1.Studies On The Relationship Between EGFR And TGF-β Signal Transduction Pathways In Epithelial Origin Tumors 2.Studies On The Tumor Multi-drug Resistance Reversal Activity And Mechanisms Of New Taxane Derivatives Syl611 And NBP071

The TGF-βand EGFR signal transduction pathways are the principal signal pathways in the genesis and development of human cancer. The EGFR and its receptor family play an important role in different tissue source of tumors, the diversity activation of EGFR signal is considered as the promotion feature of endepidermis cancer and in which overexpression of EGFR is commonly detected. TGF-β, a multifunctional polypeptide growth factor is generally considered as a regulator related with tumor cell growth, differentiation and metastbasis. Its signalling regulates cancer through mechanisms that function either within the tumor cell itself or through other signalling interactions and it has a mechanistic role in tumor suppression. In our previous research, we found that after treated the animals with anti-EGFR signal agents, lung fibrosis was observed despite of good anti-tumor effects. As the primary modulator of fibrosis, TGF-P and its signalling would have certain interaction with EGFR signalling according to the phenomenons that we had obtained.TGF-P plays suppression role in endepidermis tumors. When treated human lung adenocacinoma cell line A549 with 5ng/mL TGF-β1, distinctly growth inhibition and morphological changes were observed, the aspect of cells were similar with fibroblasts. In the mechanism research, we found that TGF-β1 protein could suppress the activities of EGFR signal transduction pathway in endepidermis tumor cell lines. A431 (Human epithelial carcinoma cell line) and SKOV3 (Human ovarian carcinoma cell line) were used as EGFR over-activated model, the activation of EGFR signalling proteins such as EGFR, ERK and AKT was depressed when these cells were contacted with TGF-β1 protein. The inhibitions of proteins were varieties in degree in different time point. The ERK, a downstream protein of EGFR signalling, had the directly interaction with the Smad2/3 which is a signal protein in TGF-P pathway. We observed that in A431 cells ERK could co-precipitated with Smad and the amount of precipitated ERK gradually raised when the treating time of TGF-β1 extended. The results that we observed demonstrated that there would be certain formulation of restraint interaction between EGFR and TGF-βsignal transduction pathway in endepidermis tumors:the legand TGF-p 1 could inhibit the activation of EGFR and its signalling pathway, this effect of TGF-βon EGFR signalling would be contribution to the directly interaction between ERK and Smad2/3.The effect of TGF-P on tumors could be explained by the theory of the function of cancer cell microenvironment, which was mainly modulated by varieties cytokines. The cancer cell microenvironment is a complicated regulation system and is regulated by multiple factors. Fibroblast is the key regulator in this microenvironment system so we used co-culture system in vitro and in vivo to investigate the function of fibroblast in cancer cell microenvironment by analyzing the growth characterization and the relationship of EGFR and TGF-βsignalling in the co-culture system. Our data showed that when the co-culture ratio of A549 and human embryonic lung fibroblast HELF was 2:3, the growth and colony formulation was highest inhibited in these two co-culture model. The amount of TGF-βin cell co-culture medium, which was detected by TGF-βElisa Kit, was highest when the co-culture ratio was 2:3. Homogeneously, the activation of EGFR signalling in co-culture model was depressed in variety degrees by immunofluorescence assay. In the co-culture model in vivo, we found that co-cultured mouse Lewis lung cancer cells and mouse epidermis fibroblasts could reduce the formation amount of tumor and affect the growth of tumor. The results of immunohistochemistry indicated that the more fibroblasts co-cultured, the more expression of TGF-β1 in tumor tissue was detected. The activation of EGFR signalling proteins were also depressed. Taking together, the suppression effect of fibroblasts in cancer cell microenvironment was contribution to the effect of TGF-βon the EGFR signalling, the restraint interaction of TGF-βand EGFR sigal transduction pathway led to the complex of cancer cell microenvironment.In summary, TGF-P and EGFR sigal transduction pathway played important regulation role in endepidermis tumors. The suppression effect of TGF-βon EGFR signalling would be located at the direct interaction of ERK and Smad2/3, but in different tissue or different tumor stage, this interaction would be another representation or absolutely opposite results. In our studies, we investigated the action mechanism of TGF-βon EGFR signalling in endepidermis tumors and fibroblasts in cancer cell microenvironment. These results would give us the clue to discovery the new inherence during cancer origin or development and to find novel anti-cancer targets. In the past five decades the usage of anticancer agents has become one of the most important ways of controlling malignant cancers. However the development of multi-drug resistance to anticancer agents among patients continues to pose a serious obstacle to cancer chemotherapy and those medical professionals and researchers who investigate it. The overexpression of P-gp, which is a drug transporting plasma membrane protein for transporting drug, is the mainly mechanical cause of MDR. The reversal of MDR would be the key for overcoming drug resistance in modern cancer therapy. Currently many MDR-reversing agents, such as synthetic chemicals and natural products have been developed for anticancer therapy, but most of these products such as Verapamil, Cyclosporine A have been abolished during clinical usage for their toxicity in crux organs. Thus finding novel interference forms on drug efflux effect from P-gp would be the indication of the next generation of P-gp inhibitors.Sinenxan A is a taxoid isolated from the callus cultures of Taxus yunnanensis. The rich resources of Sinenxan A and its taxane-skeleton provides its possibility for the semisynthesis of paclitaxel or other bioactive taxane derivatives. As in our continuing discovery for new drugs from natural products, Professor Dali Yin in Department of Chemical Synthesis of Medicinal and Professor Jungui Dai in Department of Biosynethesis of Natural Products used Sinenan A as the starting material for reorganizing the structure and got a series of new compounds to study their valuable potential for drugs, and some research results had been reported. Syl611 and NBP071 were screened out from 32 Syl compounds and 44 NBP compounds for its outstanding MDR reversing activity and wondrously low cytotoxicity.Syl611 and NBP071 could reverse the MDR of A549/Paclitaxel by the combination usage with Paclitaxel and the effects were stronger than Verapamil in the same concentration. The MDR-reversing ability was determined by Reversing Folds (RF) which was the ratio of the IC50 value in the presence of paclitaxel alone and the paclitaxel combined with Syl611 or NBP071. We also detected the reversing activities of Syl611 and NBP071 on other two drug-resistant tumor cells, the results showd that the reversing effects were different in various cells and all of them were stronger than that of verapamil: the highest RF of Syl611 on Bel7402/5-FU cells was 107.13 and of NBP071 on KB/V cells was up to 300.41, which indicated that Syl611 and NBP071 would not have the same reversal mechanisms. Low cytotoxicity would be the basic property of MDR-reversing agents because of which were combinatively used with anti-tumor agents. The foreground and development of MDR-reversing agents lied on the low cytotoxity of theirselves. Our data showed that Syl611 and NBP071 were devoid of cytotoxicity in three kinds of tumor cell lines and their related drug-resistant cell lines and one normal human cell line in vitro. The results of MTT assay demonstrated that the reduction of cell viability when Syl611 and NBP071 were used in the combination with Paclitaxel was independence on Syl611 and NBP071 themselves.Paclitaxel can stabilize microtubules and at stoichiometric concentration enhance microtubules polymerization which induce tumor cells arrested in G2/M phase and guide more cells to apoptosis. We used AO/EB double staining assay to observe the reversing effect of Syl611 and NBP071 directly. Our data about the cell apoptosis rate of treated cells showed that Syl611 and NBP071 both could enhance the apoptosis of drug-resistant cells and the effects were much stronger than that of verapamil. Meanwhile, the results showed that Syl611 and NBP071 themselves could not induce cell apoptosis.In order to find the mechanisms of reversing effect of Syl611 and NBP071, we observed the effects of Syl611 and NBP071 on the cell cycle distribution, the mRNA expression of drug efflux pump, the expression of P-gp, the Rh123 accumulation in drug-resistant cells, the Paclitaxel acculumation in drug-resistant cells and the transmemebrane ability of Paclitaxel in Caco-2 cell monolayer model. Syl611 could not alter the cell cycle distribution of drug-resistant cells but NBP071 could obviously raise the percentage of S phase and cut down that of G2/M phase in KB/V cells. Syl611 and NBP071 both could not change the mRNA expression of MDR1, MRP and BCRP when they were used alone or combined with Paclitaxel, equally, the protein expression of P-gp were observed no alteration after the treatment of Syl611 and NBP071. Little influence of Syl611 and NBP071 on the mRNA or protein expression of drug efflux pump suggested few side actions of them. In the Rh123 accumulation and Paclitaxel accumulation assay, we found that Syl611 could increase the accumulation of Rh123 and Paclitaxel in KB/V cells but NBP071 could not. The accumulation enhancement of Syl611 was stronger than that of verapamil. These results indicated that Syl611 may inhibit the drug efflux function of P-gp to reverse MDR.The Caco-2 cell monolayer model is always used in pharmacokinetics tests and defined as the standard evaluation model for transmembrane ability of drugs. As to the high expression level of P-gp in Caco-2 cells, this model would be used to test the effects of reversing agents. As contrary to our estimation, we found that Syl611 could facilitate the transmembrane of Paclitaxel either in BL to AP or AP to BL and the facilitation of Syl611 on transmembrane had dose-dependent and time-dependent features. On the other hand, the ratio of drug concentration in BL to AP and AP to BL was gradually reduced with the extension of treating time and the increase of treated Syl611. This phenomenon demonstrated that the incell accumulation of drugs increased and confirmed the results of Rh123 accumulation tests and Paclitaxel accumulation tests. Drugs crossing the intestinal membrane is a complex multi-pathway process. Passive absorption through transcellular route or paracellular route, carrier-mediated absorption, intestinal enzymes would be involved in this complex process. Taking together, Syl611 could increase the accumulation of drugs in cell by inhibiting the function of P-gp and also other process. As to the essentiality of P-gp in normal physiologic function and differing from other P-gp inhibitors, Syl611 may have low side effects.In summary, our current studies demonstrated that Syl611 and NBP071, novel semisynthetic taxane derivatives, are efficacious in reversal of MDR. The low cytotoxicity and non-interfere on the expression of P-gp and its mRNA suggest its favorable pharmacology profiles. Syl611 can inhibit the function of P-gp but also increase the accumulation of drugs in cell by other process. NBP071 would change the cell cycle distribution to raise the sensitivity of drug-resistant cells to anti-cancer agents. Our studies suggest that Syl611 and NBP071 are attractive chemotherapeutic agents for treating malignant tumors with MDR, and the researches about Syl611 and NBP071 and the derivatives of Sinenxan A in future will encourage further investigation of these kinds of compounds and finding new MDR reversal agents.