In this study primarily response to CDDP administration in terms of cell viability was examined. For demonstration of cell viability in response to CDDP treatment, MTT Assay was used in the absence or presence of the antioxidant, N-Acetyl-L-Cysteine (NAC) pretreatment. It showed around 19% decrease in cell viability for wt and 21% for p53-/- in the absence of NAC at 24h and 30μM CDDP. It also revealed around 72% decrease for wt and 31% decrease for p53-/- in the absence of NAC at 48h and 30μM CDDP, yet in the presence of NAC the cell viability was found to be favoured by almost 20% for wt. To assess the extend of apoptotic response in the absence or presence of NAC pre-treatment Flow Cytometric Analyses by Annexin-V Labelling was applied. It revealed around 1.6 fold increase for wt and 2.4 fold increase for p53-/- in the absence of NAC at 24h and 30μM CDDP, yet in the presence of NAC the apoptotic response was found to be repressed by almost 20% for wt and 40% for p53-/- It also showed around 4.7 fold increase for wt and 2.6 fold increase for p53-/- in the absence of NAC at 48h and 30μM CDDP, yet in the presence of NAC the apoptotic response was found to be repressed by almost 57% for wt. The data showed that wt and p53-/- cells differed in cell viability depending on the dose of CDDP and antioxidant pre-treatment, indicating that some fraction of the apoptotic response was due to increased ROS levels. In addition, DCFH-DA was exploited as the label for ROS in Flow Cytometric Analyses. Flow Cytometry showed around 4 fold increase for wt and 3.2 fold increase for p53-/- at 24h and 30μM CDDP yet in the presence of NAC the increase of ROS was found to be repressed by almost 50% for wt. It also revealed around 5.2 fold increase for wt and 5 fold increase for p53-/- in the absence of NAC at 48h and 30μM CDDP, yet in the presence of NAC the increase of ROS was found to be repressed by almost 66% for p53-/-. A second method, Thiobarbituric Acid Reactive Substances (TBARS) Assay was used to gain more insight, this time in terms of lipid peroxidation. TBARS Assay revealed 8.4 fold increase for wt and 5.9 fold increase for p53-/- in the absence of NAC at 24h and 30μM CDDP, yet in the presence of NAC the increase was found to be repressed by almost 75% for wt. It also showed around 1.5 fold increase for wt and 1.8 fold increase for p53-/- in the absence of NAC at 48h and 30μM CDDP, yet in the presence of NAC the increase was found to be repressed by around 13% for wt and 39% for p53-/-. Data acquired in this section indicated increased ROS and lipid peroxidation levels with CDDP treatment which could be overcome by NAC pre-treatment. In further experiments, OxyBlot™ procedure was applied to total protein isolates and also to the pro-survival proteins of the Bcl-2 family proteins, Bcl-2, Mcl-1 and Bcl-xL, fished out of the total protein suspensions via immunoprecipitation. The technique gave the picture of an increased protein carbonylation with increasing doses of CDDP (0, 30 and 60μM CDDP) and also exhibited that all three pro-survival proteins gave detectable carbonylation signal in the absence of the antioxidant and TP53 gene at 30μM CDDP, supporting the initial hypothesis of this work that loss of function of pro-survival proteins due to protein modifications contributes to apoptotic response in this experimental setup. These results were discussed in the light of intracellular signalling cascades, especially those related to apoptosis and intracellular oxidative stress. As CDDP was found to be inducing apoptosis via affecting the overall redox status of the cell and the concept of sensitization to CDDP treatment could be an interesting approach for possible future applications of this work
