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  • br In vivo an http www

    2020-08-12


    3.6. In vivo antitumor efficacy
    To evaluate the antitumor efficacy of the conjugates in gastric cancer cell line, BALB/c nude mice were xenografted with SGC-7901 tumor cells. Fig. 4A showed the body weight variations during the treatments with normal saline, DDP, free NCTD and different concentrations of CNC. Fig. 4B depicted the tumors excised from mice after treatments with various formulations. The body weights of the DDP group exhib-ited notable weight losses due to the severe side effects. Compared with control group, the body weights of free NCTD group decreased in the initial procedure, indicating the toxicity of the antitumor drug. Moreover, the body weights of mice in conjugates treated group did not clearly differ from that of the control group, suggesting the reduced toxicity and excellent biocompatibility.
    In terms of tumor inhibitory rates, DDP possessed the best antitumor efficiency as a clinical chemotherapeutic drug. Compared with control group, the inhibitory rates of DDP, free NCTD, high and low dose of CNC were 76.32%, 40.46%, 59.57% and 50.64%, respectively (Table. 2). El-evated antitumor capacity was observed with high dose of CNC treat-ment, which was higher than that of free NCTD at equal dose. Histological analysis by H&E staining showed phenomenon of cell apo-ptosis or tumor necrosis in the tumors of mice administered with differ-ent formulations. As exhibited in Fig. 5A, large, densely arranged hyperchromatic nuclei and obvious nucleolus cleavage could be ob-served in tumor tissues of control group. In addition, H&E sections disclosed that substantial amount of apoptotic tumor GSK-J4 and evident necrosis areas occurred in tumor tissues of conjugates groups, along with apparent vacuoles degeneration of tumor cells, which revealed the advantage of conjugates in improving antitumor efficacy. As a mac-romolecular derivative, CNC could accumulate at the tumor sites through enhanced permeability and retention (EPR) effect and NCTD could continuously release from the conjugates under physiological conditions in the tumor microenvironment [47]. Therefore, CNC could be considered as a prodrug. According to the above evaluation, CNC showed enhanced antitumor activity and superior security compared with free NCTD, thus it might be a promising polymer-drug conjugates for cancer therapy.
    3.7. Immunohistochemical analysis
    To further investigate the antitumor efficacy and mechanisms medi-ated by CNC, the cell apoptosis and angiogenesis in tumor tissues were evaluated by immunochemistry staining of CD34, Bcl-2, Bax and Caspase-3, respectively. Angiogenesis is the development of new capil-lary blood vessels derived from preexisting vessels and is essential for the growth and progression of neoplasms [48]. CD34 is an antigen pres-ent in hematopoietic progenitor cells and endothelial cells, which has been studied as a marker for vascular tumors [49]. As shown in Fig. 5B, CD34 staining for microvessels revealed significant differences in microvessel number and morphology among different formulations. The staining of CD34 positive microvessels were relatively abundant in tumor tissues of control group. However, treatments with CNC re-sulted in small and irregularly shaped microvessels in the tumor tissues,
    Fig. 5. Histopathological and immunohistochemical detections of tumor tissues (original magnification, 100×). (A) H&E stained sections of tumor tissues. (B) Photographs of protein expressions in tumor tissues. (C) Microvessles density of tumor tissues in different groups. (D) Relative protein expressions in different groups. (a) mice treated with normal saline; (b) mice treated with DDP at the dose of 5 mg/kg; (c) mice treated with NCTD at the dose of 6.524 mg/kg; (d) mice treated with CNC at the dose of 32.62 mg/kg; (e) mice treated with CNC at the dose of 16.31 mg/kg. Data represents mean ± SD (n = 10), * P b 0.05, ** P b 0.01 significant difference compared with control group; #P b 0.05, ##P b 0.01 significant difference compared with DDP group; △P b 0.05, △△P b 0.01 significant difference compared with NCTD group.
    indicating that CNC remarkably suppressed the angiogenesis in tumor tissues. As shown in Fig. 5C, the density of positive microvessels in DDP, NCTD, high and low dose of CNC group were decreased by 29.50%, 13.27%, 26.39 and 20.51%, respectively, compared with that of control group (P b 0.05).
    Tumor cell apoptosis has been regulated by numerous apoptosis re-lated proteins [50]. Bcl-2 proteins family plays an important role in the regulation of the mitochondria-mediated pathway of apoptosis, which consists of a network of pro-apoptotic and anti-apoptotic members, such as Bax and Bcl-2 [51]. The samples in high dose of CNC group ex-hibited a more pronounced reduction in the expression of Bcl-2 and an evident increase in Bax compared with other groups (Fig. 5B and D), demonstrating the apparent tumor apoptosis induced by CNC. Caspase-3 has been considered the central executioner protease due to its high catalytic turnover and high propensity to cleave substrates [52]. The intrinsic and extrinsic cell death pathways converge at Caspase-3, which results in dismantling of the cell structure through cleavage of specific substrates together with other effector Caspases [53]. Tumor sections stained for Caspase-3 showed that CNC treatments caused higher levels of Caspase-3 than the control and free NCTD group (Fig. 5B and D). Consistent with the in vivo antitumor efficacy analysis, the immunohistochemical analysis in high dose of CNC group showed reduced number of microvessel density (MVD) and enhanced apoptosis expressions, suggesting the elevated antitumor efficacy compared with free NCTD at equivalent dose.