Considering that the mechanism of action for CPT is DNA damage, we explored the impact of autophagy inhibition on CPT induced DNA damage as a possible mechanism for decreased sensitivity to CPT in autophagy inhibited DLM8 cells. DNA figure 2 damage as determined by phosphorylation of p53 at Ser15 was unchanged be tween autophagy competent and autophagy inhibited DLM8 cells. We also assessed the impact of au tophagy inhibition on DLM8 cell growth. Autophagy inhib Inhibitors,Modulators,Libraries ition did not significantly impact cell growth of DLM8 cells. This is relevant because the mechanism of ac tion for CPT is DNA damage that occurs during cell div ision. Had autophagy inhibition significantly reduced DLM8 cell growth, this would support the suggestion that autophagy inhibition mediated protection is due to reduced cell division.

Together, this set of data suggests Inhibitors,Modulators,Libraries that the au tophagy inhibition mediated protection Inhibitors,Modulators,Libraries observed in this study was not due to reduced DNA damage or reduced cell division. Previous reports of CPT induced oxidative stress led us to investigate the impact of autophagy inhibition on CPT induced oxidative stress as a contributing factor to the observed autophagy inhibition mediated protec tion. Oxidative stress, as determined by generation of. O2 and H2O2, was higher in autophagy competent DLM8 cells compared to autophagy inhibited Inhibitors,Modulators,Libraries DLM8 cells following CPT treatment, indicating that autophagy inhibition decreased CPT induced oxidative stress. Autophagy inhibition also reduced basal oxidative stress level.

To our knowledge, this is the first report of au tophagy inhibition mediated reduced basal oxidative stress as well as autophagy inhibition mediated reduced anticancer Inhibitors,Modulators,Libraries drug induced oxidative stress. Increased levels of CPT induced oxidative stress coupled with increased CPT induced cell death in autophagy competent DLM8 cells led us to determine if autophagy competent DLM8 cells are more sensitive to oxidative stress. The use of BSO allowed for the investi gation of the impact of oxidative stress alone on cell death and autophgay induction. Autophagy competent DLM8 cells were more sensitive than autophagy inhibited DLM8 cells to BSO induced cell death. In agreement with Martinez Outschonnra et al, BSO also induced autophagy. BSO induced cell death and BSO induced autophagy induction were reversed by NAC pretreatment indicating a link between increased oxidative stress and both cell death and autophagy induction.

Basal levels of autophagy have been previously re ported to differ among cancer cell lines and here we report varying basal levels of autophagy in two meta static murine OS cell lines. Considering these reports, it is plausible that the threshold level of autophagy induc tion that causes autopahgic cell death http://www.selleckchem.com/products/CAL-101.html also varies for dif ferent cancers or even different cell lines within the same type of cancer.