Resiniferatoxin induces death of bladder cancer cells associated with mitochondrial dysfunction and reduces tumor growth in a xenograft mouse model
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 Western countries is transitional cell carcinoma (TCC) [2]. Approximately 30–40% of patients with high-risk non-muscle-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 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], [11], [12], [13], [14], [15], [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 pre-clinical experiments.
Section snippets
Cell lines
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
RTX induces necrotic cell death of BC cells independently from TRPV1
We initially evaluated the effects of different RTX doses (0.1–50 μM) 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 μM for 5637 cells). Since T24 but not 5637 cells [17], [18] express TRPV1 channel, we used RTX in combination with 5′-iodoresiniferatoxin (I-RTX), a strong competitive antagonist of TRPV1 receptor, to verify the involvement of TRPV1 in T24 cell growth
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
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.
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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.
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Valerio Farfariello and Sonia Liberati: equal contribution.