Photodynamic therapy with toluidine blue in Jurkat cells: cytotoxicity, subcellular localization and apoptosis induction

Abstract

Toluidine blue (TBO) is a cationic thiazine dye with an affinity for neoplastic tissues in vivo. The objective of this study was to explore the in vitro photosensitizing potential of TBO and its capacity to induce apoptosis in human leukaemic T cells. Jurkat cells were incubated with TBO for one hour followed by exposure to 11 J cm(-2) of visible light from a slide projector. Cytotoxicity was assessed at 24 hours using a MTT assay. DNA fragmentation was examined at different intervals after photodynamic treatment using a DNA elution-filtration assay with [14C]-thymidine labelled cells. Caspase-3 like activation induced by photodynamic treatment was studied by measuring AC-DEVD-AMC peptide hydrolysis. The MTT assay showed a 97% decrease in optical density 24 hours following photodynamic therapy with 0.15 microg ml(-1) of TBO. Dark toxicity was absent under these conditions. DNA fragmentation was detected as early as 2 hours after photodynamic therapy and reached 68% at 6 hours. At higher TBO concentrations less DNA fragmentation and more dark toxicity was observed. An increase in caspase-3 like activity was also induced by photodynamic therapy with TBO. At the time of light exposure TBO was present in the endoplasmic reticulum and Golgi regions. In conclusion, TBO-based photodynamic therapy has a potent phototoxic effect and induces apoptosis in Jurkat cells.

Imaging chromophores with undetectable fluorescence by stimulated emission microscopy

“…4b). These imaging results support the hydrophilic delivery pathway as well as the recent hypothesis of TBO binding to cytoplasmic RNA to initiate apoptosis 23 .…”

“…In particular, we show mapping of a cationic thiazine dye toluidine blue O (TBO) at both the cellular and tissue levels. Having a selective affinity for cancer cells in vivo, TBO is a photosensitizer in photodynamic therapy 23,24 .…”

NQO1-activated phenothiazinium redox cyclers for the targeted bioreductive induction of cancer cell apoptosis

“…Phenothiazinium redox dyes have diverse applications as biological redox indicators, vital stains and diagnostic dyes [17], modulators of mitochondrial respiration [18], infusional antidotes against cyanide poisoning and methemoglobinemia [19], antiinfective agents [20,21], and photosensitizers [22][23][24], but their therapeutic potential and mechanism of action as anti-cancer redox chemotherapeutics are largely unexplored [23,25]. Compounds containing the 3,7-diaminophenothiazinium redox pharmacophore including thionine (T, 3,7-diamino-phenothiazinium acetate), methylene blue (MB, 3,7-bis (dimethylamino)-phenothiazinium chloride), and toluidine blue O (TB, 2-methyl-3-amino-7-dimethylamino-phenothiazinium chloride) are two-electron redox systems with standard reduction potentials [e.g.…”

Intracellular signaling mechanisms in photodynamic therapy

“…However, expression of the caspase-1 and caspase-8 inhibitor CrmA (viral cytokine response modifier A) did not affect apoptotic death of human adenocarcinoma HeLa cells sensitized with hypericin [20]. Also, expression of CrmA did not affect the kinetics of cytochrome C release Jurkat human lymphoma T cells [295] and procaspase-3 cleavage in a rat/mouse T cell hybridoma sensitized with hypericin [68], suggesting that caspase-8 does not play a major role in the demise process in this model. The activation of caspases in photosensitized cells leads to the cleavage of a number of other cell proteins, including Bap-31 (shuttle protein between the ER and the intermediate compartment and/or Golgi complex [71]), DNA-dependent protein kinase (catalytic subunit) (DNA-PK CS [75]), ICAD (inhibitor of caspase activated DNAse); prevents DNA fragmentation via binding to caspase-activated deoxyribonuclease [76]), focal adhesion kinase (FAK, a kinase involved in the regulation of cell adhesion [73]), lamins (structural components of the nuclear envelope [73]), PARP (poly(ADP-ribose) polymerase, a DNA repair enzyme [18,20,44,54,68,71,72,75,[77][78][79][80][81][82][83][84][85]) and Ras GTPase-activating protein (Ras-GAP, a negative regulator of the Ras signaling pathway [71]).…”