Effect of [4-valine 5-glutamine] desmopressin, a novel vasopressin analog, on human breast cancer cell growth in two-dimensional (2D) and three-dimensional (3D) cultures

GARONA J; PIFANO M; PASTRIAN B; IANNUCCI N; GOMEZ DE; ALONSO DF; RIPOLL GV

Texas

Simposio; 36th Annual San Antonio Breast Cancer Symposium; 2013

AACR

Background: Desmopressin (dDAVP), a synthetic nonapeptide derivative of arginine vasopressin, is a safe hemostatic compound that acts as a selective agonist for the vasopressin V2 membrane receptor (V2R). This receptor is expressed in endothelium and also in some tumor cells. V2R stimulation is associated with increased levels of cyclic AMP and cyclic AMP-dependent protein kinase activation. We previously reported that dDAVP can inhibit progression of residual metastatic cells in preclinical models. Among other mechanisms, the compound induces an agonist effect on V2R present in tumor cells.  This nonapeptide was substituted in positions 4 and 5 searching for compounds with improved biological activity. Such positions belong to the conformational peptide loop which has a key role in ligand-receptor interaction. After an extensive in vitro and in vivo characterization, novel peptide compound [4-valine 5-glutamine] dDAVP ([V4Q5]dDAVP) exerted a remarkable and improved antitumor effect in comparison to parental peptide dDAVP. [V4Q5]dDAVP inhibited tumor growth in both syngeneic F3II and xenogeneic MDA-MB-231 breast cancer models, modulating tumor aggressiveness and vasculature. In this study, to further understand direct cytostatic action of this novel vasopressin analog on tumor cells, we assessed the effect of [V4Q5]dDAVP on clonogenic and three dimensional growth of V2R-expressing human breast carcinoma cell lines. Methods and results: We used MCF7 and triple negative MDA-MB-231 human breast cancer cell lines to conduct the experiments. Vasopressin V2 membrane receptor expression in tumor cell lines was assessed by RT-PCR and immunofluorescence. We first examined peptide ability to modify in vitro low density cell growth by the clonogenic growth assay. Incubation with [V4Q5]dDAVP (100-1500 nM) during one week significantly reduced the number of tumor cell colonies showing a concentration dependant manner and an IC50 value of 1135 nM. To assess the effect of novel analog on three dimensional growth of cancer cells we used two different assays. The first one evaluates the ability of basement membrane extract (BME)-embedded single cells to proliferate and form spheroids. The second assay evaluates growth of single fully established spheroids (Approx. 300 μm diameter) transplanted individually on a non-adherent surface. [V4Q5]dDAVP, at a concentration of 1000 nM, was able to significantly reduce the number of BME-embedded spheroids formed from single cells after 14 days of treatment. In a similar manner, incubation of established and isolated spheroids with [V4Q5]dDAVP using the same concentration for 9 days, caused a decrease in multicellular spheroids final diameter. Cellular metabolic activity analysis by the MTS assay showed no significant differences between treated and control groups. Conclusions: We report for the first time the inhibitory effect of [V4Q5]dDAVP on colony and spheroid formation and growth of human breast carcinoma cells. Our results using three dimensional models suggest that V2R stimulation in cancer cells may affect early stages of microtumor establishment. Further analysis of involved signaling pathways in treated 3D cultures is needed.