Sphingolipids have become recognized as essential effector molecules in signal transduction with involvement in various
aspects of cell function and death, immune response and cancer treatment response. Major representatives of
sphingolipids family, ceramide, sphingosine and
sphingosine-1-phosphate (S1P), have attracted interest in their
relevance to tumor response to photodynamic therapy (PDT) because of their roles as enhancers of apoptosis, mediators
of cell growth and vasculogenesis, and regulators of immune response. Our recent in vivo studies with mouse tumor
models have confirmed that PDT treatment has a pronounced impact on sphingolipid profile in the targeted tumor and
that significant advances in therapeutic gain with PDT can be attained by combining this modality with adjuvant
treatment with ceramide analog LCL29.
The objective of this study was to determine the sphingolipid (SL) profile in autophagy-defective cells and overall cell
death after PDT with Pc 4 (PDT). Human breast cancer MCF-7 cells with downregulated autophagy protein ATG-7 and
their scrambled controls (Scr) were used. Exposure of ATG-7 knockdown cells to PDT led to defective processing of the
autophagy marker LC3, and increased overall cell killing. In both cell types PDT evoked an early (2 h) increase in
ceramides and dihydroceramides (DHceramides). When the two cell types were compared regarding time (2 and 24 h)
and treatment conditions (with and without PDT), the levels of several ceramides and DHceramides were reduced,
whereas the concentrations of C14-ceramide, C16-ceramide and C12-DHceramide were higher in ATG-7 knockdown
cells. The data imply that the SL profile might be a marker of autophagy-deficiency in cells sensitized to PDT.
The oxidative stress initiated by photodynamic therapy (PDT) results in accumulation of ceramide in various cell types undergoing apoptosis. Apoptotic responses to PDT are similar to those of the oxidative stress inducer and the Bcl-2 inhibitor HA 14-1 in murine leukemia L1210 cells. Here we tested whether de novo ceramide accumulation can be induced by HA 14-1 during apoptosis in Jurkat T human leukemia cells. Similar to photosensitization with Pc 4, treatment of Jurkat cells with HA14-1 leads to DEVDase activation and nuclear apoptosis. However, unlike Pc 4-PDT, HA 14-1 did not induce accumulation of de novo ceramide. The data suggest that, compared to HA 14-1, de novo ceramide accumulation is a distinct response to PDT in Jurkat cells.
Photodynamic treatment with the phthalocyanine Pc 4, a mitochondrially localizing photosensitizer, is an efficient inducer of cell death by apoptosis, a cell suicide pathway that can be triggered by physiological stimuli as well as by various types of cellular damage. Upon exposure of the dye- loaded cells to red light, several stress signalling pathways are rapidly activated. In murine L5178Y-R lymphoblasts, caspase activation and other hallmarks of the final phase of apoptosis are observed within a few minutes post-PDT. In Chinese hamster CHO-K1 cells, the first signs of apoptosis are not observed for 1 - 2 hours. The possible involvement of three parallel mitogen-activated protein kinase (MAPK) signalling pathways has been investigated. The extracellular- regulated kinases (ERK-1 and ERK-2), that are thought to promote cell growth, are not appreciably altered by PDT. However, PDT causes marked activation of the stress-activated protein kinase (SAPK) cascade in both cell types and of the p38/HOG-type kinase in CHO cells. Both of these latter pathways have been demonstrated to be associated with apoptosis. A specific inhibitor of the ERK pathway did not alter PDT-induced apoptosis; however, an inhibitor of the p38 pathway partially blocked PDT-induced apoptosis. Blockage of the SAPK pathway is being pursued by a genetic approach. It appears that the SAPK and p38 pathways may participate in signaling apoptosis in response to PDT with Pc 4.
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