Study On Anti-tumor Effect Of Yulangsan Polysaccharides And Its Action Mechanism

Yulangsan polysaccharides(YLSPS)is a kind of polysaccharides isolated from the foot of Millettia pulchra Kurz var-laxior(Dunn)Z.Wei,which is water-soluble.The previous study showed that YLSPS could scavenge and inhibit O₂^·-markedly.YLSPS also showed a direct protective action on the damage of the primary cultured rat hepatocytes induced by H₂O₂ or CCl₄.There is no information about the effect of YLSPS on tumor.The present study was designed to investigate the anti-tumor effect of YLSPS both in vivo and in vitro and to explore its mechanisms of action at systemic,cellular and molecular levels.Objective:1.To evaluate the anti-tumor effect and molecular mechanism of YLSPS on human tumor cell lines in vitro.2.To study the anti-tumor effect of YLSPS on sarcoma 180 in vivo and its mechanism.3.To investigate the immunological mechanism of the anti-tumor effect of YLSPS at cellular and molecular levels. Methods:1.The effects of YLSPS on four tumor cell lines including human hepatoma Bel-7404 cell line,HepG-2 cell line,gastric cancer cell line SGC-7901, and ovarian cancer cell line A2780 in vitro were assayed by MTT method.2.The effect of serum containing YLSPS on cell cycle and apoptosis of Bel-7404 were investigated by the methods of Annexin V-FITC/PI double staining FCM assay and Hoechst fluorescent staining.3.To evaluate the anti-tumor effect of YLSPS in vivo,the rates of tumor inhibition,thymus index,spleen index and peripheral white blood cell count were determined.The expression of Bcl-2,Bax and p53 in the S180 cancer cells was evaluated by immunohistochemical staining,and the ultrastructural changes of S180 cells after YLSPS treatment were examined with transmission electron microscope(TEM).4.The effect of YLSPS on spleen lymphocytes,peritoneal macrophages and cytokines were investigated by the methods of MTT,enzyme linked immunosorbant assay(ELISA)and semi-quantitative reverse transcriptase polymerise chain reaction(RT-PCR)to explore its immunopotentiating mechanisms of action at cellular levels.Results:1.YLSPS had slightly inhibitory effect with less than 30%inhibitory ratio on Bel-7404,HepG-2,SGC-7901 and A2780 cell lines at the concentration of 5-320 mg/L,While serum containing YLSPS had remarkable dose-dependent inhibitory effect on HepG-2,SGC-7901 and A2780 cell lines and higher inhibitory ratio(15.8%-43.3%%)was observed on Bel-7404.2.Serum containing YLSPS(0.75 g/kg,1.5 g/kg and 3 g/kg)could arrest cell cycle at G₂/M phase.The results also showed that the serum containing YLSPS could induce apoptosis of Bel-7404 cells at the apoptosis ratio of 7.69±0.5%,9.94±0.62%,and 10.76±0.74%,while the ratio in control group was 4.60±0.66%.This suggested that inhibitory effect of serum containing YLSPS on the growth of tumor cells was probably related to its arresting cell cycle at G₂/M phase and inducing apoptosis of tumor cells.3.YLSPS(0.15,0.3 and 0.6 g/kg,i.g.,d1to d11)could inhibit the growth of S180 tumor cells at the inhibitory ratio of 39.7%,41.3%and 52.6%,respectively. It could enhance the thymus and spleen indexes in S180 tumor bearing mice as well.In combination with YLSPS(0.15,0.3 and 0.6 g/kg,ig,d1 to d11)and CTX(10 mg/kg,ip,d1,4,7,10)treated S180 tumor in mice,q values were all from 0.85 to 1.25.Furthermore YLSPS at three doses could not only enhance the thymus.and spleen indexes,but also amelionate the decreased blood leucocyte count induced by CTX(20 mg/kg,ip,d1,4,7,10)in S180 tumor-bearing mice, This suggested that YLSPS also had synergetic effect and attenuated toxic effect in combination with CTX. The results of immunohistochemical staining indicated that YLSPS could decrease the Bcl-2 expression,but the expression of Bax was increased. Condensation of chromatin at margins of nuclei,disintegration of nucleolus, vacuoles in cytoplasm were observed in 0.6 g/kg group,and 5-FU also had the same apoptotic appearance under TEM.These results suggested that S180 cell apoptosis was induced by YLSPS in vivo.4.The experiments in vitro showed that YLSPS(50,100 and 200 mg/L) could not only enhance the proliferation response,IL-2 and IFN-γproduction of spleen lymphocytes induced by ConA(5 mg/L),but also increase TNF-a and IL-6 production of peritoneal macrophages induced by LPS(10 mg/L)from normal BALB/c mice.However,YLSPS had less influence on proliferation of spleen lymphocytes induced by LPS(10 mg/L).The results suggested that YLSPS could enhance the immune system functions,and its biological activity might mainly result from selectively activating T cells and macrophages and stimulating secretion of some cytokines,but not involved B cells.5.YLSPS(50,100 and 200 mg/L)increased the expression of IFN-γ,IL-2 mRNA in mouse spleen lymphocytes and TNF-a,IL-6 mRNA in mice peritoneal macrophages in vitro.Conclusion:YLSPS has potent inhibitory effect on the growth of implanted S180 in mice and has synergetic effect and attenuated toxic effect in combination with CTX as well.Studies on anti-tumor effect of YLSPS both in vivo and in vitro show that its mechanisms of anti-tumor activity are probably related to these as follows:(1)YLSPS can enhance the immune system functions and its biological activity may mainly result from selectively activating T cells and macrophages and stimulating secretion of some cytokines,but not involved B cells.(2) YLSPS can induce cancer cell apoptosis by down-regulated Bcl-2 and up-regulated Bax at protein level in vivo.(3)YLSPS can inhibite the growth of tumor cells directly.(4)YLSPS can arrest the cell cycle at G₀/G₁ phase,and induce the apoptosis of tumor cells.