Differential Regulation of C6 Ceramide-induced Cell Death in Different Oral Squamous Cell Carcinoma Cell Lines

Apoptosis, a cellular suicide program, is an ordered and well-orchestrated cellular process that occurs in physiological and pathological conditions.  Unlike normal cells, cancer cells are under constant apoptotic stimuli, such as oncogenic stress, genomic instability, and cellular hypoxia.  Yet cancer cells can escape apoptosis by increasing the expression of anti-apoptotic / survival factors, or by decreasing the expression of pro-apoptotic factors. During tumorigenesis, the balance between cell proliferation and apoptosis is dysregulated, leading to aberrant cell proliferation.

Ceramide plays an important role in mediating a variety of cell functions, including apoptosis, cell cycle arrest and cell senescence.  Common cancer treatment modalities, such as ionizing radiation and many chemotherapeutic agents, induce cell death via the generation of ceramide¹. Here, we report two different biological responses to C6-ceramide in two different oral squamous cell carcinoma (OSCC) cells.

Objectives: C6-ceramide is a sphingolipid metabolite, with anti-proliferative and pro-apoptotic activity in vitro and in vivo.  Its use as a therapeutic agent has been limited because of its insolubility. In this study, we have utilized liposomes as a drug carrier to deliver C6-ceramide to target cells. Survivin is a known inhibitor of apoptosis and is overexpressed in OSCC cells. Recent studies with C6-ceramide have shown the down-regulation of survivin in large granular lymphocytic leukemia². We therefore investigated the cytotoxicity and anti-survivin activity of liposomal C6-ceramide in HSC-3 and CAL-27 OSCC cells as a potential novel therapeutic agent.

Methods: Palmitoyloleoylphosphatodylcholine (POPC):dioleoylphosphatidylethanolamine (DOPE) or POPC:DOPE:C6-ceramide liposomes were added to HSC-3 and CAL-27 cells in the concentration range 0.1–50 µM C6-ceramide. After incubation for 24 h at 37°C, cellular metabolic activity was evaluated by the Alamar Blue assay. Survivin levels were measured by ELISA.

Results: HSC-3 cells treated with liposomal C6-ceramide resulted in dose-dependent, decreased cell viability, measured by the Alamar Blue assay. The viability of HSC-3 cells with plain POPC:DOPE liposomes was 93±5% of the control. For 5 and 10 µM liposomal C6 ceramide, the viability was reduced to 72±3% and 44±0% of untreated cells. Survivin levels in HSC-3 cells decreased from 1226±5 ng/mg protein in controls to 346±6 ng/mg protein at 10 µM C6-ceramide.  Contrary to the HSC-3 cells, CAL-27 cells were resistant to C6-ceramide-induced cell death.  Basal levels of survivin in CAL-27 cells were lower than that in HSC-3 cells, and did not change significantly after C6-ceramide treatment.

Conclusion: HSC-3 cells are vulnerable to liposomal C6-ceramide in a dose-dependent manner, whereas CAL-27 cells are not.   Liposomal C6-ceramide reduced cell proliferation in HSC-3 cells probably because of a decrease in the levels of the anti-apoptotic protein, survivin. The lower endogenous survivin levels in CAL-27 cells than in HSC-3 cells suggest that the resistance of CAL-27 cells to C6-ceramide-induced cell death may be regulated by mechanism(s) other than survivin.    Further studies will focus on: 1) Elucidating the mechanisms of the resistance to C6-ceramide induced cell death in CAL-27 cells, and 2) whether liposomal C6 ceramide and the reduced survivin levels will increase the susceptibility of HSC-3 cells to various anti-cancer agents such as TNF-related apoptosis-inducing ligand and paclitaxel.

References

1.         Morad SA, Cabot MC. Ceramide-orchestrated signalling in cancer cells. Nature Reviews Cancer. Jan 2013;13(1):51-65.

2.        Liu X, Ryland L, Yang J, et al. Targeting of survivin by nanoliposomal ceramide induces complete remission in a rat model of NK-LGL leukemia. Blood. Nov 18 2010;116(20):4192-4201.