Concurrent detection of lysosome and tissue transglutaminase activation in relation to cell cycle position during apoptosis induced by different anticancer drugs

Abstract

Described is the new cytometric approach do detect either stimulation or a collapse of lysosomal proton pump (lysosomes rupture) combined with activation of transglutaminase 2 (TG2) during induction of apoptosis. Apoptosis of human lymphoblastoid TK6 cells was induced by combination of 2-deoxyglucose with the isoquinoline alkaloid berberine, by DNA topoisomerase I inhibitor camptothecin, its analog topotecan, topoisomerase II inhibitors etoposide or mitoxantrone, as well as by the cytotoxic anticancer ribonuclease ranpirnase (onconase). Activity of the proton pump of lysosomes was assessed by measuring entrapment and accumulation of the basic fluorochrome acridine orange (AO) resulting in its metachromatic red luminescence (F >640 ) within these organelles. Activation of TG2 was detected in the same cell subpopulation by the evidence of crosslinking of cytoplasmic proteins revealed by the increased intensity of the side light scatter (SSC) as well as following cell lysis by detergent, by its red fluorescence after staining by sulforhodamine 101. Because at low AO concentration nuclear DNA of the lysed cells was stoichiometrically stained green (F 530 ) its quantity provided information on effects of the drug treatments on cell cycle in relation to activation of TG2. The data reveal that activation of lysosomal proton pump was evident in subpopulations of cells treated with 2-deoxyglucose plus berberine, topotecan, etoposide and mitoxantrone but not with ranpirnase. The collapse of lysosomal proton pump possibly reporting rupture of these organelles was observed in definite cell subpopulations after treatment with each of the studied drugs. Because regardless of the inducer of apoptosis TG2 activation invariably was correlated with lysosomes rupture it is likely that it was triggered by calcium ions or protons released from the ruptured lysosomes. This new methodological approach offers the means to investigate mechanisms and factors affecting autophagic lysosomes proton pump activity vis-à-vis TG2 activation that are common in several pathological states.

Microbial transglutaminase nanoflowers as an alternative nanomedicine for breast cancer theranostics

“…This points towards the mammalian transglutaminase NFs playing a role in cancer progression. 99,100 Aer the assessment, we could dene the mode of action of the two drugs tested (mammalian vs. microbial TGase), both in nanoowers. The effect of microbial MTGase NFs was pronounced as cytotoxic to cancerous cells; hence it is efficient in killing cancerous cells in lower concentrations as observed by its IC 50 .…”

Assessment of DNA Susceptibility to Denaturation as a Marker of Chromatin Structure

“…Unlike in methods that require cells to be fixed and permeabilized, in the lysosomal assay they are supravitally exposed to the fluorochrome. It was also observed that the release of AO from lysosomes upon cell treatment with anticancer drugs, reporting the disintegration of these organelles, occurs concurrently with the activation of transglutaminase 2 and subsequent crosslinking of cytoplasmic proteins; the latter is likely a result of a release of lysosome contents such as calcium ions and protons (Halicka, Li, Zhao, & Darzynkiewicz, 2019). AO in such aggregates manifests a metachromatic shift from green to red emission (Moriyama, Takano, & Ohkuma, 1982;Pierzyńska-Mach, Janowski, & Dobrucki, 2014;Traganos & Darzynkiewicz, 1994).…”

“…This approach was recently introduced to human clinical trials for treatment of radio‐resistant cancer (Kusuzaki et al., ). It was also observed that the release of AO from lysosomes upon cell treatment with anticancer drugs, reporting the disintegration of these organelles, occurs concurrently with the activation of transglutaminase 2 and subsequent crosslinking of cytoplasmic proteins; the latter is likely a result of a release of lysosome contents such as calcium ions and protons (Halicka, Li, Zhao, & Darzynkiewicz, ). It should also be noted that AO is the key marker of lysosomes used in wide‐ranging studies of autophagy (Demishtein, Porat, Elazar, & Shvets, ; SenthilKumar, Skiba, & Kimple, ; Thomé et al., ; Warnes, ).…”