Sodium 5,6-benzylidene-L-ascorbate induces oxidative stress, autophagy, and growth arrest in human colon cancer HT-29 cells
F W K Cheung1, C T Che, H Sakagami
1School of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
Abstract:
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.
Insights
Sodium ascorbate (SAA) and its derivative (SBA) show cancer cell cytotoxicity. SAA destabilizes lysosomes, while SBA induces oxidative stress, AKT phosphorylation, and p21-mediated growth arrest via distinct pathways.
Area of Science:
- Biochemistry
- Cell Biology
- Oncology
Background:
- Sodium ascorbate (SAA) and its benzaldehyde derivative (SBA) exhibit oxidative stress properties against cancer cells.
- The precise molecular mechanisms underlying their cytotoxic effects remain incompletely understood.
Purpose of the Study:
- To elucidate the distinct molecular mechanisms of cytotoxicity induced by SAA and SBA in human colon cancer cells.
- To investigate the roles of reactive oxygen species, AKT signaling, p21, and autophagy in mediating the observed effects.
Main Methods:
- Treatment of HT-29 colon cancer cells with SAA and SBA.
- Analysis of cytotoxicity, lysosomal stability, reactive oxygen species production, and protein phosphorylation (AKT).
- Assessment of p21 expression, cell cycle arrest, and autophagy markers (LC3-II) using isogenic cell lines and immunoprecipitation.
Main Results:
- Both SAA and SBA demonstrated dose- and time-dependent cytotoxicity, with SAA being more potent.
- SAA induced lysosomal destabilization and cell death.
- SBA triggered reactive oxygen species production, AKT phosphorylation, p21 expression, and transient growth arrest, involving an NAC-inhibitable pathway.
- SBA treatment led to autophagic vacuoles and increased LC3-II, suggesting AKT-mediated autophagy.
Conclusions:
- SAA and SBA induce colon cancer cell death through separate oxidative stress-mediated pathways.
- SAA's cytotoxicity involves lysosomal destabilization.
- SBA induces p21-mediated growth arrest and AKT-autophagy signaling via an oxidative stress pathway inhibited by N-acetyl-L-cysteine (NAC).
