Studies On Antitumor Effect And Molecualr Mechanism Of Ganoderma Atrum Polysaccharide

Ganoderma atrum polysaccharide (PSG-1) was extract from the mycelia ofGanoderma atrum. Sugar analysis revealed that PSG-1was composed of glucose(Glc), mannose (Man), galactose (Gal) and galacturonic acid (GalA) in molar ratioof4.91:1:1.28:0.71. Numerous studies demonstrated that PSG-1has antioxidant,immunomodulatory, myocardial protection, antidiabetic and anti-aging properties. Alot of researchers have studies the antitumor activity of PSG-1and made remarkableprogress, but the exact mechanism is still unclear. It is, therefore, necessary for themechanism to be studied further.This study was to explore the antitumor activity and mechanism through in vivoand in vitro experiments. According to the modern molecular theory and technology,Flow Cytometry, Western Blot, Enzyme Linked Immunosorbent Assay and ReverseTranscription-Polymerase Chain Reaction were used. The impact of PSG-1on thetumor growth and signaling pathways were investigated from the perspectives ofintegral level, cellular level and molecular gene level, respectively. Specificresearches are as follows:Firstly, PSG-1resistant the S180or CT26tumor cells had been establishedsuccessfully in vitro. MTT method was used to observe the effects of PSG-1on thetumor cells proliferation. The results showed that PSG-1has little effect on theproliferation of tumor cells. It is suggested that PSG-1has no cytotoxicity on thetumor cells directly. However, PSG-1-primed macrophages exhibited a highertumoricidal activity than untreated macrophages in vitro. This finding indicted thatPSG-1could stimulate the peritoneal macrophage and increase the phagocytosisfunction.In vivo, S180or CT26tumor cells were implanted subcutaneously into righthind groin of the mice and the tumor-bearing mice model was established. Aftertumors developed to approximately80-90mm3, the mice were randomized into fivegroups and administrated with PSG-1or5-fluorouracil (5-Fu). On Day15, the micewere sacrificed. Tumors, thymus and spleens were extirpated. The blood samples of mice in each group were centrifuged and the peritoneal macrophages andsplenocytes were collected for the corresponding detections. The results showedPSG-1had significant inhibitory effects on the growth of tumor in mice. The thymusand spleen indexes were significantly increased compared with control group. Thecytokines in the serum and peritoneal macrophages were increased dose-dependently,as well as NO production. Phagocytosis ability of macrophage from the mice treatedwith PSG-1increased significantly. In addition, there was a marked increase in theCon A-or LPS-induced proliferation of splenic cells in PSG-1treated groups. In vivoassay, we believed that PSG-1has the potent antitumor activity, which could notonly inhibit the tumor growth markedly, but also enhance the immunity of the host.Different concentrations of PSG-1could increase the production of TNF-α fromtumor-bearing mouse peritoneal macrophages. Anti-TLR4antibody, specific p38MAPK blocker SB203580or specific NF-κB blocker PDTC was added into thecultures to investigate the signaling pathway of PSG-1. The results showed thataddition of anti-TLR4antibody markedly, although not complete, decreased theproduction of TNF-α stimulated by PSG-1. However, specific p38MAPK inhibitorSB203580and NF-κB blocker PDTC could absolutely abrogate the stimulatoryeffect of PSG-1on the TNF-α production by macrophages from tumor-bearing mice.We also explored Western-blotting to check the expressions of TLR4, NF-κB p65,IκB and pp38related proteins. The results showed PSG-1caused a relative increasein TLR4expression. The levels of p65in nuclear fractions were also significantlyenhanced. But the cytoplasmic protein level of IκBα was decreased. So the IκBαdegradation induced NF-κB activation. Moreover, PSG-1induced the activation ofp38MAPK in a dose-dependent manner. It was suggest that PSG-1treatment mayactivate the phosphorylation of p38in macrophages. In conclusion, these resultssuggest that PSG-1could enhance the immunity, and the antitumor effect of PSG-1may be related to their potentiation of the production of TNF-α in tumor-bearingmice. The signaling mechanism by which PSG-1promotes TNF-α production maybe as follows: PSG-1acts on the TLR4receptors on macrophages, signal throughp38MAPK pathway, and then activate NF-κB. The activated NF-κB in turn initiatesthe release of TNF-α. Flow cytometry methods were used to observe the apoptosis of tumor cells. Theresults demonstrated that PSG-1induced the apoptosis of tumor cells oftumor-bearing mice. Furthermore, PSG-1significantly increased the levels ofintracellular cAMP, PKA and AC in S180cells. In contrast, cyclic GMP (cGMP),DG level and PKC activity were decreased. Similarly, Western blot also gained thesame result. The expression of PKA protein was upregulated, while PKC proteinexpression in PSG-1-treated group was lowered. Hence, we speculated that theantitumor mechanism of PSG-1may be related to the induction of tumor cellapoptosis through cAMP-PKA signaling pathway and down-regulation of DG-PKCsignal pathway.To further investigate the other signaling pathway of PSG-1, the mitochondrialmembrane potential (m) and intracellular ROS production were analyzed by flowcytometry. Experimental results suggested that PSG-1caused a very significantdepletion of mand elevated the level of ROS production. The expressions ofcytochrome c, p53, Bcl-2and Bax were detected by Western blot. The resultsrevealed that Bcl-2expression was down-regulated while Bax and p53expressionlevels were strikingly increased. In addition, level of cytochrome c protein in thecytosolic fraction increased in a dose-dependent manner. In contrast, protein levelcytochrome c decreased in the mitochondrial fraction. These results indicated thatPSG-1led to the release of cytochrome c from the mitochondria to the cytosol.Meanwhile, both caspase-3and caspase-9activities were elevated by administrationof PSG-1in the tumor-bearing mice. These results suggested that PSG-1exhibitsprominent antitumor activity in vivo, at least in part by inducing apoptosis throughthe mitochondrial pathway.In conclusion, this study confirms that PSG-1could suppress the growth oftumor. Multiple pathways are involved in the antitumor activity. One of its functionmechanisms is that PSG-1stimulates the immunocytes and immune organs, and thenactivates the whole body immune system through TLR4-mediated NF-κB andMAPK signaling pathways. Besides, PSG-1also could induce the tumor cellsapoptosis via mitochondria-mediated intrinsic apoptotic pathway or Gprotein-mediated signaling pathways.