title: Relationship between intracellular pH, metabolic co-factors and caspase-3 activation in cancer cells during apoptosis.
abstract: A complex cascade of molecular events occurs in apoptotic cells but cell-to-cell variability significantly complicates determination of the order and interconnections between different processes. For better understanding of the mechanisms of programmed cell death, dynamic simultaneous registration of several parameters is required. In this paper we used multiparameter fluorescence microscopy to analyze energy metabolism, intracellular pH and caspase-3 activation in living cancer cells in vitro during staurosporine-induced apoptosis. We performed metabolic imaging of two co-factors, NAD(P)H and FAD, and used the genetically encoded pH-indicator SypHer1 and the FRET-based sensor for caspase-3 activity, mKate2-DEVD-iRFP, to visualize these parameters by confocal fluorescence microscopy and two-photon fluorescence lifetime imaging microscopy. The correlation between energy metabolism, intracellular pH and caspase-3 activation and their dynamic changes were studied in CT26 cancer cells during apoptosis. Induction of apoptosis was accompanied by a switch to oxidative phosphorylation, cytosol acidification and caspase-3 activation. We showed that alterations in cytosolic pH and the activation of oxidative phosphorylation are relatively early events associated with the induction of apoptosis.
authors.affiliation: Nizhny Novgorod State Medical Academy, 10/1 Minin and Pozharsky Sq., 603005 Nizhny Novgorod, Russia. Electronic address: tatiana.f.sergeeva@gmail.com.
authors.forename: Tatiana F
authors.initials: TF
authors.lastname: Sergeeva
authors.affiliation: Nizhny Novgorod State Medical Academy, 10/1 Minin and Pozharsky Sq., 603005 Nizhny Novgorod, Russia. Electronic address: shirmanovam@mail.ru.
authors.forename: Marina V
authors.initials: MV
authors.lastname: Shirmanova
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, 16/10 Miklukho-Maklaya St., 117997 Moscow, Russia. Electronic address: olgazlob@yandex.ru.
authors.forename: Olga A
authors.initials: OA
authors.lastname: Zlobovskaya
authors.affiliation: Nizhny Novgorod State Medical Academy, 10/1 Minin and Pozharsky Sq., 603005 Nizhny Novgorod, Russia; Nizhny Novgorod State University, Gagarin Ave., 23, 603950 Nizhny Novgorod, Russia. Electronic address: gavrina.alena@mail.ru.
authors.forename: Alena I
authors.initials: AI
authors.lastname: Gavrina
authors.affiliation: Nizhny Novgorod State Medical Academy, 10/1 Minin and Pozharsky Sq., 603005 Nizhny Novgorod, Russia; Nizhny Novgorod State University, Gagarin Ave., 23, 603950 Nizhny Novgorod, Russia. Electronic address: orannge@mail.ru.
authors.forename: Varvara V
authors.initials: VV
authors.lastname: Dudenkova
authors.affiliation: Nizhny Novgorod State Medical Academy, 10/1 Minin and Pozharsky Sq., 603005 Nizhny Novgorod, Russia; Nizhny Novgorod State University, Gagarin Ave., 23, 603950 Nizhny Novgorod, Russia. Electronic address: kuznetsova.m.m@yandex.ru.
authors.forename: Maria M
authors.initials: MM
authors.lastname: Lukina
authors.affiliation: Nizhny Novgorod State Medical Academy, 10/1 Minin and Pozharsky Sq., 603005 Nizhny Novgorod, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, 16/10 Miklukho-Maklaya St., 117997 Moscow, Russia. Electronic address: kluk@ibch.ru.
authors.forename: Konstantin A
authors.initials: KA
authors.lastname: Lukyanov
authors.affiliation: Nizhny Novgorod State Medical Academy, 10/1 Minin and Pozharsky Sq., 603005 Nizhny Novgorod, Russia. Electronic address: ezagaynova@gmail.com.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Zagaynova
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D053148:Caspase 3 / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D003374:Coumarins / Q000737:chemistry; D004789:Enzyme Activation / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006019:Glycolysis / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D051379:Mice; D036641:Microscopy, Fluorescence, Multiphoton; D015335:Molecular Probes / Q000737:chemistry; D009249:NADP / Q000378:metabolism; D010085:Oxidative Phosphorylation / Q000187:drug effects; D015398:Signal Transduction; D019311:Staurosporine / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D003374:Coumarins; D015335:Molecular Probes; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; C505407:CASP3 protein, human; D053148:Caspase 3; D019311:Staurosporine
keywords: Apoptosis; Caspase-3; FAD; Intracellular pH; NAD(P)H