Study On The Mechanisms Of Melatonin And Mushroom Fomitopsis Officinalis Extract Inhibiting Invasion And Migration Of Cancer Cells

Molecular mechanisms of inhibitory effects of melatonin on prostate cancer cell migrationBackground: Prostate cancer(PCa)is the second largest cancer in men and the fourth most common malignant tumor in the world,making it a major public health issue for elderly men.Currently,chemotherapy plays an important role in the treatment of prostate cancer.Although prostate cancer has a high long-term survival rate,metastatic prostate cancer treated with combination therapy is still difficult to cure.Moreover,due to issues such as non-specific and high systemic toxicity,commonly used chemotherapy drugs have been limited in clinical application in prostate cancer.In addition,chemotherapy drugs leading to multidrug resistance have become the main reason for the failure of prostate cancer treatment because of tumor recurrence and metastasis.Therefore,exploring new treatment strategy is an urgent demand for prostate cancer treatment,and screening effective anti-cancer ingredients from natural compounds provides new ideas for prostate cancer treatment.Research has shown that sour cherries,bananas,grapes,rice,oats,herbs,plums,olive oil,wine,and beer all contain natural melatonin.The human pineal gland also produces melatonin,which controls the circadian rhythm.Currently,melatonin is believed to have multiple effects,such as regulating rhythms and aiding sleep and playing the role of cytokines,biological response regulators,and neuromodulators.It can also be used as an adjuvant drug for the treatment of intestinal diseases.Previous research has shown that the main function of melatonin is its strong free radical scavenging ability,which means it has antioxidant properties.In addition,melatonin plays an important role in inhibiting lung injury,breast cancer risk,myeloid leukemia,etc.However,the effect of melatonin on prostate cancer has not been reported yet.Therefore,this paper aims to explore the inhibitory effect of melatonin on prostate cancer and preliminarily reveal its underlying mechanism.Method: Different concentrations of melatonin(0.1-3.5 m M)were used to treat androgen independent prostate cancer cell lines PC3 and DU145.The cells were treated for 24 hours,48 hours,and 72 hours,respectively,with the same number of cells.The inhibitory effect of melatonin on the growth activity of prostate cancer cells was detected by MTT assay.Evaluating the anti-metastatic effect of melatonin on prostate cancer cells were by two experimental methods: scratch healing and transwell.Using RNA sequencing(RNA-seq)technology to analyze gene transcription changes in prostate cancer cells(DU145)treated with melatonin(0.5 and 0.8 m M).Using GO and KEGG analysis methods to explore the expression profile of cell composition,function,and biological process related genes in DU145 cells treated by melatonin,as well as changes in signaling pathways.Collecting total RNA and performing quantitative RT-PCR to determine the influence of melatonin on the expression of related genes in PCa cells.Meanwhile,western blotting was used to detect the effect of melatonin on the expression of proteins in related signaling pathways.In addition,gene function studies were conducted by transfecting cells with si RNA to evaluate the role of melatonin in metastasis of PCa cells.Results: MTT,wound healing and transwell assays showed that different doses of melatonin reduced the proliferation and migration ability of prostate cancer cells.In the case of the treatment of cells with low doses of melatonin(0.5 m M and 0.8 m M)for 48 hours,the IC50 results showed1.1 m M in PCa cell lines.RNA-seq data revealed that a total of 20031 genes were up and down-regulated in DU145 cells treated with melatonin.Especially,271 genes were differentially expressed,including 97 upregulated and 174 down-regulated genes.Furthermore,RNA seq results showed that melatonin treatment led to a significant increase in the expression levels of HPGD,IL2 RB,and NGFR.However,IGFBP3 and IL6 had decreased expression levels.We found that q PCR results did not completely verify RNA-seq data,but we have identified HPGD and IL2 RB as upregulated genes consistent with RNA-seq data,while NGFR,IL6 and IGFBP3 were downregulated in DU145 cancer cells.On the other hand,IL2 RB,IL6 and IGFBP3 were upregulated,and HPGD and NGFR genes were downregulated in the PC3 cell line.The immunoblot assay revealed that the expression of IL2 RB and NGFR genes was enhanced,whereas IL6 and IGFBP3 expression levels were down-regulated in DU145 cells.Besides,cell cycle and apoptosis analysis revealed that different concentrations of melatonin inhibited cell cycle progression and promoted apoptosis in prostate cancer cells.Nuclear factor-κB(NF-κB)plays an important role in cell growth,survival,angiogenesis,tumor occurrence,and metastasis progression of PCa.Our results indicated that NF-κB signaling was partially involved in the inhibitory effect of melatonin on the proliferation of PCa cells.NF-κB was activated and could result in upregulated interleukin(IL)-2 gene expression,indicating a positive interaction between activated NF-κB and IL-2 signaling in PCa cells.We found that treatment with melatonin decreased the phosphorylation of IκBα and P-65.Besides,the IκBα and p65 were increased at protein levels in independent prostate cancer cells.Furthermore,the level of P50 was decreased in the DU145 cell line,on the contrary,it was increased the level in PC3 cancer cells.Transient transfection of si IL2 RB resulted in a significant knockdown of IL2 RB protein both in the presence or absence of melatonin in the cell lines tested.Besides,the silencing of IL2 RB significantly inhibited PCa cell growth and proliferation with and without melatonin treatment.The knockdown of IL2 RB reduced the rate of wound closure and migration rate in PCa cells.Later,we tested the biological effects of IL2 RB on PCa cell growth and proliferation,and for this,we employed loss-of gene function by using IL2 RB specific si RNA on PCa cell lines.To examine whether IL2 RB could be a possible mediator of the melatonin that affects the NF-κB pathway,we utilized the silencing of IL2 RB.The knockdown of IL2 RB triggered decreased phosphorylation of IκBα and P65,and the nuclear translocation of IκBα and P65 was increased in both cancer cells.On the other hand,the P50 level significantly increased in Du145 cancer cell lines and decreased in PC3 cancer cell lines,which compared to scramble si RNA-transfected cells.Furthermore,the effect of IL2 RB silencing was enhanced remarkably in the presence of melatonin.Altogether,the derived results allude that IL2 RB performs a key role in melatonin-mediated suppression of the NF-κB pathway.Conclusion: This study used various techniques such as RNA sequencing,real-time fluorescence quantitative PCR,flow cytometry,cell growth,migration,invasion,and western blotting analysis to confirm that melatonin could effectively inhibit the proliferation and migration of human prostate cancer cells in vitro.Our results showed that melatonin was a potential novel chemotherapy drug against PCa,providing a new idea and experimental evidence for the clinical application of melatonin in prostate cancer therapy.Anti-oxidant and anticancerous effects of Fomitopsis officinalis(Vill.ex Fr.Bond.et Sing)mushroom extract on hepatocellular carcinoma cells In-Vitro through NF-κB pathwayBackground: Hepatocellular carcinoma(HCC)is a serious threat to human health,and its incidence rate and mortality remain high.Globally,the incidence rate of HCC ranks fifth among malignant tumors,and the mortality rate ranks third.Although surgical resection is still the most effective method for HCC therapy,less than 40% of HCC patients are suitable for surgical treatment.Targeted therapy and immunotherapy are commonly used for HCC patients who cannot undergo surgical resection treatment.At present,first-line targeted therapy drugs for HCC include Sorafenib and Lenvatinib.Although significant progress has been made in the development of targeted therapy and immunotherapy in recent years,studies have found that HCC patients often face the risk of high postoperative recurrence rate and poor prognosis,and the resistance of targeted therapy drugs results in a 5-year survival rate of less than 15% for HCC patients.Due to unclear pathogenesis,high malignant recurrence rate,strong metastatic ability,strong drug resistance,and tumor heterogeneity of HCC,treatment of HCC patients is difficult.Therefore,exploring new treatment strategies is of great significance for HCC treatment.Fomitopsis officinalis(Vill.ex Fr.Bond.et Sing)is a medicinal mushroom,commonly called ’Agarikon’,traditionally used to treat cough and asthma in the Mongolian population.F.officinalis(Vill.ex Fr.Bond.et Sing)(synonym: Fomes officinalis,Agaricum Officinalis,Laricifomes officinalis)is a wood-decaying fungus in the family Polyporaceae,and is commonly known as ’Agarikon’.Traditionally,Fomitopsis officinalis is considered as a unique and miraculous medicine that can effectively treat various diseases such as sweating,dizziness,rheumatism,respiratory,digestive and excretory system diseases,cancer,hemorrhoids,and dysmenorrhea.It has great medicinal development and utilization value.At the same time,Fomitopsis officinalis has the function of enhancing immunity and clearing oxygen free radicals,the biological activity of its extracts and their isolated compounds showed antiviral,anticancer,antiinflammatory,and antituberculosis activities.These indicate that it may have unique therapeutic potential in inhibiting tumors.Therefore,we aim to explore the inhibitory effect and preliminary mechanism of the extracts of Fomitopsis officinalis on HCC cells.Methods: Fresh fruiting bodies of Fomitopsis officinalis(Vill.ex Fr.Bond et Sing.)were collected in April 2015 at Khoninnuga,Mandal soum,Selenge province(N 49°01.009’,E 107°15.021’;H 1278 m),Mongolia and identified by Prof.Ch.Sanchir,Institute of Botany,Mongolian Academy of Sciences.We evaluated the antioxidant activity of 6 fractions of F.officinalis including Fo1-powder insoluble in water,Fo2-petroleum ether residue,Fo3-chloroformic,Fo4-ethylacetate,Fo5-buthanolic,and Fo6-water-ethanolic residue to determine their inhibitory effects on hepatocarcinoma cells by 2,2-diphenyl-1-picrylhydrazyl(DPPH)free radical scavenging capacity method.We performed in vitro studies of cell proliferation and viability assay by MTT assay.The anti-metastatic function Fo3 extracts of F.officinalis in PCa were evaluated by two cell migration assays,namely wound healing assay and transwell migration assay.Cell cycle distribution(G0/G1,S,and G2/M)was analyzed by flow cytometry-based on DNA content.Annexin-V and PI allowed the resolution of viable cells,early apoptotic cells,and late apoptotic cells by Annexin V-positive/PI-positive,respectively.The outcome and effect of Fo3 extracts treatment on its molecular mediator were studied by implementing treatment of cells with several chemical agents and western blotting.And finally,the related NF-κB signaling pathway hallmarks were also examined by western blotting.Results: Our findings revealed that all six fractions/extracts have antioxidant activities,and somehow,they exert anticancer effects against cancer cells.In the cell lines(Hep G2 and LO2),Fo3 chloroformic extract promoted cell apoptosis,cell viability,activated G2/M-phase cell cycle,and selectively induced NF-κB proteins,revealing as a novel antitumor extract.More or less,the antioxidant activity was shown by all six fractions,but the highest antioxidant activity was observed in chloroformic fraction(Fo3)compared to ascorbic acid(Asc.A).So,for further steps,we move forward with only one fraction of Fo3-chloroformic having the highest antioxidant activity in this study.Cell proliferation assay showed that these two cancer cell lines exhibit a sensitivity range to Fo3 treatment,and the IC50 values = 30-50 μg/ml.Fo3 fraction significantly inhibited the cell viability in hepatocellular carcinoma cell(HCC)lines(p<0.001),with the survival rate of 53.607±6.957 in the Hep G2 cell line(Figure 2A)and76.587±1.76 in LO2 cell line treated with 50 μg/ml of Fo3 fraction.We assessed cancer cell migration by wound healing assay.Cells were treated with 0,30,and 50 μg/ml Fo3-chloroformic fraction for 24 hours.The cell migration or mobility is related to the treated dose concentration,which explains Fo3 extract has an anti-cancerous effect that could inhibit HCC cell lines.Cell inhibition was higher in the Hep G2 cell line than LO2 cell line.As cell cycle arrest is another representative characteristic of ageing,therefore we examined the cell cycle distribution of cancer cells under Fo3 fraction treatment.Flow cytometry assay results demonstrated that a progressive increase of cells,retardant in S-phase,increasing G2/M phase,occurred in HCC when treated with different concentrations of Fo3 extract.Further,we investigated the possible mechanism of the inhibitory effect on cancer cells for Fo3 chloroformic fraction of Fomitopsis officinalis(Vill.ex Fr.Bond.et Sing)extract.Flow cytometry was used to detect cell apoptosis in these groups.In Hep G2 cells,the induction of cell apoptosis for 30μg/ml is 12.06%,50 μg/ml is 40.34%,and in LO2 cells,3.8% and 42.61% respectively of total cells.There are high percentage levels of apoptosis in both cell lines treated than in control,but we noticed that the LO2 cell line showed highly significant results to Hep G2 cells in50 μg/ml treatment.These results suggest that pretreatment with Fo3 fraction implies the induction of cell apoptosis in HCC.Besides,we examined the IKBα,P65,and P50 apoptosis-related proteins as these genes are grouped in NF-κB(nuclear factor-kappa B)signaling pathway,although it is reported that the NF-κB pathway is constitutively active in HCC lines.Treating the HCC line Hep G2 and LO2 with 30,50μg/ml of Fo3 fraction obtained from Fomitopsis officinalis(Vill.ex Fr.Bond.et Sing)extract significantly in a dose-dependent manner increased the expression of P65,and the IKBα expression was increased in Hep G2 cells while slightly decreased in the LO2 cells.Furthermore,the expression P50 was only slightly increased in Hep G2 but was reduced in LO2 in either of the cancer cell lines in comparison with GAPDH.These results indicated that Fo3 chloroformic fraction of Fomitopsis officinalis might have selective cytotoxic effects and induce NF-κB in a time and dose-dependent manner,it could be considered a potential anti-cancer drug of HCC.Conclusions: Nowadays,mushrooms with lower toxicity are promoted as natural product-based pharmaceuticals contributing to the treatment of many pathological processes.We found that Fo3-chloroformic extract of Fomitopsis officinalis had strong antioxidant activity and significant anti-cancer effects in HCC cells.Fo3-chloroformic extract might selectively inhibit NF-κB protein expression,promote cell apoptosis and block the cell cycle,thereby suppressing the proliferation and metastasis of HCC cells.We revealed that a new natural product(Fo3-chloroformic extract of Fomitopsis officinalis)had potential active ingredients or antioxidant effects against HCC.There are 37 figures,6 tables and 374 references in this thesis.