Anticancer Activity And Mechanisms Of Aloperine Against Bladder Cancer

The natural grasslands have been generally degraded in China for a long time under the influence of multiple natural and man-mad factors,mainly showing desertification and poisonous grassland,which has become a global environmental ecological problem.Sophora alopecuroides has formed a dominant population in desert and semi-desert degraded grassland in northwest China,especially in salinized grassland,which has a wide distribution area and a large reserve of resources in China.Sophora alopecuroides is recognized as a kind of poisonous plant from the perspective of grassland animal husbandry.Livestock are often forced to feed due to hunger,which causes poisoning and economic losses to the grassland animal husbandry.However,Sophora alopecuroides is resistant to salinization,sterility and drought from the perspective of population ecology.It is known as crucial vegetation for wind prevention sand fixation and salinization resistance,and plays an ecological role that other plants cannot replace in controlling desertification of desertification.At the same time,Sophora alopecuroides is also an important medicinal herb,its main active ingredient alkaloids have a wide range of pharmacological activities.Therefore,it is particularly important to utilize and develop Sophora alopecuroides as an available plant resource.As an important alkaloid in Sophora alopecuroides,aloperine shows to exert potent anticancer activity in multiple types of cancers.Bladder cancer is the most common malignancy in male genitourinary system in China with increasing new cases and deaths,which seriously endangers the public health.The main treatment of bladder cancer is surgery which is combined with adjuvant radiotherapy and chemotherapy.However,bladder cancer is prone to recurrence after surgery,and there are serious toxic and side effects after radiotherapy and chemotherapy.Hence,it is important for the treatment of bladder cancer to develop effective and non-toxic chemotherapeutic drugs for bladder cancer.Therefore,the potential anticancer activities,mechanisms and safeties were systematically investigated in this study.The main researches and results were as follows:(1)The anticancer activity of aloperine in bladder cancer in vitro.Aloperine was indicated to play potent anticancer activity against bladder cancer cells through screening 7alkaloid monomers including sophoridine,matrine,and the total alkaloid in Sophora alopecuroides.The IC₅₀ concentration ranges of aloperine for 48 h were 12.55?M?124.99?M.The potential anticancer activities of aloperine are identified in bladder cancer cells.The results showed that aloperine could significantly suppress cell viability,colony formation and adhesion in bladder cancer cells,induce G1 phase cycle arrest,apoptosis and autophagy.In addition,cell viability was significantly inhibited after exposed to aloperine with a short-time procedure.Aloperine played the anticancer activity mainly by inducing autophagy in bladder cancer cells.(3)Potential mechanisms of the anticancer effect of aloperine in bladder cancer cells by transcriptional analysis.Transcriptome analysis was performed to analyze the differential expressed genes alterations in aloperine-treated bladder cancer cells and to predict the potential anticancer target and signal pathways of aloperine.The results indicated that aloperine(100?M and 200?M)treatment for 24 h induced 2552 differential expressed genes alterations,regulated the expressions of 7 clinically relevant cancer genes including FLCN,CDKN1A,RET,KDR,AURKA,ERBB3 and MITF,which in turn regulated multiple biological functions of bladder cancer cells.Aloperine exerted the anticancer effects through activating the Lysosome,Inflammatory mediator regulation of TRP channels,and Glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate pathways in bladder cancer cells.Lysosome activation is the main pathway for aloperine against bladder cancer cells,and the transient receptor potential cation channel mucolipin 1(MCOLN1)may be the main target of aloperine.(4)Aloperine induced autophagy in bladder cancer cells by activating the ROS/MCOLN1-related pathways.The si RNA of MCOLN1 and the specific agonist ML-SA1 were used to evaluate the MCOLN1-dependent mechanism of aloperine induced autophagy in bladder cancer cells according to the data of transcriptome analysis.The upstream ROS regulation mechanism of aloperine induced MCOLN1 expression was assessed in bladder cancer cells.Following,the possibility of MCOLN1 as a potential target of drug therapy for bladder cancer was analyzed.The results showed that aloperine induced autophagy by promoting MCOLN1-mediated lysosomal activation and Ca²⁺release,as well as TFEB-nuclear translocation,thereby exerting its anticancer effect in bladder cancer cells.Intracellular ROS stimulated the expression of MCOLN1,which then mediated autophagy in bladder cancer induced by aloperine.In addition,the expression of MCOLN1 gene is correlated with the expressions of multiple tumor biomarkers of bladder cancer,and is also involved in improving the prognosis of patients with bladder cancer in collaboration with autophagy,suggesting the possibility that MCOLN1-mediated autophagy induction pathway may become a new target for the therapy of bladder cancer and even other cancers.(5)Aloperine suppressed mice orthotopic bladder tumor.The mice models of orthotopic bladder tumor were established using the bladder specific carcinogen BBN.The growth inhibition and autophagy induction of orthotopic bladder tumor were verified by bladder perfusion of aloperine.The si RNA for MCOLN1 interference was used to determine the mechanisms of aloperine against orthotopic bladder tumor in mice.The results showed that BBN successfully induced mice orthotopic bladder tumor with the tumor formation rate as 100%.Bladder perfusion of aloperine significantly inhibited growth and induced autophagy in orthotopic bladder tumor in mice,slowed down the weight loss of mice,and increased the expressions of MCOLN1 gene and protein.Aloperine inhibited the growth of mice orthotopic bladder tumor through the autophagy activation mechanism mediated by MCOLN1.(2)Safety of aloperine in mice.The LD₅₀ of aloperine in BALB/c mice by intraperitoneal injection was identified as 176.243 mg/kg by acute toxicity test.In the sub-acute toxicity,reversible hepatotoxicity and nephrotoxicity of intraperitoneal aloperine were observed in mice.Multiple cytochromes P450 genes,including CYP1A1,CYP1A2,CYP2C37,CYP2D9 and CYP3A1,were involved in the hepatotoxicity and nephrotoxicity induced by aloperine and in the recovery process of those toxicities.These results suggest that a recovery period is required for aloperine as a potential anticancer drug in animal and clinical researches.No obvious toxicity to the bladder tissues of mice was detected with bladder perfusion of aloperine(0?g/m L,50?g/m L,100?g/m L and 200?g/m L).In addition,no obvious influence on autophagy was detected in normal bladder by bladder perfusion of aloperine,suggesting the safety of aloperine for the development of chemotherapeutic agent of bladder cancer.In summary,aloperine exerted multiple anticancer activities in bladder cancer cells,induced autophagy in bladder cancer cells through a ROS/MCOLN1-dependent mechanism,inhibited orthotopic bladder tumor of mice,and played no obvious toxicity to normal bladder tissue.This study provided an important basis for elucidating the anticancer activities and molecular mechanisms of traditional Chinese medicines and their active ingredients as well as the resource utilization of Sophora alopecuroides.