| BackgroundCancer is a disease initiated by unfettered and unrestrained cellular growth.Cancer exists in malignant and benign forms.The cancer cells unable to move to the other body parts are named benign tumors.Almost 18.1 million new cancer cases appeared in 2020,while 10 million cancer-associated deaths were reported in the World last year.Lung cancer is the second most abundant cancer worldwide after breast cancer,and in the year 2020,the new lung cancer cases will occur 2,206,771(11.6%).Last five-year survival rate of lung cancer patients 10%-15% after the diagnosis in most countries during 2010-2014,although the higher rate of survival was reported in Japan(33%),Israel(27%)and Korea(25%).In the two-thirds of lung cancer mortality across the Globe attributable to smoking,lung cancer can be largely prevented through the effective policy of tobacco control and regulations of these policies worldwide.Liver cancer is the sixth commonly diagnosed cancer worldwide and it is counting third leading death due to liver cancer in 2020 GLOBOCAN.The incidence and mortality rate of liver cancer grows rapidly in the globe according to estimates of the World Health Organization(WHO)in 2019.Liver cancer ranks fifth in terms of global and reported is second on the basis of mortality in male population.Naturally extracted products from plants and medicinal,edible mushrooms are always suggested as the best option of therapeutics,focusing on reduced side effect and higher rate of efficiency.A mushroom has been used as medicine and food.Lentinula edodes is well-known and well-studied mushroom.Its pharmacological properties included: anti-cancer,anti-viral,anti-bacterial,anti-diabetic,anti-hyperlipidaemic,antioxidants,anti-cardiovascular,antimicrobial,and immune stimulator.Its clinical utilization as anticancer therapeutic drugs has observed to minimize the side effects when combined with traditional chemotherapeutic drugs.Although lung and liver cancer treatment have got advancements in recent past years,the effectiveness is still unsatisfactory.Chemotherapy has been used extensively for the treatment of various types of cancers with extensive side effects.Therefore,the development of novel agents with minimum side effects can enhance the chances of patient health recovery.Cancer research is getting attention due to the high scope and limitation in diagnosis and treatment.In the last few years,it was noticed that the burden of newly diagnosed lung and liver cancers has been rapidly increased,which acquired our attention and keen interest in addressing this problem efficiently.In this regard,our research team expressed and purified many secondary metabolites,including polysaccharides and polysaccharide-peptides from Lentinula edodes strain C91-3,and identified several cancer cell lines for their anticancer efficacy.The LP-7A from Lentinula edodes Strain C91-3was reported that they inhibited breast cancer cell line MDA-MB-231 and MCF-7.The LP1 and LP-7A successfully inhibited gastric cell line SGC-7901 and BGC-231 and induce apoptosis autophagy and cell cycle was arrested via inhibition PI3 K,Akt and m TOR signalling pathway.The anticancer role of LP-13 and LP-3 was noticed by arresting by cell cycle at phase and induced apoptosis by NF-κB signalling pathway in lung cancer A549 cell lines.Lp16-PSP shows an inhibitory effect anchorage-independent growth;p21 mediated at arrest the G1 phase of cell cycle and apoptosis of NF-κB in leukemia cell lines HL-60.LP-1 protein was reported to successfully inhibit lung cancer A549 cell line and its role in increasing apoptosis and induce autophagy.Our keen interest was to explore the potential role of LP-5 in liver cancer as an anti-cancer agent.The current research study is aimed to provide better and more efficient choice for dealing with lung and liver cancer.The focus of our study to find out the therapeutic effect of LP-5 on lung and liver cancer cell lines A549,H1299 and Hep G2.In this study,we discovered that LP-5 plays an important role in promoting apoptosis,autophagy,and cell cycle arrest in lung and liver cancer.These results indicate the significant anti-cancer activity of LP-5 in lung cancer as well as liver cancer cell lines.Our study discovered that LP-5 plays a significant role in apoptosis and cell cycle arrest in liver cancer cell lines Hep G2.These results disclose the significant anti-cancer efficacy of LP-5 in liver cancer and Latcripin-5(LP-5),on the other hand,has not been reported for its anti-cancer activity.Objectives1.To discover the sensitivity of LP-5 in lung and liver cancer cell lines.2.To investigate the important role of LP-5 in the inhibition of cell proliferation and cell viability.3.To determine either LP-5 induces cell cycle arrest in lung and liver cancer cell lines.4.To investigate the role of LP-5 in the induction of autophagy.5.To investigate the mechanistic pathways involvement in the stimulation of cancer cell death.6.To identify the inhibitory effect of LP-5 in the inhibition of lung cancer cells migration and invasion.MethodsPart 1:Homology modelling of LP-5 was performed via the phyre2 server.The LP-5 was synthesized from c DNA then cloned into plasmid,followed by the steps of Purification,refolding of functional domain,Dialysis,concentrated via PEG.Anti-cancer effects of LP-5 concentrated protein on liver cells were examined via CCK-8,cell cytotoxicity,cell viability,colony formation,and morphology assay.Apoptosis of LP-5 treated liver cancer cell lines Hep G2 were evaluated via q RT-PCR,flow cytometry and western blot.The Cell cycle arrestment was assessed q RT-PCR,flow cytometry and western blot.Part 2:LP-5 treated lung cancer cell lines(A549 and H1299)were assessed for cell viability,cytotoxicity,morphology,and colony formation via CCK-8 assay,acridine orange and DAPI Stain.Expressions of protein markers related to apoptosis(Bax,Cleaved Caspas-8,-9,Cytochrome-C,and Cleaved PARP.),cell cycle(CDK6,Cyclin D1 and E1,p21,MMP-2 and MMP-9),and autophagy(LC3I/II,Baclin,p62,and ATG12)were validated through Western blot analysis.Migration and invasion assays,wound healing assay were performed to analyse the LP-5 anti-migratory and anti-invasive properties,while apoptosis and cell cycle arrest analysis was performed via flow cytometry to assess its effect on cell growth.ResultsPart 1:Among seven cancerous and normal cell lines,the lowest IC50 value was reported against liver cancer line Hep G2 i.e.,58.15μg/ml.The morphological changes were cell shrinkage,blebbing formation,and cell fragmentation was observed in a dose and time-dependent manner.The apoptosis markers showed a higher level of expression of Bax,Caspass-3,-8,-9,CytochromeC,and PARP while the decreased expression level of Bcl2.Cell cycle arrest was observed that p21 and p27 were upregulated while CDK2,CDK4,CDK6,Cyclin D1 and Cyclin E1 were downregulated.Part 2:It is clearly observed that LP-5 effectively reduces migration and invasion and promotes apoptosis and autophagy in A549 and H1299 cancer cell lines.Significant upregulation was noted in apoptotic markers i.e.,Bax,Cleaved Caspas-8,-9,Cytochrome-C,and Cleaved PARP.Autophagy related marker i.e.,LC3I/II,Baclin,and ATG12 were increased while p62 was decreased.The cell cycle was arrest at G1 phase,cell cycle markers i.e.,CDK6,Cyclin D1 and E1 were downregulated while p21 was upregulated.LP-5,contributed the inhibition of migration and invasion while decreasing the expression levels of MMP-2 and MMP-9.ConclusionWe can conclude that Latcripin-5 is a promising anti-cancer therapeutic agent that can initiate intrinsic apoptosis and autophagy in lung cancer cells mediated through mitochondrial stress and arrest cell cycle at G1 phase in lung cancer cell lines A549 and H1299.It can also affect the proliferation of liver cancer cell lines Hep G2 which LP-5 induces apoptosis and arrest the cell cycle at the G1 phase.These studies findings show that LP-5 can be evaluated alongside other known anti-tumor and anti-cancer agents and details therapeutic suggestion can be investigate further study after tangible animal and preclinical trials. |
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