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  • br Determination of TNF and VEGF br Tumor necrosis factor


    3.8. Determination of TNF-α and VEGF
    Tumor necrosis factor-alpha (TNF-α) is a multifunctional proinflam-matory cytokine that possesses a wide variety of biological activities in-cluding multiple chronic inflammatory and immunomodulatory properties [54,55]. Besides, it has been described as a powerful antican-cer effector cytokine produced by immune GSK-J4 such as macrophages and lymphocytes [56]. Angiogenesis has been demonstrated to be a basic prerequisite for sustainable growth and proliferation of tumor, which is stimulated and regulated by a series of cytokines [57]. Vascular endothelial growth factor (VEGF) is the most potent and extensively studied regulator of both physiological and pathological angiogenesis [58]. Based on the above considerations, we determined the levels of TNF-α and VEGF in serum by ELISA method. Concentrations of serum TNF-α and VEGF levels were shown in Fig. 6. CNC dramatically in-creased the levels of TNF-α and showed significant differences com-pared with control group. Besides, CNC administration induced notable and simultaneous reduction in levels of VEGF in a dose-dependent manner (P b 0.05). The above results indicated that CNC displayed the antitumor efficacy by regulating the expressions of TNF-
    α and VEGF, thus achieving an enhanced immune response and anti-angiogenesis effects.
    3.9. Western blot analysis
    The effects of CNC on the metastasis and apoptosis in SGC-7901 cells were detected by western blot assay. Matrix metalloproteinases (MMPs) consist of a multigene family of zinc-dependent extracellular matrix (ECM) and play a pivotal role in controlling the degradation of extracellular matrix and promoting the invasion and metastasis of tumor cells [59]. As expressed in Fig. 7A, the expressions of MMP-2
    Fig. 7. Western blot analysis of MMP-2, MMP-9, Bcl-2 and Bax protein expressed in SGC-7901 cells after different treatments. (A) Representative protein bands of MMP-2, MMP-9, Bcl-2 and Bax. (B) The relative protein band intensity was quantified and compared using Image J software. Data represents mean ± SD (n = 6), *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 NCTD group.
    and MMP-9 were dramatically decreased after treatments with CNC, in-dicating that CNC could suppress cell metastasis by inhibiting activities of MMPs. The relatively quantitative levels of MMP-2 and MMP-9 were shown in Fig. 7B. The expressions of apoptosis related proteins were subsequently detected, such as Bcl-2 and Bax. One of the principal roles of Bcl-2 family is the regulation of the initiation of apoptosis by the mitochondrial pathway [60]. As shown in Fig. 7A and B, CNC could suppress the expression of Bcl-2 and increase the expression of Bax in tumor cells, and the effects were associated with the concentration. The results were consistent with those in immunohistochemical analy-sis. Results above indicated that CNC could inhibit metastasis and induce apoptosis of SGC-7901 cells through regulating the expressions of MMP-2, MMP-9, Bcl-2 and Bax, further illustrating the underlying mechanisms of the higher antitumor effects performed by CNC.
    4. Conclusion
    In this study, norcantharidin-conjugated carboxymethyl chitosan (CNC) was designed for GSK-J4 the delivery of NCTD, aiming to enhance its an-titumor efficiency in clinical application. Our results suggested that the conjugates could significantly inhibit cell proliferation and induce the apoptosis of SGC-7901 cells. Besides, the metastasis and tube formation of HUVECs were obviously inhibited after incubation with CNC. CNC possessed higher antitumor capacity than that of free NCTD in BALB/c nude tumor-bearing mice with a tumor inhibition rate of 59.57%. Inves-tigation for the underlying mechanisms revealed that the enhanced an-titumor effects might be mediated by the increasing expression of TNF-
    α and the reduced expressions of CD34, VEGF, MMP-2 and MMP-9. Fur-thermore, western blot assay and immunohistochemistry analysis dem-onstrated that CNC induced cell apoptosis through down-regulating the expression of Bcl-2 and increasing the expressions of Bax and Caspase-3. Overall, our results suggested the potential applicability of CNC as novel polymer therapeutics for gastrointestinal cancer treatment.
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