TRPV2 Expression and Its Role in Proliferation of Human Multiple Myeloma Cell Lines

Abstract 5003

Multiple myeloma (MM) is a disease typically characterized by repeated relapse that needs several new-drugs throughout its clinical course. Therefore, increasing knowledge of the pathways involved in the pathophysiology of MM will contribute to identified new therapeutic strategies. Specific signaling pathways as: nuclear factor kappa B (NFkB), farnelsyl-transferase (FTI), mitogenen-activated protein kinase (MAPK), proteosoma and others have been found to be disregulated in MM patients and cell lines. These findings have allowed the design of specific inhibitors that have been assessed in preclinical and clinical studies. Recent data have highlighted the contribute of ion channels (K⁺, Cl⁻, Na⁺ and Ca²⁺) in the regulation of cell proliferation, chemo-resistance, migration and invasion. Transient Receptor Potential Vanilloid type-2 (TRPV2) is a cation channel, member of the TRPV family, and its expression in human cancer cells and tissues has been reported for gliomas, prostate, bladder and pancreas, while no data were available in MM or in other hematological malignancies. TRPV2 expression has been found to influence cancer cell responses to chemo-therapeutic drugs as well as proliferation, migration and apoptosis, in part by increasing intracellular Ca²⁺ influx and/or by regulating specific signal pathways, like MAPK associated pathways. TRPV2 responds to noxious heat with an activation threshold of >52°C as well as to changes in osmolarity and membrane stretch; in addition, TRPV2 is activated by agonists such as 2-aminoethoxydiphenyl borate, as well as cannabinoids. The aim of this study was, firstly, to evaluate if TRPV2 was expressed in three human MM cell lines established models (RPMI, SKO-007, U266). Secondly we investigated on the role of TRPV2 activation in regulating the proteosoma inhibitor Bortezomib activity, in MM cell lines. By Real-Time PCR analysis we demonstrated that TRPV2 gene was expressed, with comparable relative levels, in the MM cell lines studied, and similar results were obtained at protein levels by Western blot analysis. Moreover, by immunofluorescence and FACS analysis we found that the percentage of TRPV2⁺ cells was 11% in RPMI, 3% in U266 and 2.6% in SKO-007. These data indicate that TRPV2 was expressed at low levels in MM cell lines. Since lost of TRPV2 expression was found to be associated with higher cell proliferation rate, in other tumor cell lines, and increasing of TRPV2 transcription and/or activity by specific agonists induced cell death and anti-proliferative effects, we evaluated the biological effects of TRPV2 activation in MM cell lines by MTT assay. The results showed that, after three incubation days, TRPV2 activation induced a decrease of cell viability of 70% in RPMI, 60% in U266 and 55% in SKO-007, compared to control. To evaluate if the reduced cell viability was dependent by an anti-proliferative and/or apoptotic process, the three cell lines treated with the TRPV2 agonist were analyzed by 5-bromo-2-deoxyuridine (BrdU) incorporation assay for proliferation and by Annexin-V apoptosis assays. Data shown a decrease of BrdU⁺ cells in TRPV2 agonist treated cells (40% in RPMI, 30% in U266 and 28% in SKO-007) compared to control, while no significant differences were observed by Annexin-V analysis. Moreover, to evaluate a putative role of TRPV2 in influencing Bortezomib cytotoxicity, RPMI, SKO-007 and U266, were co-treated with the TRPV2 agonist in combination or not with Bortezomib, for three days. By a dose-response MTT analysis we determined that TRPV2 activation reduced MM cell lines viability more than 40% in RPMI, 20 % in U266 and 15% in SKO-007, compared to Bortezomib (5 ng/ml) alone. Summarizing, these preliminary data demonstrated that the TRPV2 cation channel was expressed in human MM cell lines and that activation of TRPV2 could play a role in increasing Bortezomib cytotoxicity, by inhibiting cell proliferation rather than increasing apoptosis of MM cells. These data may open new perspectives in combination therapy, albeit the molecular mechanisms undergone TRPV2 activation needs to be clarified.