| Malignant tumors are severely influent to human life and healthy, recent studies have been paid close attention to the mechanism in tumor initiation and progression. Cisplatin (CDDP) was one of the most popular chemotherapeutics in clinics, which could effectively inhibit many tumor cell lines' proliferation and has higher efficiency. However, many studies proved that CDDP had many disadvantage of long half-life, low solubility, none target therapy, and especially severe side effects like nephrotoxicity, neurotoxicity, myelosuppression and stomach-intestine toxicity, those weakness restricted its application in the clinics.Biopolymers used as carriers in drug delivery were more and more popular in recent year, they can interact with compounds or drugs to increase its solubility, improve the pharmacokinetics, moderate the drug distribution in vivo, target delivery and effectively decrease side effects based on the enhanced permeability and retention effect specifically in tumor tissue (EPR effect), therefore, they can elevate stability and safety of drugs and have potential use in many fields. Poly (γ, glutamic acid) purified by fermentation was one of the most popular carriers in drug delivery, because of its many advantages, such as definite structure, simplified preparation, biocompatibility and biodegradation, and control release, poly (γ, glutamic acid, PGA) has become a good candidate in drug modification.In order to exploit a new CDDP analogue, we preparedγ-PGA-CDDP conjugate by conjugating CDDP toγ-PGA. Systematical studies compared with CDDP, carboplatin and oxaliplatin used in the clinics indicated thatγ-PGA-CDDP can significantly inhibit tumor proliferation in vivo, while dramatically decrease side effects. Meanwhile, we prepareγ-PGA-ASP conjugate (GAP460) to elevate drug loading efficiency. Our studies are summarized as follows:(1)γ-PGA-CDDP conjugate was prepared by conjugating CDDP toγ-PGA, and characterized by Fourier transformed infrared and nuclear magnetic resonance analysis. Systematical studies compared with CDDP, carboplatin and oxaliplatin in clinics indicated that y-PGA-CDDP could dramatically decrease cytotoxicity in Bcap-37, BEL-7404 and SH-SY5Y cell lines and increase the IC50 value; it could significantly inhibit tumor growth in BALB/cA nude mice, maintain animal survival and body weight, reduce oxidative damage in kidney and improve kidney functions. The WBC, platelet, GSH and GSH-Px was dramatically decareased, and CRE, BUN, MPO and MDA was strikingly increased in CDDP group, while these were all stable in y-PGA-CDDP group compared with PBS, which indicated that y-PGA-CDDP conjugate had low toxiticty. Our data suggested that y-PGA could be an effective carrier for drug delivery, and that y-PGA-CDDP conjugate may have potential therapeutic applications in human cancers that are sensitive to treatment with CDDP-based chemotherapy.(2) Linear mono-amino acid polymers compacted excessively and the drug conjugation efficiency was relatively low in practical applications. We found that the y-PGA-CDDP conjugate is only capable of conjugating 14.6% CDDP to y-PGA, requiring that high doses of drugs were accompanied by high doses of carrier. We therefore prepared a y-PGA-Asp conjugate (GAP460) through an amidate reaction between aspartate and y-PGA. The resulting material was stable in neutral pH and room temperature, nontoxic to animal, and contained more reactive groups, possessed a drug conjugation efficiency for CDDP of nearly 40%(three times higher than comparable material), and the soluble GAP460-CDDP conjugate (PACC) significantly inhibited tumor proliferation in nude mice while producing lower side effects. Cancer was one of the major diseases which severely influenced human health, the most difficulty in cancer protection and therapy was how to prohibit metastasis in malignant tumors. Breast cancer progression was a complicated network, thousands of genetic expression and cell signal pathways were involved. Distinctive system of breast cancer subtypes was established based on molecular analysis, which provided more background and information for breast cancer research. So far, more than fifty kinds of breast cancer mouse models have been generated by tissue or organ specific promoter, the efficiency of remedy is gradually elevated, and death becomes decrease.Most of animal models in breast cancer research were utilized by transgenic mouse model, the major strategy to induce breast tumor in vivo was expressing oncogenes, such as SV40, Ras and Neu, and gene's function was evaluated by measuring tumor phenotypes and molecular mechanism, however, these transgenic mouse model based on the homologous recombination could not authentically mimic tumor characteristic in human bodies. During extensive studies related to breast cancer mouse model, the RCAS-TVA system activated by retrovirus infection was more and more popular.Twist was one of the most important transcription factors in human genome, lots of studies in 1990s clearly indicated that Twist promoted mesoderm formation in early embryonic development. The relationship between Twist and tumor metastasis was firstly reported on 2004, suggesting Twist can activated EMT and promote tumor metastasis, meanwhile, Twist also has highly relationship with cell cycle and apoptosis, angiogenesis and antitumor drug resistance, however, most of the studies were performed in vitro. In vivo function of Twist related to tumor metastasis was pay close attention to when TwistF/F conditional knock out mice was generated on 2009.In order to outcome those disadvantages in transgenic mouse model, and try to authentically mimic tumor microenvironment in vivo, we established the novel breast caner mouse model based on the RCAS-TVA system, and focused on Twist function in tumor initiation, progression and metastasis. Our studies were outlined as follows: (1) Prepare RCAS-CRE-IRES-PyMT construct and retrovirusCre sequence, IRES sequence and PyMT sequence were amplified and cloned into RCAS-Y vector, the advantage of RCAS-CRE-IRES-PyMT construct was expressing Cre and PyMT separately in the same mRNA under the regulation of LTR promoter. The results indicated that Cre and PyMT can co-express separately in the same cell, which was essential for later study in gene function, and the titer of this retrovirus was approximately 1010/ml checked by Western Blot.(2) Establishment and characteristic of breast cancer mouse model based on the RCAS-TVA systemRosa26R-LacZR/+ MMTV-TVA+/- mice was generated by crossing MMTV-TVA mice and Rosa26R-LacZ reporter mice; breast tumor was induced by nipple injection; specific gene expression, tumor initiation and progression, tumor characteristic in vivo were systematically studied. The results suggested that tumors were specifically induced in luminal epithelial cells of mammary gland, solid tumors were detected at 7-9 weeks after virus infection, and invasive tumors were detected later; the characteristic of tumor cells in this mouse model was significantly different from the transgenic mouse model, which was more authentic to human tumors.(3) Twist can promote breast cancer metastasis in vivoTwistF/F-MMTV-TVA+/- mice was generated by crossing MMTV-TVA mice and TwistF/F mice; breast tumor was induced by nipple injection; tumor initiation, growth and metastasis were systematically measured. The results demonstrated that Twist had no effect in tumor initiation and progression, but significantly promoted tumor lung metastasis by regulating EMT process.We successfully established the novel breast cancer mouse model based on RCAS-TVA system, and in vivo studies suggested that Twist could promote tumor lung metastasis by regulating EMT, our research may give new insight to breast cancer mouse model and cancer gene therapy.
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