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

Chemico-Biological Interactions 224 (2014) 128–135

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Resiniferatoxin induces death of bladder cancer cells associated with mitochondrial dysfunction and reduces tumor growth in a xenograft mouse model Valerio Farfariello a,1, Sonia Liberati a,b,1, Maria Beatrice Morelli a,⇑, Daniele Tomassoni c, Matteo Santoni d, Massimo 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, Italy Department of Molecular Medicine, Sapienza University of Rome, Viale Regina Elena 291, 00161 Rome, Italy c School of Biosciences and Veterinary Medicine, University of Camerino, Via Madonna delle Carceri 9, 62032 Camerino, Italy d Department of Medical Oncology, Polytechnic University of the Marche Region, Via Conca 71, 60126 Ancona, Italy e Department of Urology and Andrology, Uro-andrology and Tissue Engineering Laboratory, University of Perugia, Piazzale Menghini 1, 06129 Perugia, Italy b

a r t i c l e

i n f o

Article history: Received 9 July 2014 Received in revised form 15 October 2014 Accepted 20 October 2014 Available online 29 October 2014 Keywords: Bladder cancer Resiniferatoxin Necrosis Xenograft Mitochondria

a b s t r a c t Bladder cancer (BC) is the fifth most common non-cutaneous malignancy and the most common form of BC in Western countries is transitional cell carcinoma. Resiniferatoxin (RTX) has found therapeutic usefulness for the treatment of bladder dysfunction but no data are available on its use as chemotherapeutic agent. The aim of this work is to evaluate the use of RTX as new anti-cancer drug in BC therapy. The effects of RTX on cell viability and cell death were evaluated on T24 and 5637 BC cell lines by MTT assay, cell cycle analysis, Annexin-V/PI staining and agarose gel electrophoresis of DNA. Mitochondrial depolarization and ROS production were assessed by flow cytometry. ADP/ATP ratio was measured by bioluminescence and caspase 3 cleavage by Western blot. For in vivo experiments, athymic nude mice, xenografted with T24 cells, received subcutaneous administrations of RTX. Tumor volumes were measured and immunohistochemistry was performed on tumor sections. Our data demonstrated that RTX influences cell cycle and induces necrotic cell death of BC cells by altering mitochondrial function, leading to depolarization, increase in ADP/ATP ratio and ROS production. Moreover, RTX is able to reduce tumor growth in a xenograft mouse model. Overall, we demonstrated that RTX induces necrotic cell death of BC cells and reduces tumor growth in a xenograft mouse model of BC, suggesting RTX as a new potential anti-cancer drug in BC chemotherapy. Ó 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Bladder cancer (BC) is among the five most common malignancies worldwide. There are over 70,000 new cases of BC each year in the United States alone [1] and the most common form of BC in

Abbreviations: Ab, antibody; BC, bladder cancer; CCCP, carbonyl cyanide chlorophenylhydrazone protonophore; DMSO, dimethyl sulfoxide; DWm, mitochondrial transmembrane potential; H&E, hematoxylin/eosin; I-RTX, 50 -iodoresiniferatoxin; PI, propidium iodide; ROS, reactive oxygen species; RTX, resiniferatoxin; TCC, transitional cell carcinoma; TRP, transient receptor potential channels; 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), Italy. Tel.: +39 737403312; fax: +39 737403325. E-mail address: [email protected] (M.B. Morelli). 1 Valerio Farfariello and Sonia Liberati: equal contribution. http://dx.doi.org/10.1016/j.cbi.2014.10.020 0009-2797/Ó 2014 Elsevier Ireland Ltd. All rights reserved.

Western countries is transitional cell carcinoma (TCC) [2]. Approximately 30–40% of patients with high-risk nonmuscle-invasive TCC of the bladder will progress to a more advanced disease within 5 years and up to 34% of them will ultimately die of bladder cancer [3]. Overall, only 20–40% of patients with advanced TCC have a 5-year survival rate, despite aggressive multimodal therapy and radical cystectomy remain the mainstay of treatment of muscle-invasive disease [4,5]. To ameliorate patient life expectancy, improvement of current chemotherapeutic regimens and development of novel chemotherapeutic strategies are necessary. Recently, different therapeutic approaches based on targeting tumor mitochondria have been proposed [6], with the expectation that this novel class of agents could reduce tumor cells viability with an acceptable therapeutic index [7]. Resiniferatoxin (RTX) is a diterpene found in the latex of the cactus Euphorbia resinifera containing a homovanillic acid ester, a

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key structural motif of capsaicin, displaying analgesic activity and functioning as an ultrapotent capsaicin analog. RTX has found therapeutic usefulness in the urologic field in the treatment of bladder dysfunctions and painful bladder [8,9]. Very few data are available on its use as chemotherapeutic agent. The anticancer activity of vanilloids such as capsaicin and dihydrocapsaicin can be mediated through both a direct pathway, independent of transient receptor potential vanilloid receptor 1 (TRPV1), the receptor for vanilloids, and an indirect pathway, through the interaction with TRPV1 and the subsequent intracellular calcium overload [10–16]. In regard to RTX, there are indications that mitochondria could be involved in the TRPV1-independent vanilloids-induced cell death. In pancreatic cancer tissue [13], squamous cell carcinoma and non-small lung cancer cell lines [14,17] RTX causes non-vanilloid receptor mediated cell death. This work is aimed to evaluate the potential use of RTX as new therapeutic strategy against BC through in vitro and in vivo preclinical experiments.

Cytometer with the CellQuest software (Becton Dickinson, San Jose, USA).

2. Materials and methods

2.5. DNA fragmentation assay

2.1. Cell lines

Cells (1.5  106) were treated with RTX (20 lM) or vehicle for 24 h and genomic DNA was extracted using DNA extraction kit (Qiagen, Milan, Italy). DNA fragmentation, used as a criterion to distinguish necrosis from apoptosis, was assessed by electrophoresis on 1.7% agarose gel and ethidium bromide staining. Ultraviolet spectroscopy at 302 nm was used to report results.

The p53 mutant T24 TCC and 5637 grade II BC cell lines, purchased from American Type Culture Collection (ATCC, Rockville, MD, USA), were maintained in RPMI-1640 medium (Lonza, Basel, Switzerland) supplemented with 10% heat-inactivated fetal bovine serum, 2.5 mM HEPES, 2 mM L-glutamine, 100 IU/ml of penicillin, 100 g/ml of streptomycin (Lonza) at 37 °C, 5% CO2 and 95% humidity. Normal human urothelial cells (NHUC) were purchased from ScienceCell Research laboratories (Carlsbad, CA, USA) and cultured in Urothelial Cell Medium supplemented with 5 ml (100x) of urothelial cell growth supplement (UCGS, ScienceCell Research laboratories) 100 IU/ml of penicillin, 100 mg/ml of streptomycin (Science Cell Research laboratories) at 37 °C, 5% CO2 and 95% humidity. 2.2. MTT assay T24 and 5637 BC cells (6  103/well) were seeded into 96-well plates and cultured with different doses of RTX (Tocris Bioscience, Bristol, UK, 0.1–50 lM) alone or in combination with the TRPV1 antagonist 50 -iodoresiniferatoxin (I-RTX) (50 nM, Tocris Bioscience), dissolved in dimethyl sulfoxide (DMSO, Sigma Aldrich, St. Louis, USA) or respective vehicles for 24 h. At the end of treatment, samples were processed as described previously [12]. Four replicates were used for each treatment and data were represented as the average of at least three separate experiments. In some experiments MTT assay was performed using NHUCs treated with RTX (50 lM) for 24 h. IC50 was mathematically determined using Graph Pad Prism 5 software. 2.3. Cell-cycle analysis and Annexin-V staining Cells (3  105/well) were plated in six-well culture dishes and treated for 12–24 h with 20 lM RTX or vehicle. Cells were fixed by adding ice-cold 70% ethanol and then washed with staining buffer (PBS, 2% FBS and 0.1% NaN3). Next, the cells were treated with 100 lg/ml ribonuclease A solution (Sigma Aldrich), incubated for 30 min at 37 °C, stained for 30 min at room temperature with Propidium Iodide (PI) 20 lg/ml (Sigma Aldrich) and finally analyzed by flow cytometry and the Cyflogic software (CyFlo Ltd, Finland). Phosphatidylserine exposure on BC cells, treated with RTX (20 lM) for 12 h was detected by Annexin-V-FITC (Enzo Life Sciences, Farmingdale, USA) and analyzed by the FACScan Flow

2.4. Western blot T24 and 5637 cells, untreated or treated with 20 lM RTX or vehicle for 12 h, were lysed and protein samples were subjected to Sodium Dodecyl Sulfate–PolyAcrylamide Gel Electrophoresis (14%) and transferred onto Hybond-C extra membranes (GE Healthcare, Uppsala, Sweden) as described [12]. Membranes were blotted with rabbit polyclonal anti-caspase 3 Ab (1:1000, Cell Signaling Technology, Denver, USA) according to the manufacturer’s instructions followed by HRP-conjugated donkey antirabbit Ab (1:2000, GE Healthcare) and with mouse monoclonal anti-GAPDH Ab (1:5000, Sigma Aldrich). GAPDH protein levels were used as loading control. Immunostaining was revealed by enhanced ECL Western Blotting analysis system (GE Healthcare). Densitometric analysis was performed by ChemiDoc using the Quantity One software (Bio-Rad).

2.6. Mitochondrial transmembrane potential (DWm) Mitochondrial transmembrane potential (DWm) was evaluated by 5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraehylbenzimidazolylcarbocyanineiodide (JC-1) staining. Cells (4  104/well) were seeded into 24-well plates and treated with 20 lM RTX or vehicle for different times and then incubated with 10 lg/ml of JC-1 [12]. Carbonyl cyanide chlorophenylhydrazone protonophore (CCCP, 50 lM, Sigma Aldrich), a mitochondrial uncoupler that collapses DWm, was used as positive control. Samples were analyzed using the FACScan cytofluorimeter with the CellQuest software. 2.7. Measurement of ADP/ATP ratio ADP/ATP ratio was measured in BC cells treated with vehicle or RTX (20 lM) by the EnzyLight ADP/ATP Ratio Assay Kit (BioAssay Systems, Hayward, CA, USA) following the manufacturer’s instructions. Bioluminescence was acquired by FluoStar OMEGA luminometer (BMG LABTECH GmbH, Ortenberg, Germany). 2.8. Reactive oxygen species (ROS) production The flow cytometric detection of ROS production was assessed by using the CellROXÒ Flow Cytometry Assay Kits (Life Technologies, CA, USA) Briefly, BC cells (4  104/well) were seeded into 24-well plates and cultured for different times with RTX (20 lM) or vehicle. Then, cells were stained for 30 min at 37 °C, 5% CO2, protected from light with the CellROXÒ Detection Reagent and analyzed by flow cytometry; fluorescence intensity was expressed in arbitrary units on a logarithmic scale. 2.9. Grafting of T24 cells into immunodeficient mice and RTX treatment protocol Athymic nude (nu/nu) 6-week-old male mice (Harlan Laboratories, San Pietro al Natisone, Italy) were housed in pathogen-free conditions on a 12 h light/dark schedule. Twenty mice

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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 are expressed as mean ± 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 are expressed as the mean ± SD of three independent experiments. One-way ANOVA, ⁄⁄p 6 0.01 RTX or RTX + I-RTX vs vehicle, n.s.: not significant. (c) Cell growth of T24, 5637 BC cells and NHUCs treated with RTX (50 lM) for 24 h as determined by MTT assay. One-way ANOVA, ⁄⁄p 6 0.01 NHUC vs T24 or 5637.

were injected subcutaneously in the right flank with 3  106 T24 cells in 0.1 mL PBS. One week after cell transplantation, tumors had 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 for 21 days. RTX was dissolved in ethanol to a concentration of 1 mM and then diluted in saline to a concentration of 10 lM. The final ethanol concentration injected into the animals was 1%. Controls were injected with saline containing 1% ethanol. Tumor volumes were monitored every day using caliper measurements. The protocol was approved by the local ethic committee (protocol n. 23/2012) and by the Italian Ministry of Health (auth n. 247/ 2013-B). 2.10. Immunohistochemistry At the end of treatments, tumors were surgically removed, fixed in a 4% buffered neutral formalin solution and embedded in a semisynthetic paraffin. Consecutive 8 lm-thick sections were stained with hematoxylin/eosin (H&E) or hematoxylin alone for assessing microanatomical changes by light microscopy using the IAS 2000 image analyzer (Delta Sistemi, Roma, Italy). Mitotic count was performed at 20 magnification in 50 fields of homogeneous tumor tissues. Paraffin-embedded sections were also stained with mouse anti-Ki-67 Ab (Clone MIB-1, 1:100, Dako, USA) following the manufacturer’s protocol. After incubation for 30 min at 25 °C with corresponding secondary biotinylated Ab (goat-anti mouse IgG, 1:200, Bethyl, USA), the immune reaction was revealed using VECTASTAINÒ EliteÒ ABC kit (Vector Laboratories, Burlingame, CA). Sections exposed to a non-immune sera were used as negative controls. For each tumor sample, Ki-67-positive cells were counted in 10 fields of 0.5 mm2 of serial consecutive sections.

2.11. Statistical analysis The statistical significance was determined by one-way Anova or by 2-way Anova with Bonferroni post-test. No statistically significant differences were found between untreated and vehicle (DMSO)-treated cells or comparing different times of vehicletreatment each other (data not shown). 3. Results 3.1. RTX induces necrotic cell death of BC cells independently from TRPV1 We initially evaluated the effects of different RTX doses (0.1–50 lM) on the viability of T24 and 5637 cell lines. As shown in Fig. 1a, RTX is able to reduce dose-dependently the growth of both cell lines at 24 h (IC50: 21.5 for T24 cells and 19.9 lM for 5637 cells). Since T24 but not 5637 cells [17,18] express TRPV1 channel, we used RTX in combination with 50 -iodoresiniferatoxin (I-RTX), a strong competitive antagonist of TRPV1 receptor, to verify the involvement of TRPV1 in T24 cell growth inhibition. I-RTX (50 nM) did not revert the 20 lM RTX-induced effects (Fig. 1b) at 24 h, thus indicating that the neurotoxin acts in a TRPV1-independent manner. In addition, NHUCs are less sensitive than BC cells to RTX-mediated cytotoxic effects (Fig. 1c). Moreover, RTX (20 lM) affected cell cycle phases (Fig. 2a) and increased the percentage of the sub-G0 population (Fig. 2b). Since the increased percentage of cells in sub-G0 phase can be addressed either to apoptosis or necrosis, we decided to investigate which type of cell death occurred in BC cells after RTX exposure by Annexin-V staining and FACS analysis and Western blot analysis using an anti-caspase 3 Ab. Neither Annexin-V+ cells nor pro-caspase 3 cleavage were

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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 PI staining and flow cytometric analysis excluding aggregates and debris. Cell percentage relative to the different cycle phases is indicated. Data are representative of one out of three different experiments. (b) Percentage of T24 and 5637 cells in the sub-G0 phase after 12–24 h of RTX (20 lM) treatment as calculated by PI staining and flow cytometry. Data are expressed as the mean ± SD of three independent experiments. Two-way Anova, ⁄⁄p 6 0.01, RTX vs vehicle-treated cells; #p 6 0.01 RTX-treated cells for 12 or 24 h vs time 0; §p 6 0.01 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 Annexin V-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 vehicle, were separated on 14% SDS–PAGE and probed with anti-caspase 3 or anti-GAPDH Abs, respectively. Data represent one out of three separate experiments. (e) DNA fragmentation was assessed in T24 and 5637 cells, treated as described in (a), by agarose gel electrophoresis, ethidium bromide staining, and acquisition with ChemiDoc. One out of three representative independent experiments is shown.

found in RTX-treated BC cells, suggesting that RTX induces a non-apoptotic cell death (Fig. 2c and d). Moreover, the agarose gel electrophoresis of DNA extracted from RTX-treated cells showed DNA smearing rather than oligonucleosomal fragmentation (Fig. 2e). Taken together, our data indicate that RTX induces necrosis of BC cells.

3.2. RTX affects the redox homeostasis in BC cells To elucidate the molecular mechanism by which RTX induced necrosis, we analyzed the DWm in BC cells. Treatment with RTX induced a time-dependent mitochondrial depolarization evident from 8 h post-treatment, indicating that RTX exerts a strong activity against mitochondrial homeostasis (Fig. 3a). Treatment of BC cells with CCCP resulted in a drop of DWm comparable with that observed in RTX-treated cells (Fig. 3a).

Changes in ADP/ATP ratio are indicative of alterations in energy metabolism and cell viability. Our data indicated a strong increase of ADP/ATP ratio in RTX-treated cells as compared with vehicle (Fig. 3b). Due to their inhibitory activity towards mitochondria, vanilloids can determine an excess of superoxide (O2–), hydroperoxide (H2O2) and hydroxyl ions (OH–) with a consequent induction of oxidative stress and cell death. We thus measured ROS production in BC cells treated with RTX for different times (0–24 h) and found a significant increase in ROS concentration from 8 to 24 h of treatment (Fig. 3c). Overall, our data confirm that RTX induces an altered redox homeostasis. 3.3. RTX reduces tumor growth in experimental tumor xenografts models Finally we tested whether RTX could reduce tumor growth in vivo. T24 cells were thus injected into athymic nude mice (tumor

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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 vehicle, 20 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 the percentage 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 only one vehicle-treated sample is shown. (b) ADP/ATP ratio in T24 and 5637 cells untreated or treated with 20 lM RTX for 12–24 h. Data are the mean ± SD of three independent experiments. Two-way Anova, ⁄⁄p 6 0.01, RTX-treated vs vehicle-treated cells; #p 6 0.01 RTX-treated cells for 24 h vs RTX-treated cells for 12 h. (c) ROS production was evaluated in T24 and 5637 cells, treated with RTX for different times, by using CellROXÒ Flow Cytometry Assay Kit and FACS analysis. Data, shown as the mean ± SD of three independent 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 Bonferroni post-test, ⁄⁄p 6 0.01, RTX-treated cells vs control; #p 6 0.01, RTX-treated cells for 24 h vs RTX-treated cells for 12 h.

take 100%) and RTX challenge started as soon as the tumor mass reached the volume of about 50 mm3. As shown in Fig. 4a, RTX treatment significantly reduced tumor growth from the 9th day after challenge (P < 0.001). After 3 weeks from RTX challenge mouse were euthanized, tumors were surgically removed and size and weight were registered. Data obtained in vivo demonstrated that RTX exerts a potent anti-tumor activity inducing a significant reduction in size (Fig. 4b) and weight (about 1.7-fold, Table 1) when compared to controls. No signs of inflammatory infiltrates were found in the epithelial and sub-epithelial tissues surrounding the tumor mass in RTX-treated animals (Fig. 4c). The side-effects of RTX were also evaluated based on the in vivo assay. No mouse died during the experimental time, and the body weight average of the mice in the RTX group was not significantly different from that of the mice in the vehicle group (Table 1).

3.4. Tumors from RTX-treated mice display reduced proliferation and necrosis Histological analysis of tumors revealed that RTX treatment significantly reduces mitotic index (Fig. 5a). Sections processed for Ki-67 showed the presence of positive-stained cells in both vehicle- and RTX-treated tumors (Fig. 5b); however, a significant

decreased number of positive cells was found in tumors from RTX-treated mice. Moreover, H&E staining showed the presence of extensive necrotic areas in the external portion of tumors from RTX-treated mice that were absent in tumors from vehicle-treated animals (Fig. 5a).

4. Discussion We demonstrate for the first time that RTX is able to induce cell cycle arrest and necrotic cell death associated with mitochondrial dysfunction in different BC cell lines and, more importantly, it reduces in vivo the growth of human BC cells xenografted into nude mice. Thus, RTX can be considered as a new potential molecule for BC therapy. These results appear to be significant if we consider that, despite several efforts have been made for the development of new effective therapies, TCC of the bladder still remains a high recurrent malignancy. Although transurethral resection (TUR) of TCC of the bladder induces an 80% early success rate, nearly 70% of these patients will develop tumor recurrence, with 25% showing progression to muscle-invasive disease within 5 years with TUR. Intravesical chemotherapy and immunotherapy are widely used as adjuvant therapies after TUR to prevent recurrence and progression of superficial disease, but meta-analysis

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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 mice that had been inoculated s.c. with human T24 cells. Tumor volumes were calculated by the formula: (D  d2)/2, where D = major diameter and d = minor diameter. Results represent the mean ± SD of 10 mice in each group. ANOVA Bonferroni post-test, ⁄⁄⁄p 6 0.001, RTX- vs vehicle-treated mice. (b) Representative image of the macroscopic appearance of heterotopic xenografts surgically removed at the end of the treatment protocol. (c) Representative sections from vehicle- and RTX-treated mice stained with hematoxylin alone. E = epithelial layer; SE = sub-epithelial layer; T = tumor. Calibration bar 100 lm.

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

Body weight (g)

Tumor incidence (%)

Tumor mass (mg)

Vehicle RTX

26.8 ± 2.1 27.2 ± 1.5

100 100

380 ± 10.8 219 ± 7.4**

Student’s t-test, ** p 6 0.01, RTX- vs vehicle-treated mice.

does not show apparent superiority of a particular treatment [20,21]. For this reason it is fundamental to develop optimal, less toxic chemotherapy regimens by incorporating novel targeted agents to improve the outcomes. The cytotoxic activity of RTX has been documented in vitro for pancreatic, lung and prostate cancer cells [11,13,14,17] but at present no studies have been conducted to test the anticancer efficacy of RTX in BC models in vitro and in vivo. Despite RTX is an ultrapotent TRPV1 agonist and even

if T24 cells express high levels of TRPV1 [19], the mechanism of RTX action we described in this work is TRPV1-independent and involves the alteration of the redox homeostasis with a significant mitochondrial depolarization, increase of ADP/ATP ratio and ROS production. Our findings are in line with those of the literature that indicate vanilloids as compounds able to interfere with the cellular redox homeostasis by directly antagonizing the coenzyme Q and affecting the electron transport chain [11,22,23]. This cell death mechanism can be potentially active in every cell: tumor cells are known to present a non-controlled cellular activity and they need to have a high quantity of energy at their disposal, thus the constitutional activation of the electron transport chain. For this reason, vanilloids can induce mitochondrial perturbation and subsequent delivery of ROS especially in proliferating cancer cells. The use of the xenograft model of BC in this study not only confirms the data obtained in vitro but also gives precious information about the potential side effects of RTX treatment. Intravesical delivery of

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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 RTXtreated mice stained with H&E (upper panels, calibration bar 100 lm) or hematoxylin alone (lower panels, calibration bar 25 lm). In the section from RTX-treated tumors is evident 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) is evident in sections from RTX-treated tumors as reported in the graph. Data are expressed as the mean ± SD of mitotic counts performed in 50 fields. ⁄⁄p 6 0.01 RTX- vs vehicle-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 graph are expressed as the mean ± SD of Ki-67 positive cells performed in 10 fields of an area of 0.05 mm2 for each tumor. ⁄p 6 0.05 RTX- vs vehicle-treated mice.

vanilloids, i.e. capsaicin, has been previously observed to induce autonomic dysreflexia, limb spasms, suprapubic discomfort and hematuria [24]. This is not the case for RTX, which shows a far more favorable ratio of desensitization to irritation than capsaicin because it is practically pungent-free [25]. Indeed, no side effects were induced by RTX treatment in our experimental model. RTX (U.S. patent number 8.338.457) is not suitable for systemic administration but subcutaneous, intraganglionic or intrathecal applications are under preclinical investigations for the treatment of pain in advanced cancer [26]. Moreover, recently, the National Institutes of Health in collaboration with Sorrento Therapeutics has started the recruitment of participants for a new clinical trials to demonstrate the safety of administering RTX directly into the human central nervous system [27]. In addition, RTX has been previously used in humans for the treatment of bladder dysfunctions and bladder pain syndrome and several clinical trials have been conducted by using different doses of RTX intravesically delivered without inducing any substantial local or systemic side effects [28–31]. 5. Conclusion Overall, we demonstrated that RTX treatment in vitro induces necrotic cell death of BC cells by affecting redox homeostasis and in vivo reduces tumor growth in a xenograft mouse model of BC. Therefore, since the high adaptability of RTX to a variety of pain problems and the relatively low toxicity when used locally (e.g. intravesical or peritumoral administration), it could represent a new 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 be found in the online version. Acknowledgements This work was supported by FIRC national grant (number 11095) and by a grant from the Italian Ministry of University and Research, PRIN 2009–2011. References [1] R. Siegel, D. Naishadham, A. Jemal, Cancer statistics, CA Cancer J. Clin. 62 (2012) (2012) 10–29. [2] D.S. Kaufman, W.U. Shipley, A.S. Feldman, Bladder cancer, Lancet 374 (2009) 239–249. [3] H. von der Maase, L. Sengelov, J.T. Roberts, S. Ricci, L. Dogliotti, T. Oliver, M.J. Moore, A. Zimmermann, M. Arning, Long-term survival results of a randomized trial comparing gemcitabine plus cisplatin, with methotrexate, vinblastine, doxorubicin, plus cisplatin in patients with bladder cancer, J. Clin. Oncol. 23 (2005) 4602–4608. [4] Advanced Bladder Cancer (ABC) Meta-analysis Collaboration, Neoadjuvant chemotherapy in invasive bladder cancer: update of a systematic review and meta-analysis of individual patient data Advanced Bladder Cancer (ABC) Metaanalysis Collaboration, Eur. Urol. 48 (2005) 202–205. discussion 205–206. [5] H.B. Grossman, R.B. Natale, C.M. Tangen, V.O. Speights, N.J. Vogelzang, D.L. Trump, R.W. deVere White, M.F. Sarosdy, D.P. Wood Jr., D. Raghavan, E.D. Crawford, Neoadjuvant chemotherapy plus cystectomy compared with

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