| Lentinus edodes(called Xianggu and Shiitake in China and Japan respectively)is a popular edible mushroom in East Asia and is frequently used in food and in folk medicine.Lentinan is the best known and most potent mushroom-derived substance isolated from L.edodes.And as a tipicalβ-1,3-glucan polysaccharide,it exhibits hypocholesterolemic and hypoglycemic properties,antifungal/antibacterial,immunomodulatory and antitumor activities.Lentinan has been used clinically in Japan since the early 1980s because of its immunomodulatory and antitumor effects.However,the mechanism underlying its antitumor activity remains unclear which results in its limited clinical application.Earlier findings suggested Lentinan exerted its antitumor effects by activating immune responses in the host rather than directly by attacking cancer cells.The antitumor activity of Lentinan was thought to require an intact T-cell component and was mediated through a thymus-dependent immune mechanism.However,recent studies in our lab have shown that Lentinan can directly inhibit proliferation of several cancer cells,which means that Lentinan exerts non-immune direct antitumor effects.Therefore,the aim of this study is to evaluate the non-immune direct antitumor activity of edible fungal polysaccharide Lentinan,rather than as a host defence potentiator,on human colon cancer and investigate its underlying mechanisms.This study will provide a new insight into the mechanisms of Lentinan-antitumor effect and contribute to its development in functional foods and cancer therapy.The dried fruit bodies of L.edodes were obtained from FangXian(Hubei,China).The polysaccharide from Lentinus edodes(SLNT)was extracted,isolated and purified as previously described.And its purity was identified by sugar content,UV scanning and determination of molecular weight analyses.We explored the direct antitumor activity of SLNT on HT-29 human colon cancer cells,HT-29 tumor-bearing athymic nude mice and HT-29 tumor-bearing NOD/SCID mice.Through these immunodeficient mice models,we could assess SLNT’s direct antitumor effects in the absence of an intact T-cell component or even entire immune system.Here,both Type I(apoptosis)and Type II PCD(autophagy)were put into consideration when we investigated the underlying mechanism of its direct antitumor effect.Part One:The extraction,purification and identification of LentinanThe polysaccharide from Lentinus edodes(SLNT)was extracted,isolated and purified via the established and optimized methods:Briefly,after soaking with 95%alcohol,the fruit body residues were subjected to water extraction followed by ethanol precipitation,decoloration and ultrafiltration.The identifications of SLNT were determined.A yield of around 1.91 g of SLNT was obtained from 250 g of L.edodes,which sugar content was approximately 98.54%and molecular weight was 623.5 kDa.UV spectrophotometer scanning showed that SLNT was absence of proteins and nucleic acids.The experiments results showed the methods of preparing SLNT were easy to repeat,control and the results were stable.Additionally,the polysaccharide isolated from Lentinus edodes had high purity,uniform molecular weight and was free of proteins and nucleic acids,which had satisfied the needs of follow-up experiment.Part Two:The research on non-immune antitumor activity of Lentinan on HT-29 human colon cancer cells in vitro and in vivoColon cancer is the second leading cause of cancer-related deaths worldwide,accounting for over 1 million new cases and about half a million deaths per year.Surgery is the primary treatment for colon cancer.For most patients with metastasis,however,systemic chemotherapy is needed to relieve symptoms and prolong life.However,the side effects associated with these chemotherapeutic strategies,which include diarrhea,nausea and vomiting,acute myocardial infarction and cerebrovascular accident,greatly decrease the patients’quality of life and can be fatal at times.There is thus a great need to screen for natural products exhibiting antitumor activities with low toxicity and high efficacy.Here,we chose primary TNMⅠhuman colon cancer HT-29 cells which had good tumorigenicity and several positive oncogene expression as our object of research.And in this part,we explored the non-immune direct antitumor effect of SLNT on HT-29 human colon cancer cells in vitro and in vivo.To investigate the effects of water-extracted Lentinan(SLNT)on the proliferation of HT-29 cells,MTT assay was performed.A concentration dependent inhibitory effect was observed when HT-29 cells were exposed to SLNT(12.5-1600μg/mL)for 48 h.SLNT(1600μg/mL)exhibited a strongest direct antitumor activity on HT-29 cells with 66.84%of inhibition rate while the inhibition rate of 5-FU was 74.24%.Then,we observed the cells morphology by optical microscope.Notably,cells-treated with SLNT detached from the plate and underwent complete loss of cellular morphology accompanied with cytoplasm contraction and nuclear shrinkage while NC cells adhered well and displayed normal morphology.We next explored whether the T-lymphocyte immune system-independent antitumor effect of SLNT was actually applicable in HT-29-bearing athymic nude mice.Thirty5-week-old male athymic nude mice(BALB/c-nu)were purchased and acclimated before the experiment.HT-29 cells were injected subcutaneously into the right flank of nude mice(17-20 g in weight).Animals were randomly divided into five groups(n=6/group)as follows:(i)sterile 0.9%saline(negative control);(ii)0.2 mg/kg SLNT;(iii)1 mg/kg SLNT;(iv)5 mg/kg SLNT;(v)20 mg/kg 5-FU,and treated by intravenous(i.v.)injection through tail vein.Ten times of injections were performed every other day.At the end of treatments,mice were euthanized;tumor xenografts and spleens were excised,weighed.The inhibition rate of tumors and spleen index of each group were calculated.And the concentration of IL-6,TNF-αin nude mice serum were measured by ELISA kits.The results showed that SLNT had no severe side effects on the mice and tumors in SLNT-receiving mice were smaller than that of NC mice.And the inhibition rates of SLNT(0.2,1 and 5 mg/kg)were17.88%,48.87%and 57.90%,respectively,while 5-FU was 67.23%,compared with the NC.These results suggested that SLNT exerted the T-lymphocyte immune system-independent antitumor effect.In addition,there were no significant changes in spleen index and IL-6level in serum of nude mice after SLNT treatments,indicating that SLNT did not improve the immune system functions.However,the results showed that SLNT significantly increased the level of TNF-αin serum,suggesting that the direct antitumor effect of lentinan might be related to tumor necrosis factor.To further explore whether Lentinan could exert non-immune direct antitumor effect in vivo,we established the HT-29-bearing combined immunodeficient NOD/SCID mice with defective T lymphocyte,B lymphocyte and NK cells and low macrophage function as model.And the same administration method and dose of SLNT were used in this experiment.Animals were randomly divided into three groups(n=5/group)as follows:(i)sterile 0.9%saline(negative control);(ii)1.0 mg/kg SLNT;(iii)5.0 mg/kg SLNT,and treated by intravenous(i.v.)injection through tail vein.Ten times of injections were performed every other day.In the end of treatments,mice were euthanized;tumor xenografts were excised,weighed.The inhibition rate of tumors of each group was calculated.And the concentration of IL-2,IL-12,IFN-γ,IL-6 and TNF-αin NOD/SCID mice serum were measured by ELISA kits.The results showed that SLNT had no severe side effects and tumors in SLNT-receiving mice were smaller than that of NC mice.And the inhibition rates of SLNT(1 and 5 mg/kg)were 45.05%and 50.61%,respectively,compared with the NC group.Additionally,there were no significant changes in spleen index and IL-2,IL-12,IFN-γ,IL-6 level in serum of NOD/SCID mice after SLNT treatments,indicating that SLNT exerted non-immune direct antitumor effect in vivo.Consistent with the result in nude mice,5 mg/kg SLNT significantly increased the level of TNF-αin NOD/SCID mice serum which suggested that TNF-αmight be involved in direct-antitumor effect of SLNT.In summary,this part of study confirmed that Lentinan exerted a significant direct antitumor effect on HT-29 cells in vitro and in vivo through non-immune pathways.Moreover,this part of study laid a foundation for the research on the molecular mechanism of the non-immune direct antitumor effect of Lentinan.Part Three:The research on Lentinan-induced HT-29 cells apoptosisOwing to apoptosis is considered as the main cell death mechanism,we next tested whether Lentinan could induce HT-29 cell apoptosis via multiple methods in this part.Through flow cytometry,we determined that SLNT significantly induced HT-29 cell apoptosis in a dose-dependent manner,with 20.26%,31.80%and 48.50%of apoptotic rate,respectively,compared with the negative control(NC,7.31%).To further confirm it,DAPI,AO/EB and Annexin V-FITC/PI staining were performed to observe the cell morphology.In DAPI staining(a nuclear stain),NC cells displayed homogenous fluorescence with no evidence of fragmentation or chromatin condensation while SLNT-treated cells had prominent DNA condensation;in AO/EB staining,the images revealed that more apoptotic cells(condensed green or orange)and less normal cells(uniform green)existed in SLNT group than NC;and Annexin V-FITC/PI staining showed more early and late apoptotic cells in SLNT group.Owing to the loss of mitochondrial membrane potential(MMP)was a key event in the early apoptosis through mitochondrial apoptotic pathway,we detected the changes of MMP by JC-1 staining.And the results showed SLNT induced the loss of MMP,suggesting that SLNT might activate mitochondrial apoptotic pathway.In conclusion,these results confirmed that SLNT induced HT-29 cell apoptosis.To further explore whether apoptosis was involved in prevention of tumor growth,we analyzed tumor tissues by H&E staining.Marked apoptotic morphologic changes such as karyorrhexis and irregular arrangement of karyomorphism were observed in SLNT group.Conversely,NC group kept good original grown status with completed,well-regulated and clearly visible karyomorphism.This suggested that SLNT also induced apoptosis in HT-29 tumor xenografts.In summary,this part of study confirmed that Lentinan could induce HT-29 cell apoptosis thereby exerted non-immune direct antitumor effect,which laid a foundation for the mechanism research in next part.Part Four:The research on the underlying molecular mechanism of Lentinan-induced HT-29 cells apoptosisApoptosis is mainly triggered via intrinsic(mitochondrial)and extrinsic(death receptor)pathways.The results in the previous part indicated SLNT might activated mitochondrial apoptotic pathway,therefore we examined the effect of SLNT on mitochondrial apoptosis-related proteins.The results showed that Lentinan significantly activated Caspase-3,Caspase-9 and increased the expression of cytosol cytochrome c Bax/Bcl-2 ratio,which suggested that SLNT indeed activated mitochondrial apoptotic pathway.As ROS generated during oxidative stress is known to activate the intrinsic apoptosis,we next determined the effect of SLNT on generation of ROS.Compared to NC cells,SLNT significantly enhanced cellular ROS,particularly in a dose of 1600μg/mL.To evaluate whether the upregulation of ROS activated intrinsic apoptosis,an antioxidant NAC was used.the apoptotic rate of cells treated with SLNT(800μg/mL)significantly decreased from 32.91±1.21%to 21.49±1.62%after pre-incubation of NAC,compared with NAC group(6.94±0.57%).Meanwhile,SLNT-induced upregulation of cytosolic cytochrome c and Bax/Bcl-2 were blocked by NAC.These results indicated that ROS might act as upstream for activating intrinsic pathway.However,interestingly,there were still about20%apoptotic cells existed when ROS was largely cleaned out.This suggested that SLNT induced apoptosis possibly not only through intrinsic pathway,but also extrinsic pathway.The extrinsic apoptotic pathway is initiated by TNF-αbinding to TNFR1 or FasL binding to Fas receptors.These associations lead to recruitment of adaptor molecules such as FADD and,in turn,activation of initiator Caspase-8,and the Caspase cascade,culminating in apoptosis.Then,we examined the effect of SLNT on death receptor apoptosis-related proteins via multiple methods.The results showed that SLNT treatment led to the activation of Caspase-8 and inhibition of nuclear translocation of NF-κB in vitro and in vivo.Owing to the enhanced level of TNF-αin both serum of nude and NOD/SCID mice after SLNT treatment,we next detected the TNF-αlevel in vitro.Results showed that SLNT also significantly enhanced level of TNF-α,indicated that SLNT activated the death receptor apoptosis which might mediate via TNF-α/TNFR1 pathway.To ascertain the role of Caspase-3 in SLNT-induced apoptosis,a Caspase-3 inhibitor(Ac-DEVD-CHO)was used.HT-29 cells were pre-incubated with Ac-DEVD-CHO before the addition of SLNT(800μg/mL).The addition of Ac-DEVD-CHO significantly prevented SLNT-induced apoptosis(from 32.91±1.21%decreased to 15.88±1.58%while NC and Ac-DEVD-CHO group were 6.45±0.96%,7.77±0.79%,respectively),indicating that SLNT-induced apoptosis depended to a large degree on the activation of Caspase-3.However,SLNT+Ac-DEVD-CHO group(15.88±1.58%)was still higher than Ac-DEVD-CHO group(7.77±0.79%),suggesting that Caspase-3-independent pathways might also exist and required further investigation.In sum,in this part,our findings demonstrated that SLNT induced apoptosis through two pathways:(i)the intrinsic pathway mediated through overproduction of ROS,loss of MMP,increased in the Bax/Bcl-2 ratio and cytosolic cytochrome c,and activation Caspase-9 and-3.(ii)the extrinsic pathway mediated through increases in TNF-α,inhibition of NF-κB,and activation of Caspase-8 and-3.Part Five:The research on the Lentinan-induced HT-29 cells autophagy and its possible molecular mechanismAutophagy(‘self-eating’)was another self-destructive process except for apoptosis(‘self-killing’).Autophagic cell death,as Type II PCD,was also a kind of pathway resulting in cell death.In this part,we further explored whether Lentinan could induce HT-29 cell autophagy and its possible molecular mechanism.Unlike the cytoplasmic localization of microtubule-associated protein light chain 3 I(LC3-I),LC3-II which associates with membranes of the autophagosome becomes an index of autophagosome.In Western blot and Immunofluorescence experiment,we observed the enhancement of LC3-II and pattern of fluorescently tagged LC3 after SLNT treatment.Additionally,the increased Beclin 1 which participated in autophagosome formation also demonstrated that SLNT significantly provoked the formation of autophagosomes.However,autophagy includes not just the increased synthesis of LC3 or formation of autophagosomes,but,most importantly,flux through the entire system containing lysosomes and the subsequent release of the breakdown products.Accordingly,three more assays were complemented to estimate the dynamic,multi-step autophagic process—autophagic flux:(1)Quantification of p62,which was incorporated into the completed autophagosome and degraded in autolysosomes,serving as an index of autophagic degradation;(2)Tandem mCherry-GFP-LC3B fluorescence microscopy,which enabled simultaneous estimation of both formation of autophagosomes and flux through autophagic compartments;(3)Transmission electron microscopy,the only tool revealed the morphology of autophagic structures included autophagosome(AVi)and autolysosomes(AVd)in the nm range.Overall,these results revealed the remarkable enhancement of autophagic flux,in other words,the excessive activation of autophagy after SLNT treatment in HT-29 cells.We next explored the effect of SLNT on autophagy-related proteins in HT-29 cells and tumors via Western blot assay.The results showed that SLNT significantly decreased the expression of p-mTOR,PI3K,p-AKT,increased the p-JNK in vitro and in vivo,and enhanced the level of p-AMPK in vitro.Therefore,we deduced that Lentinan might indueced the HT-29 cell autophagy through the pathways as followed:(1)SLNT might inhibite the PI3K/Akt and activated the AMPK pathway,which directly or indirectly inhibited the downstream negative regulatory autophagy protein mTOR,and thereby induced the autophagy.(2)SLNT might activate the JNK pathway,and therefore phosphorylated the Bcl-2/Bcl-X_L, which in turn activated the positive regulatory autophagy protein Beclin 1,then induced the autophagy.In summary,this part of study demonstrated that Lentinan could significantly induce the HT-29 cell autophagy.Additionally,we made a preliminary exploration of its underlying molecular mechanism.And the further experiments need to be preformed to explore for a more detail and comprehensive mechanism.Part Six:The effect of Lentinan-induced autophagy on its direct antitumor activityParadoxically,autophagy has two connections with cancer,one at the level of cancer development and the other at the level of cancer treatment.In this part,we further explored whether the Lentinan-induced HT-29 cell autophagy contributed to its direct antitumor effect.Hence,we inhibited autophagy by targeting the Class III PI3K involved in autophagosome formation with 3-methyladenine(3-MA)or by targeting the fusion of autophagosomes with lysosomes by using chloroquine(CQ).The results showed that after preincubation of CQ or 3-MA with HT-29 cells,the inhibitor rate of SLNT decreased from56.04%to 4.89%(SLNT+CQ)and 37.63%(SLNT+3-MA),indicating that SLNT-induced autophagy contributed to HT-29 cell death.To further test this result in vivo,we established HT-29-bearing NOD/SCID mouse model with autophagy.HT-29 cells were injected subcutaneously into the right flank of mice.Animals were randomly divided into four groups(n=5/group)as follows:(i)sterile 0.9%saline(negative control);(ii)5.0 mg/kg SLNT;(iii)5 mg/kg CQ;(iv)5 mg/kg SLNT+5 mg/kg CQ,and treated by intravenous(i.v.)injection through tail vein.Ten times of injections were performed every other day.In the end of treatments,mice were euthanized;tumor xenografts and spleens were excised,weighed.The inhibition rate of tumors of each group was calculated.The results showed that CQ and SLNT had no severe side effects,and the tumor inhibition rate of SLNT significantly decreased from 50.61%to 35.23%when treated the mice combined with CQ.This part confirmed that Lentinan-induced HT-29 cell autophagy contribute to its non-immune direct antitumor effect,which could be characterize as autophagic cell death(Type II programmed cell death).In summary,our research demonstrated that Lentinan could induce the HT-29 cell apoptosis and autophagy in vitro and in vivo,and therefore exerted its non-immune direct antitumor effect.This paper provides sufficient theoretical and experimental basis for Lentinan as a noval antitumor drug in clinical application. |
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