Sphingomyelins suppress the targeted disruption of lysosomes/endosomes by the photosensitizer NPe6 during photodynamic therapy

Abstract

Recent studies have described a biochemical pathway whereby lysosome disruption and the released proteases initiate the intrinsic apoptotic pathway. Irradiation of murine hepatoma 1c1c7 cells preloaded with the lysosomal photosensitizer NPe6 (N-aspartyl chlorin e6) caused a rapid loss of Acridine Orange staining of acidic organelles, release of cathepsin D from late endosomes/lysosomes and the activation of procaspase-3. Pretreatment of NPe6-loaded cultures with 10-50 microM 3-O-MeSM (3-O-methylsphingomyelin) caused a concentration-dependent suppression of apoptosis following irradiation. This suppression reflected a stabilization of lysosomes/endosomes, as opposed to an inhibition of the accumulation of photosensitizer in these organelles. Exogenously added sphingomyelin, at comparable concentrations, offered some protection, but less than 3-O-MeSM. Fluorescence microscopy showed that 3-O-MeSM competed with NBD-C6-sphingomyelin (6-{[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl} sphingosyl phosphocholine) for co-localization with LysoTracker Red in acidic organelles. Pre-treatment of 1c1c7 cultures with 3-O-MeSM also suppressed the induction of apoptosis by TNFalpha (tumour necrosis factor alpha), but offered no protection against HA14-1 [ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate], staurosporine, tunicamycin or thapsigargin. These results suggest that exogenously added 3-O-MeSM is trafficked to and stabilizes late endosomes/lysosomes against oxidant-induced damage, and further implicate a role for lysosomal proteases in the apoptotic processes initiated by TNFalpha and lysosomal photosensitizers.

Adaptation of neuronal cells to chronic oxidative stress is associated with altered cholesterol and sphingolipid homeostasis and lysosomal function

“…The cellular lipid composition might strongly influence lysosomal activity and protein sorting and vice versa. Sphingomyelin, for example, has been reported to stabilize lysosomes and protect them against oxidant-induced damage (Caruso et al 2005). This is consistent with our observations in stress-resistant cells, showing a higher sphingomyelin level concomitant with higher Lamp1 expression.…”

“…5c) and increased the number of large vacuoles that were positively labeled by LysoTracker Red, Lamp1 and filipin (Fig. 5a), which is consistent with the previously reported stabilization of lysosomes by sphingomyelin (Caruso et al 2005). Furthermore, 3-O-Me-SM treatment reduced the protein amount of LRP and rab7 and increased Lamp1.…”

“…Hence, increasing the amount of sphingomyelin may protect cells toward oxidative stress. To increase the sphingomyelin level, we blocked degradation of sphingomyelin by nSMase using the nSMase inhibitor 3-O-Me-SM (Caruso et al 2005). 3-O-Me-SM treatment of HT22 WT cells (20 lM for 24 h) decreased the activity of nSMase as expected (Fig.…”

Aryl Hydrocarbon Receptor Modulation of Tumor Necrosis Factor-α-induced Apoptosis and Lysosomal Disruption in a Hepatoma Model That Is Caspase-8-independent

“…C, 1c1c7 cultures were treated with nothing (ϫ), TNF␣ (F), various doses of CHX (OE) or TNF␣ ϩ CHX (f) as described in A and were harvested 9 -11 h after TNF␣ addition for analyses of viability. Data represent means Ϯ S. tosensitizer NPe6 (45). Strong protection was afforded by 50 M 3-OMeSM in the PDT protocol (45).…”

Lysosomal membrane permeabilization in cell death

“…Sphingomyelin and some of its derivatives (for example, 3-O-methylsphingomyelin, an inhibitor of neutral sphingomyelinase) can stabilize lysosomes against the lysosomal photosensitizer, N-aspartyl chlorin e6, which causes rapid loss of AO staining of acidic organelles, release of CD from late endosomes/lysosomes and the activation of procaspase-3, as well as TNF-a-induced LMP and cell death (Caruso et al, 2005). These results point to the possibility that proteins, as well as non-proteinaceous molecules, act as endogenous regulators of LMP.…”