The Effect And Molecular Mechanism Of Curcumin Combined With FAPα Vaccine On Melanoma

Tumor stromal cells constitute the majority of microenvironment for the growth, invasion and metastasis of tumor cells. It has become an important and effective target for clinical cancer treatment. The inhibition of fibroblast activation protein alpha(FAPα), which express in tumor stromal cells specifically, can promote the release of anti-tumor factors IFN-γ and TNF-α to kill tumor cells. However, the clinical outcomes of most immunotherapeutic strategies including the targets of FAPα have unsatisfactory results. One of the main reasons is IFN-γ produced by immune effector cells can induce the expression of IDO which leaded to tumor immune tolerance. Furthermore, immune effector molecular TNF-α can induce epithelial-mesenchymal transitions(EMT) which is believed to trigger tumor cells invasion and metastasis. This study demonstrated that curcumin has dual inhibitory effects on IDO expression induced by IFN-γ and TNF-α-induced EMT in B16 cells. Therefore, the first objective of the present proposal is to clarify these functions induced by curcumin. Then the curcumin and FAPα vaccine will be combined to investigate the effects of melanoma prevention and treatment, which proposed to inhibit tumor growth at immune activation and immune effector phase. This new approach will eliminate the adverse effects of immune tolerance and tumor cell metastasis induced by the immune effector molecules IFN-γ and TNF-α, ultimately provides a new and effective method for melanoma prevention and treatment.The main aims of this study:Firstly, we investigate the effect of curcumin on IDO expression induced by IFN-γ and TNF-α-induced tumor EMT in vitro, then, curcumin combined FAPα vaccine were used for the treatment of melanoma in vivo, ultimately provides a new and effective method for melanoma prevention and therapy.The main methods of this study:The main methods used in this study include a variety of molecular biology and immunology methods such as cell culture, ELISA, Western blot, immunohistochemistry, prokaryotic expression and purification of protein and animal experiments.The main contents of this study:1) The expression of prokaryotic vector pET28a-FAPαc containing the core sequence fragments of FAPα was induced by IPTG, then the polypeptide of FAPαc were purified to take as the tumor vaccine for subsequent animal immunization.2) To demonstrate the effection of curcumin on IDO expression induced by IFN-γ and TNF-α-induced EMT in melanoma in vitro.3) Evaluated he efficacy and related molecular mechanisms of curcumin combined FAPa vaccine in melanoma in vivo.The results of this study:1) The expression of FAPα core polypeptides in BL21 engineering bacteria is in the form of inclusion body, a number of highly purified FAPα core polypeptides were obtained after the processes of washing, purification and renaturation, the purity degree is >99%.2) Western blot results demonstrated that the expression of IDO induced by IFN-γ could be significantly decreased by curcumin, furthermore, curcumin can downregulate the expression of vimentin induced by TNFα.3) No statistically significant between the experimental groups of mice in body weight(p >0.05), high-valence FAPα antibodies were produced after immunization with FAPα vaccine compared with other groups(P<0.01), curcumin combined with FAPα vaccine could significantly prolong the survival time and significantly inhibited tumor growth in mice implanted with B16 cells(P <0.01).The conclusion of this study:1) FAPα inclusion bodies were successfully obtained and their purity degree reach to the requirement of vaccine for subsequent experiments.2) Curcumin not only can inhibit expression of IDO induced by IFN-γ, but also can inhibit EMT induced by TNF-α in B16 cells.3) Curcumin combined FAPα vaccine could inhibit tumor growth and significantly prolonged the survival of mice implanted with B16 cells, which provides a new and effective method of prevention and treatment for melanoma.