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. 2013 Jun;5(6):1854-1858.
doi: 10.3892/ol.2013.1305. Epub 2013 Apr 16.

Paclitaxel Disrupts Polarized Entry of Membrane-Permeable C6 Ceramide Into Ovarian Cancer Cells Resulting in Synchronous Induction of Cell Death

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Free PMC article

Paclitaxel Disrupts Polarized Entry of Membrane-Permeable C6 Ceramide Into Ovarian Cancer Cells Resulting in Synchronous Induction of Cell Death

Charles Best et al. Oncol Lett. .
Free PMC article

Abstract

Exogenous cell-permeable C6 ceramide has been demonstrated to act synergistically with chemotherapeutic drugs, including paclitaxel, cisplatin, doxorubicin and the histone deacetylase inhibitor, trichostatin A, to induce cell death in a variety of cancer cells. We previously demonstrated that C6 ceramide and paclitaxel function synergistically to induce ovarian cancer cell death via modulation of the PI3/AKT cell survival pathway. In the present study, the entry pattern of C6 ceramide into ovarian cancer cells was investigated using fluorescent short chain C6-NBD sphingomyelin (C6-NBD). Confocal microscopy revealed that C6-NBD enters the cells in a polarized pattern, characterized by marked signals at one cellular end, representing a likely mitosis initiation site. Pretreatment of the cells with filipin, an inhibitor of the lipid raft/caveolae endocytosis pathway, decreases C6-NBD entry into the cells. A pretreatment with the water channel inhibitor, CuSO4, was also found to reduce the entry of C6-NBD. Notably, the pretreatment with paclitaxel was shown to disrupt the polarized entry of C6-NBD into the cells, resulting in an even distribution of C6-NBD in the cytoplasm. In addition, the pretreatment of the cells with paclitaxel destabilized the cytoskeletal proteins, releasing an increased number of short tubulin fragments. The results of the present study indicate that C6 ceramide preferentially enters the cells via a predetermined initiation site of mitosis. In addition to diffusion, short chain C6 ceramide may also enter cells via water channels and caveolae-mediated endocytosis. Paclitaxel disrupts the cell cytoskeleton and induces an even distribution of C6 ceramide in the cytoplasm resulting in synergistic ovarian cancer cell death.

Keywords: AKT; C6 ceramide; ovarian cancer; paclitaxel.

Figures

Figure 1
Figure 1
Polarized distribution of C6-NBD ceramide in ovarian cancer cells. (A and C) CaOV3 cells were cultured in 8-well chamber slides and treated with C6-NBD ceramide (6 μg/ml). (B) The cells were fixed with formaldehyde at various times following treatment or were treated with the indicated doses of C6-NBD-ceramide, with or without fixation. The cells were processed and observed under a confocal microscope. Fluorescent short chain C6-NBD sphingomyelin, C6-NBD; Green, C6-NBD-ceramide; blue, nuclear staining.
Figure 2
Figure 2
Effect of inhibitors on polarized entry of C6-NBD-ceramide into ovarian cancer cells. CaOV3 cells were cultured in 8-well chamber slides and treated with or without (A) a lipid raft/caveolae inhibitor or with (B) water channel inhibitors, CuSO4 or NiCl2. The cells were fixed with formaldehyde, processed and observed under a confocal microscope. Fluorescent short chain C6-NBD sphingomyelin, C6-NBD; Green, C6-NBD-ceramide; blue, nuclear staining.
Figure 3
Figure 3
Effect of chemotherapeutic agents on C6-NBD-ceramide polarized entry into ovarian cancer cells. (A) CaOV3 cells were cultured in 8-well chamber slides, pretreated with taxol or doxorubicin for 1 h and then treated with C6-NBD-ceramide for 5 or 15 min. The cells were fixed with formaldehyde and processed for confocal microscopy. (B) The cells were pretreated with taxol and then C6-NBD-ceramide and processed for confocal microscopy without fixation. Fluorescent short chain C6-NBD sphingomyelin, C6-NBD; Green, C6-NBD-ceramide; blue, nuclear staining.

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