Reated with carboplatin, a DNA alkylating agent (Figure 2C). The response amongst K-Ras independent cells to DNA damaging agents was variable, despite the fact that general they had been practically 4 occasions far more responsive to etoposide and twice as responsive to SN-38 as K-Ras dependent cells (Figures 2A and 2B). When cells have been treated using the microtubule disruption agent, paclitaxel, some cell lines in both the K-Ras dependent and independent sub-groups responded, with no important distinction in response amongst groups (Figure 2D). Several tumor cells inactivate DNA harm induced apoptotic pathways, suggesting a attainable Catalase References mechanism for the resistance of K-Ras dependent cells to apoptosis. TP53, which encodes the tumor suppressor protein p53, is mutated in about 50 of NSCLC, and mutation of TP53 predicts resistance to chemotherapeutic drugs in lung and other types of cancer (29). Evaluation of TP53 mutations by means of the COSMIC (http://cancer.sanger.ac.uk/ cosmic) and UMD TP53 mutation databases (http://p53.fr) indicates that 4/7 K-Ras independent cell lines have mutant TP53 (H157, H1792, H1155 and CaLu-6), though all ten K-Ras dependent cell lines have TP53 mutations (30). Certain TP53 mutations are summarized in Table S1. As not all TP53 mutations are inactivating, we verified the functional status of p53 by analyzing p53 stability, Ser15 phosphorylation, and expression with the p53 target gene, p21. In a handful of cell lines with mutant TP53 (see H2009 and H727, Figure 2E), remedy with etoposide elevated p53 protein stability and/or Ser15 phosphorylation, also as p21 expression, indicating a partially functional p53 protein. Notably, H2009 and H727 cells are amongst the least PKC dependent of the K-Ras dependent NSCLC cells (see Figure 1C). In the K-Ras independent cell lines, these with WT TP53 (A549, H460 and SW-1573) are among by far the most sensitive to etoposide, nonetheless some K-Ras independent cells with mutant TP53, specifically H157 cells, nevertheless showed sensitivity. Hence, even though mutations in TP53 correlate with resistance to apoptosis and having a pro-tumorigenic function for PKC in K-Ras dependent cells, inside the K-Ras independent sub-group, wild form TP53 alone does not predict sensitivity to apoptosis. Our information suggests that the pro-apoptotic function of PKC, especially in the context of topoisomerase inhibitors, is lost or suppressed in K-Ras dependent NSCLC cells, relative to K-Ras independent cells. To Direct Inhibitors targets explore this directly, we analyzed etoposide-induced apoptosis in cells depleted of PKC. Depletion of PKC with either 193 or 203 resulted inAuthor Manuscript Author Manuscript Author Manuscript Author ManuscriptOncogene. Author manuscript; readily available in PMC 2017 October 03.Ohm et al.Pagesuppression of apoptosis in K-Ras independent A549 and H460 cells when compared with cells expressing scr (Figure 3A). In contrast, depletion of PKC had little effect on etoposideinduced apoptosis, or synergized with etoposide to further raise DNA fragmentation in K-Ras dependent H2009 and HCC-44 cells (Figure 3C). We next explored the hypothesis that PKC is differentially linked to survival or apoptotic pathways in K-Ras dependent and independent NSCLC cells. We found no consistent variations in basal ERK or Akt activation between these NSCLC subsets (data not shown). Even so, our prior studies recommended that ERK activation is differentially regulated by PKC depletion in K-Ras dependent versus independent NSCLC cells (9). In our present study we explored this additional applying our pan.
