AZA and DAC were supplied by the Research and Production Center “HimFarmSintez”, Institute of Bioorganic Chemistry of the National Academy of Sciences of Belarus, Republic of Belarus; 4-amino-1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-1,3,5-triazine-2(1H)-one (2’F-araAC) was synthesized according to the method described in [32] . HCT-116 human colorectal carcinoma cell line, L-1210 mouse lymphocytic leukemia cell line and P-388 murine lymphoid neoplasm cell line were obtained from the Russian Collection of Cell Cultures (Institute of Cytology, Russian Academy of Sciences).

HCT-116 cancer cells were grown to 70% - 80% confluence in McCoy’s 5A (Sigma M4892) medium, 10% fetal bovine serum (Capricorn FBS-12A) and 1% Antibiotic Antimycotic solution (Sigma A5955), and then removed with 0.25% trypsin/EDTA solution (Capricorn TRY-2B), washed from trypsin with potassium phosphate buffer centrifuging for 5 minutes at 1000 rpm [36] .

The nuclear protein was isolated using the Nuclear Extraction Kit (Abcam, ab113474). Cell pellet (31 × 10⁶) was resuspended in a pre-extraction buffer (containing DTT and 1:1000 protease inhibitors), incubated for 10 minutes on ice and vortexed vigorously for 10 seconds and centrifuged for 1 minute at 12,000 rpm. The supernatant was removed and an extraction buffer was added to the precipitate. It was incubated on ice for 15 minutes and vortexed for 5 seconds every 3 minutes. Then it was sonicated for 10 seconds. It was then centrifuged for 10 minutes at 14,000 rpm. A supernatant containing the nuclear protein was collected.

The amount of nuclear protein was quantified using the Bradford Assay Kit (Abcam, ab102535) according to manufacturer’s instructions. Absorbance was measured at 595 nm using a Tecan Infinite M200 plate reader. The DNMTs Activity Quantification Kit (Fluorometric) (Abcam, ab113468) was used to analyze DNA methyltransferases activity. The analysis was performed according to manufacturer’s instructions.

The stock solution of the studied compounds of 2’F-araAC, DAC and AZA was diluted to final concentrations of 1 µM and 10 µM with buffer for analysis. The concentration of the initial solvent in the reaction medium was no more than 1%. The reaction medium contained 7 µg of nuclear protein, 1 µM and 10 µM of the studied compounds, and the Adomet reaction buffer containing S-adenosylmethionine coenzyme. The control reaction medium contained 7 µg of nuclear protein and the Adomet reaction buffer, as well as 25 ng and 50 ng of DNMT enzyme and the Adomet reaction buffer. The reaction was carried out at 37˚C, 120 min in filmed plate strips. Fluorescence was measured on the Tecan Infinite M200 plate reader at Ex/Em = 530/590 nm. The degree of DNA methyltransferases activity inhibition was calculated by the equation:

Inhibition (%) = [1 − ((RFU, inhibitor-containing sample – RFU, blank)/(RFU, non-inhibitor-containing sample - RFU, blank))] × 100%.

where RFUs are relative fluorescence units.

L1210 and P388 cells were placed in a 6-well plate with a density of 3 × 10⁵ per well. The tested compounds were added at concentrations equal to IC₅₀ (prespecified) for 48 hours. Then the cells were washed from the culture medium by centrifugation at 1000 rpm for 5 minutes, rewashed with PBS, and fixed with ice ethanol under permanent shaking and incubated at −20˚C for 24 hours. The samples were afterwards washed from alcohol by centrifugation, rewashed with PBS, and a solution containing 100 µg/ml of RNase A and 50 µg/ml of propidium iodide (PI) was added; incubated for 40 minutes in the dark and measured on the Cytomics FC 500 Beckman Coulter flow cytometer (FL4 channel for PI). The results were analyzed using Kaluza 2.0 software (Beckman Coulter) [37] .

L1210 and P388 cells were placed in a 6-well plate in quantities of 3 × 10⁵ per well. Test compounds were added at concentrations equal to IC₅₀ (prespecified in the MTT assay for L1210 ‒ 2’F-araAC, DAC and AZA at a concentration of 0.03 μM, 0.1 μM and 2.5 μM, respectively; for P388 2’F-araAC, AZA and DAC at a concentration of 0.3 μM, 0.3 μM and 5.0 μM, respectively) for 48 hours. The cells are then washed from the culture medium by centrifugation at 1000 rpm for 5 minutes, re-washed with a phosphate buffer, and the Annexin V-AlexaFluor488 and PI dyes were added from the Dead Cell Apoptosis Kit with Annexin V for Flow Cytometry (Invitrogen, V13245) were added as described in the manufacturer’s instructions; they were incubated for 15 minutes in the dark and measured on the Cytomics FC 500 Beckman Coulter flow cytometer (FL1 for Annexin V-AlexaFluor488 and FL4 for PI) [38] .

The level of DNMTs inhibition was determined in the nuclear extract of the HCT-116 human colorectal cell line because the aberrant gain of DNA methylation at CpG islands is frequently observed in colorectal tumors [39] . The nuclear extract was exposed to varying concentrations of 2’F-araAC, DAC and AZA for 120 minutes. Protein extracts were analyzed according to the DNMT Activity Quantification Kit (Fluorometric) (Abcam, ab113468) protocol. Known DNA methyltransferase inhibitors DAC and AZA were used as comparators.

Figure 2 shows calculated data on the inhibitory activity of AZA, DAC and 2’F-araAC at concentrations of 1 µM and 10 µM with respect to DNMT contained in the HCT-116 cell nuclear extract. The experimental results showed that the DNMT activity was remarkably inhibited by the tested compounds. At 10 μM concentration of 2’F-araAC the level of DNMT activity vs. control was the lowest. At the same time, AZA and DAC inhibited DNMT activity by 75% and 86%, respectively, of the value of the inhibition by 2’-fluorodeoxy-arabino analog at a concentration of 10 µM. It should be added that the ability of the studied compounds to diminish the DNA methylation activity can be achieved at low doses. Azacitidine at a concentration of 1 µM showed a 2-fold weaker DNA methylation activity compared to 2’F-araAC, while for DAC it was 1.7-fold weaker compared to fluoro analog.

We assume that the compound may have metabolism similar to decitabine. In particular, once incorporated into DNA, decitabine is recognized as a substrate by DNMTs, namely DNMT1, but due to the presence of an N5 rather than C5 atom, irreversible formation of a covalent bond between DNA and DNMT occurs. Under low concentrations, this activity depletes DNMTs and results in global DNA hypomethylation while under high concentrations, it additionally results in double-strand DNA breaks and cell death [40] [41] .

Experimental results indirectly allowed us to suggest the binding between the carbon-6 atom of the cytosine ring and the catalytic site of DNMT resulting in enzyme inactivation.

The inhibition of the cell cycle resulting in the delay of the cancer cells proliferation is an underlying mechanism of action of antitumor agents.

As previously shown, 2’F-araAC demonstrated antiproliferative activity against several types of human hemoblastosis cell lines (KG-1, TPH-1, HL-60, K562) [32] . It is interesting that 2’F-araAC was strongly cytotoxic against acute myelogenous leukemia cells at a concentration of 0.1 µM, whereas the threshold limit for AZA was 1.0 µM. The saturation effect was achieved with increased concentration of the tested nucleoside to 1.0 µM. The Aza saturating concentration was much greater (10 µM) compared to 2’F-araAC [32] .

The effect of the novel 2’-fluoro analog on the cell cycle was examined in comparison AZA and DAC. Cell cycle experiments were performed using P388 cells treated with 2’F-araAC, DAC and AZA at concentrations of 0.3 µM, 0.3 µM and 5.0 µM, respectively. L1210 murine lymphoma cells were treated with 2’F-araAC, DAC and AZA at concentrations of 0.03 µM, 0.1 µM and 2.5 µM based on their IC₅₀. The cells were exposed to the compounds for 48 h. Treated cells were harvested, fixed in ethanol, stained with propidium iodide and analyzed for cell cycle distribution by flow cytometry.

As seen from the results ( Table 1 and Table 2 ), the novel 2’-fluoro analog significantly disrupted the proliferation of P388 and L1210 cells.

2’F-araAC significantly changed the cell cycle distribution of cells P388, causing a 4-fold decrease in the fraction of G1 cells compared to control. Whereas, AZA and DAC slightly increased the fraction of G1 cells in concentrations of 5.0 μM and 0.3 μM and concomitantly reduced for all other cell cycle phases, respectively.

Unlike AZA and DAC, 2’F-araAC increased the percentages of G2/M phase almost three times with a concomitant decrease in the S-phase population ( Table 1 , Figure 3(a) ). This experiment suggested that new fluoro analog induces G2/M-phase cell cycle arrest at concentration of 0.3 µM.

The L1210 mouse lymphocytic leukemia cell cycle analysis is shown in Figure 3(b) . The results are summarized in Table 2 . For L1210 cells, lower concentrations of the studied compounds were used to diminish cytotoxic effects of the drugs. Treatment for 48 hours with 2’F-araAC decreased fraction of G1 cells and caused the increase in the percentages of cells in the G2/M phase from 9.57% (DMSO) to 18.72% (0.03 µM). AZA at a concentration of 2.5 µM had no significant effect on the cell cycle. Similar to 2’F-araAC, however, at a lower concentration, DAC (0.1 µM) increased the fractions of G2/M cells to 15.79%. Moreover, DAC prolonged the G1 phase with a concomitant decrease in the S phase ( Table 1 ).

Our results have shown that P388 and L1210 cells treated with 2’F-araAC at a dose of 0.3 and 0.03 µM, respectively, contributed to a G2/M phase arrest suggesting that the inhibition of cell cycle progression could be one of the mechanisms through which fluoro analog might influence cancer cell proliferation. Inhibition of cell proliferation of rapidly growing cancer cells is a compelling argument for the development of an anticancer drug on the basis of 2’F-araAC.

Apoptosis is crucial in the elimination of mutated neoplastic and hyperproliferating neoplastic cells from the system and is therefore considered a protective mechanism impeding cancer progression [42] .

The fact that 2’F-araAC induces P388 and L1210 cell cycle disruption prompted us to examine the effects produced by this compound on cell apoptosis level. In the case of P388 the tested concentrations were 0.3 µM for 2’F-araAC, 5 µM for AZA and 0.3 µM for DAC. L1210 cells were treated with 0.03 µM of 2’F-araAC, 2.5 µM of AZA and 0.1 µM of DAC. The concentrations were chosen on the bases of there IC₅₀. Both cell lines were treated for 48 hours. Apoptotic cells were determined by flow cytometry using Annexin V-FITC/PI double labeling. On the histograms at Figure 4 and Figure 5 , we can see that all investigating compounds in the effective concentrations (IC₅₀) cause early (B4 quadrant) and late apoptosis (B2 quadrant), whereas the amount of necrotic cells is small.

Apoptotic cells percentages were 29.41% for AZA, 42.16% for DAC and 54.53% for 2’F-araAC vs. 2.88% for the control P388 cell culture. Less than 1% of cell population treated with all compounds showed necrotic signs ( Figure 4 ).

Apoptotic cells percentages were 30.21% for AZA, 43.00% for DAC and 43.35% for 2’F-araAC vs. 5.25% for the control L1210 cell culture. Less than 2% of cell population treated with all compounds showed necrotic signs ( Figure 5 ).

The P388 and L1210 cells showed different viability under the influence of 2’F-araAC, which could be species-specific. A greater cell death of P388 cell line caused by 2’F-araAC vs. AZA and DAC was due to a greater percentage of cells undergoing early apoptosis ( Figure 6 ). Early apoptotic cells percentages were 38.7% for 2’F-araAC vs. 15.2% and 25.2% for AZA and DAC, respectively. For L1210 cells fluoro analog and DAC were similar in their effects on apoptosis level. Of note, the small quantity of necrotic cells appeared after the 2’F-araAC treatment of both cell lines is not more than 1.44% suggesting no significant cytotoxic effect of 2’F-AZA on these sublines.

The obtained data suggest common cell death mechanisms of action of 2’F-araAC, AZA and DAC and clearly different effect on the cell cycle. It can be assumed that the novel azacitidine 2’-fluorodeoxy-arabino analog inhibits the proliferation of P388 and L1210 cells through G2/M cell cycle arrest and apoptosis induction.