Study Of Purified Sulfated Polysaccharide-protein Complex On Tumor Inhibition

Objective: To investigate the inhibitory effect of PSPPC on tumor growth in H22tumor-bearing mice in vivo and on human hepatoma SMMC-7721 cells growth in vitro, and explore its possible mechanisms of antitumor activity.Materials and methods: 1.SPPC was purificated by ethanol fractionation, taking off the dissociative protein, and decoloring with charcoal. 2. Taking the human hepatoma SMMC-7721 cells as our experimental object, the SMMC-7721 cell's proliferation inhibitiontreated with PSPPC was observed by the way of MTT-colorimetric assay and Flow cytometric analysis. Hepatocellular carcinoma cells H22 were subcutaneously injected into mice and PSPPC was administered to the H22 tumor-bearing mice (i.p.). The tumor growth were measured, tumor growth inhibition rate was calculated, and the alterations of histomorphologism of H22 tumor were taken and examined by H&E staining to assess the inhibitory effects of PSPPC on tumor growth. 3. In order to explore the possible mechanisms of antitumor activity of PSPPC, further bioactivities were detected, which included: In vitro, free radical scavenging activities of PSPPC; In vivo, the functions of modulating the activities of antioxidase and inhibiting the production of MDA of PSPPC were detected. 4. To study the apoptosis effects of PSPPC on human hepatoma SMMC-7721 cells.5.The effects of PSPPC onthe H22 tumor-bearing mice immunofunction were also observed in vivo. Based on the same H22 xenograft tumor models, the effects of PSPPC on the immune function were surveyed in mice from the following aspects: the thymus glands and spleens in the H22 tumor-bearing micewere excised and weighted and the thymus gland index and spleen index were caculated, respectively, which help to indicate whether PSPPC can affect the immune organs'function or not; the effects of PSPPC on the proliferation of peripheral blood lymphocyte (PBLC)separated from spleen of the H22 tumor-bearing mice in vitro evaluated using MTT assay; the alternations of concentration of murine serum IL-2 and IL-12 were examined by ELISA.Results: 1. After purification, the total available components of PSPPC were obviously increased, the contents of sulfate, total polysaccharide and protein in PSPPC were 26.47%, 37.63% and 2.84% respectively. 2. PSPPC exhibited a suppressive effect on SMMC-7721 cells growth in concentration and time-dependent manners. PSPPC at concentrations (0.4,2.0 and 10.0 mg·ml-1) dose-dependently increased the cell number at G1 phase and decreased the number at S phase and G/M phase in SMMC-7721 cells,that means, PSPPC could disrupt celldivision by blocking cell cycle at the checkpoint G1à2/M, ( which resulting in the inhibitionof cell proliferation. Marked inhibitory effect of PSPPC on the transplanted hepatocellularcarcinoma H22 was observed in the tumor-bearing mice. The inhibitory rates were 33.38% and30.48％in the groups treated with high and low dosage of PSPPC, respectively (P <0.01 vs.control group). Histopathological examination revealed widespread necrosis in the tumors.Numerous apoptotic bodies were observed in the tumors under the electron microscope.3.Theresults from the free radical-scavenging systems (Superoxide Anion and Hydroxyl Radical)revealed that PSPPC had significant free radical-scavenging activity in vitro. PSPPC couldmodulate the activities of antioxidase and inhibite the production of MDA, that means, PSPPCshowed significant antioxidant activity. 4. PSPPC can induce the apoptosis of SMMC-7721cells: PSPPC dose-dependently decreased the expression of the relative apoptotic proteins(procaspase-3); The treatment of PSPPC resulted in the induction of DNA fragmentation;Annexin assay discovered , after being treated with 0.4,2.0 and 10.0 mg·ml-1 PSPPC for 48h,the cells'early stage apoptosis rate were 11.13％,26.05％and 29.56％, respectively, higherthan that of control(P <0.01).5. Moreover, PSPPC could improve the immune function intumor-bearing mice. The NK activity and proliferation ability of T lymphocytes were increasedin tumor-bearing mice.Conclusion: The components of sulfated polysaccharide-protein complex (SPPC) arecomplicated. It is undoubtedly a promising and singnificant work to purificate the SPPC andexplore the bioactivities of PSPPC. After purification, the total available components of thesamples were obviously increased. Collectively, the bioactivities of PSPPC were also enhanced.Our research had proved that PSPPC could significantly inhibit the proliferation of the humanhepatoma SMMC-7721 cells. Hepatocellular carcinoma cells H22 were subcutaneously injectedinto mice and PSPPC was administered to the H22 tumor-bearing mice (i.p.). The experiment inH22 tumor-bearing mice documented PSPPC had obvious effects on tumor inhibition. PSPPCshowed significant free radical-scavenging activity in vitro, and could modulate the activitiesof antioxidase and inhibite the production of MDA In vivo. PSPPC could induce apoptosis ofSMMC-7721 cells with a dose-dependent manner. Moreover, PSPPC-induced apoptosis waspartially attributed to the activation of caspase-3. Further investigation proves that PSPPCcould improve the immune function in tumor-bearing mice. Based on the conclusionsmentioned above, some possible mechanisms of antitumor activity of PSPPC can be proposedas: Inducing the apoptosis of tumor cells; scavenging free radical; modulating the activities ofantioxidases; improving the immune function.