Table 1.
HLM006474 IC50 values across various cell types.
Figure 1.
Expression of common E2F-regulated genes increases in early time point treatments with HLM006474.
A. The NSCLC cell lines H1299 and H292 were treated with 60 µM HLM006474 for 0, 3, 6, 9, 12, and 24 hours, then harvested and examined through Western blots. Modest increases in E2F1 and more dramatic increases in E2F3a and b protein levels were noted at early time point in both cell lines. B–D. mRNA was harvested from cells that were treated as described in 1A, converted to cDNA using RT-PCR, and analyzed for expression levels of E3F3 (B), E2F1 (C), and E2F4 (D) using real-time PCR and tubulin as a control. Short treatments with 6474 altered mRNA expression levels of E2F3 and E2F1, but not E2F4. E–G. mRNA expression of well-known genes commonly regulated by E2Fs were analyzed in a manner similar to the previous description in 1B–D. Expression levels of MCM10 (E), MCM2 (F), and CCNE2 (G) mRNA were analyzed with tubulin as a control and were all noted to be more highly expressed following short treatments with 6474. Notes: n.s. represents not significant, * represents p<0.05, ** represents p<0.01.
Figure 2.
HLM006474 synergizes with paclitaxel, but not with cisplatin or gemcitabine.
A–C. H1299 cells were subjected to viability assays in the presence of 6474 (HLM) combined with cisplatin (A, CisPt), gemcitabine (B, Gem) and paclitaxel (C, Pac), as indicated (see methods). Results reveal synergy with paclitaxel (CI average 0.98) and antagonism with cisplatin and gemcitabine (CI average 1.40 and 1.39, respectively). D. Western blotting reveals that in H1299 cells treated for 72 hours with 20 µM 6474 alone, 5 nM paclitaxel alone, or the combination of the two, 6474 and paclitaxel synergize in the induction of PARP cleavage. E–G. H292 cells were tested in similar viability assays to determine the efficacy of 6474 (HLM) combined with cisplatin (E, CisPt), gemcitabine (F, Gem) and paclitaxel (G, Pac). As seen in H1299 cells, 6474 synergizes with paclitaxel (CI average 0.96), but is antagonistic with cisplatin (CI average 1.51) and gemcitabine (CI average 1.46).
Figure 3.
Sensitivity to paclitaxel correlates with E2F3 levels in NSCLC.
A. cDNA from ten NSCLC cell lines were utilized in real-time PCR to detect endogenous E2F3 expression levels (as compared to GAPDH as a control). The expression levels were then compared to the paclitaxel logIC50 of each line and graphed as shown. B. Lysates from ten NSCLC cell lines were prepared and ran in a Western blot to detect endogenous E2F3 levels. C. Densitometrically analyzed values of the protein expression levels (as compared to β-actin as a control) were then graphed against the paclitaxel logIC50 for each line as shown.
Figure 4.
Depletion of E2F3 alters sensitivity to paclitaxel.
A. H1299 cells were transiently transfected with 200 pmol control, E2F3a, or E2F3b siRNA and harvested after 24 hours. Western blots of these lysates demonstrate the extent of E2F knockdown. B. MTS assays were used to determine the sensitivity of each cell line to 50 nM paclitaxel. Cells with diminished E2F3 levels were more viable in the presence of paclitaxel than control cells. Notes: * represents p<0.05, ** represents p<0.01.
Figure 5.
Model of HLM006474 synergy with paclitaxel.
In untreated cells, E2F/dimerization partner (DP)/Rb complexes predominate, and thus, E2F3 levels are relative low (as well as many other E2F-regulated genes). Treatment with HLM006474 disrupts the E2F/DP/Rb repressive complexes from binding DNA, resulting in increased transcription of E2F3 (as well as many other E2F-regulated genes). In this model, the elevated levels of E2F3 sensitize cells to taxane treatment, as previously demonstrated, through an unknown mechanism.