Induction of Apoptosis and Autophagy of Oral Squamous Cell Carcinoma Cells by Safingol

Atsushi Masui DDS, Oral and Maxillofacial Surgery II, Osaka University Graduate School of Dentistry, Suita, Japan
Masakazu Hamada DDS, PhD, Oral and Maxillofacial Surgery II, Osaka University Graduate School of Dentistry, Suita, Japan
Ken Wakabayashi DDS,PhD, Oral and Maxillofacial Surgery II, Osaka University Graduate School of Dentistry, Suita, Japan
Soichi Iwai , Oral and Maxillofacial Surgery II, Osaka University Graduate School of Dentistry, Suita, Japan
Yoshiaki Yura DDS, PhD, Oral and Maxillofacial Surgery II, Osaka University Graduate School of Dentistry, Suita, Japan
Molecular target therapy is a promising field in chemotherapy for cancer. Protein kinase C (PKC) family consists of up to 12 distinct isotypes and plays diversity of roles depending on cell cycle, development phase or stimulus. They are linked to lysosomal, endosomal and degenerative process, and also involved in the MAPK and Akt pathways that influence autophagy. They are considered to be a molecular target for chemotherapeutic drugs. Safingol is the synthetic L-threo-sphingosine and an established PKC inhibitor. We have shown that safingol induced caspase 3-independent and endnuclease G (endo G) -mediated apoptotic cell death of oral squamous cell carcinoma (SCC) cells [1]. In the present study, we examined whether safingol induced autophagy in oral SCC cells and whether drugs that inhibit or induce autophagy could modify the effect of safingol on cell viability. 

3-methyladenine (3MA) that blocks autophagosome formation and bafilomycin A1 that blocks autolysosome formation were used. The human oral SCC cell line SAS was cultured in DMEM supplemented with 5% fetal bovine serum. Cell viability was measured by MTT assay. After drug exposure, cells were harvested, washed with ice-cold PBS and stained with annexin V-FITC/ propidium iodide (PI) for 15 min and analyzed on the flow cytometer, FACSCalibur. The expression of light chain 3 (LC3) in the cytoplasm was examined by fluorescence antibody staining.

Results are reported as means±SD. The statistical analyses were performed using the software Statcel3. A value of P<0.05 was considered to be statistically significant.

When SAS cells were cultured in the presence of 25 micromolar safingol for 24 h, floating cells appeared and cell viability was decreased to 75% of the control. annexin V-FITC and PI staining of treated cells revealed marked increase of apoptotic cells. Nuclear fragmentation was also observed, indicating apoptosis by safingol. The expression of LC3 was increased and it accumulated in the cytoplasm. Immunoblot analysis showed increase of LC3 protein. Acidic vesicular organelles that were stained with the lysosome-tropic agent, acridine orange, appeared in the cytoplasm, showing the induction autophagy by safingol. Combination of safingol with inhibitors of autophagy, 3MA and bafilomycin A1, further suppressed cell viability. Treatment of cells with reactive oxygen species (ROS) scavenger, N-acetyl-L-cysteine, prevented apoptosis and autophagy by safingol. Ling et al. [2] reported that safingol induced necrosis and autophagy of human breast cancer cells and colon cancer cells. They found that ROS was the mediator of safingol-induced cell death. Previously, we reported that that cell death by safingol is dependent on the translocation of apoptogenic mitochondrial factor endo G. Recently, production of ROS by treatment of SAS cells by safingol was confirmed. In the present study, we demonstrated that safingol induced autophagy as well as apoptosis of oral SCC cells. It can be stated that autophagy occurs in the situation where endo G-mediated apoptosis is induced.

These results indicated that safingol induced apoptosis and autophagy in SCC cells. Since autophagy acts for cell survival, inhibition of autophagy would result in increase of cell death. Combination of inhibitors for autophagy may be useful to enhance the cytotoxic effect of safingol on oral SCC.

[1] Hamada M, Sumi T, Iwai S, Nakazawa M, Yura Y. Induction of endonuclease G-mediated apopotosis in human oral squamous cell carcinoma cells by protein kinase C inhibitor safingol. Apoptosis 2006;11:47-56.

[2] Ling LU, Tan KB, Lin H, Chiu GNC The role of reactive oxygen species and autophagy in safingol-induced cell death. Cell Death Dis. 2011;2:e129.