FIGURE 2.

Rhamnetin and cirsiliol in combination with radiation reduce cell proliferation through the inhibition of Notch-1 expression in NSCLC cells. A, chemical structures of rhamnetin and cirsiliol. B, effects of rhamnetin and cirsiliol on the viability of NCI-H1299 and NCI-H460 cells were measured by a triazolyl blue tetrazolium bromide assay. Cells were treated for 4 h with the rhamnetin or cirsiliol at concentrations up to 25 μm. Absorbance was measured at 570 nm. C, effects of the rhamnetin and cirsiliol on proliferation of the NSCLC cells were measured with a colony forming assay. *, p < 0.05 for nonirradiated cells versus irradiated cells; **, p < 0.05 for irradiated cells versus irradiated cells treated with each drug. D, effects of rhamnetin and cirsiliol on radiation-induced NOTCH-1 mRNA expression in NSCLC cells were evaluated with a Northern blot analysis. Total RNA was analyzed using a α-³²P-labeled NOTCH-1 cDNA probe. GAPDH was used for normalization. E, effects of rhamnetin and cirsiliol on the radiation-induced expression of Notch-1, and downstream target proteins were detected by Western blot analysis. F, effects of radiation, rhamnetin, and cirsiliol on the expression of Notch-2 and Notch-3 were detected by Western blot analysis. G, effects of rhamnetin and cirsiliol on radiation-induced increase of cleaved-Notch-1 and γ-secretase complex proteins were assessed by Western blot analysis.