Alexandra Bogdanova1, Martin Kello1, Alexandra Macejova1, Natalia Nosalova1, Peter Petik2, Peter Takac3, Miroslava Martinkova4, Eva Mezeiova4, Ladislav Mirossay1, Peter Gal5,6,7,8, Martina Bago Pilatova9. 1. Department of Pharmacology, Faculty of Medicine, P.J. Šafárik University, Košice, Slovak Republic. 2. Department of Pathology, Faculty of Medicine, P.J. Šafárik University, Košice, Slovak Republic. 3. Department of Pharmacology and Toxicology, University of Veterinary Medicine and Pharmacy, Košice, Slovak Republic. 4. Institute of Chemical Sciences, Department of Organic Chemistry, Faculty of Science, P.J. Šafárik University, Košice, Slovak Republic. 5. Center of Clinical and Preclinical Research MEDIPARK, Faculty of Medicine, P.J. Šafárik University, Košice, Slovak Republic. 6. Department of Biomedical Research, East-Slovak Institute of Cardiovascular Diseases, Inc., Košice, Slovak Republic. 7. Prague Burn Center, Third Faculty of Medicine, Charles University, Prague, Czech Republic. 8. Department of Pharmacognosy and Botany, Faculty of Pharmacy, Comenius University, Bratislava, Slovak Republic. 9. Department of Pharmacology, Faculty of Medicine, P.J. Šafárik University, Košice, Slovak Republic; martina.pilatova@upjs.sk.
Abstract
BACKGROUND/AIM: A series of experiments on HeLa cells were conducted to provide new information concerning the anti-cancer properties of jaspine B hydrochloride (JBH). MATERIALS AND METHODS: HeLa cells treated with 0.5 μmol/l JBH for 24, 48, and 72 h underwent flow cytometric analysis of the cell cycle, and measurement of phosphatidylserine externalization, mitochondrial membrane potential (MMP), casp-3 activation, cleavage of PARP, ceramide levels, aSMase activity, and Bcl-2 release. nSMase activity was measured by a colorimetric assay. Gene expression was determined by qRT-PCR. Immunocytochemistry was performed to detect p21 and p27 expression. RESULTS: JBH-induced apoptosis in HeLa cells associated with externalization of phosphatidylserine, reduced MMP, activation of casp-3, and cleavage of PARP as well as up-regulation of TNF-α, FasL, and casp-8. Significant increase in nSMase activity, ceramide levels, Bcl-2 release (predominantly in the inactive form), and pro-apoptotic nuclear localization of p21 and p27 were also detected. CONCLUSION: JBH-induced apoptosis in HeLa cells is associated with disrupted sphingolipid homeostasis resulting in increased ceramide levels.
BACKGROUND/AIM: A series of experiments on HeLa cells were conducted to provide new information concerning the anti-cancer properties of jaspine B hydrochloride (JBH). MATERIALS AND METHODS:HeLa cells treated with 0.5 μmol/l JBH for 24, 48, and 72 h underwent flow cytometric analysis of the cell cycle, and measurement of phosphatidylserine externalization, mitochondrial membrane potential (MMP), casp-3 activation, cleavage of PARP, ceramide levels, aSMase activity, and Bcl-2 release. nSMase activity was measured by a colorimetric assay. Gene expression was determined by qRT-PCR. Immunocytochemistry was performed to detect p21 and p27 expression. RESULTS:JBH-induced apoptosis in HeLa cells associated with externalization of phosphatidylserine, reduced MMP, activation of casp-3, and cleavage of PARP as well as up-regulation of TNF-α, FasL, and casp-8. Significant increase in nSMase activity, ceramide levels, Bcl-2 release (predominantly in the inactive form), and pro-apoptotic nuclear localization of p21 and p27 were also detected. CONCLUSION:JBH-induced apoptosis in HeLa cells is associated with disrupted sphingolipid homeostasis resulting in increased ceramide levels.