treatment, then, to extract RNA in each group after48hours, reverse transcription into cDNA. Using PCR experiments to detect the expression of estrogen receptor gene ER-a36, ER-a66, apoptosis gene Bax, tumor suppressor gene P27and cell cycle-related gene Cyclin D1.3. To explore the effect of Icaritin anti-tumor at animal level:Frist, solid tumor-bearing mice model successfully established. Second, the Mice were divided into six treatment groups: high/low dose groups, cyclophosphamide alone group, Icaritin combined with cyclophosphamide group, model group and the blank control group. To weight mice daily, observed weight changes. After15days, to anatomy mice, to retent solid tumors, spleen, thymus, weighing, calculating the indexs, blood test biochemical indexes.Results1. Cellular level:Icaritin has the anti-tumor effect on endometrial cancer cell lines.Ishikawa cells MTT assay results showed that:Icaritin treated in Ishikawa cells for24h, the cells showed morphological changes. After48and72h, the cytoplasmic concentration, cell membrane ruptured. Its inhibition gradually increased with the increase of drug concentration and treatment time, at a concentration of5μmol/L the inhibit rate back ward and forth, concentrations greater than5μmol/L, the inhibition rate gradually increased with the increase of concentration and treatment time. Using a regression equation to calculate the IC50value of72h was11.54μmol/L.In Hec-lb cells in MTT assay results showed that:Icaritin treatment in Hec-lb after24h, cell morphological changes, After48and72h, the cytoplasmic concentration, cell membrane ruptured. Its inhibitory effect gradually increased with the increase of drug concentration and treatment time.Using a regression equation to calculate the72h IC50value was determined5.26μmol/L.Siha cells MTT assay results showed that:Icaritin treatment in Siha after24h, cell morphological changes, After48and72h, the cytoplasmic concentration, cell membrane ruptured. Its inhibitory effect gradually increased with the increase of drug concentration and treatment time. Treatment after24h,48h,72h the maximum inhibition rate reaches about40%, expressed the cell has a low sensitivity in Icaritin, the inhibition of cell growth is not obvious.Hela cells MTT assay results showed that:Icaritin treatment in Hela after24h, cell morphological changes, After48and72h, the cytoplasmic concentration, cell membrane ruptured. Its inhibition enhanced gradually with the increase of drug concentration and treatment time. However, the highest inhibition rate reached around30%, and it no longer has the tendency to increase. This result showed that the Hela cell line has the resistance of the Icaritin, and the treatment of inhibition in cell growth is not obvious.2. Molecular level:Icaritin alone or it combined with anti-tumor drugs have anti-tumor effect.Ishikawa cells tested by RT-PCR experiment results show that:in the low concentrations of Icaritin, it set up estrogen receptor ER-a36, the ER-a66expression (P>0.05) slightly, significantly reduced the expression of apoptotic genes Bax (P<0.05), which indicated that low concentrations of Icaritin can reduce the cell line proliferation and its possible mechanism is to set down the expression of apoptotic genes. At the same time, from the results of the study we also observed that low concentration of Icaritin combined with the common anticancer drug cisplatin (DDP), tamoxifen (TAM), can significantly reduce the expression of ER-a36, ER-a66(P<0.05), significantly increase apoptotic gene Bax and the tumor suppressor gene P27in (P<0.05). These results suggest that Icaritin can be used as a supplementary drug to achieve synergistic effect. In Hec-lb cells tested by RT-PCR results show that:Icaritin significantly lowered estrogen receptor ER-a36and ER-a66expression (P<0.05), significantly up-regulated the expression of tumor suppressor gene P27(P<0.05), which indicated that Icaritin played the role of inhibition in Hec-lb cells by down-regulating the estrogen receptor and up-regulating of tumor suppressor genes. At the same time, from the results of the study,we also observed that Icaritin combined with the common anticancer drug cisplatin (DDP), tamoxifen (TAM) can significantly reduce the estrogen receptor ER-a36,ER-a66expression (P<0.05). These results suggest that Icaritin assisted the anticancer drugs in curing estrogen-dependent tumors.Siha cells tested by RT-PCR results show that Icaritin significantly lowered estrogen receptor ER-a36and ER-a66expression (P<0.05), significantly up-regulated apoptotic gene Bax and the tumor suppressor gene P27expression (P<0.05), slightly lowered the cell cycle related genes cyclin D1expression, these effects may be related in the inhibition of cell proliferation. However, the mechanism of Siha cells has low sensitivity to Icaritin is not clear yet, it may be related in other tumor gene.Hela cells tested by RT-PCR results show that:Icaritin significantly increased in the estrogen receptor ER-a36and ER-a66expression (P<0.05), significantly up-regulated the expression of apoptotic gene Bax and the tumor suppressor gene P27(P<0.05), slightly set down the cell cycle related genes cyclin D1expression.These effects may be associated with the cell proliferation inhibition. But, Hela cells has the ability to resist Icaritin is not clear, it presumably associated with multidrug resistance gene MDR1.3. Animal level:The effect of Icaritin, Icaritin combined with anti-tumor drugs is not obvious, but Icaritin, Icaritin combined with anti-tumor drugs have some improvement in tumor-bearing mice immune function.H22hepatoma solid tumor mice experimental results:(1) Compared with model group, high/low doses of Icaritin, cyclophosphamide groups, tumor weight index was significantly increased (P<0.05), indicates that the inhibition Icaritin in tumor growth at animal level is not obvious.(2) After treatment of each drug group, as the immune organs:spleen and thymus, as well as the biochemical indicators showed that Icaritin or Icaritin combined with anti-tumor drugs have the ability to improve the immune function of tumor-bearing mice.S180solid tumor mice experimental results:(1) Compared with model group, except for Icaritin combined with cyclophosphamide, cyclophosphamide alone group, the tumor mass index drug were significantly decreased (P<0.05), high/low doses of Icaritin group, tumor weight index does not reduce (P>0.05), this result indicated that the Icaritin in the inhibition of tumor growth in animals is not obvious.(2) After treatment of each drug group, as the immune organs, spleen and thymus, as well as the biochemical indicators showed that Icaritin or Icaritin combined with anti-tumor drugs have the ability to improve the immune function of tumor-bearing mice.Conclusion1. Cellular level:Icaritin has the anti-tumor effect on endometrial cancer cell lines.2. Molecular level:Icaritin alone or it combined with anti-tumor drugs have anti-tumor effect in the related gene expression.3. Animal level:The effect of Icaritin, Icaritin combined with anti-tumor drugs to cure tumor is not obvious, but Icaritin, Icaritin combined with anti-tumor drugs have some abilities to improve tumor-bearing mice in immune function. |
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