1. Intracellular determination of activated caspases (IDAC) by flow cytometry using a pancaspase inhibitor labeled with FITC
Sundararajan Jayaraman Cytometry A . 2003 Dec;56(2):104-12. doi: 10.1002/cyto.a.10094.
Background:Flow cytometry-based methods of simultaneous detection of multiple activated caspases in single cells are of diagnostic value and remain to be fully explored.Methods:Genomic DNA fragmentation was determined by agarose gel electrophoresis and flow cytometry. Whole cells were incubated with the cell-permeable pancaspase inhibitor, FITC-Val-Ala-Asp-fluoro-methyl-Rerone (FITC-VAD-FMK), and propidium iodide (PI) and analyzed for intracellular activated caspases and plasma membrane integrity, respectively, by flow cytometry.Results:Two distinct populations of apoptotic cells were identified by flow cytometry: an early apoptotic population indicated by FITC-VAD-FMK binding and the late apoptotic cells characterized by FITC-VAD-FMK staining and permeability to PI. The binding of FITC-VAD-FMK to apoptotic cells was time dependent and concurred with DNA fragmentation. Pretreatment with the pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-methyl-fluoro-methyl-kerone (Z-VAD (OMe)-FMK), coordinately attenuated apoptosis induction and activation of caspases. The pancaspase inhibitor also competitively blocked the binding of FITC-VAD-FMK to early apoptotic cells in vitro. Significantly, the use of FITC-VAD-FMK permitted apoptosis determination in a number of cell lines including the caspase-3 gene-deleted MCF-7 responding to various apoptotic stimuli.Conclusions:Apoptosis detection based on FITC-VAD-FMK binding is specific and easily performed by flow cytometry in a variety of cells. This novel technique has implications for detecting apoptosis in pathophysiologic conditions.
2. Antifungal Effects and Potential Mechanisms of Benserazide Hydrochloride Alone and in Combination with Fluconazole Against Candida albicans
Xueqi Chen, Shan Su, Jiyong Wu, Shujuan Sun, Jing Nie, Lei Sun Drug Des Devel Ther . 2021 Nov 16;15:4701-4711. doi: 10.2147/DDDT.S336667.
Purpose:The resistance ofC. albicansto traditional antifungal drugs brings a great challenge to clinical treatment. To overcome the resistance, developing antifungal agent sensitizers has attracted considerable attention. This study aimed to determine the anti-Candidaactivity of BEH alone or BEH-FLC combination and to explore the underlying mechanisms.Materials and methods:In vitro antifungal effects were performed by broth microdilution assay and XTT reduction assay. InfectedGalleria mellonellalarvae model was used to determine the antifungal effects in vivo. Probes Fluo-3/AM, FITC-VAD-FMK and rhodamine 6G were used to study the influence of BEH and FLC on intracellular calcium concentration, metacaspase activity and drug efflux ofC. albicans.Results:BEH alone exhibited obvious antifungal activities againstC. albicans. BEH plus FLC not only showed synergistic effects against planktonic cells and preformed biofilms within 8 h but also enhanced the antifungal activity in infectedG. mellonellalarvae. Mechanistic studies indicated that antifungal effects of drugs might be associated with the increasement of calcium concentration, activation of metacaspase activity to reduce virulence and anti-biofilms, but were not related to drug efflux.Conclusion:BEH alone or combined with FLC displayed potent antifungal activity both in vitro and in vivo, and the underlying mechanisms were related to reduced virulence factors.
3. Protective autophagy or autophagic death: effects of BEZ235 on chronic myelogenous leukemia
Yan Zheng, Xiongpeng Zhu, Chuntuan Li, Qunyi Peng, Tingjin Zheng, Wenqian Xu, Pengliang Xin, Wenzhao Cheng Cancer Manag Res . 2019 Aug 22;11:7933-7951. doi: 10.2147/CMAR.S204472.
Purpose:To investigate the effects of BEZ235 on chronic myeloid leukemia (CML) cells.Methods:MTS assay was used to detect the proliferation of CML cells. The proteins expression were detected by Western blot assay. The effects of BEZ235 on autophagy in CML cells were verified through transmission electron microscopy and evaluated by laser confocal microscopy. Annexin V-FITC/PI double staining flow cytometry was used to detect apoptosis. A xenograft model was established to observe the therapeutic effect of BEZ235 in vivo.Results:BEZ235 could inhibit the proliferation of CML cells; CQ and 3-MA could increase the proliferation inhibition and Z-VAD-FMK can reduce the proliferation inhibition of BEZ235 on CML cells (P<0.05). Results of TEM showed that the autophagosomes of CML cells treated with BEZ235 increased (P<0.05). The results by confocal microscopy showed that the autophagic activity of K562 cells increased with BEZ235 treatment. When BEZ235 combined with CQ, BEZ235-induced autophagic flow was blocked. FCM results showed that BEZ235 could induces apoptosis in CML cells. Z-VAD-FMK could decrease the apoptosis of CML cells induced by BEZ235. CQ increased the apoptosis of CML cells induced by BEZ235 (P<0.05). Western blot showed that BEZ235 inhibited the phosphorylation of AKT and S6K. BEZ235 alone could upregulate the expression of cleaved caspase-3 and LC3II. When combined with Z-VAD-FMK, the expression of cleaved caspase-3 was lower than that of BEZ235 alone. When combined with CQ, the expression of cleaved caspase-3 and LC3II were higher than those of BEZ235 alone (P<0.05). BEZ235 could inhibit the growth of xenografts of CML cell line.Conclusion:BEZ235 can inhibit the proliferation of CML cells, induce apoptosis, and enhance autophagy activity. It induces protective autophagy. The combination of CQ can enhance the apoptosis and proliferation inhibition of CML cells induced by BEZ235.