Study On Anti-bladder Cancer Activity,Metabolic Characteristics And Structure-Activity Relationship Of Resveratrol In Vitro

Background and Objectives Bladder cancer(BC)is a very common malignancy of the urinary tract.In clinic,around 80%of BCs are transitional cell carcinoma,while,70～80%of TC are superficial.The standard treatment for this type of cancers is transurethral resection of bladder combined with adjuvant intravesical chemotherapy or immunotherapy.DDP is one of the first-generation chemotherapy agents approved by FDA as early as in 1978.However,this treatment may cause non-or reduced sensitivity of chemotherapy,such as primary drug resistance and acquired drug resistance,and adverse side effect,including hemorrhagic cystitis or even suppression of bone marrow,which limit the antitumor efficacy in patients.Therefore,it is necessary to explore more effective approach with less or not toxic to BC patients.Resveratrol(RV)is one of those natural product candidates,which has been regarded as a polyphenolic compound found in grapes,peanuts,Polygonum cuspidatum,and berries.Till now,it has been found that RV has multiple biological activities,such as anti-inflammatory,anti-tumor,antioxidative.Moreover,both in animal models and clinic study show that RV is non-toxic,which become a rational candidate for a combination with chemotherapy.Besides,several studies have shown that the synergistic inhibition effect of combination treatment with RV and other chemotherapy agents,such as DDP,didox,gefitinib,paclitaxel on different types of cancers,such as leukemias,breast cancers,prostate cancers and so on.Although RV is the promising natural compound could be considered in clinic in the future,there are still some questions needed to be answered.Is there combination efficacy of RV and DDP to inhibit drug resistant on bladder cancers?What is the metabolism pattern of RV,and the bioactive form in cell metabolism?How is the structure-activity relationship of RV chemical structure?Therefore,explore the efficacy,pharmacokinetics of combination therapy is expected to provide the foundation evidence for individual therapy of BC.Materials and methods Human BC cell lines T24 and EJ were cultured in Dulbecco’s modified Eagles medium(DMEM)with 10%fetal bovine serum.To mimic intravesical drug instillation in vitro,both of the cells were treated with different concentrations of DDP,or RV,or the combination of DDP and RV,and observed the morphological changes under microscope and H&E staining,MTT assay,cell migration and flow cytometry.In terms of figuring out molecular mechanism behind this,multiple approaches,including Western blot,RT-PCR,qRT-PCR,were used to determine the RNA and protein levels change.Secondly,the metabolites in T24 and EJ were detected and quantified,also qualified the real active form of RV by HPLC,LC/MS/MS,HRMS,separately.Certain concentration of RV was also treated the normal bladder epithelial cells extract from rats to detect the toxic effect.Lastly,to figure out the structure-activity relationship of RV,we determined the active group in the structure of RV both in supernatant and cell lysate by using the above techniquesResults1.Determination the inhibitory effect of RV and its potential mechanism on DDP-different sensitivity BC cellsDifferent sensitivity of T24 and EJ cells to RV and DDP was determined,T24 cells are more sensitive than EJ cells to both RV and DDP.The combination treatment of RV and DDP on T24 cells showed the effect of reduced dose of DDP was improved by RV to inhibit cell proliferation and reduce cell migration.The synergistic effect of DDP and RV on primary drug-resistant EJ cells to induce cell proliferation and reduce cell migration to reverse the drug resistant of EJ to DDP.The synergistic effect of DDP and RV on both cells were exerted through reduced drug resistant protein MRP2,induced DNA damage,activited ATM/ATR and its relevant downstream signaling pathway PARP-1,p53 and p21,increased cleaved Caspase-32.Metabolic bioactive pattern of RV in BC cellsHigh liquid performance chromatography,liquid chromatography mass spectroscopy and high resolution mass spectroscopy detected that different sensitive of those two cells generated the same RV metabolites,RVS and both exhibited upregulation of the RV-associated metabilic enzyme SULT1 A1.Yet T24 cells exhibited no sensitivity to an RVS mixture.No impact effect of phenol red in media was detected on the inhibitory effect or metabolic pattern of RV.Primary rat bladder epithelial cells showed no adverse effects when exposed to a therapeutic dose(100 μM)of RV.The differences in RV sensitivity between the two HBC cell lines did not reflect differences in the RV metabolic profile or SULT1A1 expression3.Metabolic profile and structure-activity relationship of RV and its analogues in human BC T24 cellsRV,oxyresveratrol(ORV)and acetylresveratrol(ARV)inhibit bladder cancer cells growth in a dose-and time-dependent manner,except polydatin(PD),and exert the anti-tumor potency to T24 cells in order of ORV>ARV>RV>PD.Meanwhile,similar metabolic profiles of the above compounds are found not only in cell supernatant and lysate,but also in dead and alive T24 cells after drug treatment,and the main metabolites of RV,ORV and PD are their prototypes,but ARV is mainly metabolized to RV.Conclusion1.The combination of RV and DDP have synergistic effect on sensitive T24 cells and improve inhibitory effect on DDP-resistant EJ cells;2.The synergistic effect of RV and DDP through regulated MRP2,directly damaged the DNA and regulated its relavant signal pathway to improve sensitivity of T24 cells to DDP,and reverse DDP-resistant in EJ cell;3.Different sensitivity of T24 and EJ cells generated same metabolites,and the main one is RV monosulfate(RVS);RV itself is responsible for antitumor activity;4.RV’s upregulated SULT1A1 expression and the estrogen-like hormones of RV were not correlated with T24 and EJ’s RV sensitivities;5.The therapeutic dosage of RV showed almost no side-effect to the primary rat cultured normal bladder epithelial cells;6.The number and position of free phenolic hydroxyl groups play a prominent role in antitumor activities,especially 3-OH and 4’-OH.Protecting phenolic hydroxyl groups,and inhibiting drug metabolism to keep phenolic hydroxyl groups free could increase or maintain RV anticancer activity.