An autophagy inhibitor
ROC-325 is an autophagy inhibitor.1 It inhibits autophagic flux and induces lysosomal deacidification and apoptosis in 786-O renal cell carcinoma cells when used at a concentration of 5 ?M. ROC-325 decreases viability in a panel of 12 cancer cell lines, including renal, lung, pancreatic, and prostate cancer cells, with IC50 values ranging from 4.9 to 11 ?M. It reduces tumor growth in a 786-O mouse xenograft model when administered at a dose of 50 mg/kg. ROC-325 also decreases the cytopathic effect of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in infected Vero E6 cells (EC50 = 3.28 ?M).2
1.Carew, J.S., Espitia, C.M., Zhao, W., et al.Disruption of autophagic degradation with ROC-325 antagonizes renal cell carcinoma pathogenesisClin. Cancer Res.23(11)2869-2879(2017) 2.Gorshkov, K., Chen, C.Z., Bostwick, R., et al.The SARS-CoV-2 cytopathic effect is blocked by lysosome alkalizing small moleculesACS Infect. Dis.(2020)
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°C 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]. |
参考文献: [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. | |
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