Zebularine significantly sensitises MEC1 cells to external irradiation and radiopharmaceutical therapy when administered sequentially in vitro

Abstract

Zebularine is a cytidine analogue incorporated into DNA during replication, inhibiting DNA methyltransferase 1 (DNMT1), resulting in demethylation and changes in gene expression. Such modification may improve radiosensitivity in resistant lymphoma cells. The hypothesis of this study was that zebularine and radiation would synergistically inhibit cell growth and viability. Human MEC1 malignant B cells were incubated with 0-200 µM zebularine for 48 h. Media containing zebularine was removed, and the cells were irradiated with 0-2 Gy of either external beam irradiation or (177) Lu-DOTA-TATE, a radiolabelled somatostatin analogue. Concentration and viability were measured over 48-72 h. The proportion of apoptotic cells was identified using an active Caspase 3/7 assay. Zebularine inhibited growth of cells in a dose-dependent manner during exposure. No residual growth inhibition occurred following removal of the drug. Zebularine and external irradiation inhibited cell proliferation in a dose-dependent, synergistic interaction, but the effect on viability was additive. Treatment with zebularine and (177) Lu-DOTA-TATE resulted in less inhibition of proliferation (P = 0.0135), but a synergistic decrease in viability. Apoptotic fraction was much higher in cells irradiated with (177) Lu-DOTA-TATE than external irradiation. External irradiation induces growth arrest rather than apoptosis. Apoptosis is the primary effect of radiopharmaceutical therapy on tumour cells. Treatment with the methylation inhibitor, zebularine, appears to synergistically augment these natural effects in vitro, which could be exploited clinically.

Keywords: demethylation; lymphoma; radiopharmaceuticals; radiosensitivity; somatostatin analogue.

Figure 1

Proportion of MEC1 growth is inhibited by zebularine in a dose-dependent and linear manner (P<0.001, r=0.904, r²=0.819). No change in cell viability was observed over the concentrations. IC₂₀=75μM. IC₅₀=195μM.

Figure 2

Western blot of DNMT1 protein (~240 kd) with GAPDH protein as a loading control. The lane on the left is the control lane. DNMT 1 was significantly reduced by treatment with zebularine at all concentrations from 50 μM to 200 μM.

Figure 3

Cell proliferation after external irradiation (in 10⁴ cells/mL over time). Proliferation over a three day period was decreased significantly by increasing dose of radiation (P=0.0026).

Figure 4

Cell viability measured by WST-1 assay of MEC1 cells relative to controls following treatment with 2 Gy external irradiation, 200 μM zebularine, or the combination of the two. Zebularine and the combination of zebularine and external irradiation decreased cell viability compared to control (P<0.0001), but in an additive, not synergistic, manner.

Figure 5

Apoptotic proportion scaled relative to cell population and control apoptosis. Cells were pre-treated with zebularine at 100 μM and 200 μM (Zeb 100 or Zeb 200) and ¹⁷⁷Lu-DOTA-TATE (Lu-177) or external beam radiation (EBR). Relative apoptotic proportion was increased from baseline for combined zebularine and both forms of radiation (P<0.001). At high doses of zebularine and radiation, relative apoptotic proportion was much greater for the radiopharmaceutical-treated cells (P=0.005).

Figure 6

Cell proliferation after 2Gy radiation presented by zebularine dose including cells exposed to zebularine without radiation (in 10⁴ cells/mL over time). Note the progressive, dose-dependent decrease in proliferation observed at increasing concentrations of zebularine treatment up to approximately 50% for the cells treated with 200μM zebularine. Contrast this with the lack of zebularine dose-effect in cells without radiation exposure.

Figure 7

Cell proliferation of MEC1 cells at increasing doses of zebularine treated with 2Gy radiation by ¹⁷⁷Lu-DOTA-TATE (in 10⁴ cells/mL over time). In the analysis, the effect of day interacted with dose, suggesting less direct impact of the radiopharmaceutical on cell proliferation than was observed following irradiation by the external source.

Figure 8

Cell viability over time for MEC1 cells treated with 2Gy radiation from ¹⁷⁷Lu-DOTA-TATE or external beam radiation at a 200μM dose of zebularine (in 10⁴ cells/mL over time). The maximal decrease using ¹⁷⁷Lu-DOTA-TATE was over 16% relative to controls, compared to only 6% decrease in cells receiving external beam irradiation, a >2-fold difference (P = 0.005).