Studies On The Drug Design, Anti-tumor Activity, Genotoxicity And Reprodutive Toxicity Of Novel Isoflavone Derivatives F11

The prevention of tumor has been the focus of the enormous amount of research both in china and oversea countries. Chemotherapy plays a significant, positive and definite role in the treatment of tumor and improvement of living quality of patients with tumor. Epidemiology studies have demonstrated that the incidence of suffering rectal cancer,breast cancer and carcinoma of prostate is much lower in eastern people than western people, which may be due to the higher proportion of bean production in daily intake of food in the former. The effective ingredient has been proved to be, suggesting a pivotal role of isoflavone in decreasing the incidence of cancer. Lee et al pointed out that soybean has a remarkable preventive effect against breast cancer that occurs before menopause in women. Important biological roles played by isoflavone promoted researchers to do large amount of work which revealed that soybean isoflavone is a kind of Polyphenols mixture including genistein,daidzein and glycitein. Since they can bind to estrogen receptor (ER) and have weak estrogen like function, they are called phytoestrogen. Studies in recent years found that isoflavone can effectively suppress the growth of tumor in experimental models through interaction with highly specific tyrosine protein kinase, implicating a potential mechanism for its anti-tumor effect. However, most of the isoflavone compound fail to display anti-tumor effect unless administered in high dose. For example, daidzein exhibits anti-hypoxia activity in the dose of 250-500mg/kg. Thus, it is inadequate for natural intake of food to accumulate certain amount of compound with anti-tumor effect. One solution is to synthesize compound with similar biological effects with more potent activity. More importantly, the phenomenon that little difference in the structure of several isoflavone compounds render them sharp different biological activities. These basic facts promote us to make modifications to the structure of isoflavone compounds so as to obtain potential anti-tumor drugs. We synthesized a series of isoflavone compounds through modification of their structure, and examined their biological anti-tumor effect upon the proliferation of tumor cells. It is found that compound F11 exhibited excellent anti-tumor activity with more potential for further modification. Our study aims to assess F11 through performing experiments in drug design, anti-tumor activity, genetoxic and reprodutive toxicity. We also studied the mechanism underlying its anti-tumor activity, providing experimental data for the development of new anti-tumor drugs.Our study consisted of three parts:1. Analyze the relationship between structure modification and anti-tumor activity through related experiments. Find out possible effective groups and investigate the potential mechanism with an emphasis on the biological activity of F11.2. The anti-tumor activity of F11 and mechanism(1) In vivo anti-tumor activity: By establishing the heterologous graft models of human cancer in nude mice with MCF-7 and HeLa cell lines, we administered F11 to tumor mice in the dose of 20mg/kg and 40mg/kg, with genistein and Cytoxan as control treatment, and observed the anti-tumor activity and toxicity of F11.(2) Anti-tumor mechanism: We stained cell nucleus with DAPI and tried to determine whether F11 might induce apoptosis in tumor cells. Western-blot of apoptosis related proteins was performed to find out the signaling pathway of apoptosis. Then, caspase specific antagonist was administered to tumor cells to prove, in a opposite way, that the right signaling pathway was involved.3.The study on genetoxic and reprodutive toxicity of F11(1) Assessment of genetoxic of F11: three experimental models were used i.e. reversible mutation,chromosomal aberration (CA) and micronuclei (MN), to evaluate the genetoxic of F11.(2) Assessment of reprodutive toxicity: we used sperm shape abnormality analysis to examine the potential mutagenicity of F11 on germ cell.By analysis the three researches above, the following conclusions can be obtained:1. Structure and function analysis(1) When isoflavones were substituted with methyl, ethyl or other alkyl at 2-point, the proliferation of Hela cells was suppressed. With the elongation of carbon chain in 2-point alkyl substituted group, this suppressive effect was enhanced. It could be predicted that the 2-point alkyl substituted group in isoflavones structure is an essential group in inhibiting the proliferation of Hela cells, the longer the carbon chain in alkyl substituted group the more potent activity.(2) When isoflavones were substituted with hydroxy, methoxy or other electron-rich groups at 4'-point, the proliferation of Hela cell was suppressed. We thus postulated that the 4'-point ectron-rich substituted group in isoflavones structure in indispensable for the suppressive effect.(3) When isoflavones were substituted with Piperidine or other strong alkaline groups at 7-point, the proliferation of Hela cell was suppressed. Thus we predicted that the 7-point strong alkaline substituted group in isoflavones structure is necessary for the suppression of Hela cells' proliferation.(4) By examining the suppressive effect of isoflavones on the proliferation of Hela cell line, we preliminarily thought that the isoflavone F11 has an ideal anti-tumor activity in vitro. The introduction of alkaline side chain of Piperidine at 7-point enhances the Partition Coefficient of F11 and contributes to the absorption and distribution of compound. Further, Piperidine can be changed into the salts, improving its solubility and helpful for the production of compound's crystal. Taken together, we chose F11 as a potential drug for further analysis.2.The anti-tumor activity of F11 and possible mechanism(1) Results from in vivo in nude mice modelThe tumors progressed rapidly in both saline and genistein treated groups, the former more apparent; whereas, animals in F11 (20mg/kg), F11 (40mg/kg) and Cytoxan treated groups had tumors whose growth were suspended, indicating an obvious anti-tumor effect exerted by F11 and Cytoxan. Pathological studies found that large areas of zone of necrosis appeared in F11 and Cytoxan treated groups. In the mean time, no observable differences were found between F11 (20mg/kg), F11 (40mg/kg) and Cytoxan treated groups in terms of their anti-tumor effects, suggesting a similar anti-tumor effect exhibited by F11 with Cytoxan.We also observed the pathological changes in the major organs such as heart, spleen, liver, kidney, intestine, uterus, and ovary in F11 treated groups. No obvious damages to tissues or cells were observed. Especially, we did not found overt growth of endometrium or the induction of hysteromyoma. By contrast, we found apparent renal toxicity in Cytoxan treated groups. These results indicated that F11 had weaker estrogen less toxic effect(2) Mechanism underlying anti-tumor effect of F11Within 48h after the treatment of F11 upon Hela cells, DAPI staining revealed the emergence of large amount of cells undergoing apoptosis, with densedyeing nucleus, suggesting that F11 can induce the apoptosis of tumor cells in vito. Western blot analysis revealed that the expression of cleaved caspase-3 was increased as the time went after administration of F11, accompanied by effective cutting of caspase-7 and PARP. We thus proposed that F11 may activate the caspase-7 and caspase-3 pathway, cleave PARP, and finally induce apoptosis, though the molecules in upstream of caspase-3 remained to be determined. MTT demonstrated that antagonist of caspase-3 can attenuate the suppressive effect of F11 on the proliferation of tumor Hela cells.3. The result on genetoxic and reprodutive toxicity of F11 (1) The result on genetoxic of F11Ames test: we examined the mutagenic action of F11 on TA97, TA98, TA100 and TA102. Results indicated that F11 has no effect on the four strain in the range from 1 to 5000ug/dish, at S9 active and unactive situation, suggesting that F11 does not have observable gene mutation effect on TA97,TA98,TA100 and TA102.Chromosome aberration test of Cricetulus griseus lung fibroblast: Our results demonstrated that chromosome aberration rate in either blank,dissolvant control,S9 mixed liquor control fluctuated in normal level, whether cells were harvested in 24h or 48h. At the S9 activated and S9 non-activated situation, F11 in the concentration of 410～4100ug/ml, chromosome aberration rate remained in the control level. Thus, in our experimental set-up, increase in CHL cell chromosome aberration rate induced by F11 in 410～4100ug/ml were not found.Micronucleus test of bone marrow cell in mice: Within 24h after administration, F11 in 2.5,0.5,0.25,0.05g/kg did not have significant influence on the micronuclear rates. Micronuclear rates for each group was, 2.67±1.11,2.50±0.96,2.20±1.07 and 2.67±1.11‰, respectively, exhibiting no difference compared with 1.83±0.69‰in control group. This result indicated that, in our experimental setup, F11 does not exert marked influence on the micronuclear rates. (2) The result on reprodutive toxicity of F11Sperm shape abnormality test: Compared with negative control, animals in the high and middle dose groups of F11 displayed no difference in the frequency of sperm abnormalities; whereas, high dose of F11 and Cytoxan groups exhibited increased the frequency of sperm abnormalities. This result suggested that F11 in high dose can have mutagenesis on mice sperm; while F11 in low dose (0.25g/kg) or middle dose (0.5g/kg) did not.Conclusion1. The 2-point alkyl substituted group in isoflavones structure is an essential group for the anti-tumor activity, the longer the carbon chain in alkyl substituted group the more potent activity. Moreover, both the 4'-point ectron-rich substituted group in isoflavones structure and the 7-point strong alkaline substituted group in isoflavones structure are necessary for the inhibition of proliferation of Hela cells.2. In vivo the heterologous graft models of human cancer in nude mice revealed that the two dose group of F11 has profound suppressive effect on the growth of the transplantation tumor, compared with normal saline and genistein group. F11 also displayed weak toxicity compared with Cytoxan, which can also effectively suppress the growth of transplanted tumor, as indicated by little damage to the major organs in F11 treated animals. Examination of apoptosis related proteins suggested that F11 can induce the apoptosis of tumor cells through activation of members in caspase family, though further studies have to be done to delineate the details.3. Within the range of selected concentrations in this study, F11 did not induce obvious mutation effect as assessed by Ames test, Chromosomal aberration test and Micronucleus test . High abnormal rate in mouse sperm was observed only in large dose group.