Decytabine enhances cytotoxicity induced by oxaliplatin and 5-fluorouracil in the colorectal cancer cell line Colo-205

<p>Abstract</p> <p>Background</p> <p>DNA methylation is an epigenetic phenomenon known to play an important role in the development of cancers, including colorectal cancer (CRC). Aberrant methylation of promoter regions of genes is potentially reversible, and if methylation is important for cancer survival, demethylation should do the opposite. To test this we have addressed the hypothesis that DNA methyltransferase inhibitors (DNMTi), decytabine and zebularine, potentiate inhibitory effects of classical anti-CRC cytostatics, oxaliplatin and 5-fluorouracil (5-FU), on survival of CRC cells <it>in vitro</it>.</p> <p>Results</p> <p>Isobole and median effect analysis revealed that decytabine shows potent synergistic interaction with oxaliplatin and 5-FU and that this is probably not the class effect of DNMTi as zebularine shows strong antagonistic interaction with oxaliplatin. The synergistic combination treatment was also applied to the cultures to investigate their mechanisms of action. We have shown that combinations of decytabine with cytostatics produced dose-dependent growth inhibition and treatment-induced apoptosis.</p> <p>Conclusion</p> <p>The observed synergism between decytabine and cytostatics is most probably related to the augmented apoptotic signal and allowed for significant (both biologically and statistically) reduction of the cytotoxic doses of cytostatics used.</p

Combinations of chemotherapeutics with DNMTi agents induce major signaling checkpoints in response to DNA damage.

<p><b>A.</b> Quantification of ATR and ATM phosphorylation level following 72(MFI) was used. Data represented the mean ±SD (n = 3). Significant difference at <i>P</i>≤0.05 is indicated by an asterisk (*). <b>B.</b> Analysis of protein phosphorylation levels using Western blotting method. The detection of β-actin was used as a gel loading control. OXA, oxaliplatin; 5-FU, 5-fluorouracil; DAC, decitabine; ZEB, zebularine; a.u., arbitrary units.</p

Induction of apoptosis in colorectal cancer cells following 72 h incubation with chemotherapeutic agents, DNMT inhibitors and their combinations.

<p><b>A.</b> Detection of apoptosis by the annexin V-fluorescein isothiocyanate (FITC)/porpidium iodide (PI) analysis. Data are expressed as the means ±S.D. (n≥4). An asterisk (*) indicates that the induction of apoptosis by the evaluated agents was significant in comparison with the combination of chemotherapeutic agents and DNMT inhibitors <i>versus</i> chemotherapeutic agents applied alone (<i>P</i><0.05). <b>B.</b> Western blotting analysis of pro-apoptotic protein levels. The β-actin was used as a gel loading control. <b>C.</b> Representative cytograms of flow cytometry experiments demonstrating changes in Δ<i>Ψ_m</i> in CRC cells lines after 72 h of incubation with chemotherapeutic agents, DNMT inhibitors and their combinations. Cells were stained with JC-1. Cells in the R2 quadrant were counted as cells deprived of mitochondrial membrane potential. An asterisk (*) indicates a significant difference between experimental groups and control group at <i>P</i><0.05. OXA, oxaliplatin; 5-FU, 5-fluorouracil; DAC, decitabine; ZEB, zebularine.</p

Chemotherapeutic agents, DNMTi and their combinations influence the cell cycle progression of colorectal cancer cells.

<p><b>A.</b> Changes in the cell cycle distribution of SW48 and HT-29 cells after 72 h of treatment with the evaluated agents. The cells were stained with propidium iodide (PI) and then analyzed by flow cytometry. The percentage of cells in each phase of the cell cycle was determined using ModFit LT™ (version 3.0). Each bar represents the mean ±S.D. (n≥4). Significant difference at <i>P</i><0.05 are indicated by asterisk (*). <b>B.</b> Analysis of <i>CCNE1</i> and <i>ATM</i> mRNA levels by semi-quantitative RT-PCR method after 72 h incubation of CRC cells with chemotherapeutic agents, DNMTi and their combinations at concentrations as indicated. M, marker [bp]; <i>GAPDH</i>, transcript encoding glyceraldehyde-3-phosphate dehydrogenase, a constitutively expressed gene, used as an internal control. <b>C.</b> Western blotting analysis of the cell cycle regulatory proteins. The β-actin was used as a gel loading control. OXA, oxaliplatin; 5-FU, 5-fluorouracil; DAC, decitabine; ZEB, zebularine.</p

Effects of chemotherapeutic agents and DNMT inhibitors on cell viability of colorectal cancer cell lines.

<p>The cells were treated singly or in combinations with the indicted doses of the agents for 72; 5-FU, 5-fluorouracil; DAC, decitabine; and ZEB, zebularine. Each point represents the mean ±SD (n = 5), asterisks indicate a significance at <i>P</i><0.05 for comparison with oxaliplatin or 5-FU alone.</p

Interactions of standard chemotherapeutics with DNMT inhibitors in the human colorectal cancer cell lines SW48 and HT-29.

<p><b>A.</b> Isobolograms at a 50% effect level. Concentrations of particular drugs are indicated on x and y axis. The isobolograms were constructed by connecting the IC₅₀ values of demethylating agents (on the ordinate) with the IC₅₀ of oxaliplatin or 5-FU plotted on the abscissa. When the doses of two agents in combination are lower or higher than the additive doses, the synergy or antagonism is present, respectively. <b>B.</b> Combination index values (CI) with a 95% confidence interval at all effect levels as calculated by the CalcuSyn software. A CI value significantly less than 1 indicates synergy, a CI value insignificantly different from 1 indicates addition, and a CI significantly higher than 1 indicates antagonism.</p