The Inhibiting Effect And Its Mechanism Of Isorhamnetin On Cancer In Vitro Or In Vivo

OBJECTIVE: To study the inhibiting effect of isorhamnetin on human tumor cells in vitro,and the inhibiting effect of this compound on animal model in vivo, and also to elucidate the molecular mechanisms involving in anti-cancer effect of this compound -isorhamnetin. METHODS: 1) In vitro cancer cells were treated with 10-320μg/ml isorhamnetin, morphologic characteristic were observed, and analyzed by light-microscope and electronic microscopy. Growth inhibition was observed and analyzed using an MTT (microculture tetrazolium assay), growth curve, clone formation test, 3H-TdR assay(3H-thymidine uptake assay). 2) Tumor model was set up by transplantating Lewis lung cancinoma cells into C57BL/6 mice. To investigate the effect of isorhmnetin on tumor growth, cell proliferation and apoptosis in transplantation tumor of lung cancer of Lewis cell line in C57BL/6 mice. Lewis cells were inoculated into the the C57BL/6 mice to establish Lewis lung cancer model, then fifty six C57Bl/6 mice with Lewis tumor were randomized four groups: control group, isorhmnetin group, treated in advanced with isorhmnetin group, cisplatin group. After 7 days treatment, samples of tumor were collected. The sections of tumor were observed under light microscope and electron microscope. The expression of PCNA, bcl-2 and bax were detected by immunohistochemistry. 3) The anticancer molecular mechanisms of isorhamnetin were researched in Vitro and in Vivo. Apoptosis and expression-associated gene peaks were detected by flow cytometry (FCM), agarose gel electrophoresis, immunohistochemical method, immunofluorescence method, comet assay and western blot. The catalase activities of cancer cells were analyzed with UV-visible Recording Spectrophotometer. 4) IL-6 secretion by the Lung cells was assayed by using RT-PCR. RESULTS: Isorhamnetin markedly inhibited the growth of cancer cells, especiasly lung cancinomar and apoptosis of those cells was observed. The cells showed characters of apoptsis and differentiation observed by light and electronic microscopy, agarose gel electrophoresis of DNA showed change of "Ladder" pattern. The rates of apoptosis increased after treated with isorhamnetin, and the cell was inhibited in G0/G1 phase, the cellular number in S phase decreased (P<0.05). We observed that the tumor weight was significantly lighter and the tumor size smaller compared to the control group. The results of apoptosis-related genes and proteins expression levels of transplanted Lewis cells were the same as those of A549 cells in vitro. Immunohistochemical stainng of PCNA showed that the PCNA lable index displayed significally difference between negative control group and medicine groups, the expression of PCNA of medicine groups lower than that of negative control, and so did the staining of bcl-2; but the expression of bax of medicine groups higher than that of control group (P<0.05). 20μg/ml isorhamnetin can induce cancer cell apoptosis and up-regulate the expression of apoptosis genes Bax, c-fos, Caspase-3, P53 and down-regulate the expression of the anti-apoptotic gene Bcl-2, c-myc, H-ras oncogene and PCNA protein.Isorhamnetin can inhibit the expression of IL-6, so inhibit the anti-apoptosis effect of IL-6. CONCLUSION: The results once more confirmed that isorhamnetin can inhibit growth, proliferation and induce differentiation of different cancer cells, especialy in human A549 cells. Isorhamnetin can induce apoptosis in human A549 cells and transplanted Lewis lung carcinoma cells , and plays a role in the down-regulation of anti-apoptotic genes and the up-regulation of apoptotic genes.. The mechanism might relate to modulation of gene expressions associated proliferation and apoptosis, the results provide a guide for laying down the chemotherapy for human tumors, when isorhmnetin was used as anti-cancer agent.