Figure 8

The effects of ALDH inhibitors DEAB and TT on ALDH activity, cell proliferation and gene expression. A. Dose-response effect of DEAB and TT on cell proliferation of A549 and H522 lung cancer cell lines. A colorimetric non-radioactive (MTT based) assay was used as described in Methods to quantify cell proliferation. The results are expressed as the mean ± SD of absorbance and reflect 8 measurements from 2 experiments. Overall, significant decrease (P < 0.023) in cell proliferation was seen with both inhibitors. B &C. H1299 lung cancer cell line with no measurable ALDH activity and A549 and H522 cells were cultured with or without ALDH inhibitors and cell proliferation and ALDH activity were measured as described in Methods. The results are expressed as % decrease (mean ± SD of at least 2 experiments) in cell proliferation (B) or ALDH activity (C) in comparison to untreated controls. The results demonstrate that the effect of ALDH inhibitors (1 and 100 μM) on H1299 cell proliferation is significantly (P < 0.029) less than that on A549 and H522. These results suggest that the effect on cell proliferation is mostly due to the inhibition of ALDH activity. D. After 48 hr incubation of A549 cells with 5 μM of DEAB (dotted bars) or TT (clear bars), RNA was extracted and real-time RT-PCR was performed, as described in Methods, for the 3 genes shown in Figure 7. The results are expressed as the log 10 of relative change in gene expression when compared to untreated WT cells (see Figure 7 legend). These results confirm that the increase in CXXC5 and RAB20 expression is most likely related to the decrease in ALDH activity. The effect on LHX8 was different by the two inhibitors and therefore non specific to ALDH activity inhibition.