Synergistic Effect Of Polysaccharide From Lentinus Edodes With Anticancer Drugs And Its Mechanism

Lentinus edodes,named as xianggu and shiitake,is famous as a source of medicine and food.It has been cultivated for 4 thousands of years and is applicable to aid in the treatment of cancer.Lentinan is the most effective biological active ingredient in Lentinus edodes.Since the polysaccharide with antitumor effect were extracted from lentinus edodes by the Japanese cholar Chihara at 1969,the study of the antitumor activity of lentinan has become a hot research on natural medicine and health food.Some studies indicated that lentinan has many pharmacological activity,such as antitumor,immunoregulation,antioxidation,reducing blood fat.Lentinan now has many verieties listed,but its antitumor mechanism is still not clear.At present,it is said that the antitumor effect of lentinan is through enhancing the immune system of the body.Therefore,lentinan is used as an immunomodulator in the therapy of cancer.Our previous studies found that lentinan not only can enhance the body immunity,but also by inducing tumor cell apoptosis to kill cancer cell.And lentinan almost no toxic side effects.Therefore,to explore the combination effect of lentinan and other chemotherapy drugs and it potential mechanisms is valueable to expand the medicinal value of lentinan and is important to the clinical treatment of cancer.In the present study,the polysaccharide JLNT were isolated and purified from the fruiting bodies of Lentinus edodes.And the combination effect of JLNT with oxaliplatin or ifosfamide on liver cancer and lymphoma were studied and the molecular mechanisms involved were investigated.This work will make contribution to the clinical application of Lentinan.Part Ⅰ.The isolation and purification of the polysaccharide JLNT from Lentinus edoeds.The crude polysaccharide from Lentinus edodes were extracted by dilute alkali solution extraction method.And the purified Lentinan polysaccharide fraction JLNT was purified by H2O2 decolorization,ultrafiltration and freeze drying.UV scanning showed that JLNT contained almost no nucleic acid and protein.The molecular weight was 550 kDa by high performance gel permeation chromatography(HPGPC).In conclusion,these results showed that the polysaccharides extracted by these methods were suitable for the follow-up experiment.Part Ⅱ.The synergistic effect of lentinan combined with oxaliplatin on hepatocellular carcinoma in vitro and its underlying mechanism.The inhibitory effects of lentinan and oxaliplatin on the proliferation of human hepatocellular carcinoma cell line HepG2 and mouse hepatocarcinoma cell line H22 were investigated by MTT assay.The results showed that lentinan and oxaliplatin inhibited the proliferation of tumor cells in a dose-dependent manner.And the inhibition effect was increased after the combination of the two drugs.The combined drug index of lentinan and oxaliplatin was calculated by Chou-Talalay combined drug index method and the results showed that lentinan combined with oxaliplatin had synergistic effect on HepG2 cells,and had synergistic effect on H22 cells except for the lowest concentration.Apoptotic cells were detected by flow cytometry.The apoptotic rated of lentinan(800 μg/mL),oxaliplatin(20 μM)and combination group(800 μg/mL + 20 μM)were 16.24%20.01%and 30.60%respectively.Results showed that the mechanism of lentinan and oxaliplatin on the proliferation of tumor cells may be related to the induction of apoptosis.The molecular mechanism of lentinan and oxaliplatin induced apoptosis of HepG2 cells was performed and the expression of caspase-3,NF-κB and survivin was detected by Western blot assay.Results showed that the expression of Caspase-3 and cleaved caspase-3 were significantly increased after lentinan and oxaliplatin administration,while the expression of survivin was significantly decreased.Oxaliplatin can upregulate the expression of NF-κB,which may increase the resistance of cells to apoptosis,and the combination administration can reduce the expression of NF-κB.To ascertain the role of survivin in mechanism of lentinan and oxaliplatin induced apoptosis,the cell transfection of survivin overexpression plasmid assay was performed.The results indicated that the expression of Caspase-3 and Cleaved caspase-3 was not significantly affected by the combination of lentinan and oxaliplatin after the transfection of survivin overexpression plasmid,suggesting that survivin may be one of the targets of lentinan combined with oxaliplatin-induced apoptosis of HepG2 cells.Part Ⅲ.The antitumor effect of lentinan and oxaliplatin in H22 tumor bearing mice and its underlying mechanism.The antitumor activity and mechanism of lentinan and oxaliplatin in vivo were investigated in H22 tumor bearing mice model.The mice were divided into negative group(intraperitoneal injection of glucose solution),lentinan group(intraperitoneal injection of lentinan 25 mg/kg),oxaliplatin group(intraperitoneal injection Oxaliplatin of 10 mg/kg)and combination group(intraperitoneal injection of lentinan and Oxaliplatin 25 mg/kg + 10 mg/kg).The results showed that the inhibitory rates of lentinan,oxaliplatin and combined administration on H22 solid tumors were 31.24%,59.09%and 80.98%,respectively.The expression of CD31 and VEGF in H22 tumor tissues was detected by immunohistochemistry to investigate the effects of lentinan and oxaliplatin on tumor angiogenesis.The results showed that the expression of CD31 and VEGF were significantly decreased after combination administration,which indicated that lentinan and oxaliplatin could inhibit the angiogenesis by decreasing the expression of VEGF.H&E staining and Tunel staining were used to investigate the apoptosis of H22 tumor tissue.Results showed that lentinan and oxaliplatin could induce the apoptosis of H22 tumor cells,and the combination administration could enhance the induction of apoptosis.Apoptosis-related proteins were detected by Western blot and immunohistochemistry.The results showed that the expression of Bcl-2 was significantly decreased and the expression of Bax was significantly increased.The Bax/Bcl-2 also increased significantly.The expression of cleaved caspase-8 was significantly increased after combination administration.The expression of caspase-3 and cleaved caspase-3 was also increased.The expression of NF-κB,STAT3 and p-STAT3 was decreased after combination administration.The mechanism of lentinan combined with oxaliplatin induced tumor cell apoptosis were concluded as follow:(1)down-regulated the expression of STAT3 and p-STAT3,directly or indirectly inhibit the expression of survivin,thus activating caspase-3,promoting tumor cell apoptosis;(2)up-regulate the expression of pro-apoptotic protein Bax and down-regulate the expression of Bcl-2,thereby increasing the ratio of Bax/Bcl-2,initiating mitochondrial apoptosis,and finally promoting the expression of caspase-3,inducing tumor cell apoptosis;(3)down-regulate the expression of NF-κB,directly or indirectly promote the activation of caspase-8,initiating caspase cascade reaction,promoting caspase-3 expression,and ultimately promote tumor cell apoptosis.Part Ⅳ.Effects of Lentinan on the side effects of OxaliplatinThe effects of oxaliplatin and lentinan on the body weight of mice were recorded during the administration of H22 tumor-bearing mice.Results showed that the body weight were significantly decreased after administration of oxaliplatin.and lentinan was able to relieve the weight loss caused by oxaliplatin after 2 weeks.The diarrhea of H22 tumor-bearing mice was recorded and the effects of lentinan and oxaliplatin on small intestine and liver were investigated by H&E staining.Results showed that oxaliplatin caused severe diarrhea,and lentinan can alleviate the incidence of diarrhea caused by oxaliplatin,which may through protecting the intestine damage caused by oxaliplatin.Liver H&E staining showed that the liver was injured after oxaliplatin administration.And lentinan was able to effectively relieve the hepatotoxicity of oxaliplatin.The effects of oxaliplatin on the immunity of mice were investigated by spleen index,carbon particle clearance and flow cytometry.Results showed that oxaliplatin could significantly reduce the spleen index,and lentinan could effectively alleviate the cytotoxicity of oxaliplatin and increase the spleen index of mice.Oxaliplatin also significantly reduced the phagocytosis of mouse macrophages.However.lentinan could also alleviate the cytotoxicity of oxaliplatin.Oxaliplatin reduced the number of CD4 lymphocytes and CD8 lymphocytes,but the ratio of CD4/CD8 was increased.After combination administration,the number of CD4 and CD8 lymphocytes increased and the ratio of CD4/CD8 also increased,but there was no significant difference.Part Ⅴ.Synergistic effect of lentinan combined with ifosfamide on mouse lymphomaThe inhibitory effect of JLNT on human lymphoma cell line Raji and mouse lymphoma cell line EL4 was investigated by MTT assay.The results showed that lentinan had a direct inhibitory effect on EL4 cells in a dose-dependent manner,but had no significant inhibitory effect on Raji cell proliferation.The antitumor effect of lentinan combined with ifosfamide was investigated on EL4 tumor bearing mice.Results showed that lentinan and ifosfamide had a certain inhibitory effect on the tumor growth of EL4 tumor-bearing mice.As the large difference in individual and the low rate of tumor formation,the effect of lentinan combined with ifosfamide was still unclear.