Rhythms in the pseudo-steady state (PSS) levels of reactive species (RS), particularly superoxide and hydroxyl radicals, exist in cancer cells. The RS rhythm characteristics, particularly frequency and amplitude, are entrained (reset) by the anticancer compounds/drugs. In this work, we show for the first time that the phase of the RS rhythm at which the drug is added is significantly important in determining the cytotoxicity of anticancer compounds/drugs such as menadione and curcumin, in two different cancer cell lines. Curcumin, the more effective of the two drugs (IC 50 = 15 µM, SiHa; 6 µM, HCT116) induced reset of superoxide and hydroxyl rhythms from 15.4 h to 9 h, and 25 h to 11 h respectively, as well as caused increases in these radical levels. However, menadione (IC 50 = 20 µM, SiHa; 17 µM, HCT116) affected only the superoxide levels. Drug treatment at different time points/ phase of the RS rhythm resulted in a maximum of 27% increase in cytotoxicity, which is significant. Further, we report for the first time, an unexpected absence of a correlation between the intracellular PSS RS and antioxidant levels; thus, the practice of using antioxidant enzyme levels as surrogate markers of intracellular oxidative stress levels may need a re-consideration. Therefore, the RS rhythm could be a fundamental/generic target to manipulate for improved cancer therapy.Reactive oxygen species (RS) such as superoxide and hydroxyl radicals seem to be an important set of molecular mediators of the effectiveness of many anticancer therapies; they are also important determinants of the cellular redox status and conditions such as hypoxia 1-7 . RS are also known to regulate cellular rhythms, and the components of the cellular redox system such as glutathione, glutathione reductase and NAD + have been shown to be rhythmic [8][9][10][11] . Such rhythms along with their crosstalk with the timekeeping mechanisms control the metabolic, transcriptional and translational machinery in the cells 12,13 . Further, the rhythms and their alterations have been linked to immune gating responses, lipid peroxidation levels as well as to drug resistance observed in some treatments 4,14-19 . However, the rhythms in the pseudo-steady state (PSS) levels of the more fundamental molecules, the RS themselves, have not been reported in the above context, probably due to an incorrect perception of their utility, given their high reaction rates.The cellular antioxidant levels have been used as an indirect measure of the oxidative stress in the cells 20 , and oxidative stress is caused by an imbalance in the rates of production and consumption of reactive species 21 . The indirect measure seems to arise from an expectation based on the molecular interactions between enzymatic antioxidants and the relevant RS -e.g. superoxide dismutase (SOD) and superoxide. However, the dynamic aspects of the cell system do not seem to be considered in that expectation. For example, the rate constants of the RS reactions are many orders of magnitude higher than the synthesis...
