p53-Independent Inhibition of Proliferation and p21^Waf1/Cip1-Modulated Induction of Cell Death by the Antioxidants N-acetylcysteine and Vitamin E¹

Epidemiological evidence has suggested an association between diets rich in antioxidants and diminished risks of various types of cancer. Proposed mechanisms for protective effects of antioxidants have involved inhibition of free radical-mediated DNA damage. Recent data suggest that antioxidants may prevent or eliminate cancerous cells through their ability to inhibit proliferation or to induce programmed cell death (PCD). To begin to identify cell cycle and cell death regulatory factors involved in antioxidant-induced growth arrest and PCD, we have studied colorectal carcinoma cells (CRCs) that differ in expression of the tumor suppressor protein p53, and of the cyclin-dependent kinase (CDK) inhibitor p21^Waf1/Cip1. The antioxidants, N-acetylcysteine (NAC) and vitamin E either inhibited proliferation in a p53-independent manner without affecting cell viability or induced cell death. Growth arrest was not associated with upregulation of the CDK inhibitors p21^Waf1/Cip1, p18^lnk4c or p16^lnk4a, but was associated with a decrease in reactive oxygen species (ROS). In contrast to previous observations, the absence of p21^Waf1/Cip1 increased susceptibility of CRCs to antioxidant-induced PCD. NAC decreased levels of retinoblastoma protein (Rb) phosphorylation in all cells tested, but Rb was cleaved only in cells which underwent NAC-induced death. Although NAC decreased ROS in all cells studied, cell lines in which PCD occurred had higher baseline levels of ROS than cell lines in which proliferation was blocked. These observations suggest that expression of p21^Waf1/Cip1 and basal levels of ROS are important determinants of outcome after antioxidant treatment.