Figure 1.
Inhibition of cytidine deaminase (CDA) contributes to improved gemcitabine sensitivity.
A. Levels of CDA mRNA in pancreatic and lung carcinoma cell lines were evaluated by real-time PCR for Panc-1, MIAPaCa-2, BxPC-3, H322, H1299, and H441. Normalized δCt was calculated as 35-(CDA Ct-GAPDH Ct). B. Three pancreatic and three lung cancer cell lines were cultured with just gemcitabine (GEM) alone, or with GEM and 100 mM tetrahydrouridine (THU). IC50 was determined using a colorimetric assay as described in the experimental procedures. Values are means ±SD, and results are representative of 3 independent experiments.
Figure 2.
Tetrahydrouridine (THU) inhibits cell growth in MIAPaCa-2, H1299, and H441.
Tetrahydrouridine (THU) significantly reduced cell growth by colorimetric assay in MIAPaCa-2, H441, and H1229 cell lines as compared with the respective control. Values are means of three independent experiments performed in triplicate. An error bar is presented as S.D. Day4 data were used for the statistical analysis.
Figure 3.
Metabolic pathway of gemcitabine.
Gemcitabine (2′,2′-difluorodeoxycytidine, dFdC) is taken in DNA through the active diphosphate (dFdCDP) and triphosphate (dFdCTP) by deoxycytidine kinase (dCK). dFdCTP can inhibit ribonucleotide reductase (RR). On the contrary, Cytidine deaminase (CDA) inactivates dFdC to 2′,2′-difluorodeoxyuridine (dFdU) by deamination. Tetrahydrouridine (THU) prevents dFdC from being inactivated by binding CDA directly. THU also leads to the potential for cell growth inhibition.
Figure 4.
THU (Tetrahydrouridine) influences the cell cycle in MIAPaCa-2, H441 and H1299.
A. After 3 days of THU exposure, the cell cycle was analyzed using flow cytometry. In accordance with cells whose growth was inhibited by THU, MIAPaCa-2, H441, and H1229 cell lines exhibited an increased rate of the G1-phase and a decrease in the S-phase. B. Cells were processed for immuno-cytochemistry with an antibody against Ki-67. Microscopic images of Ki-67-stained cells are shown in THU-treated cells and non-treated cells. Nuclei were stained with DAB. Data represents the mean ± S.D. of 3 independent experiments and significance values relate to comparisons between medium and THU treated cells.
Figure 5.
Cytidine deaminase (CDA) inhibition does not participate in cell proliferation.
A. After transfecting siRNA against CDA, real-time PCR analysis of CDA expression in MIAPaCa-2, H441, and H1299 exhibited cell growth inhibition by THU. Statistical significance was confirmed using a t-test at Day4. B. After knocking down CDA, a proliferation assay was performed. Down-regulated CDA did not affect cell growth in MIAPaCa-2, H441, and H1229 cell lines. C. A combination therapy of CDA knockdown and THU did not show any differences compared to THU alone.
Figure 6.
Tetrahydrouridine (THU) inhibits E2F1 at the protein level which is associated with the G1/S transition.
Panc-1, MIAPaCa-2, H441, and H1229 cells were treated with THU (100 µM) for 96 hrs as indicated. Subsequently, E2F1 protein levels were analyzed by western blotting. THU could down-regulate E2F1 expression. However, THU did not affect E2F1 mRNA expression level.