title: Synergy of 2-deoxy-D-glucose combined with berberine in inducing the lysosome/autophagy and transglutaminase activation-facilitated apoptosis.
abstract: Utilizing a variety of flow cytometric methods evidence was obtained indicating that a combination of the glucose analog 2-deoxy-D-glucose (2-dG) and the plant alkaloid berberine (BRB) produces synergistic effect in the induction of apoptosis in human lymphoblastoid TK6 cells. The synergistic effect is seen at concentrations of the drugs at which each of them alone shows no cytotoxicity at all. The data suggest that the combination of these drugs, which are known in terms of their overall toxicity, side effects and pharmacokinetics may be considered for further studies as chemopreventive and cancer treatment modalities. Of interest are results indicating that rapamycin, which similarly to BRB, suppresses mTOR signaling, when combined with 2-dG shows no synergistic properties. Metformin, on other hand, requires much higher concentration to show the synergy with 2-dG. Also of interest are the findings pertaining to the methodology of the present study. Specifically, dynamic assessment of cellular viability was performed by using the DRAQ7 cell exclusion fluorochrome present in cultures from 0 to 72 h. Concurrent measurement of lysosomal proton pump using acridine orange as the probe shows activation of lysosomes in the cells treated with 2-dG or BRB alone as well as with the drugs combined. Apoptosis was assessed by measuring DNA fragmentation, cell cycle, activation of caspase-3 and tissue transglutaminase (Tgase). A novel cytometric method was developed based on analysis of lysosomal (acidic vesicles) proton pump in live cells followed by cell lysis with detergent and fluorochrome labeling of proteins and DNA to analyze Tgase activation concurrently with cell cycle, in same population of cells. The data show that the cell subpopulation undergoing apoptosis has increased side (right-angle) light scatter likely due to the presence of the crosslinked (solid state) proteins, the consequence Tgase activation.
authors: Department of Pathology, Brander Cancer Research Institute, New York Medical College, BSB 438, Valhalla, NY, 10595, USA.
authors: H Dorota
authors: HD
authors: Halicka
authors: Department of Pathology, Brander Cancer Research Institute, New York Medical College, BSB 438, Valhalla, NY, 10595, USA.
authors: Jorge
authors: J
authors: Garcia
authors: Department of Pathology, Brander Cancer Research Institute, New York Medical College, BSB 438, Valhalla, NY, 10595, USA.
authors: Jiangwei
authors: J
authors: Li
authors: Department of Pathology, Brander Cancer Research Institute, New York Medical College, BSB 438, Valhalla, NY, 10595, USA.
authors: Hong
authors: H
authors: Zhao
authors: Department of Pathology, Brander Cancer Research Institute, New York Medical College, BSB 438, Valhalla, NY, 10595, USA. darzynk@nymc.edu.
authors: Zbigniew
authors: http://orcid.org/0000-0002-2040-7081
authors: Z
authors: Darzynkiewicz
mesh_terms: D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D001343:Autophagy / Q000187:drug effects / Q000235:genetics; D001599:Berberine / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003847:Deoxyglucose / Q000494:pharmacology*; D004357:Drug Synergism; D005434:Flow Cytometry; D006801:Humans; D008247:Lysosomes / Q000187:drug effects / Q000235:genetics; D008687:Metformin / Q000494:pharmacology; D017494:Proton Pumps / Q000235:genetics; D011503:Transglutaminases / Q000235:genetics*
chemical_list: D017494:Proton Pumps; D001599:Berberine; D008687:Metformin; D003847:Deoxyglucose; D011503:Transglutaminases
keywords: Acridine orange; Apoptosis; Chemoprevention; DNA content; Flow cytometry; Proton pump