Kinase experiment:

Renal cancer cells are incubated with ROC-325 for 24 hours. Cells are harvested and then lysed. Approximately 50 μg of total cellular protein from each sample are subjected to SDS-PAGE, proteins are transferred to nitrocellulose membranes, and the membranes are blocked with 5% nonfat milk in a Tris-buffered saline solution containing 0.1% Tween-20 for 1 hour. The blots are then probed overnight at 4℃ with primary antibodies, washed, and probed with species-specific secondary antibodies coupled to horseradish peroxidase. Immunoreactive material is detected by enhanced chemiluminescence[1].

Cell experiment:

Cell viability is estimated by the MTT assay. Cells are seeded into 96-well microcultureplates at 10,000 cells per well and allowed to attach for 24 hours. Cells are then treated with ROC-325 for 72 hours. Following ROC-325 treatment, MTT is added and formazan absorbance is quantified using a microplate reader. The estimated cell viability under each experimental condition is calculated by normalizing the respective formazan optical density to the density of control cells. Proapoptotic effects following in vitro ROC-325 exposure are quantified by propidium iodide (PI) staining and fluorescence-activated cell sorting (FACS) analysis of sub-G0/G1 DNA content and by measurement of active caspase-3 by flow cytometry using a commercial kit[1].

Animal experiment:

786-0 renal cancer cells (5×106) are suspended in a mixture of HBSS and Matrigel and subcutaneously implanted into female nude mice. Tumor-bearing animals from each cell line xenograft are randomized into treatment groups. Mice are treated with vehicle (water), ROC-325 (25, 40, and 50 mg/kg PO) QD×5 for 6 weeks. Mice are monitored daily and tumor volumes are measured twice weekly. At study completion, tumors from representative animals are excised from each group, formalin-fixed, and paraffin-embedded for immunohistochemical analysis[1].

ROC-325 is a novel inhibitor of autophagy.

ROC-325 is a novel inhibitor of autophagy. Treatment with ROC-325 results in a significant loss of acridine orange fluorescence. ROC-325 triggers a highly significant increase in cathepsin D (CTSD) levels. ROC-325 treatment yields pharmacodynamic effects that are consistent with inhibition of autophagy. Treatment with 5 μM ROC-325 for 24 hours leads to the formation of LC3B punctae and a robust increase in LC3B levels in both A498 and 786-0 RCC cells. Immunoblotting analysis conducted in both A498 and 786-0 cells demonstrates that ROC-325 promotes a dose-dependent increase in LC3B expression in a manner that correlated with a corresponding increase in the levels of p62 and cathepsin D[1].

ROC-325 treatment leads to significant, dose-dependent inhibition of disease progression. ROC-325 is well tolerated and no notable toxicities are observed other than a very modest, nonsignificant reduction in mean body weight at the highest dose. Immunohistochemical analysis of specimens collected from animals treated with ROC-325 demonstrates significant, dose-dependent increases in the autophagic markers LC3B and p62 and increases apoptosis[1].

[1]. Carew JS, et al. Disruption of Autophagic Degradation with ROC-325 Antagonizes Renal Cell Carcinoma Pathogenesis. Clin Cancer Res. 2017 Jun 1;23(11):2869-2879.