| Background and Objectives: Bladder cancer is the most common malignancy ofthe urinary tract. It is three～four times more common in male than female. The risk fordeveloping this disease increases with age, with a peak between50and70years,especially for65-year-old people. About386000new bladder tumors were diagnosedworldwide in2008. It is responsible for over130000annual deaths worldwide eachyear. Moreover, bladder cancer patients have the highest lifetime treatment cost perpatient of all cancers, which has brought a serious economic burden to people.Nowadays bladder cancers have become a public health problem across the world.Transitional cell carcinoma (TCC, also known as urothelial cancer) representsabout90%of bladder cancers. Less common types include squamous cell carcinoma(3%～7%) and adenocarcinoma (2%). Around70%～85%of TCCs are superficial (Ta,T1and carcinoma in situ) and are also termed non-muscle invasive bladder cancer(NMIBC). The remaining15%～30%of TCCs are muscle invasive bladder cancer(T2-T4) and the outcomes of patients with this type are less favourable, nearly50%willdie from bladder cancer within5years of diagnosis.In clinical practice transurethral resection of bladder tumor (TURBT) is thestandard treatment for non-muscle invasive bladder cancers. However, the tumors recurin approximately50%～80%of patients and10%～25%of the recurrent tumors will progress to muscle invasive bladder cancers. Recurrence and tumor progression afterTURBT are the leading death causes of bladder cancer patients. To reduce the risk ofpost-TURBT tumor recurrence, adjuvant intravesical chemotherapy or immunotherapyis recommended for treatments of NMIBC. The commonly used chemotherapy agentsinclude cisplatin, mitomycin C (MMC), Bacillus Calmette-Guerin (BCG) and TNF-α.However, such treatments often have limited efficacy and adverse side effects, forexample, suppression of bone marrow, immunity function and hemorrhagic cystitis,which pose serious adverse effects on life qualities of patients. It is therefore in urgentneed to explore lesser toxic and more effective approach for better management ofTCCs.Resveratrol has been regarded as a non-toxic polyphenolic compound found ingrapes, berries, peanuts and red wine, which possesses varieties of biological activitiesincluding anti-tumor, anti-aging, anti-inflammatory, antioxidative, cardioprotective andneuroprotective effects and so on. Its non-toxic property is the greatest advantage ofresveratrol compared with other chemotherapy agents. A body of evidence shows thatresveratrol is able to inhibit the growth of many cancers such as retinoblastomas,leukemias, breast cancers, primary brain tumors, prostate cancers and melanomas. Theanti-tumor mechanisms of resveratrol were related with many signaling pathwaysinvolved in cell proliferation and apoptosis, for example, JAK/STAT3, Wnt/β-catenin,Notch, NF-κB and Sirt1signaling pathways. In the case of bladder cancers, resveratroleffectively decreases cell viability and induces apoptosis of human and murine bladdercancer cells. So resveratrol is likely to become a rational candidate for an idealintravesical agent.In vitro and in vivo experimental models are usually required to investigate theefficacy of novel antineoplastic drugs on bladder cancers. So far cultured bladdercancer cell lines are the most frequently used in vitro bladder cell model not only tostudy the mechanism involved in bladder cancer formation and development but alsoto evaluate new antitumor drug efficacy. The advantages of in vitro models applicationare that in vitro models offer the possibility to maintain cells in completely controlled environmental and experimental conditions, being less expensive and lesstime-consuming. The limitation of in vitro models is that the cell line consists of singlecell type and cells grow in vitro, which cannot simulate exactly the in vivocounterparts. Bladder tumors are composed of not only tumor cells but also stroma andblood vessels. In addition, in vitro models do not predict the adverse effects of drugs.Therefore prediction of drug efficacy in patients with bladder cancers based only on invitro studies is not completely reliable, and in vivo experimental models are essentialto investigate the efficacy of antitumor drug. Xenograft transplanted tumor mouse ornude mouse models are most employed for in vivo experimental models. Theadvantages of mice as animal models are small size, high reproductive rate, cleargenetic background and physiological and biochemical similarities to humans.Therefore bladder cancer animal models are indispensable to better evaluate theefficacy, safety and pharmacokinetics.For the above reasons, the efficacy and safety of resveratrol as an intravesicalchemotherapy agent for bladder cancer were evaluated by bladder cancer clinicallyrelated in vitro experimental model, heterotopic and orthotopic TCC nude mousemodels in our study, which is expected to provide preclinical proof-of-principle forresveratrol in clinical treatment of bladder cancer.The research contents include:(1) Cultured human TCC cell line, EJ, was firstly treated in short term byresveratrol to mimic clinical intravesical drug instillation, then the cellular andmolecular responses of EJ cells to the treatments were analyzed by multiple approaches.Meanwhile, the anti-tumor molecular mechanism of resveratrol was investigated invitro.(2) The anti-tumor efficacy and mechanism of resveratrol were evaluated in theestablished subcutaneous TCC transplanted tumor nude mouse model by multipleapproaches.(3) An orthotopic TCC nude mouse model was established by injecting EJ cellsinto the sub-urothelial layer and treated by intravesical resveratrol instillation. The cellular and molecular responses to those treatments and the antitumor mechanism ofresveratrol were evaluated thereafter.(4) Compared with the routine chemotherapy agent MMC, the local effects onnormal bladder mucosa of resveratrol were investigated in mice.Materials and methods: Human TCC EJ cell line is a high-grade, invasive humanTCC cell line that was established in1970from a patient undergoing cystectomy forhigh-grade invasive bladder cancer. Female BALB/c-nude mice and ICR mice wereprovided by Experimental Animal Centre of Dalian Medical University. Human TCC EJcells were cultured in Dulbecco’s modified Eagles medium (DMEM) containing10%fetal bovine serum. Firstly cultured EJ cells were exposed to different concentrations ofresveratrol in short term to mimic clinical intravesical drug instillation. The cellular andmolecular responses of EJ cells to the treatments were analyzed by H&E staining, MTTassay, TUNEL staining and flow cytometry. Meanwhile, in terms of the anti-tumormolecular mechanism of resveratrol, STAT3, p-STAT3and their downstream targets(c-Myc、 survivin、 cyclinD1and VEGF) were investigated in vitro by multipleapproaches of selective inhibitor of signaling pathway (AG490), RT-PCR,Western-blotting and immunocytochemical (ICC) staining. Secondly, the subcutaneoustransplanted tumor model was established through the method of injecting EJ cellssubcutaneously in the flanks of nude mice. Certain concentration of resveratrol wasdirectly injected into the peritumoral area to inhibit transplanted tumor growth. Thegrowth curve was plotted to compare the growth rate of control group and Res group.According to the data from the weight of excised tumors and body weight, the scatterdiagram about tumor weight and tumor burden of nude mice were plotted to exactlyreflect the tumor growth rates of both control and Res groups. Immunohistochemical(IHC) stainings were performed to detect the changes of STAT3signaling pathway(STAT3, p-STAT3and their downstream targets c-Myc、survivin、cyclinD1and VEGF).Thirdly, the orthotopic TCC transplanted tumor nude mouse model was established byinjecting EJ cells into the sub-urothelial layer and treated by resveratrol in the manner ofintravesical drug instillation. The cellular and molecular responses to resveratrol treatment and the antitumor mechanism of resveratrol were evaluated by multiplemethods. H&E staining was initially performed on bladder tissues to detect the successrate of tumor implantation. The tumor burdens were determined by weighing the wholebladders harvested from control and Res groups. TUNEL staining was performed onbladder tissues treated by resveratrol to detect cell apoptosis. IHC staining was used toevaluate the changes of STAT3signaling pathway in both control and Res groups in theorthotopic TCC nude mouse model. Finally, compared with routine chemotherapy agentMMC, the effects of resveratrol on normal bladder mucosa were investigated in ICRmice by H&E staining.Results1. The antitumor effects of short-term resveratrol in TCC cells and its molecularmechanism1.1Short-term resveratrol treatment suppressed TCC cell growth and induced cellapoptosisMTT assay revealed that EJ cell growth was suppressed by short-term resveratroltreatments in dose-and time-related fashions. H/E staining and TUNEL assay showedfrequent apoptotic death in2h150μM and200μM Res-treated groups. Flow cytometryshowed that2h150μM and200μM Res treatments caused S-phase arrest and apoptosisin EJ cell populations.1.2Resveratrol inhibited STAT3transcription and activationRT-PCR results showed that STAT3was expressed in normally cultured EJ cellsand down-regulated after2h200μM Res treatment for72hours. ICC staining performedon the same experimental groups revealed strong STAT3staining (+++) in eithercytosolic space or nuclei of normally cultured EJ cells, which apparently weakened (+)after72hours of2h200μM Res treatment. It was also found that p-STAT3was mainlylocalized in the nuclei of EJ cells and was diminished after2h200μM Res treatment for72hours. In accordance with the results of RT-PCR and ICC, Western blotting showedthat2h200μM Res treatment caused distinct STAT3and p-STAT3reduction.The levels of STAT3downstream genes (c-Myc, survivin, cyclinD1and VEGF) in normally cultured and2h200μM Res treated EJ cells were examined by the methods ofRT-PCR, ICC and Western blotting. The results revealed that the expression levels ofc-Myc, survivin, cyclinD1and VEGF were respectively down regulated in2h200μMRes-treated EJ cells.1.3Inhibitory effects ofAG490on EJ cellsICC staining demonstrated that STAT3phosphorylation was inhibited inAG490-treated cells in terms of reduction of STAT3and p-STAT3nuclear labeling. Theproliferation of EJ cells was suppressed by AG490in dose-and time-related fashion.Flow cytometry analysis revealed that48hour50μM and80μM AG490treatmentscaused S-phase arrest without inducing distinct apoptosis. These results are inconsistence with those of short-term resveratrol treatments. Only the apoptosispercentage was less than short-term resveratrol treatments. In comparison with normallycultured EJ cells, c-Myc, cyclinD1, survivin and VEGF expressions were individuallydown-regulated inAG490-treated cells.2. The antitumor effects of resveratrol and its molecular mechanism insubcutaneous TCC transplanted tumor nude mouse model2.1The subcutaneous TCC transplanted tumor nude mouse model was successfullyestablished by injecting subcutaneously EJ cells in the flanks of nude mice.2.2Injection of200μM resveratrol into subcutaneous peritumoral area inhibited thegrowth of subcutaneous bladder tumorsThe growth curve of subcutaneous bladder tumors showed that there were nodifferences in tumor volumes between control and Res groups (P>0.05) in the first8days after tumor implantation, but the tumor growth of control group was faster thanthat of Res group from days12to28(P<0.05). Further analysis of gross weight ofexcised tumors revealed significant reductions in Res treatment group compared withcontrol group (P=0.000). The ratios of tumor to body weight were much lower in Restreatment group (P=0.000) showing a significantly reduced tumor burden.2.3The activity of STAT3signaling pathway was inhibited by resveratrol in thesubcutaneous TCC transplanted tumor nude mouse model IHC stainings of STAT3, p-STAT3and their downstream target genes (c-Myc,cyclinD1and survivin) were performed in paraffin-embedded transplantedsubcutaneous bladder tumor tissues. IHC results of STAT3showed that the strongpositive staining of STAT3existed in nuclei and cytoplasm of EJ cells in control group,but denser （+++）in cell nuclei than in cell cytoplasm (++). In Res treatment group, thepositive staining of STAT3became apparently reduced in the nuclei（-）. MeanwhileIHC results of p-STAT3also revealed that the positive staining was strong in cell nucleiof tumor cells in control group (+++), and became significantly reduced in cell nucleitreated by resvertrol (-). In parallel, IHC results of STAT3downstream target genesshowed that c-Myc, cyclinD1and survivin were distributed in the nuclei and cytoplasmof EJ cells in control group and their expression levels were significantlydown-regulated after intravesical Res treatment group.3. The antitumor effects of intravesical instillation resveratrol and its molecularmechanism in orthotopic TCC transplanted tumor nude mouse model3.1The orthotopic TCC transplanted tumor nude mouse model was successfullyestablished by injecting EJ cells into sub-urothelial layer of bladder wallH/E staining was performed on the inoculated sites and confirmed that thesuccessful rate of orthotopic tumor implantation was100%(20/20).3.2Intravesical resveratrol treatment inhibited orthotopic tumor growth andinduced tumor cell apoptosisThe orthotopic tumor burdens were determined by weighing the whole bladdersharvested from control and Res groups, which revealed the greater mean bladder weightin NaCl-treated control group than that in resveratrol-treated group (0.03362g vs0.01625g, P＜0.01). TUNEL assay performed on orthotopic tumor samples with andwithout short-term resveratrol instillation demonstrated remarkable apoptotic death(apoptotic index=23.2%) in the former cases.3.3The activity of STAT3signaling pathway was suppressed by intravesicalinstillation resveratrol in the orthotopic TCC nude mouse modelIHC stainings of STAT3, p-STAT3and their downstream target genes (c-Myc, cyclinD1, survivin and VEGF) were performed in paraffin-embedded transplantedorthotopic bladder tumor tissues. IHC results of STAT3showed that the positivestaining of STAT3existed in nuclei and cytoplasm of EJ cells in control group, butdenser （+++）in cell nuclei than in cell cytoplasm (++). In Res treatment group, thepositive staining of STAT3became apparently reduced, especially in the nucle（i-）. IHCresults of p-STAT3revealed that the positive staining existed mainly in cell nuclei oftumor cells in control group (++), and became significantly reduced in cell nuclei oftumor cells after resveratrol treatment (-). The positive stainings of c-Myc, cyclinD1,survivin and VEGF were distributed in the nuclei and cytoplasm of EJ cells of controlgroup and significantly down-regulated, especially in cell nucle of tumor cells in Restreatment group.4. Effects of intravesical resveratrol instillation on normal bladder mucosa in ICRmice and nude mice compared with routine chemotherapy agent MMCBy the end of the experiment, the whole bladders were removed and fixed in10%neutral buffered formalin for H/E staining-based cystitis examination. The distinct bodyweight loss was found in MMC-treated group (18.1g in average) in comparison withthat in Res-treated group (27.2g in average; P＜0.01). The transitional epithelia ofurinary bladder walls were intact, and neither distinct capillary congestion norinflammatory lymphocyte infiltration was observed in the sub-mucosal space ofresveratrol-treated cancer-free ICR mice. Similar situation was also found in the tumorsurrounding bladder tissues of tumor-bearing nude mice treated by intravesicalresveratrol instillation. In contrast, severe epithelial damage, capillary congestion andhemorrhage could be observed in the MMC-treated urinary bladders of ICR mice.Conclusions1. Short-term resveratrol treatment effectively inhibited TCC cell growth andinduced tumor cell apoptosis in vitro.2. The inhibition of STAT3signaling activity was the main molecular mechanismfor the antitumor effects of resveratrol in vitro.3. Resveratrol could pose inhibitory effects on TCC transplanted tumor growth and induced tumor cell apoptosis in heterotopic and orthotopic nude mouse models ofbladder cancer.4. The inhibition of STAT3signaling activity as the main molecular mechanism forthe antitumor effects of resveratrol was confirmed in heterotopic and orthotopic nudemouse models of bladder cancer.5. Activated STAT3signaling was involved in bladder cancer formation andprogression.6. Because of the organ specificity of bladder, the instilled resveratrol can be easilyretained in the bladder and directly exerts its effects on the tumor tissue/cells withoutsystemical administration, avoiding the low intracellular bioavailability problem ofresveratrol in vivo. This is a great advantage of resveratrol used for clinical practice ofbladder cancer.7. No obvious local irritation of resveratrol-treated bladder mucosa was observedin mice and our data provide in vivo evidence of cancer-targeting property of resveratrolin the urinary bladder and further suggest the suitability of resveratrol in clinicaltreatment of human TCCs.In conclusions, for the first time the safety and efficacy of resveratrol in clinicaltreatment of bladder TCCs have been systematically validated in our study by in vitroexperimental model, heterotopic and orthotopic TCC transplanted tumor nude mousemodels. Our findings provide valuable preclinical proof-of principle for resveratrol asan ideal intravesical chemotherapy agent. |
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