Reated with carboplatin, a DNA alkylating agent (Figure 2C). The response among K-Ras independent cells to DNA damaging agents was variable, even though general they have been nearly 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 had been treated with the microtubule disruption agent, paclitaxel, some cell lines in both the K-Ras dependent and independent sub-groups responded, with no considerable difference in response among groups (Figure 2D). Numerous tumor cells inactivate DNA damage induced apoptotic pathways, suggesting a doable 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 also other sorts of Benzyl-PEG8-t-butyl ester PROTAC cancer (29). Evaluation of TP53 mutations by way 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), when all ten K-Ras dependent cell lines have TP53 mutations (30). DAP Inhibitors MedChemExpress Distinct 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 from the p53 target gene, p21. Inside a couple of cell lines with mutant TP53 (see H2009 and H727, Figure 2E), remedy with etoposide enhanced p53 protein stability and/or Ser15 phosphorylation, at the same time as p21 expression, indicating a partially functional p53 protein. Notably, H2009 and H727 cells are amongst the least PKC dependent from the K-Ras dependent NSCLC cells (see Figure 1C). On the K-Ras independent cell lines, those with WT TP53 (A549, H460 and SW-1573) are among one of the most sensitive to etoposide, on the other hand some K-Ras independent cells with mutant TP53, particularly H157 cells, nevertheless showed sensitivity. As a result, while mutations in TP53 correlate with resistance to apoptosis and with a pro-tumorigenic function for PKC in K-Ras dependent cells, inside the K-Ras independent sub-group, wild form TP53 alone will not predict sensitivity to apoptosis. Our data suggests that the pro-apoptotic function of PKC, in particular within the context of topoisomerase inhibitors, is lost or suppressed in K-Ras dependent NSCLC cells, relative to K-Ras independent cells. To explore this straight, 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; offered 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 small impact on etoposideinduced apoptosis, or synergized with etoposide to further enhance DNA fragmentation in K-Ras dependent H2009 and HCC-44 cells (Figure 3C). We subsequent explored the hypothesis that PKC is differentially linked to survival or apoptotic pathways in K-Ras dependent and independent NSCLC cells. We located no consistent differences in basal ERK or Akt activation among these NSCLC subsets (information not shown). Nonetheless, our prior studies suggested 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 using our pan.
