TRPV2 Activation Induces Apoptotic Cell Death In Human T24 Bladder Cancer Cells: A Potential Therapeutic Target For Bladder Cancer

Main Category: Cancer / Oncology
Also Included In: Urology / Nephrology
Article Date: 11 Aug 2010 - 0:00 PDT

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Bladder carcinoma is the second most common malignancy of the urinary tract and nearly 90% of all primary tumors of the bladder are urothelial carcinomas (UCs). Superficial UCs can be "shaved off" using an electrocautery device attached to a cystoscope. Immunotherapy in the form of bacillus Calmette-Guerrin (BCG) instillation is also used to treat and prevent the recurrence of superficial UCs, but there are still patients, whose UCs recurred after treatment with BCG. Therefore, the development of new drugs that target UC cells is desirable.

In cancer cells, many proteins exhibit increased or decreased expression compared to their levels of expression in normal cells. Some of the proteins are mainly encoded by oncogenes and tumor suppressor genes, others are involved in intracellular calcium ion ([Ca2+]i) homeostasis. Since transient receptor potential (TRP) channels are regulators of [Ca2+]i and several members of the TRP family (TRPV1, TRPM8, TRPV6 and TRPM1) have been reported to show altered expression in cancer cells, TRP channels present in UCs could be potential target molecules for the development of novel strategies for antitumor therapeutics.

The present study focused on TRPV2 channel (1), because a previous study showed that expression levels of TRPV2 mRNA and protein directly correlated with stage and grade of malignancy in human UC tissues (2). Starting from superficial pT1 towards the more invasive pT2 and pT3 stages, progressive up-regulation of TRPV2 expression was found, reaching very high levels in muscle-invasive pT4 stage. Unfortunately, the authors examined the gene and protein expressions by RT-PCR, western blot and immunohistochemical analyses, but it was still unknown whether TRPV2 in UCs is functional or not.

There are two phases in this experiment. In the first phase, functional TRPV2 expression in two types of human UC cell lines (RT4, a well-differentiated cell line and T24, a poorly differentiated cell line) was assessed by investigating the effect of agonists and an antagonist of TRPV2 on [Ca2+]i mobilization. In addition, siRNA silencing of TRPV2 was also performed. In the Ca2+ influx assay, most of T24 cells (95%) responded to 2-aminoethoxydiphenyl borate (2-APB) (an agonist for TRPV1, TRPV2 and TRPV3) and cannabidiol (CBD) (a TRPV2 agonist), but not to capsaicin (a TRPV1 agonist), carvacrol (an agonist of TRPV3 and TRPA1) or menthol (a TRPM8 agonist). In contrast, RT4 cells did not show any response to 2-APB and CBD, implying that functional TRPV2 is abundantly expressed in high-grade T24 cells, but not in low-grade RT4 cells.

In the second phase, we examined the effect of TRPV2 activation on cellular viability in T24 UC cells. The lower concentration of CBD slightly increased cellular viability, whereas the higher concentration dramatically reduced it. A growing number of studies have demonstrated that increases in [Ca2+]i regulate various signaling mechanisms that control proliferation, metabolism and gene transcription; yet, under certain condition, increase in [Ca2+]i are cytotoxic. Although TRPV2 may be involved in cell proliferation and migration in T24 cells, as reported in other TRP channels in cancer cells, we were interested whether TRPV2 can be used to kill cancer cells. Since apoptotic cells are silently removed from tissues in contrast to necrotic cell death that leads to the spillage of intracellular proteins that activates unwished damage responses like inflammation. Thus, an annexin-V assay was combined with siRNA strategy to detect apoptosis in T24 cells. Most significant finding of this study is that continuous CBD treatment induced apoptotic cell death in T24 cells and this effect is predominantly mediated by TRPV2 activation.

This investigation gives support to a previous report in human specimens (2) and further reveals that TRPV2 in high-grade UC cells is indeed functional, suggesting that TRPV2 channel could be a potential therapeutic target for the grade of UC. However, several points should be addressed before clinical investigation. First, it has been reported that TRPV2 mRNA is expressed in the normal human bladder urothelium. Although the expression level is very low (2), it should be determined whether normal human bladder urothelial cells induce calcium influx in response to CBD or other TRPV2 agonists, such as probenecid and tetrahydrocannabinol (THC). In mouse urothelial cells, TRPV2 protein is predominantly expressed in the cytoplasmic compartments and during Ca2+ imaging experiments, the onset of responses to THC was very slow and highly variable between cells (3). It suggests that in normal urothelial cells, TRPV2 protein resides in the cytoplasmic organelle and is translocated by an unknown mechanism, as detected in pancreatic beta cell. Second, there is lack of selective pharmacologic tools specific to TRPV2. CBD used in the present study was previously shown to activate human TRPV1 receptor, to act as an allosteric modulator of mu- and delta opioid receptors, and to be an antagonist of CB1 and CB2 cannabinoid receptors. Many researchers are expectant of a TRPV2 selective agonist. Finally, the pathophysiological roles of TRPV2 in UC cells should be examined. In prostate cancer cells, TRPV2 has been shown to be involved in cell migration (4). It is possible that TRPV2 plays a significant role in cellular migration in UCs as well, because the channel is abundantly expressed in high-grade muscle-invasive pT3 and pT4 of UC. The present study provides a starting point for a number of exciting follow-up investigations into the physiological and pathological roles of TRPV2 in the bladder urothelial cells.

References

1. Yamada T, Ueda T. et al. "TRPV2 Activation Induces Apoptotic Cell Death in Human T24 Bladder Cancer Cells: A Potential Therapeutic Target for Bladder Cancer." Urology in press.

2. Caprodossi S. et al. "Transient Receptor Potential Vanniloid Type 2 (TRPV2) Expression in Normal Urothelium and in Urothelila Carcinoma of Human Bladder: Correlation with the Pathologic Stage." Eur. Urol. 54 (2008): 612-20.

3. Everaerts W. et al. "Functional Characterization of Transient Receptor Potential Channels in Mouse Urothelial Cells." Am. J. Physiol. Renal Physiol. 298 (2010): F692-701.

4. Monet M. et al. "Role of Cationic Channel TRPV2 in Promoting Prostate Cancer Migration and Progression to Androgen Resistance." Cancer Res. 70 (2010): 1225-35.

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