A photodynamic pathway to apoptosis and necrosis induced by dimethyl tetrahydroxyhelianthrone and hypericin in leukaemic cells: possible relevance to photodynamic therapy

Abstract

SummaryThe mechanism of cell death induction by dimethyl tetrahydroxyhelianthrone (DTHe), a new second-generation photodynamic sensitizer, is analysed in human leukaemic cell lines in comparison with the structurally related hypericin. DTHe has a broad range of light spectrum absorption that enables effective utilization of polychromatic light. Photosensitization of HL-60 cells with low doses of DTHe (0.65 µM DTHe and 7.2 J cm -2 light energy) induced rapid apoptosis of ≥90% of the cells. At doses ≥2 µM, dying cells assumed morphological necrosis with perinucleolar condensation of chromatin in HL-60 and K-562 cell lines. Although nuclear fragmentation that is characteristic to apoptosis was prevented, DNA digestion to oligonucleosomes proceeded unhindered. Such incomplete apoptosis was more prevalent with the related analogue hypericin throughout most doses of photosensitization. Despite hypericin being a stronger photosensitizer, DTHe exhibited advantageous phototoxic properties to tumour cells, initiating apoptosis at concentrations about threefold lower than hypericin. Photosensitization of the cells induced dissociation of the nuclear envelope, releasing lamins into the cytosol. DTHe also differed from hypericin in effects exerted on the nuclear lamina, causing release of an 86-kDa lamin protein into the cytosol that was unique to DTHe. Within the nucleus, nuclear envelope lamin B underwent covalent polymerization, which did not affect apoptotic nuclear fragmentation at low doses of DTHe. At higher doses, polymerization may have been extensive enough to prevent nuclear collapse. Hut-78, CD4 + cells were resistant to the photodynamically activated apoptotic pathway. Beyond the tolerated levels of photodynamic damage, these cells died exclusively via necrosis. Hut-78 cells overexpress Bcl-X L as well as a truncated Bcl-X L tr isoform that could contribute to the observed resistance to apoptosis.Keywords: photodynamic therapy; hypericin; dimethyl tetrahydroxyhelianthrone; Bcl-X; Bax; lamin; apoptosis 423British Journal of Cancer (1999) 79(3/4), 423-432 © 1999 Cancer Research Campaign Article no. bjoc.1998 Received Efforts to identify novel efficacious agents for photodynamic therapy led us to evaluate the photodynamically induced cytotoxicities of a newly designed photosensitizer 10,13-dimethyl 1,3,4,6-tetrahydroxyhelianthrone (DTHe) (Figure 1), in leukaemic cell lines. DTHe was chosen because of its dibenzperylenequinone chromophore (seven aromatic rings) that has absorption spectral properties virtually identical to hypocrellins, potent anti-tumoral photosensitizers isolated from the parasitic fungus Hypocrella bambuase which grows in China and Tibet (Diwu, 1990;Diwu, 1995; Miller, 1997). DTHe also shares structural similarities with HY and was anticipated to be a potent photosensitizer owing to its considerable light absorbance in the visible range of the spectrum. The phototoxicity profiles and mechanisms of cell death induction of DTHe were analysed in comparison with those of HY in HL-60, K-562 and Hut-...

Photochemical destruction of the Bcl-2 oncoprotein during photodynamic therapy with the phthalocyanine photosensitizer Pc 4

“…First, western blots of proteins from the treated cells but not from the control cells reveal multiple high-molecular weight bands recognized by the anti-Bcl-2 antibody, suggestive of the chemical crosslinking of Bcl-2 to itself or other proteins. Crosslinking of lamin B and the generation of high molecular weight slowly migrating protein bands has been observed in studies of the response of HL-60 cells to PDT photosensitized by hypericin or an anthraquinone (Lavie et al, 1999). Second, protease inhibitors do not prevent the loss of Bcl-2 protein.…”

Antimetastatic activity of the photodynamic agent hypericin in the dark

“…13 The mode of cell death that is induced in the dark also differs from the phototoxic activities. While the light-mediated photodynamic effects of HY lead to apoptosis at low light doses and to necrosis at higher light doses, 18 the dark effects have been found to elicit mitotic cell death. 16 A snowball process of disrupting cell cycle signaling pathways in tumor cells has been found to begin with ubiquitinylation of the Hsp90 chaperone.…”

“…18 HY was dissolved in 70% aqueous ethanol to a stock solution of 3 mg/ml with subsequent dilutions in sterile double distilled H 2 O.…”

Synthesis and in vitro/vivo Evaluation of Iodine-123/124 Labelled Hypericin Derivatives

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