TRPV6 Calcium Channel Targeting by Antibodies Raised against Extracellular Epitopes Induces Prostate Cancer Cell Apoptosis

Abstract

The TRPV6 calcium channel is known to be up-regulated in various tumors. The efforts to target the TRPV6 channel in vivo are still ongoing to propose an effective therapy against cancer. Here, we report the generation of two antibodies raised against extracellular epitopes corresponding to the extracellular loop between S1 and S2 (rb79) and the pore region (rb82). These antibodies generated a complex biphasic response with the transient activation of the TRPV6 channel. Store-operated calcium entry was consequently potentiated in the prostate cancer cell line LNCaP upon the treatment. Both rb79 and rb82 antibodies significantly decreased cell survival rate in a dose-dependent manner as compared to the control antibodies of the same isotype. This decrease was due to the enhanced cell death via apoptosis revealed using a sub-G1 peak in a cell cycle assay, TUNEL assay, and a Hoechst staining, having no effects in the PC3Mtrpv6−/− cell line. Moreover, all TUNEL-positive cells had TRPV6 membrane staining as compared to the control antibody treatment where TRPV6-positive cells were all TUNEL negative. These data clearly demonstrate that TRPV6 channel targeting using rb79 and rb82 antibodies is fatal and may be successfully used in the anticancer therapies.

Trpv6 channel targeting using monoclonal antibody induces prostate cancer cell apoptosis and tumor regression

“…In order to evaluate the consequences of mAb82 binding to TRPV6, electrophysiological recordings using whole cell configuration were performed in HEK cells transiently transfected with vEF1ap-5’UTR-TRPV6_CMVp-mGFP vector. Incubation in the physiological solution HBSS was used to stabilize HEK cell and inhibit constitutive TRPV6 activity due to the presence of 2 mM Ca 2+ in the solution [ 28 ]. Following acquisition of TRPV6 activity under basal conditions, the extracellular (bath) solution was replaced by a divalent-cation-free (DVF) solution, known to prevent Ca 2+ -induced TRPV6 inactivation [ 29 ] and thus allowing monovalent currents passing through TRPV6 to be seen (Fig.…”

“…In this work, the generation and the use of a mAb, called mAb82, against the highly selective calcium channel TRPV6 has been reported. The epitope used in this work is identical to that used for the generation of the rabbit polyclonal antibody, rb82, which was published previously [ 28 ]. Despite the use of the same peptide against which these mAb82 and rb82 were generated, the actions of these two antibodies are different.…”

News and views on ion channels in cancer: is cancer a channelopathy?

“…One such calcium-dependent gene expression is the transcription factor Nuclear Factor of Activated T cell (NFAT). Elevated TRPV6 has been seen in several cancers (e.g., breast, ovarian, prostate, and pancreatic) and its activity is linked to the calcium-dependent transcription of NFAT which has been identified as a key pathway in these cancers ( Haustrate et al, 2023 ; Lehen’kyi et al, 2012 ; Nilius and Szallasi, 2014 ; Tran Quang et al, 2015 ).…”

Characterization of the TRPV6 calcium channel-specific phenotype by RNA-seq in castration-resistant human prostate cancer cells

“…The plasmids vEF1ap-5′UTR-TRPV6_CMVp-mCherry vector and control vEF1ap-5′UTR_CMVp-mCherry vector (e-Zyvec, Loos, France) were obtained as previously described (Haustrate et al, 2023b). The cells were transfected using the Lipofectamine 3000 transfection reagent (Thermofisher, Massachusetts, United States) following the manufacturer's instructions and were selected with the selective antibiotic G418 at 200 μg/mL.…”