L-ascorbic acid (L-AA; vitamin C) induces apoptosis in cancer cells. This study aimed to elucidate the molecular mechanisms of L-AA-induced apoptosis in human laryngeal epidermoid carcinoma Hep-2 cells. L-AA suppressed the viability of Hep-2 cells and induced apoptosis, as shown by the cleavage and condensation of nuclear chromatin and increased number of Annexin V-positive cells. L-AA decreased Bcl-2 protein expression but upregulated Bax protein levels. In addition, cytochrome c release from the mitochondria into the cytosol and activation of caspase-9, -8, and -3 were enhanced by L-AA treatment. Furthermore, apoptosis-inducing factor (AIF) and endonuclease G (EndoG) were translocated into the nucleus during apoptosis of L-AA-treated Hep-2 cells. L-AA effectively inhibited the constitutive nuclear factor-κB (NF-κB) activation and attenuated the nuclear expression of the p65 subunit of NF-κB. Interestingly, L-AA treatment of Hep-2 cells markedly activated Akt and mitogen-activated protein kinase (MAPK; extracellular signal-regulated kinase 1/2, p38, and c-Jun N-terminal kinase [JNK]) and and LY294002 (Akt inhibitor), SB203580 (p38 inhibitor) or SP600125 (a JNK inhibitor) decreased the levels of Annexin V-positive cells. These results suggested that L-AA induces the apoptosis of Hep-2 cells via the nuclear translocation of AIF and EndoG by modulating the Bcl- 2 family and MAPK/Akt signaling pathways.

Introduction
Materials and Methods
    1. Materials
    2. Cell culture and cell viability assay
    3. 4′,6-diamidino-2-phenylindole (DAPI) staining
    4. Flow cytometry
    5. Caspase activity and inhibition of apoptosis with acaspase inhibitor
    6. Protein extraction and Western blot analysis
    7. Extraction of nuclear protein
    8. Electrophoretic mobility shift assay (EMSA)
    9. Statistical analyses
Results
    1. L-AA induced apoptotic cell death in humanlaryngeal Hep-2 cancer cells
    2. L-AA induced the release of cytochrome c frommitochondria into the cytosol and upregulated AIFand EndoG levels
    3. L-AA-induced apoptosis was caspase dependentin Hep-2 cells
    4. L-AA-induced apoptosis is mediated via NF-κB,MAPK, and Akt signal pathways
Discussion
Acknowledgements
Conflicts of Interest
References

• Jung-Sun Park(Department of Oral Physiology, Dental Science Research Institute, School of Dentistry, Chonnam National University)
• Yoon-Jung Kim(Department of Oral Physiology, Dental Science Research Institute, School of Dentistry, Chonnam National University, 2Hard Tissue Biointerface Research Center, School of Dentistry, Chonnam National University)
• Sam Young Park(Department of Oral Physiology, Dental Science Research Institute, School of Dentistry, Chonnam National University, 2Hard Tissue Biointerface Research Center, School of Dentistry, Chonnam National University)
• Kyung-Yi Chung(Department of Dental Hygiene, School of Health Science, Honam University)
• Sang-Jin Oh(School of Biological Sciences and Technology, College of Natural Sciences, Chonnam National University)
• Won-Jae Kim(Department of Oral Physiology, Dental Science Research Institute, School of Dentistry, Chonnam National University) Correspondence to
• Ji-Yeon Jung(1Department of Oral Physiology, Dental Science Research Institute, School of Dentistry, Chonnam National University, Hard Tissue Biointerface Research Center, School of Dentistry, Chonnam National University) Correspondence to