OBJECTIVE: Cigarette smoking is the main risk factor for bladder cancer development. Among the mediators of this effect of smoking is nuclear factor-kappa B. Curcumin suppresses cellular transformation by downregulating the activity of nuclear factor-kappa B. Prima-1 is a compound that induces apoptosis in human tumor cells, restoring the function of mutant p53. Our study aimed to evaluate the effects of curcumin and prima-1 in an animal model of bladder cancer. METHODS: Tumor implantation was achieved in six- to eight-week-old female C57BL/6 mice by introducing MB49 bladder cancer cells into the bladder. Intravesical treatment with curcumin and Prima-1 was performed on days 2, 6, 10, and 14. On day 15, the animals were sacrificed. Immunohistochemistry was used to determine the expression of cyclin D1, Cox-2, and p21. Cell proliferation was examined using PCNA. RESULTS: Animals treated with curcumin exhibited a higher degree of necrosis than animals in other groups. Immunohistochemistry showed reduced expression of cyclin D1 in the curcumin-treated group. All of the cells in mice treated with curcumin were p21 positive, suggesting that the p53 pathway is induced by this compound. Prima-1 did not induce any change in tumor size, necrosis, cell proliferation, or the expression of proteins related to the p53 pathway in this animal model. CONCLUSION: Curcumin showed activity in this animal bladder cancer model and probably acted via the regulation of nuclear factor-kappa B and p53. Therefore, curcumin is a good choice for the use in clinical trials to treat superficial bladder cancer as an alternative to bacillus Calmette-Guerin. In contrast, Prima-1 does not seem to have an effect on bladder cancer

Chade, Daher C.

Dall&#8217

Leite, Katia R. M.

Piantino, Camila

Reis, Sabrina T.

Srougi, Miguel

Watanabe, Fábio T.

English

Cadernos Espinosanos (E-Journal)

BASIC RESEARCHCurcumin, but not Prima-1, decreased tumor cellproliferation in the syngeneic murine orthotopicbladder tumor modelFa´bio T. Watanabe,I Daher C. Chade,II Sabrina T. Reis,I Camila Piantino,I Marcos Francisco Dall’ Oglio,IIMiguel Srougi,I Katia R. M. LeiteII Faculdade de Medicina da Universidade de Sa˜o Paulo, Laboratory of Medical Investigation, Department of Urology (Lim55), Sa˜o Paulo/SP, Brazil.II Faculdade de Medicina da Universidade de Sa˜o Paulo, Instituto do Caˆncer do Estado de Sa˜o Paulo (ICESP), Department of Urology, Sa˜o Paulo/SP, Brazil.OBJECTIVE: Cigarette smoking is the main risk factor for bladder cancer development. Among the mediators of thiseffect of smoking is nuclear factor-kappa B. Curcumin suppresses cellular transformation by downregulating the activityof nuclear factor-kappa B. Prima-1 is a compound that induces apoptosis in human tumor cells, restoring the function ofmutant p53. Our study aimed to evaluate the effects of curcumin and prima-1 in an animal model of bladder cancer.METHODS: Tumor implantation was achieved in six- to eight-week-old female C57BL/6 mice by introducing MB49bladder cancer cells into the bladder. Intravesical treatment with curcumin and Prima-1 was performed on days 2, 6,10, and 14. On day 15, the animals were sacrificed. Immunohistochemistry was used to determine the expression ofcyclin D1, Cox-2, and p21. Cell proliferation was examined using PCNA.RESULTS: Animals treated with curcumin exhibited a higher degree of necrosis than animals in other groups.Immunohistochemistry showed reduced expression of cyclin D1 in the curcumin-treated group. All of the cells inmice treated with curcumin were p21 positive, suggesting that the p53 pathway is induced by this compound.Prima-1 did not induce any change in tumor size, necrosis, cell proliferation, or the expression of proteins related tothe p53 pathway in this animal model.CONCLUSION: Curcumin showed activity in this animal bladder cancer model and probably acted via the regulationof nuclear factor-kappa B and p53. Therefore, curcumin is a good choice for the use in clinical trials to treatsuperficial bladder cancer as an alternative to bacillus Calmette-Guerin. In contrast, Prima-1 does not seem to havean effect on bladder cancer.KEYWORDS: Bladder cancer; Treatment; Curcumin, Prima-1; Apoptosis.Watanabe FT, Chade DC, Reis ST, Piantino C, Dall’ Oglio MF, Srougi M, et al. Curcumin, but not Prima-1, decreased tumor cell proliferation in thesyngeneic murine orthotopic bladder tumor model. Clinics. 2011;66(12):2121-2124.Received for publication on July 7, 2011; First review completed on August 8, 2011; Accepted for publication on August 10, 2011E-mail: katiaramos@uol.com.brTel.: 55 11 30617183INTRODUCTIONCancer of the urinary bladder is diagnosed in approxi-mately 71,500 people in the United States, and approximately14,500 individuals die of the disease each year.1 The conditionaffects individuals 60 to 70 years of age, and racial and ethnicvariations in its incidence have been observed.2-4 Currenttreatment for superficial urothelial carcinoma includes endo-scopic tumor resection, followed by intravesicular adminis-tration of bacillus Calmette-Guerin (BCG), a treatment thathas well-known side effects.5 Topical chemotherapy withmitomycin, thiotepa, and epirubicin has been used as analternative, but this treatment has no impact on long-termsurvival or disease progression. The toxicity and inefficacy ofthese intravesicular agents prompted us to explore newtreatments for superficial urothelial carcinoma of the bladder.The use of tobacco is one of the main causes of bladdercancer development, which is due to the presence of certaincompounds present in cigarette smoke (CS). Exposure tosome of these compounds is associated with the induction ofnuclear factor-kappa B (NF-kB). We previously studied theeffects of curcumin in an orthotopic murine bladder tumormodel, demonstrating that curcumin has an inhibitory effecton bladder urothelial cancer, possibly by downregulating NF-kB-related genes,6 Therefore, curcumin could be an option forthe treatment of urothelial neoplasms.7 The tumor suppressorprotein p53 inhibits tumor growth primarily through itsability to induce apoptosis. Mutations in p53 occur in at least50% of human tumors, and p53 is characteristically mutatedin high-grade, invasive urothelial carcinoma. Prima-1 is a low-molecular-weight compound able to induce apoptosis inCopyright  2011 CLINICS – This is an Open Access article distributed underthe terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided theoriginal work is properly cited.No potential conflict of interest was reported.CLINICS 2011;66(12):2121-2124 DOI:10.1590/S1807-593220110012000192121human tumor cells by restoring the transcriptional transacti-vation function of mutant p53. In the presence of Prima-1,mutant p53 proteins regain their sequence-specific DNAbinding ability and their active conformations in vitro and inliving cells. In vivo studies in mice revealed that Prima-1 hasan anti-tumor effect with no apparent toxicity.8Now, using the same animal model,9 we assessedwhether curcumin function is enhanced by Prima-1 or ifthere is a specific role of curcumin that could be used totreat bladder cancer.MATERIALS AND METHODSOrthotopic Tumor ImplantationSix- to- eight-week-old female C57BL/6 mice were putunder general anesthesia with an intraperitoneal injection of150 mL of a mixture of xylazine-ketamine-PBS (15158). Then,a 24-gauge Teflon intravenous IV catheter was insertedthrough the urethra into the bladder using xylocaine gel. Toprepare the bladder for tumor implantation, a chemical lesionon the bladder wall was made by intravesicular administra-tion of 0.15 M AgNO3 (10 mL). This promoted an adequateand controlled diffuse cauterization of the bladder wall. After10 seconds, the contents were washed out by transurethralinfusion of PBS. Then, a suspension of 56105 viable tumorcells of the murine transitional cell carcinoma cell line MB49(MB49) was introduced into the bladder. The day of theadministration of tumor cells was considered to be Day 1.TreatmentTreatment was performed by administering the testeddrugs four times. Twenty-four hours following tumor cellintroduction (Day 2), therapy with 100 mL intravesicularcurcumin (100 mM - Sigma) or 100 mL Prima-1 (100 mM)was initiated. The ‘‘curcumin+Prima-1’’ treatment groupreceived 100 mL of a 151 mixture of curcumin (200 mM) andPrima-1 (200 mM). Mice were assigned to 1) the controlgroup, which received 100 mL saline (n = 10); 2) the ‘‘onlytumor’’ group (n = 10), which received tumor cells and werenot handled again until sacrifice; and 3) treatment groupsreceiving curcumin (n = 10), Prima-1 (n = 10) or a mixture ofboth (n = 10). The other three drug applications occurred ondays 6, 10, and 14.Assessment of the tumorsThe mice were evaluated on a daily basis for viability andgross hematuria. At Day 15, they were sacrificed by CO2inhalation. The bladder, lungs, kidneys, and liver wereresected for histological examination.Histopathology analysisThe specimens were fixed in 10% buffered formalin andsectioned for histological examination. For the bladderslides, tumors were measured, and the degree of necrosiswas recorded. Sections of the lungs, kidney, and liver werealso analyzed to identify the presence of metastases.ImmunohistochemistryThree-micrometer sections from the paraffin block wereplaced on adhesive-coated slides. For antigen retrieval, theslides were placed in citrate buffer (1 mM, pH 6.0) andheated for 30 min in the steamer. The slides were thenincubated overnight at 4 C˚ with monoclonal antibodiestargeting cyclin D1 (Spring, 15100 dilution), Cox-2 (Spring,15100), p21 (Millipore, 1550), and PCNA (Dako, 15200). TheLSAB system was used for immunostaining. The color wasdeveloped using a 3,39-diaminobenzidine substrate-chromo-gen solution, followed by counterstaining with Harrishematoxylin. Samples were then dehydrated, preservedwith a cover slip and reviewed using light microscopy.Statistical analysisStatistical analyses were performed using ANOVA andchi-squared tests for the evaluation of histological char-acteristics and immunohistochemistry findings betweendifferent groups of animals using the GraphPad Prism 5.0software.RESULTSInitially, the study included 50 animals distributed amongthe groups. Five animals in the control (saline) group wereexcluded from the study due to a possible bladder infectionobserved by microscopy. Two animals died prematurely,probably as the result of the anesthesia. Five mice died later,possibly due to the neoplasm. In four animals, the tumorimplantation was not successful. These animals were alsoexcluded from the study. After these exclusions, the studyincluded 34 mice.The results are shown in Table 1. We found that animalstreated with curcumin showed a higher degree of necrosisthan the mice in all other groups. The mean percentage oftumor necrosis in mice treated with curcumin was 67%,whereas in the other groups, the mean was only 27%(p,0.001).The mean tumor sizes at the end of the study in theuntreated and saline-injected groups were 4.2 mm and6.2 mm, respectively. Although these sizes seem muchlarger than that in the curcumin-treated group (mean size2.9 mm), there were no statistically significant differences.We next compared the expression of genes regulated by NF-kB. Cyclin D1 was expressed in fewer cells in the group treatedwith curcumin, with a mean percentage of 23% compared with34%, 53%, 50%, and 60% in the Prima-1, curcumin and Prima-1, saline control, and untreated control groups, respectively(p,0.001). There was no statistically significant difference inCox-2 expression between groups (p= 0.064).We also analyzed the expression of p21, which isregulated by p53, a frequently mutated gene in invasivebladder cancer. Because Prima-1 restores the activity of themutated p53 protein, we expected to find some difference inthe expression of p21 among these groups of animals.However, there were no differences in the expression of p21between the different groups of mice (p= 0.55).In addition, proliferative activity, as measured usingPCNA, was similar among the treatment groups.DISCUSSIONOur results confirm our previous finding that intravesi-cular injection of curcumin was able to promote bladdertumor necrosis. Although there was no statistically sig-nificant difference in tumor size between the differentgroups, tumors treated with curcumin were smaller,especially when compared with those exposed to salinealone (mean 2.85 mm vs. 6.23 mm).Recently, we showed that curcumin promotes apoptosisin MB49 mouse bladder tumor cells in vitro and causes aCurcumin and Prima-1 in an in vivo bladder cancer modelWatanabe FT et al.CLINICS 2011;66(12):2121-21242122decrease in tumor size in vivo in the syngeneic orthotopicmurine bladder cancer model.7,8 Garg and Aggarwal10previously discovered that curcumin functions by modulat-ing TNF-induced NF-kB activation. NF-kB activates thetranscription of genes related to cell proliferation, includingcyclin D1 and Cox-2. We confirmed that curcumin affectsNF-kB function in our animal model, reducing the expres-sion of cyclin D1 in mice treated with this substancecompared to untreated animals, in agreement with ourprevious results. The mean percentage of cyclin D1-positivetumor cells was 23% in animals treated with curcumin and49% in untreated mice (p,0.01). However, we did not finddifferences in the regulation of other genes related to theNF-kB pathway among the groups of animals. The Cox-2expression profile varied widely in animals treated withcurcumin, Prima-1, or both. In addition, there was nodifference in terms of cell proliferation.Chen et al.11 were able to show that BCG activatesmultiple intracellular signaling pathways, including NF-kB,in MB49 cultures. BCG is the standard of care for thetreatment of low-grade pTa and pT1 urothelial carcinomasand reduces tumor recurrence without affecting specificcancer survival rates. However, one-third of patients fail torespond to BCG, and BCG can cause many side effects. Inthis in vivo mouse model, we demonstrated that curcuminaffects tumor behavior in a manner similar to that of BCGwithout side effects. Curcumin, a compound derived fromCurcuma longa Linn, is not easily absorbed, and thepossibility of using it as an intravesicular anti-cancer drugis very attractive.The rationale for testing Prima-1 in this animal model wasthat Prima-1 can restore p53 transcriptional activity. Weexpected to observe an effect of Prima-1 on tumor size,apoptotic index or p21 protein expression either whenPrima-1 was used alone or when it was used together withcurcumin. The transcription of p21/WAF1 is controlled byp53, and the loss of its expression has been related to poorprognosis in bladder cancer.12 However, we saw nodifferences in these studies between animals treated withor without Prima-1. We can speculate that p53 plays no rolein this tumor model.However, 100% of tumor cells in mice treated withcurcumin were p21 positive, suggesting that p53 can beinduced by this compound, as previously observed in invitro and in vivo models of bladder cancer13 and in othertumors, such as neuroblastomas14 and adenocarcinomas ofthe prostate15 and colon.16We conclude that curcumin is a good choice for use inclinical trials as an alternative to BCG to treat superficialbladder cancer because curcumin controls the geneticpathways important in the carcinogenesis of this neoplasia.In contrast, Prima-1 does not seem to have an effect on thistumor.AUTHOR CONTRIBUTIONSWatanabe FT conceived and designed the study and was also responsiblefor the draft of the manuscript. Chade DC was responsible for the datacollection. Reis ST conceived and designed the study and was alsoresponsible for the statistical analysis. Piantino C was responsible for theadministrative support. Dall’Oglio MF was responsible for the datacollection, critical revision and important intellectual content of themanuscript. Leite KRM conceived and designed the study and wasresponsible for the critical revision and important intellectual content of themanuscript. Srougi M supervised the study.REFERENCES1. Jemal A, Siegel R, Xu J, Ward E. Cancer statistics. 2010. CA Cancer J Clin.2010;60:277-300, Epub 2010 Jul 7, doi: 10.3322/caac.200732. Lynch CF, Cohen MB. Urinary system. Cancer. 1995;75:316-29, doi: 10.1002/1097-0142(19950101)75:1+,316::AID-CNCR2820751314.3.0.CO;2-T.3. Scosyrev E, Noyes K, Feng C, Messing E. Sex and racial differences inbladder cancer presentation and mortality in the US. Cancer. 2009;115:68-74, doi: 10.1002/cncr.23986.4. Howe HL, Wu X, Ries LA, Cokkinides V, Ahmed F, Jemal A, et al.Annual report to the nation on the status of cancer, 1975-2003, featuringcancer among U.S. Hispanic/Latino populations. Cancer. 2006;107:1711-42, doi: 10.1002/cncr.22193.5. Herr HW, Wartinger DD, Fair WR, Oettgen HF. Bacillus Calmette-Guerin therapy for superficial bladder cancer: a 10-year followup. J Urol.1992;147:1020-3.6. Leite KR, Chade DC, Sanudo A, Sakiyama BY, Batocchio G, Srougi M.Effects of Curcumin in an Orthotopic Murine Bladder Tumor Model. IntBraz J Urol. 2009;35:599-607, doi: 10.1590/S1677-55382009000500012.7. Chade DC, Andrade PM, Borra RC, Leite KR, Andrade E, Villanova FE,et al. Histopathological characterization of a syngeneic orthotopicmurine bladder cancer model. Int Braz J Urol. 2008;34:220-6; discussion226-9, doi: 10.1590/S1677-553820080002000138. Bykov VJ, Issaeva N, Shilov A, Hultcrantz M, Pugacheva E, ChumakovP, et al. Restoration of the tumor suppressor function to mutant p53 by alow-molecular-weight compound. Nat Med 2002;8:282-8, doi: 10.1038/nm0302-282.9. Gu¨nther JH, Jurczok A, Wulf T, Brandau S, Deinert I, Jocham D, et al.Optimizing syngenic orthotopic murine bladder cancer (MB49). CancerRes. 1999;59:2834-7.10. Garg A, Aggarwal BB. Nuclear transcription factor kB as a target forcancer drug development. Leukemia. 2002;16:1053-68, doi: 10.1038/sj.leu.2402482.11. Chen F, Zhang G, Cao Y, Hessner MJ, See WA. MB49 Murine UrothelialCarcinoma: Molecular and Phenotypic Comparison to Human Cell Linesas a Model of the Direct Tumor Response to Bacillus Calmette-Guerin.J Urol. 2009;182:2932, doi: 10.1016/j.juro.2009.08.018.12. Korkolopoulou P, Konstantinidou AE, Thomas - Tsagli E, ChristodoulouP, Kapralos P, Davaris P. WAF1/p21 protein expression is anindependent prognostic indicator in superficial and invasive bladdercancer. Appl. Immunohistochem. Mol. Morphol. 2000;8:285-92, doi: 10.1097/00022744-200012000-00005.13. Tian B, Wang Z, Zhao Y, Wang D, Li Y, Ma L, et al. Effects of curcuminon bladder cancer cells and development of urothelial tumors in a ratbladder carcinogenesis model. Cancer Lett. 2000;264:299–308, doi: 10.1016/j.canlet.2008.01.041.Table 1 - Summary of the findings for the different groups of animals treated with curcumin, Prima-1, orcurcumin+Prima-1 and the control animals.Morphological aspects ImmunohistochemistryGroupPercentage of necrosis(Mean) Size (mm)PCNA(Mean)Cyclin D1%(Mean)Cox-2%(Mean)p21%(Mean)Curcumin (N=6) 67% 2.88 66% 23% 33% 100%Prima-1 (N=8) 28% 3.46 47% 34% 25% 43%Curcumin+Prima-1 (N=8) 26% 3.10 69% 53% 75% 63%Control (saline) (N=4) 38% 6.23 66% 50% 0% 67%Control (N=8) 23% 4.94 71% 60% 28% 67%p=0.001 p.0.05 p.0.05 p,0.001 p=0.064 p=0.55CLINICS 2011;66(12):2121-2124 Curcumin and Prima-1 in an in vivo bladder cancer modelWatanabe FT et al.212314. Liontas A, Yeger H. Curcumin and resveratrol induce apoptosis andnuclear translocation and activation of p53 in human neuroblastoma.Anticancer Res. 2004;24:987–98.15. Kawamori T, Lubet R, Steele VE, Kelloff GJ, Kaskey RB, Rao CV,et al. Chemopreventive effect of curcumin, a naturally occurringanti-inflammatory agent, during the promotion/progression stages ofcolon cancer. Cancer Res. 1999;59:597–601.16. Chauhan DP. Chemotherapeutic potential of curcumin for colorectalcancer. Curr Pharm Des. 2002;8:1695-706, doi: 10.2174/1381612023394016.Curcumin and Prima-1 in an in vivo bladder cancer modelWatanabe FT et al.CLINICS 2011;66(12):2121-21242124

Curcumin, but not Prima-1, decreased tumor cell proliferation in the syngeneic murine orthotopic bladder tumor model

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Curcumin, but not Prima-1, decreased tumor cell proliferation in the syngeneic murine orthotopic bladder tumor model

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