Study On The Anti-tumour Effects And Mechanisms Of Effective Compounds From Ganoderma Lucidum

Ganoderma lucidum(GL),called "Lingzhi" in China,has a long history of use for the treatment of many different diseases.Chemical,pharmacological and clinical studies have testified that GL possesses various therapeutic functions including anti-tumor, hepatoprotective,anti-senility,anti-viral,anti-inflammatory,anti-hypertensive and hypocholesterolemic activity.In recent years,the anti-cancer effects of GL have become a major focus of research activity involving this mushroom.However,the chemical composition of GL is very complex,and obtaining purified material for research into the pharmaceutical effects and mechanisms of GL is often difficult.In this study,two compounds(GLIS-Ganoderma lucidum immunity stimulator,and GLAI-Ganoderma lucidum apoptosis inducer) have been isolated from GL fruit bodies and their bioactivities determined.GLIS stimulates immunity and GLAI induces apoptosis of tumor cells.The mechanisms underlying these activities have been investigated in order to establish a basis for future research.1.Immunomodulating activity of GLISGLIS is a proteoglycan isolated from GL fruit bodies using successive chromatographic steps.Previous research has shown that GLIS stimulated the proliferation of lymphocytes,and that most of the activated lymphocytes were B cells.GLIS also stimulates the activity of NK cells.In this study,the mechanism(s) involved in macrophage stimulation investigated using GLIS modulation of RAW264.7 cells(a mouse macrophage-like cell line),bone marrow-derived macrophages(BMMs) and mouse spleen lymphocytes(MSLs).After exposure to GLIS,RAW264.7 cells exhibited altered morphology,increased in size and formed numerous pseudopodia.GLIS also triggered the respiratory burst in RAW264.7 cells,and stimulated the macrophages to produce superoxide anion and NO. GLIS therefore activates the Oxygen-Dependent Kill System of macrophages.GLIS also regulated related cytokine expression at the mRNA level in activated RAW264.7 cells. Interleukin-1β(IL-1β),IL-12p35 and IL-12p40 gene expression was markedly increased within six hours after exposure whereas no changes were recorded in IL-6,IL-18 and TNF-a gene expression.Large increases in IL-1β,IL-12 and TNF-a protein production were also recorded.After exposure to GLIS,BMMs also exhibited altered morphology,increased in size and formed numerous pseudopodia.GLIS also triggered the respiratory burst in BMMs and stimulated the macrophages to produce superoxide anion and NO.Therefore,GLIS also activates the Oxygen-Dependent Kill System of macrophages.GLIS also regulated related cytokine expression at the mRNA level in activated BMMs.IL-6,IL-12p35 and IL-12p40 gene expression was markedly increased,and increases in IL-1β,IL-18 and TNF-a gene expression was also observed.Large increases in IL-1β,IL-12 and TNF-a protein production was also recorded.GLIS stimulated the proliferation of lymphocytes,and induced the respiratory burst in MSLs.The data indicate that induction of the antitumor effect of macrophages by GLIS involves activation of the Oxygen-Dependent Kill System and regulation of related cytokine expression.2.Apoptosis-inducing activity of GLAIA triterpene fraction,GLAI,was isolated from GL fruiting bodies using successive separation steps combined with activity tracking.The triterpene content of GLAI was 62.8%(using ganodermanondial as standard).HPLC revealed 8 peaks including those corresponding to ganodermanondial and lucidumol A.GLAI inhibited the proliferation of several tumor cell lines including L1210(mouse lymphocytic leukemia),K562(human chronic myelogenous leukemia),SW620(human lymph node metastasis,colon carcinoma) and MCF7(human breast adenocarcinoma,pleural effusion).Marked changes in the morphology of SW620 cells occurred following exposure to GLAI.Cells became more rounded and crimped,and many apoptotic bodies were observed.Hoechst 3258 fluorescence staining combined with annexin V-FITC/PI staining confirmed that GLAI induced apoptosis in SW620 cells.Exposure of SW620 cells to GLAI also increased the activity of caspase-3,the key enzyme associated with apoptosis.At the mRNA level, resulted in down-regulation of Bcl-2 gene expression,and also in XIAP protein production (both Bcl-2 and XIAP are associated with apoptosis inhibition).Conversely,increasing GLAI concentrations and exposure times up-regulated expression of apoptosis enhancer Bax gene and production of apoptosis enhancing p53 protein.In summary,multiple pathways are involved in the antitumor activity GL including the stimulation of macrophages by GL polysaccharide to effectively eliminate tumor cells,and the induction of tumor cell apoptosis by GL triterpenes.