Sodium 5,6-benzylidene-L-ascorbate induces oxidative stress, autophagy, and growth arrest in human colon cancer HT-29 cells.
Our previous studies have demonstrated the oxidative stress properties of sodium ascorbate (SAA) and its benzaldehyde derivative (SBA) on cancer cell lines, but the molecular mechanisms mediating their cytotoxicity remain unclear. In this study, we treated human colon cancer HT-29 cells with SAA and SBA, and found a significant exposure time-dependent increase of cytotoxicity in both treatments, with a higher cytotoxicity for 24 h with SAA (IC(50) = 5 mM) than SBA (IC(50) = 10 mM). A short-term treatment of cells with 10 mM SAA for 2 h revealed a destabilization of the lysosomes and subsequent induction of cell death, whereas 10 mM SBA triggered a remarkable production of reactive oxidative species, phosphorylation of survival kinase AKT, expression of cyclin kinase-dependent inhibitor p21, and induction of transient growth arrest. The crucial role of p21 mediating this cytotoxicity was confirmed by isogenic derivatives of the human colon carcinoma HCT116 cell lines (p21(+/+) and p21(-/-)), and immunoprecipitation studies with p21 antibody. The SAA cytotoxicity was blocked by co-incubation with catalase, whereas the SBA cytotoxicity and its subsequent growth arrest were abolished by N-acetyl-L-cysteine (NAC), but was not affected by PI3K phosphorylation inhibitor LY294002, or catalase, suggesting two separated oxidative stress pathways were mediated by these two ascorbates. In addition, neither active caspase 3 nor apoptotic bodies but autophagic vacuoles associated with increased LC3-II were found in SBA-treated HT-29 cells; implicating that SBA induced AKT phosphorylation-autophagy and p21-growth arrest in colon cancer HT-29 cells through an NAC-inhibitable oxidative stress pathway.
Cheung
F W K
FW
School of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
Che
C T
CT
Sakagami
H
H
Kochi
M
M
Liu
W K
WK
D000970:Antineoplastic Agents; D000975:Antioxidants; D001205:Ascorbic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects*; D001597:Benzylidene Compounds / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects*; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000473:pathology; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D006801:Humans; D018384:Oxidative Stress / Q000187:drug effects*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D013997:Time Factors
D000970:Antineoplastic Agents; D000975:Antioxidants; D001597:Benzylidene Compounds; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D051057:Proto-Oncogene Proteins c-akt; D001205:Ascorbic Acid; C070680:zilascorb