C16-Ceramide Analog Combined with Pc 4 Photodynamic Therapy Evokes Enhanced Total Ceramide Accumulation, Promotion of DEVDase Activation in the Absence of Apoptosis, and Augmented Overall Cell Killing

Abstract

Because of the failure of single modality approaches, combination therapy for cancer treatment is a promising alternative. Sphingolipid analogs, with or without anticancer drugs, can improve tumor response. C16-pyridinium ceramide analog LCL30, was used in combination with photodynamic therapy (PDT), an anticancer treatment modality, to test the hypothesis that the combined treatment will trigger changes in the sphingolipid profile and promote cell death. Using SCCVII mouse squamous carcinoma cells, and the silicone phthalocyanine Pc 4 for PDT, we showed that combining PDT with LCL30 (PDT/LCL30) was more effective than individual treatments in raising global ceramide levels, as well as in reducing dihydrosphingosine levels. Unlike LCL30, PDT, alone or combined, increased total dihydroceramide levels. Sphingosine levels were unaffected by LCL30, but were abolished after PDT or the combination. LCL30-triggered rise in sphingosine-1-phosphate was reversed post-PDT or the combination. DEVDase activation was evoked after PDT or LCL30, and was promoted post- PDT/LCL30. Neither mitochondrial depolarization nor apoptosis were observed after any of the treatments. Notably, treatment with the combination resulted in augmented overall cell killing. Our data demonstrate that treatment with PDT/LCL30 leads to enhanced global ceramide levels and DEVDase activation in the absence of apoptosis, and promotion of total cell killing.

C6-pyridinium ceramide sensitizes SCC17B human head and neck squamous cell carcinoma cells to photodynamic therapy

“…Cell survival was assessed using clonogenic assay according to the modified pre-plating protocol, as we described in [22]. Cells were resuspended in growth medium containing Pc4 (20 nM) and seeded (250 cells/well) in a 6-well plate (Thermo Fisher Scientific).…”

Combining anticancer agents photodynamic therapy and LCL85 leads to distinct changes in the sphingolipid profile, autophagy, caspase-3 activation in the absence of cell death, and long-term sensitization

“…For PDT experiments, after overnight incubation with Pc 4 (250 nM) at 37°C, cells were irradiated with red light (2 milliwatts/cm 2 ; λ max ~ 670 nm) using a light-emitting diode array light source (EFOS) at the fluence of 200 mJ/cm 2 at room temperature. The PDT dose (250 nM Pc 4 + 200 mJ/cm 2 ) used in our studies was chosen based on our previous findings [16]. Pc 4 alone had no effect on any biological outcome tested in the presented studies.…”

“…We have demonstrated that combining Pc 4-PDT with the cationic C16-ceramide analog LCL30 led to augmented overall killing of mouse SCCVII squamous carcinoma cells (SCCVII cells) [16]. LCL85, a cationic B13 SL analog, is a promising anticancer agent [17].…”

Cationic ceramides and analogues, LCL30 and LCL85, as adjuvants to photodynamic therapy of tumors

“…This appears not to be the case with LCL30 since similar percentages of apoptotic cancer cells were found in SCCVII tumors treated by PDT alone or PDT combined with LCL30. Our in vitro studies with SCCVII cells revealed that combining PDT with LCL30 enhanced global ceramide level increase, caspase-3 activation, and overall cell killing in the absence of apoptosis [21]. However, these in vitro results were obtained with Pc4-PDT and it remains to be seen to what extent they are mimicked by Photofrin-PDT.…”

“…Two additional SL modulators, LCL30 (C16-ceramide analogue)[19] and LCL85 (acid ceramidase inhibitor B13 analogue)[20] were examined in vitro combined with PDT and were both found to augment PDT-mediated killing of SCCVII cells [21,22]. These cationic, mitochondrially-targeted compounds were originally developed as potential anticancer agents [23 24…”