School of Chinese Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong.
J Cell Biochem. 2010 Oct 1;111(2):412-24. doi: 10.1002/jcb.22717.
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.
我们之前的研究已经证明了抗坏血酸钠（SAA）及其苯甲醛衍生物（SBA）对癌细胞系的氧化应激特性，但介导其细胞毒性的分子机制仍不清楚。在这项研究中，我们用 SAA 和 SBA 处理人结肠癌细胞 HT-29，发现两种处理方式均存在显著的暴露时间依赖性细胞毒性增加，SAA（IC50=5mM）的细胞毒性高于 SBA（IC50=10mM），24 小时的细胞毒性更高。用 10mM SAA 短时间处理细胞 2 小时会导致溶酶体不稳定，随后诱导细胞死亡，而 10mM SBA 则会引发大量活性氧的产生、生存激酶 AKT 的磷酸化、细胞周期蛋白依赖性抑制剂 p21 的表达以及短暂的生长停滞诱导。p21 介导这种细胞毒性的关键作用通过人结肠癌细胞系 HCT116 的同源衍生物（p21(+/+)和 p21(-/-))得到了证实，并且通过 p21 抗体的免疫沉淀研究也得到了证实。SAA 的细胞毒性可以通过与过氧化氢酶共孵育来阻断，而 SBA 的细胞毒性及其随后的生长停滞可以通过 N-乙酰-L-半胱氨酸（NAC）来消除，但不受 PI3K 磷酸化抑制剂 LY294002 或过氧化氢酶的影响，这表明这两种抗坏血酸通过两条独立的氧化应激途径来介导。此外，在 SBA 处理的 HT-29 细胞中没有发现活性 caspase 3 或凋亡小体，但发现了与 LC3-II 增加相关的自噬空泡；这表明 SBA 通过 NAC 抑制的氧化应激途径诱导 AKT 磷酸化自噬和 p21 生长停滞。
