5). We also determined the effects of EGCG analogues, including EC, ECG, and EGC, on DNR-mediated growth inhibition (Fig. 3C). ECG, which like EGCG also inhibits CBR1 GDC-0941 order in vitro, showed significant enhancement of DNR-mediated cell growth inhibition in both HepG2 (P < 0.01) and SMMC7721 (P < 0.05), whereas EGC and EC, which weakly inhibited CBR1 in vitro, did not show an obvious synergic effect with DNR. Thus, there is a

correlation between the inhibition of CBR1 and the enhancement of DNR-mediated tumor cell growth by EGCG and its analogues. We next examined the effect of EGCG on DNR-induced G2/M cell cycle arrest by fluorescence-activated cell sorting analysis. As shown in Fig. 3D and Supporting Information Fig. 6A, DNR treatment of cells induced a reduction of the cell number in the G1 phase and a corresponding increase in the G2/M phase population. In contrast, 10 μM EGCG alone had no effect on the cell cycle progression. However, a combination of 10 μM EGCG and 0.04 μM DNR resulted in an increase in the percentage of G2/M cells from 52.8% (DNR alone) to 62.4% (EGCG and DNR) in HepG2 LY294002 cost cells. For SMMC7721 cells, EGCG and 0.03 μM DNR induced a 10.4% increase in cells in the G2/M

phase versus DNR alone. EGCG was thus capable of enhancing the DNR-induced G2/M cell cycle arrest, and this reflected the ability of EGCG to enhance the inhibition of cell proliferation by DNR. We also examined the effect of EGCG on DNR-induced apoptosis with flow cytometry (Fig. 3E and Supporting

Information Fig. 6B). EGCG alone at 20 μM did not induce apoptosis. However, EGCG at the same concentration increased DNR-induced apoptosis from 36.4% to 45.2% in HepG2 cells. For SMMC7721 cells, the percentage of apoptosis increased from 12.8% (DNR alone) to 17.2% (DNR and EGCG). These results strongly suggest that EGCG is capable of enhancing the antitumor activity of DNR. To further verify that the synergic effect of EGCG with DNR is mediated by CBR1, we generated Hep3B-CBR1 cells stably expressing CBR1 and control Selleckchem Rucaparib Hep3B cells stably transfected with empty pcDNA3.1(-)/myc-HIS vector (pcDNA). The ectopic expression of CBR1 was confirmed by western blotting in Hep3B-CBR1 cells (Fig. 4A). Hep3B-pcDNA cells and Hep3B-CBR1 cells were treated with DNR, EGCG, or EGCG and DNR. As shown in Fig. 4B, the treatment of Hep3B-pcDNA cells with 0.4 μM DNR led to 34.4% cell viability in comparison with the untreated cells, whereas the cell viability of Hep3B-CBR1 was 52.9%. Hep3B-CBR1 cells were more resistant to DNR than Hep3B-pcDNA, whereas no differences were observed for these two lines in their resistance to 5-fluorouracil (5-FU; P > 0.05; Fig. 4C). The treatment of Hep3B-CBR1 cells with EGCG and 0.4 μM DNR decreased the cell viability from 52.9% to 39.0% (P < 0.01; Fig. 4B).