Expression of Bcl-2 contributes to 5-fluorouracil resistance in Huh-7 pre-S2Δ cells.

<p>(A) Huh-7 V, Huh-7 pre-S2Δ and Huh-7 pre-S cells (1×10³) were seeded in 6-well plates. Cells were treated with 5-fluorouracil with or without Bcl-2 inhibitor, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028977#s2" target="_blank">Materials and Methods</a>, and then incubated for another 10 days, and the colony formation ability of three cell lines were evaluated using a crystal violet assay determined by crystal violet in response to 5-fluorouracil. (B) A quantitative measure of colony formation was determined by crystal violet and the number of colonies in the graphs was representative of three independent experiments (lower panel). Data represent the mean ± SD (n = 3). Significant differences (**, <i>P</i><0.01 and ***, <i>P</i><0.001) between the control and experimental group are marked with an asterisks. (C) The morphologic changes after a 72-hour 0.25 mM 5-fluorouracil with or without Bcl-2 inhibitor treatment of Huh-7 pre-S2Δ cells. (D) The effect of 5-fluorouracil or combination with Bcl-2 inhibitor on caspase-3 activity in Huh-7 pre-S2Δ cell line. Enzymatic activity of caspase-3 was determined by caspase-3 antibody, as described under “materials and methods”. (E) The relative caspase-3 activities, normalized to DMSO control, at the indicated 0.25 mM 5-fluorouracil or with 5 µM Bcl-2 inhibitor. <i>Columns</i>, mean of three independent experiments; bars, SD (n = 3). Significant differences (***, <i>P</i><0.001) between the control and experimental group are marked with an asterisks. (F) Both apoptosis and necrosis were involved in 5-Fluorouracil or combination with Bcl-2 inhibitor-induced cell death. Huh-7 pre-S2Δ cells were treated with 5-fluorourail with or without Bcl-2 inhibitor and cells were analyzed by annexin V assay. (G) (H) The ratio of apoptotic and necrotic cells, normalized to DMSO control, at the indicated 0.25 mM 5-fluorouracil or with 5 µM Bcl-2 inhibitor. <i>Columns</i>, mean of three independent experiments; bars, SD (n = 3). Significant differences (*, <i>P</i><0.05; ***, <i>P</i><0.001) between the control and experimental group are marked with an asterisks. (I) Downregulation of Bcl-2 family expression by combination 5-fluorouracil with Bcl-2 inhibitor. Cells were treated with 5-fluorourail with or without Bcl-2 inhibitor and cell lysates were obtained by RIPA lysis buffer. The cell lysates were determined by Western blotting using antibodies specific for Bcl-2, Bcl-xL, Mcl-1, Bax, Bak and β-actin. (J) The effect of 5-fluorouracil or combination with Bcl-2 inhibitor on Akt, ERK and NF-κB phosphorylation in Huh-7 pre-S2Δ cell line. The total lysates were analyzed by Western blotting using ERK, p-ERK, Akt, p-Akt, p65, p-ser²⁷⁶-p65, p-ser³¹¹-p65 and β-actin antibodies.