#4157 A NOVEL STRATEGY TO TARGET V2R IN RENAL CARCINOMA
Authors: Zucaro Laura , Suzumoto Yoko , D’apolito Luciano , De Rosa Giuseppe , Capasso Giovambattista , Trepiccione Francesco 
Published: June 2023

Abstract

Background and Aims
The vasopressin V2 receptor (V2R) is a G-protein Coupled Receptor (GPCR) selectively expressed in the kidney. V2R is located at basolateral membrane of principal cells of the distal nephron and thick ascending limb cells and in response to circulating levels of vasopressin (AVP), promotes sodium and water reabsorption. Ectopic expression of V2R plays a role in clear cell renal carcinoma (ccRCC). A recent study shows that tolvaptan, a V2R-antagonist, inhibits cancer growth in an experimental model of ccRCC. However, polyuria remains a serious side effect, limiting the repositioning of this drug in clinical practise. Recent evidences demonstrate that ccRCC have also an up regulation of the transferrin receptor 1 (TfR1). Based on this, we developed Transferrin-conjugated liposomes (Tf-LPs) encapsulating tolvaptan to allow a site-specific delivery to the tumor site, in the attempt to reduce drug side effects. We performed in vitro studies to evaluate the effect of Tf-LPs on cell proliferation and the immunoblotting of cyclin A2, an essential regulator of the cell cycle that participates in the regulation of S phase as well as mitotic entry.

Method
Liposome formulation and characterization. Liposomes composed of DPPC/DSPEPEG2000/DSPEPEG-MAL were mixed with tolvaptan and prepared by hydration of a thin lipid film, followed by extrusion. Transferrin was conjugated to liposomes at room temperature, overnight. The resulting liposomes were then purified by ultracentrifuge (80000 rpm, 40 min, 4°C). Liposomes were characterized in terms of colloidal dimensions, polydispersity index and surface charge by using dynamic light scattering (DLS) (Zetasizer Nano Z, Malvern, UK). The encapsulation efficiency of Tolvaptan was measured by spectrophotometer (Epoch, Biotek) at the wavelength of 266 nm. In vitro studies. CAKI-1 cells were cultured until the exponentially growing phase. Cells were then incubated with Tf-LPs with or without tolvaptan in growth medium with 0.2%FBS for 24h. Cell viability was assessed by measuring the mitochondrial activity by the 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay quantified by spectrophotometry at 570nm. Cell lysate were immunoblotted and Cyclin A2 primary antibody tested as a marker of cell proliferation.

Results
The liposomes we have generated have an average diameter of about 100 nm, a low polydispersity index (&lt;0.2) and a 50% encapsulation efficiency. MTT assay showed that, compared with control, cells treated with Tf-LPs encapsulating tolvaptan have a reduction in cell proliferation. Interestingly, this reduction starts at lower concentrations and timescales than in a previous study performed with free drug. To further confirm the previous result, we also performed immunoblotting of cyclin A2 which is usually linked to cell proliferation and as such is often found expressed at a high level in human cancers. The expression level of this protein is significantly reduced when cells are treated with Tf-LPs+TOLV while it remains the same in the treatment with Tf-LPs.

Conclusion
In this study we developed and tested Tf-LPs encapsulating tolvaptan with cytotoxic activity on tumor cells. The preliminary results of this study will be confirmed by subsequent in vivo studies to demonstrate the advantages of this system: site-specific delivery to the tumor site and reduction of drug side effects.