The Effects Of Podophyllic Acid Piperidyl Hydrazone Nidroxide Radical On Biological Characteristis Such As Proliferation,Invasion Of Human Colorectal Carcinoma SW620Cells

Background and ObjectThe colorectal cancer is one of the most common gastrointestinal cancers; there is a high morbidity and high mortality in Europe and other developed countries. For the past few years, the morbidity and mortality of colorectal cancer in Chinese resident is increasing year by year with the improvement of the economy, living standards. Etiology and mechanism of colorectal cancer has not yet been elucidated, the common determinant may include genetic, lifestyle and other factors related. As a specific tumor type, besides the cancer’s common characteristics (such as unrestricted growth, invasion damage, depletion of the body and distant metastasis), there is personality trait in colorectal carcinoma (such as delay the best healing time by confused with colitis, hemorrhoids, simple anemia). Therefore, the infinite hyperplasia and early metastasis are the most impact of colorectal cancer on human. Currently, the treatment for colorectal cancer is based on surgery and chemotherapy. Five-year survival rate for colorectal cancer has greatly improved after the optimization of surgical approach, the continuous development of chemotherapy and use of radiotherapy. But there is no denying that still a considerable number of patients had early postoperative metastases lesions, the effect of chemotherapy and radiotherapy on normal cells influence the patients’ quality of life. Therefore, therapeutic effect of tumor can be improved significantly according to reduce the infinitely proliferation and superior invasive ability of tumor cell. Podophyllotoxin is a lignin compounds extracted from podophyllum peltatum, sinopodophyllum hexandrum, dysosma versipellis, umbrellaleaf and other plants. It has been demonstrated that many derivatives synthesized from its basic skeleton showed different biological activity (including anti-inflammatory, antioxidant and anti-tumor effects) due to their different structural features. It has been proven, the components above showed effect in a wide variety of tumor, such as lung cancer, liver cancer, stomach cancer, breast cancer, cervical cancer, leukemia and lymphoma and other cancers. Among these components, Podophyllic acid piperidyl hydrazone nidroxide radical (GP-1) has strong anticancer activity, GP-1can block cell cycle in G2/M phase and induce apoptosis in many tumors. Traditional research has reminded that the possible mechanism of the proliferation inhibition and apoptosis-inducing effect of GP-1include destruction of microtubules metabolic balance and the function of DNA topoisomerase. But there is less study on the effects and mechanisms of GP-1on the migration and invasion of tumor cell, especially for colorectal cancer.Present study demonstrated that, matrix metalloproteinase (matrix metalloproteinase, MMPs) played an extremely important role in the process of tumor invasion and metastasis. MMPs are proteolysis enzymes which are included in the same category, and its catalytic activity depends on the metal ions Zn2+. Among the24members of the family of MMPs, MMP-2,MMP-9are closely associated with colorectal tumor.Research shows that some small molecules extracts from natural plant is of anti-angiogenesis activity, thus whether GP-1is of this feature is also worth studying on.In the present study I evaluated the effects of GP-1on the growth, migration, invasion of human colorectal cancer cell SW620and apoptosis-inducing, try to explain its role in the anti-angiogenesis and investigated its possible mechanism involved in the apoptosis and invasion induced by GP-1,to find a new direction for drug treatment of colorectal cancer.Method1、After interferenced by different concentration GP-1for24hours or48hours, the growth inhibiting of colorectal cancer SW620cells were determined by CCK-8assay to get its IC50and select some appropriate conditions for follow-up experiments.2、The cell migration and invasion ability of human colorectal cancer SW620cells after interferenced by GP-1were evaluated by transwell chamber migration and artificial basement membrane invasion assay.3、The morphological changes of human colorectal cancer S W620cells after interferenced by GP-1were observed by inverted microscopy. The apoptosis rate of human colorectal cancer SW620cells after treated by different concentrations of GP-1was detected by Annexin-V/PI assay.4、The level of matrix metalloproteinases MMP-2, MMP-9(invasion-related factor) in cultural supernatant of human colorectal cancer SW620cells interferenced by different concentration GP-1were determined by ELISA assay.5、 Expression level of collagenase-IV MMP-2、 MMP-9and vascular endothelial growth factor (VEGF) were analyzed by RT-PCR assay in order to explore the possible mechanism of GP-1on invasion and metastasis of human colorectal cancer SW620cells.Results 1、Our study demonstrated that GP-1significantly inhibited the proliferation of SW620cells in a concentration-and time-dependent manner(P<0.01) and the IC50were,24h:IC50=30.09ug/ml,48h:IC50=18.87ug/ml, concentration of12.5ug/ml,25.0ug/ml,50.0ug/ml and24hours were selected for the other experiments.2、Transwell chamber migration and artificial basement membrane invasion assay showed that after the cells were treated with GP-1The migration and invasion ability of SW620cells was decreased significantly (P<0.01). Treated by Minimum test concentrations12.5ug/ml for48h the inhibition rate was16.84%inhibition, and maximum test concentrations50.0ug/ml for48h the inhibition rate up to47.45%.3、After the cells were treated with GP-1inverted microscope cell mass getting smaller,the number of cells was reduce,some cell rounding and cell nuclear enrichment suggesting apoptosis possibly. The results of FCM showed that GP-1could affect the cell apoptosis of SW620but in a feeble way. Its early apoptotic ratio and late apoptotic ratio rise with drug concentration slightly, but there was no statistical significance compared with the control group.4、ELISA results suggest that the expression level of matrix metalloproteinases MMP-2, MMP-9in cultural supernatant of SW620cells was decreased after treating by GP-1and in a concentration-dependent manner.5、In addition, RT-PCR analysis showed that the expression of MMP-2mmp-9and VEGFmRNA in SW620treated by GP-1were lower significantly than those in SW620negative control group(P<0.05), and in a concentration-dependent manner.Conclusion1、In vitro, GP-1could inhibit the growth of colorectal cancer cells, but the ability of inducing apoptosis was not obvious.2、GP-1may be breakdown the migration、invasiveness of colorectal cancer cells by decreased the expression of MMP-2MMP-9.3、GP-1can reduce the expression of VEGF in colorectal tumor cells, suggesting that GP-1may have the ability of anti-angiogenesis.