Antitumor Activities And Associated Mechanisms Of The Total Extract And An Active Compound Osmundacetone From Sanghuangporus Vaninii

Part 1 Apoptosis induction of the total extract of Sanghuangporus vaninii in A375 human melanoma cellsMelanoma is one of the most aggressive and drug-resistant cancers with high malignant.Although the proportion of melanoma in skin cancers is low,its mortality rate is as high as 80%.For the treatment of advanced or unresectable melanoma,the frequent adverse reactions and short-term resistance of drugs such as dacarbazine(DTIC),dabrafenib,trametinib and velofenib reduce the clinical benefits in these drug treatments.It is also necessary to develop new therapeutic methods and drugs.Natural products have received more and more attention because of their safe,effective,and low-toxic properties.There is a famous medicinal fungus,Sanghuangprous vaninii,in China and other East Asian countries.It's also named "Sanghuang" in Chines.It has been used to treat various diseases including cancers."Sanghuang" has been recorded as "Sangchen" and "Sang'er" in many classical documents of Chinese medicine According to the descriptions in these documents,"Sanghuang" is considered to be an effective treatment of tumors,leucorrhea,malignant ascites and immunocompromisedObjective:The purpose of this research is to prepare the total extract of S.vaninii(ESV)and explore its anticancer effects on the melanoma,and the mechanisms that might be involved.So as to provide part of the theoretical basis for the development of S.vaninii.Methods:The ESV was prerared with 95%ethanol and water.Cell viabilities of ESV at doses of 0,25,50,100,200 and 400 ?g/mL were evaluated by CCK-8 assay in six human cancer cell lines including MDA-MB-231,HCT116,U87,U251 and A375 The morphology of cells and nuclei in A375 cells after treatment of ESV at doses of 100,200,and 400 ?g/mL for 24 h were observed by an optical microscope and a fluorescence microscope,respectively.Flow cytometry was used to detect the effects of ESV on cell cycle distribution by using PI as a probe in A375 cells.The Annexin-V/PI double staining was used to detect the relative proportions of normal cells,early apoptotic cells,middle and late apoptotic cells,and necrotic cells after treatment with ESV for 24 h in A375 cells.Flow cytometry was used to detect the effects of ESV on mitochondrial membrane potential(MMP)by using JC-1 as a probe.Western blot was used to detect the expression levels of apoptosis-related proteins including Bcl-2,Bax,cleaved caspase-3 and cleaved PARP after A375 cells were treated with ESV at different concentrations for 24 h.The subcutaneous xenograft in BALB/c nude mice were used to detect the effect of ESV on tumor growth in vivo.H&E staining,TUNEL staining and immunohistochemical(IHC)staining were used to analyze the pathological characteristics of tumor tissues.In addition,wound healing assay and transwell invasion assay were used to detect the effects of ESV on the migration and invasion in A375 cellsResults:CCK-8 assay exhibited that ESV strongly inhibited viability of A375 melanoma cancer cells in a dose-and time-dependent manner.The IC50 value is 64.9?g/mL with treatment of ESV for 48 h.The results of cell cycle assy showed that A375 cells were blocked in the S phase of the cell cycle by ESV.The proportion of cells in S phase in the control group was 32.3%.After treatment with ESV at 100,200,and 400?g/mL,the proportions of cells in S phase were increased to 33.0%,46.7%and 71.7%,respectively.It was observed that the numbers of cells were decreased with increasing concentrations of ESV.And the cells were observed to be shranked and shattered Hoechst staining assay showed that the nuclei were shrinked,the blue fluorescence intensity was increased,and the cells were broken.Flow cytometry analysis indicated that ESV signifcantly induced A375 cells apoptosis.Apoptosis rate increased from 5.8%to 13.4%,21.7%and 39.4%after treatment with 100,200,and 400 ?g/mL ESV,respectively.And the proportion of cells with decreased MMP were increased from 0.98%to 13.57%,53.47%and 92.8%.Western blot analysis showed that ESV increased the expression of Bax,and decreased Bcl-2 in A375 cells.ESV remarkably caused MMP collapse and induced a mitochondrial-dependent apoptosis in A375 cells evidenced by caspase-3 activation,followed by PARP cleavage.The tumor inhibition rates of ESV were 46.52%,49.57%,and 65.92%at 300,600,1200 mg/kg,respectively.And the tumor inhibition rate at high-dose is close to that of the DTIC.H&E staining showed that the cells in the control group were tightly arranged with nuclear fission indicating rapid growth.While the cells in the ESV groups were sparsely arranged with many fissures between cells.And the cells exhibited nuclear pyknosis.Western blot showed that ESV increased the expression of pro-apoptotic protein Bax and cleaved caspase-3 in a dose-dependent manner,and decreased the expression of anti-apoptotic protein Bcl-2 of tissue.TUNEL staining showed that the intensity of green fluorescence was positively correlated with concentration of ESV.IHC staining showed the expression of cleaved caspase-3 were increased compared to the control.More importantly,ESV showed a strong inhibitory effect on the migration and aggression on A375 cells through the wound healing and transwell invasion assayConclusion:ESV has obvious antitumor effects on A375 human melanoma cells The associated mechanisms of its antitumor effects may be related to a variety of pathways,including apoptosis induction,inhibition of cell proliferation,migration and invasion.S.vaninii is a great potential natural product for melanoma with rapid proliferation and high metastasis characteristicsPart 2 Antitumor activities and associated mechanisms of an active compound osmundacetone from Sanghuangporus vaniniiAccording to a number of reports at home and abroad and several years of studies by our research team,the total extract of Sanghuangporus vaninii(ESV)has remarkable antitumor effects.It is necessary to further explore the material basis of its effect.The Part 1 of this study shows ESV induces apoptosis in A375 human melanoma cells in vivo and in vitro.In our team's previous studies,S.vaninii has antitumor effects on SMMC-7721 hepatoma cells and SiHa cervical cancer cells in vitro and in vivo with different meshanisms.In fact,S.vaninii can inhibit proliferation or induce death in many kinds of tumor cells.It plays an anti-cancer role with multiple targets and multiple ways.In view of the better development of S.vaninii,23 compounds were extracted and isolated from S.vaninii in our team's previous study.In the previous study of high-throughput screening of 23 compounds in 41 cell lines of 11 cancers,many compounds exhibited obvious cytotoxicity on various cancer cells,while had no obvious cytotoxic effect on two normal hamster cells.In this study,based on the high-throughput screening,7 compounds with effective activity are selected to effect on more cells.These compounds are found to have no effective activity against A375 cells.While compound 15(Osmundacetone,OSC)has effective antitumor activity against H460 and A375 non-small cell lung cancer(NSCLC)cells.According to search the reports,no scholars have studied antitumor effects on NSCLC cells with OSC treatment.In this study,the antitumor effects of an active compound OSC from S.vaninii on NSCLC and associated mechanisms will be deeply explored,thus providing a natural product origins for new drug discovery against cancerLung cancer is one of the most malignant tumors all over the world.In recent years,the morbidity and mortality rates have remained high.NSCLC accounts for 85%of the total lung cancers.Although current treatment methods and drugs are constantly increasing,the morbidity and mortality have not been alleviated.Existing methods and drugs of treatment always have various limitations in the clinic.Study of antitumor activities of small molecules is still an indispensable direction for the development of anti-cancer drugsObjective:The purpose of this study is to explore the inhibitory activity on NSCLC cells proliferation of an active compound osmundacetone(OSC)and the mechanisms that might be involvedMethods:MTT assays were used to detect viabilities of 7 kinds of compound isolated from S.vaninii at the concentrations of 0,6.25,12.5,25,50 and 100 ?M on 8 kinds of human tumor cells including A375,SiHa,SK-OV-3,A2780,SH-SY5Y,H460,A549 and PC-3.Colony formation assay was used to observe the effects of different concentrations of OSC(0,10,20,40 ?M)on the colony formation abilities of NSCLC H460 and A549 cells.Flow cytometry was used to detect the effects of OSC on cell cycle distribution by using PI as a probe in H460 and A549 cells.The morphology of H460 and A549 cells after treatment with OSC at doses of 0,10,20,40 ?M for 48 h was observed by an optical microscope.The subcutaneous xenograft in BALB/c nude mice were used to detect the effect of OSC on tumor growth in vivo.H&E staining and IHC staining were used to analyze the pathological characteristics of tumor tissues Serum metabolomics analyses in nude mice were conducted to observe differential metabolites and metabolic pathways influenced with treatment of OSC.NSCLC cell models in vitro was used to verify the expression levels of related metabolites and proteins in the glutamate metabolism pathway.The regulatory effect of OSC on glutamate dehydrogenase 1(GLUD1),a key protein related to the glutamate metabolism was verified by knocking down or over-expressing GLUD1.The morphology of mitochondria in H460 and A549 cells after treatment with OSC at doses of 0 and 20 ?M for 48 h was observed by a transmission electron microscope.Laser confocal microscopy was used to detect the effects of OSC on MMP by using JC-1 as a probe.ELISA assays were conducted to study the levels of ?-KG and NADH,which are glutamate-and mitochondrion-dependent metabolites in the NSCLC cells with OSC treatment,and the changes in GLUD1 silenced or overexpressed cells with and without OSC treatment.The Agilent Seahorse XF ATP real-time rate kit was used to detect the effect of OSC on the ATP rates produced by mitochondrial oxidative phosphorylation(OXHPOS)and glycolysis,respectively,and the total ATP rate.GLUD1 silenced and overexpressed cell models were used to test the regulatory effect of OSC on GLUD1 Proteomics analyses of timor tissues was conducted to verify the antitumor effects of OSC in vivoResults:The results of MTT assays showed that 7 compounds had no obvious inhibitory effects on the proliferation of A375 human melanoma cells.Compound 15(OSC)had good proliferation inhibitory effects on A2780,H460,A549 and PC-3 cells with treatment for 48 h.Among them,the proliferation inhibitory effect on H460 and A549 cells was the most obvious,with an IC50 values at 20.69 ± 1.04 and 21.20 ± 0.16?M,respectively.Moreover,OSC inhibited viabilities of H460 and A375 cells in a dose-and time-dependent manner.OSC also inhibited abilities of clonal formation of cells in a dose-dependent manner.The proportion of cells in G2/M phase in the control group was 2.86%.After treatment with OSC at 10,20,and 40 ?M,the proportions of cells in G2/M phase were increased to 7.49%,11.78%and 21.93%,respectively.Cell morphology was changed and even broken with OSC tretment.The tumor inhibition rates of OSC were 53.17%,53.49%and 68.51%at 20,40,80 mg/kg,respectively Futhermore,compared with the significant decrease in body weight of mice in the cisplatin(DDP)group,the body weight of mice in the OSC groups did not decrease and was similar to that in the normal group.H&E staining showed that the cells in the control group were tightly arranged with nuclear fission indicating rapid growth.While the cells in the OSC groups were sparsely arranged with many fissures between cells.And the cells exhibited nuclear pyknosis.Serum metabolomics analyses showed that the glutamine and glutamate metabolism pathways and tricarboxylic acid(TCA)cycle of the model group mice treated with OSC were affected.The levels of key metabolites including ?-KG and citric acid in the model mice were nearly decreased to the normal mice after treatment with OSC.qRT-PCR and western blot showed treatment with OSC down-regulated mRNA and protein levels of GLUD1.Such a result was further verified with GLUD1 silenced and overexpressed cell models.IHC staining and western blot analysis of tumor tissues showed that GLUD1 could also be down-regulated in tumor tissues after treatment with OSC.ELISA assays showed that the intracellular levels of?-KG,the catalytic product of GLUD1,was decreased with the increase of the concentrations of OSC.The results of TEM showed mitochondrial structures of H460 and A549 cells were destroyed after treatment with OSC.The measurement of MMP with JC-1 as a probe verified this conclusion.The ELISA results showed that NADH,which transfered hydrogen to electronic respiratory chain,was decreased with the increase of the concentration of OSC.The ATP produced by the OXHPOS in mitochondria decreased significantly after treatment with OSC.While the ATP produced by glycolysis did not change significantly.Proteomics analysis of tumor tissues showed that proteins related to mitochondrial energy production,cell proliferation and metastasis were regulated after treatment with OSC.These changes are beneficial to inhibit tumor growth,cell metastasis and energy metabolism.The antitumor effects of OSC were verified from the perspective of proteomics in vivo.Conclusion:The key role of OSC in mitochondrial energy metabolism of NSCLC cells is to inhibit cell proliferation and tumor growth by down-regulating GLUD1 to inhibit glutamine-glutamate-?-KG axis metabolism and OXPHOS.