Resiniferatoxin induces death of bladder cancer cells associated with mitochondrial dysfunction and reduces tumor growth in a xenograft mouse model

Resiniferatoxin induces death of bladder cancer cells associated withmitochondrial dysfunction and reduces tumor growth in a xenograftmouse model

Valerio Farfariello a1 Sonia Liberati ab1 Maria Beatrice Morelli a Daniele Tomassoni c Matteo Santoni dMassimo Nabissi a Antonella Giannantoni e Giorgio Santoni a Consuelo Amantini c

a School of Pharmacy Experimental Medicine Section University of Camerino Via Madonna delle Carceri 9 62032 Camerino Italyb Department of Molecular Medicine Sapienza University of Rome Viale Regina Elena 291 00161 Rome Italyc School of Biosciences and Veterinary Medicine University of Camerino Via Madonna delle Carceri 9 62032 Camerino Italyd Department of Medical Oncology Polytechnic University of the Marche Region Via Conca 71 60126 Ancona Italye Department of Urology and Andrology Uro-andrology and Tissue Engineering Laboratory University of Perugia Piazzale Menghini 1 06129 Perugia Italy

a r t i c l e i n f o

Article historyReceived 9 July 2014Received in revised form 15 October 2014Accepted 20 October 2014Available online 29 October 2014

KeywordsBladder cancerResiniferatoxinNecrosisXenograftMitochondria

a b s t r a c t

Bladder cancer (BC) is the fifth most common non-cutaneous malignancy and the most common form ofBC in Western countries is transitional cell carcinoma Resiniferatoxin (RTX) has found therapeutic use-fulness for the treatment of bladder dysfunction but no data are available on its use as chemotherapeuticagent The aim of this work is to evaluate the use of RTX as new anti-cancer drug in BC therapy Theeffects of RTX on cell viability and cell death were evaluated on T24 and 5637 BC cell lines by MTT assaycell cycle analysis Annexin-VPI staining and agarose gel electrophoresis of DNA Mitochondrialdepolarization and ROS production were assessed by flow cytometry ADPATP ratio was measured bybioluminescence and caspase 3 cleavage by Western blot For in vivo experiments athymic nude micexenografted with T24 cells received subcutaneous administrations of RTX Tumor volumes weremeasured and immunohistochemistry was performed on tumor sections Our data demonstrated thatRTX influences cell cycle and induces necrotic cell death of BC cells by altering mitochondrial functionleading to depolarization increase in ADPATP ratio and ROS production Moreover RTX is able to reducetumor growth in a xenograft mouse model Overall we demonstrated that RTX induces necrotic celldeath of BC cells and reduces tumor growth in a xenograft mouse model of BC suggesting RTX as anew potential anti-cancer drug in BC chemotherapy

2014 Elsevier Ireland Ltd All rights reserved

1 Introduction

Bladder cancer (BC) is among the five most common malignan-cies worldwide There are over 70000 new cases of BC each year inthe United States alone [1] and the most common form of BC in

Western countries is transitional cell carcinoma (TCC) [2]Approximately 30ndash40 of patients with high-risk non-muscle-invasive TCC of the bladder will progress to a moreadvanced disease within 5 years and up to 34 of them will ulti-mately die of bladder cancer [3] Overall only 20ndash40 of patientswith advanced TCC have a 5-year survival rate despite aggressivemultimodal therapy and radical cystectomy remain the mainstayof treatment of muscle-invasive disease [45] To amelioratepatient life expectancy improvement of current chemotherapeuticregimens and development of novel chemotherapeutic strategiesare necessary Recently different therapeutic approaches basedon targeting tumor mitochondria have been proposed [6] withthe expectation that this novel class of agents could reduce tumorcells viability with an acceptable therapeutic index [7]

Resiniferatoxin (RTX) is a diterpene found in the latex of thecactus Euphorbia resinifera containing a homovanillic acid ester a

httpdxdoiorg101016jcbi2014100200009-2797 2014 Elsevier Ireland Ltd All rights reserved

Abbreviations Ab antibody BC bladder cancer CCCP carbonyl cyanidechlorophenylhydrazone protonophore DMSO dimethyl sulfoxide DWmmitochondrial transmembrane potential HampE hematoxylineosin I-RTX50-iodoresiniferatoxin PI propidium iodide ROS reactive oxygen species RTXresiniferatoxin TCC transitional cell carcinoma TRP transient receptor potentialchannels TRPV TRP vanilloid TUR transurethral resection Corresponding author at School of Pharmacy Experimental Medicine Section

University of Camerino Via Madonna delle Carceri 9 62032 Camerino (MC) ItalyTel +39 737403312 fax +39 737403325

E-mail address beatricemorellihotmailcom (MB Morelli)1 Valerio Farfariello and Sonia Liberati equal contribution

Chemico-Biological Interactions 224 (2014) 128ndash135

Contents lists available at ScienceDirect

Chemico-Biological Interactions

journal homepage wwwelsevier comlocate chembioint

key structural motif of capsaicin displaying analgesic activity andfunctioning as an ultrapotent capsaicin analog RTX has found ther-apeutic usefulness in the urologic field in the treatment of bladderdysfunctions and painful bladder [89] Very few data are availableon its use as chemotherapeutic agent The anticancer activity ofvanilloids such as capsaicin and dihydrocapsaicin can be mediatedthrough both a direct pathway independent of transient receptorpotential vanilloid receptor 1 (TRPV1) the receptor for vanilloidsand an indirect pathway through the interaction with TRPV1 andthe subsequent intracellular calcium overload [10ndash16] In regardto RTX there are indications that mitochondria could be involvedin the TRPV1-independent vanilloids-induced cell death In pancre-atic cancer tissue [13] squamous cell carcinoma and non-smalllung cancer cell lines [1417] RTX causes non-vanilloid receptormediated cell death

This work is aimed to evaluate the potential use of RTX as newtherapeutic strategy against BC through in vitro and in vivo pre-clinical experiments

2 Materials and methods

21 Cell lines

The p53 mutant T24 TCC and 5637 grade II BC cell lines pur-chased from American Type Culture Collection (ATCC RockvilleMD USA) were maintained in RPMI-1640 medium (Lonza BaselSwitzerland) supplemented with 10 heat-inactivated fetal bovineserum 25 mM HEPES 2 mM L-glutamine 100 IUml of penicillin100 gml of streptomycin (Lonza) at 37 C 5 CO2 and 95 humid-ity Normal human urothelial cells (NHUC) were purchased fromScienceCell Research laboratories (Carlsbad CA USA) and culturedin Urothelial Cell Medium supplemented with 5 ml (100x) of uro-thelial cell growth supplement (UCGS ScienceCell Research labora-tories) 100 IUml of penicillin 100 mgml of streptomycin (ScienceCell Research laboratories) at 37 C 5 CO2 and 95 humidity

22 MTT assay

T24 and 5637 BC cells (6 103well) were seeded into 96-wellplates and cultured with different doses of RTX (Tocris BioscienceBristol UK 01ndash50 lM) alone or in combination with the TRPV1antagonist 50-iodoresiniferatoxin (I-RTX) (50 nM TocrisBioscience) dissolved in dimethyl sulfoxide (DMSO Sigma AldrichSt Louis USA) or respective vehicles for 24 h At the end oftreatment samples were processed as described previously [12]Four replicates were used for each treatment and data were repre-sented as the average of at least three separate experiments Insome experiments MTT assay was performed using NHUCs treatedwith RTX (50 lM) for 24 h IC50 was mathematically determinedusing Graph Pad Prism 5 software

23 Cell-cycle analysis and Annexin-V staining

Cells (3 105well) were plated in six-well culture dishes andtreated for 12ndash24 h with 20 lM RTX or vehicle Cells were fixedby adding ice-cold 70 ethanol and then washed with staining buf-fer (PBS 2 FBS and 01 NaN3) Next the cells were treated with100 lgml ribonuclease A solution (Sigma Aldrich) incubated for30 min at 37 C stained for 30 min at room temperature withPropidium Iodide (PI) 20 lgml (Sigma Aldrich) and finally ana-lyzed by flow cytometry and the Cyflogic software (CyFlo LtdFinland) Phosphatidylserine exposure on BC cells treated withRTX (20 lM) for 12 h was detected by Annexin-V-FITC (Enzo LifeSciences Farmingdale USA) and analyzed by the FACScan Flow

Cytometer with the CellQuest software (Becton Dickinson SanJose USA)

24 Western blot

T24 and 5637 cells untreated or treated with 20 lM RTX orvehicle for 12 h were lysed and protein samples were subjectedto Sodium Dodecyl SulfatendashPolyAcrylamide Gel Electrophoresis(14) and transferred onto Hybond-C extra membranes (GEHealthcare Uppsala Sweden) as described [12] Membranes wereblotted with rabbit polyclonal anti-caspase 3 Ab (11000 CellSignaling Technology Denver USA) according to the manufac-turerrsquos instructions followed by HRP-conjugated donkey anti-rabbit Ab (12000 GE Healthcare) and with mouse monoclonalanti-GAPDH Ab (15000 Sigma Aldrich) GAPDH protein levelswere used as loading control Immunostaining was revealed byenhanced ECL Western Blotting analysis system (GE Healthcare)Densitometric analysis was performed by ChemiDoc using theQuantity One software (Bio-Rad)

25 DNA fragmentation assay

Cells (15 106) were treated with RTX (20 lM) or vehicle for24 h and genomic DNA was extracted using DNA extraction kit(Qiagen Milan Italy) DNA fragmentation used as a criterion todistinguish necrosis from apoptosis was assessed by electrophore-sis on 17 agarose gel and ethidium bromide staining Ultravioletspectroscopy at 302 nm was used to report results

26 Mitochondrial transmembrane potential (DWm)

Mitochondrial transmembrane potential (DWm) was evaluatedby 550660-tetrachloro-110330-tetraehylbenzimidazolylcarbocy-anineiodide (JC-1) staining Cells (4 104well) were seeded into24-well plates and treated with 20 lM RTX or vehicle for differenttimes and then incubated with 10 lgml of JC-1 [12] Carbonyl cya-nide chlorophenylhydrazone protonophore (CCCP 50 lM SigmaAldrich) a mitochondrial uncoupler that collapses DWm was usedas positive control Samples were analyzed using the FACScan cyto-fluorimeter with the CellQuest software

27 Measurement of ADPATP ratio

ADPATP ratio was measured in BC cells treated with vehicle orRTX (20 lM) by the EnzyLight ADPATP Ratio Assay Kit (BioAssaySystems Hayward CA USA) following the manufacturerrsquosinstructions Bioluminescence was acquired by FluoStar OMEGAluminometer (BMG LABTECH GmbH Ortenberg Germany)

28 Reactive oxygen species (ROS) production

The flow cytometric detection of ROS production was assessedby using the CellROX Flow Cytometry Assay Kits (LifeTechnologies CA USA) Briefly BC cells (4 104well) were seededinto 24-well plates and cultured for different times with RTX(20 lM) or vehicle Then cells were stained for 30 min at 37 C5 CO2 protected from light with the CellROX Detection Reagentand analyzed by flow cytometry fluorescence intensity wasexpressed in arbitrary units on a logarithmic scale

29 Grafting of T24 cells into immunodeficient mice and RTXtreatment protocol

Athymic nude (nunu) 6-week-old male mice (HarlanLaboratories San Pietro al Natisone Italy) were housed in patho-gen-free conditions on a 12 h lightdark schedule Twenty mice

V Farfariello et al Chemico-Biological Interactions 224 (2014) 128ndash135 129

were injected subcutaneously in the right flank with 3 106 T24cells in 01 mL PBS One week after cell transplantation tumorshad grown to an average volume of 50 mm3 (tumor take 100)Mice were then randomly assigned to the control or RTX (10 lM)group and received a 50 ll peritumoral injection every 3 days for21 days RTX was dissolved in ethanol to a concentration of1 mM and then diluted in saline to a concentration of 10 lM Thefinal ethanol concentration injected into the animals was 1Controls were injected with saline containing 1 ethanol Tumorvolumes were monitored every day using caliper measurementsThe protocol was approved by the local ethic committee (protocoln 232012) and by the Italian Ministry of Health (auth n 2472013-B)

210 Immunohistochemistry

At the end of treatments tumors were surgically removed fixedin a 4 buffered neutral formalin solution and embedded in a semi-synthetic paraffin Consecutive 8 lm-thick sections were stainedwith hematoxylineosin (HampE) or hematoxylin alone for assessingmicroanatomical changes by light microscopy using the IAS 2000image analyzer (Delta Sistemi Roma Italy) Mitotic count was per-formed at 20 magnification in 50 fields of homogeneous tumortissues Paraffin-embedded sections were also stained with mouseanti-Ki-67 Ab (Clone MIB-1 1100 Dako USA) following the man-ufacturerrsquos protocol After incubation for 30 min at 25 C with cor-responding secondary biotinylated Ab (goat-anti mouse IgG 1200Bethyl USA) the immune reaction was revealed usingVECTASTAIN Elite ABC kit (Vector Laboratories BurlingameCA) Sections exposed to a non-immune sera were used as negativecontrols For each tumor sample Ki-67-positive cells were countedin 10 fields of 05 mm2 of serial consecutive sections

211 Statistical analysis

The statistical significance was determined by one-way Anovaor by 2-way Anova with Bonferroni post-test No statistically sig-nificant differences were found between untreated and vehicle(DMSO)-treated cells or comparing different times of vehicle-treatment each other (data not shown)

3 Results

31 RTX induces necrotic cell death of BC cells independently fromTRPV1

We initially evaluated the effects of different RTX doses(01ndash50 lM) on the viability of T24 and 5637 cell lines As shownin Fig 1a RTX is able to reduce dose-dependently the growth ofboth cell lines at 24 h (IC50 215 for T24 cells and 199 lM for5637 cells) Since T24 but not 5637 cells [1718] express TRPV1channel we used RTX in combination with 50-iodoresiniferatoxin(I-RTX) a strong competitive antagonist of TRPV1 receptor to ver-ify the involvement of TRPV1 in T24 cell growth inhibition I-RTX(50 nM) did not revert the 20 lM RTX-induced effects (Fig 1b) at24 h thus indicating that the neurotoxin acts in a TRPV1-indepen-dent manner In addition NHUCs are less sensitive than BC cells toRTX-mediated cytotoxic effects (Fig 1c) Moreover RTX (20 lM)affected cell cycle phases (Fig 2a) and increased the percentageof the sub-G0 population (Fig 2b) Since the increased percentageof cells in sub-G0 phase can be addressed either to apoptosis ornecrosis we decided to investigate which type of cell deathoccurred in BC cells after RTX exposure by Annexin-V stainingand FACS analysis and Western blot analysis using an anti-caspase3 Ab Neither Annexin-V+ cells nor pro-caspase 3 cleavage were

Fig 1 RTX reduces BC cells viability (a) Cell growth of T24 and 5637 cell lines treated with different doses of RTX for 24 h as determined by MTT assay Data shown areexpressed as mean plusmn SE of three separate experiments (b) Cell growth of T24 cells treated with vehicle or RTX (20 lM) alone or in combination with I-RTX (50 nM) Data areexpressed as the mean plusmn SD of three independent experiments One-way ANOVA fraslfraslp 6 001 RTX or RTX + I-RTX vs vehicle ns not significant (c) Cell growth of T24 5637 BCcells and NHUCs treated with RTX (50 lM) for 24 h as determined by MTT assay One-way ANOVA fraslfraslp 6 001 NHUC vs T24 or 5637

130 V Farfariello et al Chemico-Biological Interactions 224 (2014) 128ndash135

found in RTX-treated BC cells suggesting that RTX induces anon-apoptotic cell death (Fig 2c and d) Moreover the agarosegel electrophoresis of DNA extracted from RTX-treated cellsshowed DNA smearing rather than oligonucleosomal fragmenta-tion (Fig 2e) Taken together our data indicate that RTX inducesnecrosis of BC cells

32 RTX affects the redox homeostasis in BC cells

To elucidate the molecular mechanism by which RTX inducednecrosis we analyzed the DWm in BC cells Treatment with RTXinduced a time-dependent mitochondrial depolarization evidentfrom 8 h post-treatment indicating that RTX exerts a strong activ-ity against mitochondrial homeostasis (Fig 3a) Treatment of BCcells with CCCP resulted in a drop of DWm comparable with thatobserved in RTX-treated cells (Fig 3a)

Changes in ADPATP ratio are indicative of alterations in energymetabolism and cell viability Our data indicated a strong increaseof ADPATP ratio in RTX-treated cells as compared with vehicle(Fig 3b) Due to their inhibitory activity towards mitochondriavanilloids can determine an excess of superoxide (O2ndash) hydroper-oxide (H2O2) and hydroxyl ions (OHndash) with a consequent inductionof oxidative stress and cell death We thus measured ROS produc-tion in BC cells treated with RTX for different times (0ndash24 h) andfound a significant increase in ROS concentration from 8 to 24 hof treatment (Fig 3c) Overall our data confirm that RTX inducesan altered redox homeostasis

33 RTX reduces tumor growth in experimental tumor xenograftsmodels

Finally we tested whether RTX could reduce tumor growthin vivo T24 cells were thus injected into athymic nude mice (tumor

Fig 2 RTX induces cell cycle arrest and necrosis in BC cells (a) Cell cycle analysis of T24 and 5637 cells treated with 20 lM RTX or vehicle for 12 h was performed by PIstaining and flow cytometric analysis excluding aggregates and debris Cell percentage relative to the different cycle phases is indicated Data are representative of one out ofthree different experiments (b) Percentage of T24 and 5637 cells in the sub-G0 phase after 12ndash24 h of RTX (20 lM) treatment as calculated by PI staining and flow cytometryData are expressed as the mean plusmn SD of three independent experiments Two-way Anova fraslfraslp 6 001 RTX vs vehicle-treated cells p 6 001 RTX-treated cells for 12 or 24 h vstime 0 sectp 6 001 RTX-treated cells for 24 h vs RTX-treated cells for 12 h (c) T24 and 5637 BC cells treated with RTX (20 lM) or vehicle for 12 h were stained with AnnexinV-FITC and analyzed by FACS Data represent one out of three separate experiments (d) Lysates from T24 and 5637 BC cell lines treated with RTX (20 lM) for 12 h or vehiclewere separated on 14 SDSndashPAGE and probed with anti-caspase 3 or anti-GAPDH Abs respectively Data represent one out of three separate experiments (e) DNAfragmentation was assessed in T24 and 5637 cells treated as described in (a) by agarose gel electrophoresis ethidium bromide staining and acquisition with ChemiDoc Oneout of three representative independent experiments is shown

V Farfariello et al Chemico-Biological Interactions 224 (2014) 128ndash135 131

take 100) and RTX challenge started as soon as the tumor massreached the volume of about 50 mm3 As shown in Fig 4a RTXtreatment significantly reduced tumor growth from the 9th dayafter challenge (P lt 0001) After 3 weeks from RTX challengemouse were euthanized tumors were surgically removed and sizeand weight were registered Data obtained in vivo demonstratedthat RTX exerts a potent anti-tumor activity inducing a significantreduction in size (Fig 4b) and weight (about 17-fold Table 1)when compared to controls No signs of inflammatory infiltrateswere found in the epithelial and sub-epithelial tissues surroundingthe tumor mass in RTX-treated animals (Fig 4c) The side-effects ofRTX were also evaluated based on the in vivo assay No mouse diedduring the experimental time and the body weight average of themice in the RTX group was not significantly different from that ofthe mice in the vehicle group (Table 1)

34 Tumors from RTX-treated mice display reduced proliferation andnecrosis

Histological analysis of tumors revealed that RTX treatment sig-nificantly reduces mitotic index (Fig 5a) Sections processed forKi-67 showed the presence of positive-stained cells in bothvehicle- and RTX-treated tumors (Fig 5b) however a significant

decreased number of positive cells was found in tumors fromRTX-treated mice Moreover HampE staining showed the presenceof extensive necrotic areas in the external portion of tumors fromRTX-treated mice that were absent in tumors from vehicle-treatedanimals (Fig 5a)

4 Discussion

We demonstrate for the first time that RTX is able to induce cellcycle arrest and necrotic cell death associated with mitochondrialdysfunction in different BC cell lines and more importantly itreduces in vivo the growth of human BC cells xenografted intonude mice Thus RTX can be considered as a new potential mole-cule for BC therapy These results appear to be significant if weconsider that despite several efforts have been made for the devel-opment of new effective therapies TCC of the bladder still remainsa high recurrent malignancy Although transurethral resection(TUR) of TCC of the bladder induces an 80 early success ratenearly 70 of these patients will develop tumor recurrence with25 showing progression to muscle-invasive disease within5 years with TUR Intravesical chemotherapy and immunotherapyare widely used as adjuvant therapies after TUR to prevent recur-rence and progression of superficial disease but meta-analysis

Fig 3 RTX alters mitochondrial homeostasis in BC cells (a) Time course analysis of DWm changes in T24 and 5637 cells treated for different times (8 12 or 24 h) with vehicle20 lM RTX or 50 lM CCCP used as positive control was evaluated by JC-1 staining and biparametric FL1(green)FL2(red) flow cytometric analysis Numbers indicate thepercentage of cells showing a drop in DWm-related red fluorescence intensity Data are representative of one out of three separate experiments For the sake of simplicity onlyone vehicle-treated sample is shown (b) ADPATP ratio in T24 and 5637 cells untreated or treated with 20 lM RTX for 12ndash24 h Data are the mean plusmn SD of three independentexperiments Two-way Anova fraslfraslp 6 001 RTX-treated vs vehicle-treated cells p 6 001 RTX-treated cells for 24 h vs RTX-treated cells for 12 h (c) ROS production wasevaluated in T24 and 5637 cells treated with RTX for different times by using CellROX Flow Cytometry Assay Kit and FACS analysis Data shown as the mean plusmn SD of threeindependent experiments are expressed as fold change with respect to vehicle The dotted line represents ROS basal level (time 0) used as control One-way Anova Bonferronipost-test fraslfraslp 6 001 RTX-treated cells vs control p 6 001 RTX-treated cells for 24 h vs RTX-treated cells for 12 h

132 V Farfariello et al Chemico-Biological Interactions 224 (2014) 128ndash135

does not show apparent superiority of a particular treatment[2021] For this reason it is fundamental to develop optimal lesstoxic chemotherapy regimens by incorporating novel targetedagents to improve the outcomes The cytotoxic activity of RTXhas been documented in vitro for pancreatic lung and prostatecancer cells [11131417] but at present no studies have been con-ducted to test the anticancer efficacy of RTX in BC models in vitroand in vivo Despite RTX is an ultrapotent TRPV1 agonist and even

if T24 cells express high levels of TRPV1 [19] the mechanism ofRTX action we described in this work is TRPV1-independent andinvolves the alteration of the redox homeostasis with a significantmitochondrial depolarization increase of ADPATP ratio and ROSproduction Our findings are in line with those of the literature thatindicate vanilloids as compounds able to interfere with the cellularredox homeostasis by directly antagonizing the coenzyme Q andaffecting the electron transport chain [112223] This cell deathmechanism can be potentially active in every cell tumor cellsare known to present a non-controlled cellular activity and theyneed to have a high quantity of energy at their disposal thus theconstitutional activation of the electron transport chain For thisreason vanilloids can induce mitochondrial perturbation and sub-sequent delivery of ROS especially in proliferating cancer cells Theuse of the xenograft model of BC in this study not only confirms thedata obtained in vitro but also gives precious information about thepotential side effects of RTX treatment Intravesical delivery of

Fig 4 RTX significantly reduces the growth of T24 TCC xenografts (a) 10 lM RTX or vehicle were injected peritumorally once every 3 days during 3 weeks into nude micethat had been inoculated sc with human T24 cells Tumor volumes were calculated by the formula (D d2)2 where D = major diameter and d = minor diameter Resultsrepresent the mean plusmn SD of 10 mice in each group ANOVA Bonferroni post-test fraslfraslfraslp 6 0001 RTX- vs vehicle-treated mice (b) Representative image of the macroscopicappearance of heterotopic xenografts surgically removed at the end of the treatment protocol (c) Representative sections from vehicle- and RTX-treated mice stained withhematoxylin alone E = epithelial layer SE = sub-epithelial layer T = tumor Calibration bar 100 lm

Table 1RTX exerts a marked anti-tumor activity in BC xenografts

Group Body weight (g) Tumor incidence () Tumor mass (mg)

Vehicle 268 plusmn 21 100 380 plusmn 108RTX 272 plusmn 15 100 219 plusmn 74

Studentrsquos t-test p 6 001 RTX- vs vehicle-treated mice

V Farfariello et al Chemico-Biological Interactions 224 (2014) 128ndash135 133

vanilloids ie capsaicin has been previously observed to induceautonomic dysreflexia limb spasms suprapubic discomfort andhematuria [24] This is not the case for RTX which shows a farmore favorable ratio of desensitization to irritation than capsaicinbecause it is practically pungent-free [25] Indeed no side effectswere induced by RTX treatment in our experimental model RTX(US patent number 8338457) is not suitable for systemic admin-istration but subcutaneous intraganglionic or intrathecal applica-tions are under preclinical investigations for the treatment ofpain in advanced cancer [26] Moreover recently the NationalInstitutes of Health in collaboration with Sorrento Therapeuticshas started the recruitment of participants for a new clinical trialsto demonstrate the safety of administering RTX directly into thehuman central nervous system [27] In addition RTX has been pre-viously used in humans for the treatment of bladder dysfunctionsand bladder pain syndrome and several clinical trials have beenconducted by using different doses of RTX intravesically deliveredwithout inducing any substantial local or systemic side effects[28ndash31]

5 Conclusion

Overall we demonstrated that RTX treatment in vitro inducesnecrotic cell death of BC cells by affecting redox homeostasis andin vivo reduces tumor growth in a xenograft mouse model of BCTherefore since the high adaptability of RTX to a variety of painproblems and the relatively low toxicity when used locally (egintravesical or peritumoral administration) it could represent anew promising strategy for the treatment of patients with BC

Conflict of Interest

The authors declare that there are no conflicts of interest

Transparency Document

The Transparency document associated with this article can befound in the online version

Acknowledgements

This work was supported by FIRC national grant (number11095) and by a grant from the Italian Ministry of University andResearch PRIN 2009ndash2011

References

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[2] DS Kaufman WU Shipley AS Feldman Bladder cancer Lancet 374 (2009)239ndash249

[3] H von der Maase L Sengelov JT Roberts S Ricci L Dogliotti T Oliver MJMoore A Zimmermann M Arning Long-term survival results of a randomizedtrial comparing gemcitabine plus cisplatin with methotrexate vinblastinedoxorubicin plus cisplatin in patients with bladder cancer J Clin Oncol 23(2005) 4602ndash4608

[4] Advanced Bladder Cancer (ABC) Meta-analysis Collaboration Neoadjuvantchemotherapy in invasive bladder cancer update of a systematic review andmeta-analysis of individual patient data Advanced Bladder Cancer (ABC) Meta-analysis Collaboration Eur Urol 48 (2005) 202ndash205 discussion 205ndash206

[5] HB Grossman RB Natale CM Tangen VO Speights NJ Vogelzang DLTrump RW deVere White MF Sarosdy DP Wood Jr D Raghavan EDCrawford Neoadjuvant chemotherapy plus cystectomy compared with

Fig 5 RTX treatment reduces the mitotic index Ki-67 positive staining and induces necrosis of BC xenografts (a) Representative section of tumors from vehicle- and RTX-treated mice stained with HampE (upper panels calibration bar 100 lm) or hematoxylin alone (lower panels calibration bar 25 lm) In the section from RTX-treated tumors isevident an extensive necrotic area in the external portion of the tissue On the other hand at higher magnification a decrease in the presence mitotic figures (black arrows) isevident in sections from RTX-treated tumors as reported in the graph Data are expressed as the mean plusmn SD of mitotic counts performed in 50 fields fraslfraslp 6 001 RTX- vsvehicle-treated mice (b) Ki-67 immunoreaction in sections from vehicle- and RTX-treated tumors processed with non immune serum (upper panels calibration bar 25 lm)or with the specific Ab (lower panels calibration bar 25 lm) In the section from RTX-treated tumors is evident a decrease of Ki-67 positive cells Data reported in the graphare expressed as the mean plusmn SD of Ki-67 positive cells performed in 10 fields of an area of 005 mm2 for each tumor fraslp 6 005 RTX- vs vehicle-treated mice

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cystectomy alone for locally advanced bladder cancer N Engl J Med 349(2003) 859ndash866

[6] L Biasutto LF Dong M Zoratti J Neuzil Mitochondrially targeted anti-canceragents Mitochondrion 10 (2010) 670ndash681

[7] L Galluzzi N Larochette N Zamzami G Kroemer Mitochondria astherapeutic targets for cancer chemotherapy Oncogene 25 (2006) 4812ndash4830

[8] F Cruz P Dinis Resiniferatoxin and botulinum toxin type A for treatment oflower urinary tract symptoms Neurourol Urodyn 26 (2007) 920ndash927

[9] PK Matsuoka JM Haddad AM Pacetta EC Baracat Intravesical treatment ofpainful bladder syndrome a systematic review and meta-analysis IntUrogynecol J 23 (2012) 1147ndash1153

[10] G Santoni V Farfariello C Amantini TRPV channels in tumor growth andprogression Adv Exp Med Biol 704 (2011) 947ndash967

[11] F Ziglioli A Frattini U Maestroni F Dinale M Ciufifeda P CortelliniVanilloid-mediated apoptosis in prostate cancer cells through a TRPV-1dependent and a TRPV-1-independent mechanism Acta Biomed 80 (2009)13ndash20

[12] C Amantini P Ballarini S Caprodossi M Nabissi MB Morelli R LucciariniMA Cardarelli G Mammana G Santoni Triggering of transient receptorpotential vanilloid type 1 (TRPV1) by capsaicin induces FasCD95-mediatedapoptosis of urothelial cancer cells in an ATM-dependent mannerCarcinogenesis 30 (2009) 1320ndash1329

[13] M Hartel FF di Mola F Selvaggi G Mascetta MN Wente K Felix NA GieseU Hinz P Di Sebastiano MW Buumlchler H Friess Vanilloids in pancreaticcancer potential for chemotherapy and pain management Gut 55 (2006)519ndash528

[14] N Hail Jr Mechanisms of vanilloid-induced apoptosis Apoptosis 8 (2003)251ndash262

[15] K Ito T Nakazato K Yamato Y Miyakawa T Yamada N Hozumi K SegawaY Ikeda M Kizaki Induction of apoptosis in leukemic cells by homovanillicacid derivative capsaicin through oxidative stress implication ofphosphorylation of p53 at Ser-15 residue by reactive oxygen species CancerRes 64 (2004) 1071ndash1078

[16] C Amantini M Mosca M Nabissi R Lucciarini S Caprodossi A Arcella FGiangaspero G Santoni Capsaicin-induced apoptosis of glioma cells ismediated by TRPV1 vanilloid receptor and requires p38 MAPK activation JNeurochem 102 (2007) 977ndash990

[17] A Athanasiou PA Smith S Vakilpour NM Kumaran AE Turner DBagiokou R Layfield DE Ray AD Westwell SP Alexander DA KendallDN Lobo SA Watson A Lophatanon KA Muir DA Guo TE Bates Vanilloidreceptor agonists and antagonists are mitochondrial inhibitors how vanilloidscause non-vanilloid receptor mediated cell death Biochem Biophys ResCommun 354 (2007) 50ndash55

[18] S Caprodossi C Amantini M Nabissi MB Morelli V Farfariello M SantoniA Gismondi G Santoni Capsaicin promotes a more aggressive geneexpression phenotype and invasiveness in null-TRPV1 urothelial cancer cellsCarcinogenesis 32 (2011) 686ndash694

[19] Z Shen T Shen MG Wientjes MA OrsquoDonnell JL Au Intravesical treatmentsof bladder cancer review Pharm Res 25 (2008) 1500ndash1510

[20] JB Shah DJ McConkey CP Dinney New strategies in muscle-invasivebladder cancer on the road to personalized medicine Clin Cancer Res 17(2011) 2608ndash2612

[21] S Gupta A Mahipal Role of systemic chemotherapy in urothelial urinarybladder cancer Cancer Control 20 (2013) 200ndash210

[22] ZH Yang XH Wang HP Wang LQ Hu XM Zheng SW Li Capsaicinmediates cell death in bladder cancer T24 cells through reactive oxygenspecies production and mitochondrial depolarization Urology 75 (2010) 735ndash741

[23] N Hail Jr R Lotan Examining the role of mitochondrial respiration invanilloid-induced apoptosis J Natl Cancer Inst 94 (2002) 1281ndash1292

[24] A Giannantoni SM Di Stasi RL Stephen P Navarra G Scivoletto E MeariniM Porena Intravesical capsaicin versus resiniferatoxin in patients withdetrusor hyperreflexia a prospective randomized study J Urol 167 (2002)1710ndash1714

[25] K Sugimoto I Kissin G Strichartz A high concentration of resiniferatoxininhibits ion channel function in clonal neuroendocrine cells Anesth Analg107 (2008) 318ndash324

[26] MJ Iadarola AJ Mannes The vanilloid agonist resiniferatoxin forinterventional-based pain control Curr Top Med Chem 11 (2011) 2171ndash2179

[27] Clinical trialsgov Identifier NCT00804154[28] M Lazzeri M Spinelli P Beneforti A Zanollo D Turini Intravesical

resiniferatoxin for the treatment of detrusor hyperreflexia refractory tocapsaicin in patients with chronic spinal cord diseases Scand J UrolNephrol 32 (1998) 331ndash334

[29] C Silva ME Rio F Cruz Desensitization of bladder sensory fibers byintravesical resiniferatoxin a capsaicin analog long-term results for thetreatment of detrusor hyperreflexia Eur Urol 38 (2000) 444ndash452

[30] HC Kuo HT Liu WC Yang Therapeutic effect of multiple resiniferatoxinintravesical instillations in patients with refractory detrusor overactivity arandomized double-blind placebo controlled study J Urol 176 (2006) 641ndash645

[31] A Giannantoni SM Di Stasi RL Stephen V Bini E Costantini M PorenaIntravesical resiniferatoxin versus botulinum-A toxin injections for neurogenicdetrusor overactivity a prospective randomized study J Urol 172 (2004)240ndash243

V Farfariello et al Chemico-Biological Interactions 224 (2014) 128ndash135 135