Access Type

Open Access Thesis

Date of Award

January 2015

Degree Type

Thesis

Degree Name

M.S.

Department

Pharmaceutical Sciences

First Advisor

Duska Separovic

Abstract

The ceramide generated via the de novo sphingolipid biosynthesis has been shown to regulate apoptosis and cell death. The de novo sphingolipid biosynthesis includes ceramide synthase (CERS)-dependent acylation of dihydrosphingosine, giving rise to dihydroceramide, which is then converted to ceramide by a desaturase-dependent reaction. The mitochondrial pathway of apoptosis, characterized by induction of Bax associated with mitochondria and cytochrome c release/redistribution. Bcl2, an anti-apoptotic protein blocks apoptosis by inhibiting Bax. Elucidating the role of de novo sphingolipid biosynthesis and mitochondrial apoptosis in PDT, PDT+4HPR and PDT+LCL29 could help in improving the effectiveness on these anti-cancer treatments. The objective of this study was to determine the involvement of de novo sphingolipid biosynthesis and mitochondrial apoptosis in PDT-induced cell killing and testing whether combining LCL29 and 4HPR with PDT enhances cell killing via de novo sphingolipid biosynthesis and mitochondrial apoptosis. Silicon phthalocyanine Pc4 was used as a photosensitizer for PDT and SCC17B human head and neck squamous cell carcinoma cells were used in this study.

Results indicate that ceramide synthase, caspases and Bcl2 were involved in PDT-induced cell killing. PDT-induced ER and mitochondrial ceramide/dihydroceramide accumulation was inhibited by fumonisin B1 (FB) indicating involvement of ceramide synthase. PDT led to increase in majority of individual ceramides and C16-dihydroceramide. FB inhibited mitochondrial apoptotic events like PDT-induced Bax associated with mitochondria, cytochrome c redistribution and caspase-3 activation. The data suggests that PDT-induced cell killing is mediated by ceramide synthase-dependent ceramide/dihydroceramide accumulation and mitochondrial apoptosis. Combining PDT with 4HPR led to enhanced cell killing via ceramide synthase, caspases and Bcl2. PDT+4HPR enhanced ceramide synthase-dependent ER ceramide/dihydroceramide accumulation. Combining PDT with 4HPR enhances C16-dihydroceramide levels but leads to attenuation of total and individual ceramide levels compared to PDT. PDT+4HPR-induced enhanced FB-sensitive Bax associated with mitochondria and cytochrome c redistribution. These data indicate that combining PDT with 4HPR enhanced cell killing via de novo sphingolipid biosynthesis and mitochondrial apoptosis. Combining PDT with LCL29 resulted in enhanced cell killing via ceramide synthase and caspases. PDT+LCL29-induced enhanced ceramide/dihydroceramide accumulation in the mitochondria was inhibited by FB. PDT+LCL29 enhanced cytochrome c redistribution and caspase-3 activation were inhibited by FB. Taken together, this study has shown that de novo sphingolipid biosynthesis and mitochondrial apoptosis play a key role in killing of SCC17B human head and neck squamous cell carcinoma cells after PDT, PDT+4HPR and PDT+LCL29.

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