The Immunoregulation And Anti-tumor Effect Of Derivatives Of Chitin

Renal cell carcinoma(RCC) is a common malignant tumor which is originatedin urinary tubular epithelial system in renal parenchyma layer. Both radiotherapyand chemotherapy have no obvious treatment effect on the renal cell carcinoma. Thebiological treatment technology becomes one of the most popular cancer treatmentmethods. The principle of biological therapy is by improving the immune ability torealize the “auto anti-tumor”. As the main drugs of biological treatment, chitin andchitosan get more and more attention.Chitosan is a polysaccharide and is primarily obtained from chitin bydeacetylation. Carboxymethyl chitin (CMCT) and carboxymethyl chitosan (CMCTS)could be obtained from chitin and chitosan by carboxymethylation. N-acetylglucosamine (NAG) and chitosan oligosaccharide (COS) could be obtained fromchitin and chitosan by hydrolyzation. A number of water-soluble derivatives ofchitin and chitosan is more useful in biomedical industries for their goodbiocompatibility and biodegradation. A number of water-soluble derivatives of chitinand chitosan are getting more and more attention in biomedical industries for theirgood biocompatibility, no toxicity and no immunogenicity of biodegraded product.Some researchers reported that chitosan could stimulate the growth of nerve cell, butthere have been few reports about the effect of chitin and chitosan on theproliferation and differentiation of peripheral blood mononuclear cells (PBMCs). Inantineoplastic immune response, the cellular immune plays a main role. Mostmononuclear cells (PBMCs) participate in the cellular immune cells, including: Tlymphocytes,NK cells,mononuclear macrophages, etc. If the derivatives of chitinand chitosan could promote the proliferation and differentiation of mononuclearcells, including the numbers of the immune cells, they could also bring significantimprovement of the body immune adjustment and antitumor function.In this study, the effect of derivatives of chitin (carboxymethyl chitosan, carboxymethyl chitin, chitooligosaccharides, N-acetyl-D-glucosamine) on theproliferation, differentiation, apoptosis and cytokine secretion of peripheral bloodmononuclear cells (PBMCs) before and after medication in vitro was evaluated.Then the optimum dose of the derivatives of chitin on the inhibition of renalcarcinoma OS-RC-2in vitro was evaluated. At the same time, the renal carcinomaOS-RC-2was transplanted into animals and the optimum dose of the derivatives ofchitin on the inhibition of renal carcinoma OS-RC-2in vivo was evaluated.provide theoretical foundation for their clinical application.To discuss theimmunoregulation mechanism of the derivatives of chitin.MTT, AO-EB staining,flow cytometry, Elisa, RT-PCR, immunohistochemistry and transmission electronmicroscope were done respectively.The results of MTT and AO-EB staining about the PBMCs cultured with fourdifferent kinds of carbohydrate in vitro showed that, CMCTS and CMCT withmedium and low dose, COS and NAG with medium and high dose promote theproliferation and inhibit the apoptosis of PBMCs obviously(P<0.05). The result offlow cytometry showed that, CMCT could promote the differentiation of NK cellsby promoting the expression of CD3-CD56subgroup and the optimum dose ofCMCT is300μg/ml; CMCTS could promote the differentiation of both NK cells andT cells by promoting the expression of CD3-CD56and CD3-CD8subgroup and theoptimum dose of CMCTS is300μg/ml. Both COS and NAG could promote thedifferentiation of T cells by promoting the expression of CD3-CD4subgroup and theoptimum dose of COS and NAG are both300μg/ml. The result of Elisa showed that,CMCTS and CMCT with medium and low dose could promote the secretion ofIFN-γ and IL-2obviously(P<0.05), but100μg/ml CMCTS could only promote thesecretion of IFN-γ(P<0.01); COS and NAG with dose of100μg/ml,300μg/ml couldpromote the secretion of IFN-γ and IL-2obviously(P<0.01), and500μg/ml NAGalso had obvious effect on the secretion of IL-2.Renal carcinoma OS-RC-2cells was cultured in vitro and the influence of thederivatives of chitin and chitosan on the OS-RC-2was observed. The result of MTT, AO-EB staining showed that, CMCTS, CMCT with medium and low dose and COS,NAG with medium and high dose could inhibit the proliferation and promote theapoptosis of renal carcinoma OS-RC-2cells obviously(P<0.05). The result of flowcytometry showed that, CMCTS, CMCT with medium and low dose and COS, NAGwith medium and high dose could make more tumor cells be detained in G0/G1phase and less tumor cells be detained in S phase and G2/M phase to prevent theDNA composition and the apoptosis of tumor cells.Results of experiments in vivo showed that, CMCT、CMCTS with medium andhigh dose and COS, NAG with high dose could both inhibit the growth of tumor andprotect immune organs. The optimum dose of CMCTS is50mg/kg, and the inhibitoryrate of it is48.03%±1.05%；the optimum dose of CMCTS is100mg/kg, and theinhibitory rate of it is50.87%±1.03%；the optimum dose of COS is100mg/kg, and theinhibitory rate of it is53.54%%±1.11%；the optimum dose of NAG is500mg/kg, andthe inhibitory rate of it is58.66%±1.43%(P<0.01). The result of RT-PCR andimmunohistochemistry showed that,50mg/kgCMCTS and100mg/kg CMCT,300mg/kg COS and500mg/kg NAG could inhibit the expression of VEGF mRNAobviously (P<0.01). The result of transmission electron microscope showed that,CMCT, CMCTS with medium and high dose and COS, NAG with medium and highdose could promote the ultrastructural change of renal carcinoma OS-RC-2cells, andthe pyconsis of chromatin in tumor cells cultured with CMCT and NAG could beobserved. The result of NK activity and detection of the IFN-γand IL-2in the serumshowed that, CMCT, CMCTS and COS with medium and high dose and NAG withmedium and high dose could promote both NK activity and the secretion of IFN-γ andIL-2. The derivatives of chitin could promote the cell immunity and antibodyimmunity.In the process of immunoregulation, CMCT with appropriate dose could promotethe immunity by promoting the proliferation of NK cells and the secretion of IFN-γand IL-2; CMCTS with appropriate dose could promote the immunity by promotingthe proliferation of NK cells and T cells and the secretion of IL-2; COS and NAG with appropriate dose could promote the immunity by promoting the pro-liferation ofNK cells and Th cells and the secretion of IFN-γ and IL-2to promote the immunity ofthe body. There are data demonstrating that the proliferation and differentiation of NKcells and T cells could stimulate the secretion of IFN-γ and IL-2, which couldstimulate the proliferation and differentiation of NK cells and T cells in return. So thetwo aspects interact and both could promote the immunity of the body. This four kindsof carbohydrate could inhibit the angiogenesis of the tumor by inhibiting theexpression of VEGF mRNA and its proteins. There has reports showing that VEGFcould upregulate the expression of Bcl-2mRNA and Bcl-2protein to inhibit theapoptosis of tumor cells. So the expression of VEGF inhibited could promote theapoptosis of tumor cells.In conclusion, these four kinds of carbohydrate all had good immunoregulateeffect and could inhibit the proliferation and differentiation of RCCs, and couldpromote the apoptosis of tumor cells. CMCT and CMCTS with medium and lowdose, COS and NAG with medium and high dose had obvious effect. To sum up, theimmunogregulation effect of NAG is best, and COS takes the second place, andCMCTS is superior to CMCT. The antitumor effect of NAG is still the best, andCOS takes the second place, and CMCT is superior to CMCTS. The optimumconcentration of these four kinds of carbohydrate is different, and it is probablyrelated with molecular weight and other elements.