| Backgroud:Urothelial bladder cancer(UBC),also called as transitional bladder cancer,is a relatively common malignancy and remains the leading cause of urinary disease-reelated death.UBC is the 9th most commonly diagnosed cancer in men and the 17th in women worldwide,and the mortality rate of UBC ranks in the 13th among all maliganant tumors.UBC is a heterogeneous disease which can lead to different clinical outcomes.Non-muscle-invasive bladder cancer(NMIBC)is the major histological subtype,which apprpxomately accounts for 70%of primary bladder tumor diagnoses and typically managed with local therapy,including transurethral resection of the bladder tumor(TURBT)and intravesical perfusion of Bacillus Calmette Guerin(BCG)or chemotherapeutic drugs.Unfortunately,NMIBC has the highest rate of recurrence among solid tumors,and after initial treatmen,50%of NMIBC patients tends to recur within five yeaes.This disease requires a number of treatments and brings a huge economic burden to patients and sociery.Furthermore,as many as 20%of NMIBCs eventually develop in muscle-invasive bladder cancer(MIBC)with local lymph node and distant metastas,which predicts poor prognosis.The latest researches finds that the incidence of bladder cancer(BCa)is increasing every year.Although current treatments can reduce the risk of tumor metastasis and improve the prognosis of patients,there are still many patients who can’t respond to cisplatin or tolerate its side effects,and drug resistance will occur as the disease progresses.Accordingly,it is significant to urgently find new anti-tumor drugs for clinical treatment against patients with bladder cancer.Chinese traditional herbs,widely distributed in China,are the traditional medicine resources,and have a wide range of pharmacological effects for clinical applications,such as anti-inflammatory,lipid modulation,antivirus,and antitumor.Due to advantages of their low toxicity and less-adverse reaction,Chinese traditional herbs are considered as ideal anti-cancer drugs.At present,More than 50%of chemotherapeutic drugs approved by the U.S.Food and Drug Administration(FDA)are derived from terrestrial plants.Etoposide,paclitaxel and vincristine have been used in anti-tumor therapies.Berbamine(BBM),initially identified as an effective antileukemic agent extracted from the herbal medicine Berberis,garnered much attention.Its structure formula is C37H40N2O6.Subsequent researches further corroborate the antitumor properties of berbamine and its derivatives in various carcinomas,such as colon cancer,ovarian cancer,prostate cancer,and liver cancer.Meanwhile,berbamine has no obvious cytotoxicity to normal cells,which reaveals its great potential in anti-tumor therapy.Nonetheless,there have been no reports so far involving the effects of berbamine in bladder cancer.ObjectiveThe aim of this study was to determine the inhibitory effects of berbamine on bladder cancer in vitro and in vivo and to clarify the molecular mechanisms involved in the regulation of biological activities.Our research provides a basis for further comprehensive study and presents a potential candidate for clinical treatment strategies against bladder cancer.Methods1.Regulation of berbamine on the growth of bladder cancer cells in vitro(1)BCa cells(5637 and T24)were selected in our study.After BCa cells were incubated with different concentration of berbamine for 24 h or 48 h in vitro,we use CCK-8 assay to evaluate the regulation of berbamine on the cell viability,and 50%inhibitory concentration(IC50)values of berbamine for different cells at 24 h or 48 h were calculated,respectively.(2)After treatment for 48 h,colony formation assay was conducted to evaluate the influence of berbamine in the formation abilities of BCa cells,EdU+assay and Ki-67 immunofluorescence assay were applied to explore the effects of berbamine on the proliferation of BCa cells.(3)After treatment for 48 h,collect cells for RNase A/PI staining,then flow cytometry was used to detect the regulation of berbabmine on the cell cycle of BCa cells,meanwhile the difference in the protein levels of cell cycle regulators involving P21,P27,CyclinD,CyclinA2 and CDK2 were examined using western blot.2.Effects of berbamine on the metastasis of bladder cancer cells in vitro(1)After treatment for 48 h,wound healing assay was performed to detect the regulation of berbamine on the migratory ability of BCa cells,and Transwell assay was used to evaluate the regulation of berbamine on the migratory and invasive capacities of BCa cells,then western blot was applied to detect the influence of berbamine in the the expression of N-cadherin,E-cadherin,Vimentin and MMP-9 proteins of epithelia-mesenchymal transition(EMT)markers in BCa cells.(2)After treatment for 48 h,phalloidin staining was performed to visualize the effects of berbamine on F-actin cytoskeleton arrangement.3.The role of the NF-κB pathway in berbamine-mediated the biological activities of bladder cancer cells(1)After treatment for 48 h,western blot experiment was performed to examine the effects of berbamine on the difference in the protein expression of key proteins P-65,P-P65 and P-IκB in nuclear factor kappa-B(NF-κB)signaling pathway of BCa cells.(2)After the NF-κB pathway-specific inhibitor Bay-11-7082 alone or in combination with berbamine treated BCa cells,CCK-8 assay was used to detect the cell viability.(3)P65 overexpression plasmid(pcDNA-3.1-P65)was constructed and transfected into BCa cells,then qRT-PCR and western blot were performed to detect the transfection efficiency.(4)After berbamine treated BCa cells for 48 h,or combined with pcDNA-3.1-P65 transfection,the influence in the total P-65 and P-P65 protein expression of BCa cells was detected using western blot.EdU+assay and Transwell assay were performed to evaluate the regulation of NF-κB inhibition mediated by berbamine on the proliferative and metastatic abilities of BCa cells,respectively.4.Regulation of berbamine on ROS generation and cell apoptosis of bladder cancer cells(1)After berbamine treatment for 24 h,MitoSOX fluorescence was used to explore the effects of berbamine on the reactive oxygen species(ROS)levels of BCa cells,and western blot was conducted to determine the difference in the protein expression of several antioxidative genes(Nrf2,HO-1,SOD2 and GPX1)of BCa cells.(2)After berbamine treatment for 24 h,collect cells for Annexin V-FITC/PI double staining,then flow cytometry was applied to examine the regulation of berbabmine on the apoptosis of BCa cells,meanwhile the difference in the protein levels of apotosis-related genes(Bcl-2 and Bax)were determine using western blot.5.The role of ROS/NF-κB axis in anti-tumor activities of berbamine in bladder cancer(1)Add N-acetylcyseine(NAC),a ROS scavenger,to the berbamine treatment group for 2 h in advance,MitoSOX fluorescence was used to detect the generation of ROS in BCa cells,furthermore,the regulation of berbabmine on the apoptosis of BCa cells was examined using flow cytometry,meanwhile western blot was applied to determine the difference in the expression of Bcl-2 and Bax proteins in BCa cells.(2)After NAC pre-treatment for 2 h in the berbamine treatment group,the difference in the levels of P-65 and P-P65 proteins was determine using western blot.6.The effects of berbamine on growth of bladder cancer tumors in vivo(1)A subcutaneous BCa xenograft model was constructed,and the mice were intraperitoneally injected with berbamine at 35 mg/kg body weigh every three days.During the experiment period,the weight and volume of implanted tumors as well as mice were weighed and recorded,and the growth curve was drawn.(2)Extract the implanted tumor and imbed it in paraffin for slices,Ki-67 and P-65 were stained using Immunohistochemistry(IHC)Results1.Berbamine suppressed the growth of bladder cancer in vitro(1)Berbamine inhibited the viability of both 5637 and T24 cells in a concentration and time-dependent manner.The 50%inhibitory concentration(IC50)values of berbamine for 5637 and T24 cells at 24 h were 31±3.259 μM and 41±0.37μM,respectively.Furthermore,the IC50 values of berbamine at 48 h were 15.58±2.489 μM and 19.09±0.68 μM,respectively.Therefore,we applied the suitable administration(8 μM and 16 μM of berbamine for 48 h or 16 μM and 32 μ2M of berbamine for 24 h)to explore the regulation of berbamine on the biological activities of BCa.(2)Berbamine dose-dependently decreased the number of colonies in 5637 and T24 cells.Following berbamine treatment,the percentages of EdU-positive BCa cells were dramatically reduced compared to that in the control group.Immunofluorescence assay indicated that mean gray values of Ki-67,a vital marker of cell proliferation,were notably decreased in the berbamine treatment group.(3)Berbamine increased the percentage of cells in S phase and exhibited a dose-dependent trend,but the proportion of cells in G0/G1 phase and G2/M phase did not change significantly.Furthermore,the expression of cyclin-dependent kinase inhibitors(CKIs)p21 and p27 was clearly upregulated upon berbamine treatment.In contrast,berbamine dramatically downregulated the levels of CyclinD,CyclinA2 and CDK2.2.Berbamine inhibited the metastasis of bladder cancer in vitro(1)In scratch test,berbamine obviously inhibited the migratory ability of BCa cells.In transwell assays with or without Matrigel,berbamine induced significant decreases in the numbers of cells that metastasize to the lower chamber.After berbamine treatment,the level of E-cadherin expression was augmented,and the expression of N-cadherin,Vimentin and MMP-9 was concomitantly downregulated in both cell lines,which indicated that EMT was suppressed by berbamine.To sum up,berbamine inhibited the metastatic ability of BCa cells through reversing EMT process.(2)Phalloidin staining indicated that berbamine reduced cellular protrusion and contact surfaces between cancer cells.Moreover,berbamine transformed the spindle-and fibroblast-like appearance of BCa cells into a cobblestone-like morphology.3.Berbamine inhibited the biological activities of bladder cancer by suppressing the NF-κB pathway(1)Western blot results found that berbamine significantly downregulated the levels of total P-65,P-P65 and P-IκB proteins in BCa cells,which indicated the NF-κB pathway was inhibited by berbamine.(2)CCK-8 assay demonstrated that the NF-κB pathway-specific inhibitor BAY-11-7082 alone exhibited superior inhibitory activity,and had a synergistic effect to exacerbate cytotoxicity mediated by berbamine.(3)qRT-PCR and western blot results showed that compared to the control group,the P-65 expression of BCa cells dramatically increased,indicating successful(4)After pcDNA3.1-P65 transfection,the levels of P-65 and P-P65 inhibited by berbamine were up-regulated.In the rescue experiments,P-65 overexpression partially abolish the inhibitory effects of berbamine on proliferation and metastasis of BCa cells.4.Berbamine triggered ROS generation and cell apoptosis in bladder cancer(1)MitoSOX assay demonstrated that berbamine significantly accelerated ROS accumulation in BCa cells,which is associated with downregulation of antioxidative gene(Nrf-2,HO-1,SOD2 and GPX-1)expression.(2)Berbamine increased the expression of proapoptotic Bax protein,while significantly inhibiting the level of antiapoptotic Bcl-2 protein in BCa cells,thus inducing the apoptosis of BCa cells.5.Berbamine exerted anti-tumor activity against bladder cancer cells by modulating the ROS/NF-κB axis(1)Pretreatment with NAC inhibited berbamine-mediated mitochondrial ROS generation,followed by a decrease in BCa cell apoptosis.Consistent with the flow cytometry assay results,western blot analysis showed NAC partially reversed the regulations of berbamine on the protein levels of Bcl-2 and Bax in BCa cells.(2)After NAC was added to the berbamine-treated group,the levels of total P-65 and P-P65 proteins,to some extent,were up-regulated in BCa cells.6.Anti-tumor effects of berbamine in vivo(1)The in vivo experiment showed that tumor volume and weight in berbamine treatment group were grew much more slowly than that in the control group.However,there was no significant difference in the average weight of mice between the two groups.(2)IHC analysis proved that berbamine inhibited the positive expression of Ki-67 and P-65 in BCa cells,which indicated that berbamine suppressed the tumor growth and NF-κB pathway in vivo.ConclusionOur study elucidated for the first time that berbamine could exert anti-tumor activities in bladder cancer by inhibiting cell proliferation as well as metastasis and inducing cell cycle arrest at S phase in vitro.Further analysis highlighted that berbamine suppresses the aberrantly active NF-κB pathway to interfere with the progression of bladder cancer.In addition,ROS accumulation induced by berbamine contributes to the intrinsic apoptosis of bladder cancer cells and inhibits the NF-κB pathway to some degree.Therefore,ROS/NF-κB axis plays a vital role in the antitumor activity of berbamine against bladder cancer.Consistent with our in vitro experiments,berbamine blocked the NF-κB signaling pathway,and inhibited the tumor growth in our xenograft model,while having no evident side effects in vivo.Based on the above results,it can be concluded that berbamine has potential clinical applications for patients with bladder cancer.Futher studies are encouraged to ensure its drug safety and clarify its broader mechanisms. |
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