Western blot analyses revealed that Doxo and Gem treatment alone increased p53 this website levels (Figure 3A). When NQO1-knockdown-KKU-100 cells were treated with chemotherapeutic agents, p53 level was enhanced further by all 3 agents (Figure 3A). Then, we examined the expression levels of some p53 downstream proteins, i.e. p21, cyclin D1, and Bax protein. Similar to p53, p21 and Bax were over-expressed by the drug treatments (Figure 3B, 3D). In contrast, in the NQO1 knockdown cells, treatment with chemotherapeutic agents strongly suppressed the cyclin D1 level (Figure 3C). In the non-target siRNA transfected KKU-100 cells, Doxo and Gem, but not 5-FU, treatments increased cyclin D1 expression

(Figure 3C). Figure 3 Altered expressions of proteins related to cell proliferation and apoptosis pathways. A-D, Expressions of proteins related to cell proliferation and apoptosis pathways. KKU-100 with NQO1 knocked down cells were exposed Trichostatin A chemical structure click here to chemotherapeutic agents; 5-FU (3 μM), Doxo (0.1 μM), and Gem (0.1 μM) for 24 hr. Whole cell lysates were prepared after indicated treatment and Western blot analysis was conducted using anti-p53 (A), -p21 (B), -cyclin D1 (C), -Bax (D) and -β-actin antibodies. The relative bars that were normalized with β-actin as a loading control of each band is shown below the Western blot images. Data represent mean ± SEM, each from three separated experiments. *p < 0.05

vs the treated non-targeting knocked down cells. **p < 0.05 vs the untreated non-targeting knocked down cells. Over-expression of NQO1 in CCA cells induces drug resistance against chemotherapeutic agents Since KKU-M214 cells naturally express relatively low level of NQO1, effects of NQO1 over-expression by transient transfection with NQO1 expression vector on the susceptibility of cells to chemotherapeutic agents was evaluated. After transfection, the NQO1 enzyme activity in the transfected

cells was elevated approximately 2.5-fold and the NQO1 protein level was 2.25-fold higher than the control vector (Figure 4A-B), indicating Phospholipase D1 that NQO1 construct was efficiently expressed in KKU-M214 cells. Then, NQO1-over-expressed KKU-M214 cells were exposed to 5-FU and Gem for 48 hr, and to Doxo for 24 hr. The results showed that the cytotoxicity of 5-FU, Doxo, and Gem were markedly decreased for NQO1-over-expressed KKU-M214 cells (Figure 4C-E), indicating the protective effect of NQO1. Figure 4 Effects of NQO1 over-expression on the susceptibility of KKU-M214 cells to chemotherapeutic agents (5-FU, Doxo, and Gem). A-B, Effect of NQO1 over-expression on mRNA and protein levels of NQO1 in KKU-M214 cells. The pCMV6-XL5-NQO1 (wild type NQO1) or pCMV6-XL5 (control vector) was transfected to KKU-M214 for 24 hr. The whole cells were collected for NQO1 enzyme activity assay (A) and Western blot analysis (B). The data represent mean ± SEM, each from three experiments. *p < 0.05 vs the control vector transfected cells.