Repurposing ketoconazole as an exosome directed adjunct to sunitinib in treating renal cell carcinoma

Abstract

Renal Cell Carcinoma (RCC) is the most common form of kidney cancer, with clear cell RCC (ccRCC) representing about 85% of all RCC tumors. There are limited curable treatments available for metastatic ccRCC because this disease is unresponsive to conventional targeted systemic pharmacotherapy. Exosomes (Exo) are small extracellular vesicles (EVs) secreted from cancer cells with marked roles in tumoral signaling and pharmacological resistance. Ketoconazole (KTZ) is an FDA approved anti-fungal medication which has been shown to suppress exosome biogenesis and secretion, yet its role in ccRCC has not been identified. A time-course, dose-dependent analysis revealed that KTZ selectively decreased secreted Exo in tumoral cell lines. Augmented Exo secretion was further evident by decreased expression of Exo biogenesis (Alix and nSMase) and secretion (Rab27a) markers. Interestingly, KTZ-mediated inhibition of Exo biogenesis was coupled with inhibition of ERK1/2 activation. Next, selective inhibitors were employed and showed ERK signaling had a direct role in mediating KTZ’s inhibition of exosomes. In sunitinib resistant 786-O cells lines, the addition of KTZ potentiates the efficacy of sunitinib by causing Exo inhibition, decreased tumor proliferation, and diminished clonogenic ability of RCC cells. Our findings suggest that KTZ should be explored as an adjunct to current RCC therapies.

Combination of Tipifarnib and Sunitinib Overcomes Renal Cell Carcinoma Resistance to Tyrosine Kinase Inhibitors via Tumor-Derived Exosome and T Cell Modulation

“…The study protocol was conducted in accordance with current guidelines and federal/local regulations. The normal plasma samples (the control group) were purchased from BioIVT elevating science (Westbury, NY, USA) [ 21 ].…”

“…Tipifarnib and sunitinib were obtained from Selleckchem (Houston, TX, USA). All additional chemicals used in this study were obtained from Sigma (St. Louis, MO, USA) [ 21 ].…”

“…Sunitinib-resistance (SR) was induced in these cell lines (786-0-SR, Caki-2-SR, and A498-SR) by treating sensitive cells with increasing dosages of sunitinib for six months. Once SR was confirmed at high dosages, cultures were maintained by adding 5 μM sunitinib to cell media for more than 10 passages as previously described [ 21 ]. Full images for Jurkat T cells detected with triple staining can be found in Supplementary Figure S1 .…”

“…Cells were then allowed to grow for 48 h. Subsequently, media was extracted from the plate, and the supernatant was isolated for exosome isolation as previously described. Finally, exosomes were purified by using differential ultracentrifugation (DU) as described previously [ 21 ].…”

“…Thereafter, RCC cells were treated with various concentrations of tipifarnib and sunitinib dissolved in DMSO for 48 h at 37 °C, 5% CO 2 in a humidified CO 2 incubator [ 23 ]. Cell viability was measured by the MTT (methylthiazolyldiphenyl-tetrazolium bromide) (Sigma-Aldrich, St. Louis, MO, USA) cell cytotoxicity assay according to the manufacturer’s protocol as we described [ 21 ]. Cell viability was determined by incubating the treated cells with MTT solution (5 mg/mL) for 1 h at 37 °C.…”