The Contrasting Delayed Effects of Transient Exposure of Colorectal Cancer Cells to Decitabine or Azacitidine

Abstract

(1) Background: Decitabine and azacitidine are cytosine analogues representing the class of drugs interfering with DNA methylation. Due to their molecular homology and similar clinical application, both drugs are often regarded as interchangeable. Despite their unique mechanism of action the studies designed for observation and comparison of the prolonged activity of these drugs are rare. (2) Methods: The short-time (20-72 h) and long-term (up to 20 days) anti-cancer activity of decitabine and azacitidine has been studied in colorectal cancer cells. We observe the impact on cell culture's viability, clonogenicity, proliferation, and expression of CDKN1A, CCND1, MDM2, MYC, CDKN2A, GLB1 genes, and activity of SA-β-galactosidase. (3) Results: Decitabine has much stronger anti-clonogenic activity than azacitidine. We show that azacitidine, despite significant immediate toxicity, has negligible long-term effects. Contrary, decitabine, which does not exert initial toxicity, profoundly worsened the condition of the cells over time. On the 13th day after treatment, the viability of cells was decreased and proliferation inhibited. These functional changes were accompanied by up-regulation of expression CDKN1A, CCND1, and CDKN2A genes and increased activation of SA-β-galactosidase, indicating cellular senescence. (4) Conclusions: Our head-to-head comparison revealed profound differences in the activities of decitabine and azacitidine important in their anti-cancer potential and clinical application. The effects of decitabine need relatively long time to develop. This property is crucial for proper design of studies and therapy concerning decitabine and undermines opinion about the similar therapeutic mechanism and interchangeability of these drugs.

Keywords: azacitidine; cellular senescence; chemotherapy; colon cancer; decitabine; epigenetic drug; vidaza.

Figure A1

Detailed scheme of treatments for colony formation assay.

Figure A2

Detailed scheme of treatments for 3 day long MTS assay.

Figure A3

Detailed scheme of treatments for western blotting.

Figure A4

Detailed scheme of treatments for 13 day long MTS assay.

Figure A5

Detailed scheme of treatments for microscopic staining.

Figure A6

Detailed scheme of treatments for proliferation assay.

Figure A7

Detailed scheme of treatments for (A) gene expression assay, (B) 20 day long western blotting and (C) assesment of activity of SA-$\beta$-galactosidase.

Figure A8

Clonogenic assay of HT-29 cells.

Figure A9

Clonogenic assay of RKO cells.

Figure A10

Whole western blots of CHOP assayed on DLD-1 cells (Figure 3), along with densitometric quantification.

Figure A11

Whole western blots of CHOP assayed on HT-29 cells (Figure 3), along with densitometric quantification.

Figure A12

Whole western blots of p21 assayed on DLD-1 cells (Figure 7B), along with densitometric quantification.

Figure 1

Decitabine (DAC), but not azacitidine (AZA), inhibits the growth of DLD-1 colorectal cancer cells in a clonogenic assay. (A) Average ± standard deviation of colonies number are shown on the graph; ns—not significant, p > 0.05; * p < 0.05, ** p < 0.01, *** p < 0.001 in t-Welch test. (B) representative photographs of plates with the colonies.

Figure 2

Azacitidine (AZA), but not decitabine (DAC), impairs the viability of several colorectal cancer cells in 3 days long MTS assay. Average viabilities and standard deviations of viabilities are shown on the graph. Cells treated with 0.01% of DMSO (solvent) were set as a 100% control. The dashed line represents 50% viability.

Figure 3

Azacitidine (AZA), but not decitabine (DAC), induces expression of CHOP, an ER stress marker on DLD-1 and HT-29 cells. Analysis was performed 20 h after treatment with specified drug. Representative blots are shown. The uncropped blots are presented on Figure A10 and Figure A11.

Figure 4

Comparison of viabilities of colorectal cancer cells on 3rd and 13th-day post-treatment with decitabine (DAC) or azacitidine (AZA). Average viability ± standard deviation are shown on the graph, ns: non-significant, p > 0.05; * p < 0.05; ** p < 0.01.

Figure 5

Inhibition of proliferation by decitabine (DAC) or azacitidine (AZA) on the 13th day after treatment. Average percentage of cells with inhibited proliferation ± standard deviation are shown on the graph; ns: non-significant, p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001 (t-Welch test). Below, representative histograms are shown. Empty histogram—negative control, unstained cells; gray histogram—cells stained on day of the measurement, positive control; black histogram—untreated control, green histogram—cells treated with decitabine (DAC), orange histogram cells treated with azacitidine (AZA).

Figure 6

Expression of CDKN1A, CCND1, MDM2, MYC, GLB1 genes in DLD-1 or HT-29 cells upon treatment with decitabine (DAC) or azacitidine (AZA). Real-time RT-PCR analysis. Untreated cells served as a control. Average RQ ± standard deviation are shown on the graphs; ns: non-significant, p > 0.05, * p < 0.05, ** p < 0.01, (t-Welch test). Range of expression changes by less than half of the PCR cycle was marked as gray and regarded as insignificant.

Figure 7

Expression of cell cycle inhibitors CDKN2A gene and p21 in DLD-1 cells upon treatment with decitabine (DAC) or azacitidine (AZA). (A) Real-time RT-PCR analysis. Untreated cells served as a control. Average RQ ± standard deviation are shown on the graphs; ns: non-significant, p > 0.05, * p < 0.05, (t-Welch test). Range of expression changes by less than half of the PCR cycle was marked as gray and regarded as insignificant. (B) Western blot analysis of p21 expression on the 20th day after exposure to DAC or AZA. Representative blots are shown. The uncropped blots are presented on Figure A12.

Figure 8

Changes of the morphology of DLD-1 cells on the 13th day after treatment with decitabine (DAC) or azacitidine (AZA). CB indicates cells with large bodies, and arrows indicate large intracellular vehicles. Representative microphotographs are shown.

Figure 9

Cytohistochemistry based assay of SA-$\beta$-galactosidase in HT-29 cells on 13th day after treatment with decitabine (DAC) or azacitidine (AZA). Increase in green color indicates activity of SA-b-galactosidase. Representative microphotographs are shown.

Figure 10

Activity of SA-$\beta$-galactosidase after treatment with decitabine (DAC) or azacitidine (AZA). (A) Time course of activity of SA-$\beta$-galactosidase in HT-29 cells. (B) Activity of SA-$\beta$-galactosidase in DLD-1 and HCT116 cells on the 13th day after exposure to DAC or AZA. Averages ± standard deviation are shown on the graph. ns: non-significant, p > 0.05, * p < 0.05 (t-Student test).