Fig 1.
Chemical structure of the small molecule inhibitors.
The chemical structures of the NSC666715 and its analogs NSC661073, NSC666713, NSC666717 and NSC666719 have been drawn using the ChemDraw software.
Table 1.
Effect of PFTα on the cell cycle phases of HCT116 cells treated with TMZ and NSC666715 either alone or in combination.
Fig 2.
NSC666715 and its analogs block Pol-β-directed LP-BER activity.
The LP-BER was determined using an in vitro reconstituted assay system. Panel A shows the experimental protocol. The assay details are given in Materials and Methods. Panel B shows an autoradiogram of LP-BER, which is representative of three different experiments. Lane 1 shows 32P-labeled 63-mer F-DNA and Lane 2 shows the 23-mer product after APE1 incision. Lane 3 shows Pol-β-mediated strand-displacement synthesis. Lane 4 shows Pol-β-mediated strand-displacement synthesis stimulated by Fen1 (Lane 4). Lane 5 shows the complete repair of the 63-mer F-DNA through the LP-BER pathway in the presence of APE1, Pol-β, Fen1 and DNA ligase I. Lanes 6–9, 10–13, 14–17, 18–21 and 22–25 show the effect of different concentrations of the Pol-β inhibitors NSC 661073, 666713, 666715, 666717 and 666719 on LP-BER activity.
Fig 3.
NSC666715 and its analogs cause the accumulation of TMZ-induced AP sites in the HCT116 cell line.
Cells were pretreated for 2 h with 25 μM of the small molecule inhibitors NSC666715, NSC666717 and NSC666719, followed by treatment with 500 μM of TMZ for an additional 48 h. Genomic DNA was isolated and processed for AP site determination as described in Materials and Methods. Panel A shows the slot blot analysis of AP sites in HCT116 cells treated with 25 μM of NSC666715, NSC666717 and NSC666719 and 500 μM TMZ. Panel B shows the quantitative analysis of AP sites. Results are presented as mean ± SE of three different determinations. * = Significantly different than the untreated control group (P<0.05).
Fig 4.
PFTα inhibits p53-mediated expression of p21 in HCT116 cells treated with TMZ and NSC666715.
The HCT116 cells were pretreated with different concentrations of PFTα and 50 μM NSC666715 for 2 h and then with 500 μM TMZ alone or in combination for an additional 48 h. Cells were harvested, and cellular lysates were prepared and processed for Western blot analysis. Panel A shows immunoblots of the p53, p21, GAPDH and β-actin proteins. Panel B is the quantitative representation of p53 and p21 protein levels after normalization with GAPDH. Lane 1 shows p53 and p21 protein levels from the untreated control group and in Lane 2 and 3 after treatment with 50 and 500 μM NSC666715 and TMZ, respectively. Results in Lane 4 are from combination treatment with 50 μM NSC666715 and 500 μM TMZ. Results in Lane 5 are from the combination treatment with 50 μM NSC666715 and 30 μM PTFα. Results in Lanes 6–8 show p53 and p21 levels after treatment with 10, 20 and 30 μM PFTα, respectively. Results in Lanes 9–11 and 12–14 are from combination treatments with either 500 μM TMZ and 10, 20 and 30 μM PFT, or with 50 μM NSC666715, 500 μM TMZ and 10, 20 and 30 μM PFT, respectively. The data are presented as mean ± SE of three different experiments. * and # = Significantly different than the untreated control (P<0.05).
Fig 5.
Effect of TMZ on senescence in HCT116 cells.
HCT116 cells were treated with different concentrations of TMZ for 48 h and then processed for SA-βgal staining. Panel A shows SA-βgal staining. Panel B represents the quantitative analysis of SA-βgal staining. Data are presented as mean ± SE of four different estimations. * = Significantly different than the untreated control (p<0.05).
Fig 6.
NSC666715 enhances TMZ-induced senescence in HCT116 cells.
The HCT116 cells were pretreated with different concentrations of NSC666715 for 2 h followed by treatment with 250 μM of TMZ for an additional 48 h. After treatment, the cells were processed for SA-βgal staining. Panel A shows SA-βgal staining. Panel B represents the quantitative analysis of SA-β gal staining. Data are presented as mean ± SE of four different estimations. * and # = Significantly different than the untreated control or the 10, 25, 50 and 100 μM NSC666715 treated groups, respectively, (p<0.05).
Fig 7.
TMZ-induced senescence in HCT116 cells is dependent upon the p53/p21 pathway.
HCT116 cells with or without p53 and p21 expression were treated with 500 μM TMZ for 48 h, and then processed for SA-βgal staining. Panel A shows the involvement of p53 and p21 in TMZ-induced senescence. Panel B represents quantitative analysis of the data, which are presented as mean ± SE of four different estimations. * = Significantly different than the untreated control (p<0.05).
Fig 8.
PFTα decreases TMZ and NSC666715-induced senescence in HCT116 cells.
HCT116 cells were pretreated with different concentrations PFTα (10–30 μM) and/or 50 μM of NSC666715 for 2 h followed by the treatment with 500 μM TMZ for an additional 48 h. Panel A shows SA-βgal staining of the cells. Panel B represents the quantitative analysis of the number of SA-βgal positive cells. Data are presented as mean ± SE of four different estimations. ¶ = Significantly different than the untreated control (p<0.05). # = Significantly different than the 50 μM NSC666715 alone treated group (p<0.05). § and §§ = Significantly different than the 10 and 20 μM PFTα alone treated groups, respectively, (p<0.05). *, ** and *** = Significantly different than the 500 μM TMZ in combination with 10, 20 and 30 μM PFTα treated groups, respectively, (p<0.05).
Fig 9.
Effect of NSC666715 and PFTα on TMZ-induced levels of apoptosis-related proteins.
The HCT116 cells were pretreated with different concentrations of PFTα and 50 μM NSC666715 for 2 h and then with 500 μM TMZ alone or in combination for an additional 48 h. Cells were harvested and the cellular lysates were prepared and processed for Western blot analysis. The Western blot analysis data are representative of two different experiments.