2′-Deoxy-5-Azacytidine Enhances Cytotoxic Effect Of Other Anti-Leukemic Agents Synergistically In Acute Lymphoblastic Leukemia Cell Line

Advances in chemotherapy have improved the outcome of childhood acute lymphoblastic leukemia (ALL). However, leukemia cells in refractory ALL are often resistant to anti-leukemic agents. Although recent studies have focused on the epigenetic changes in refractory leukemia, the relationship between the demethylating agent 2′-deoxy-5-azacytidine (decitabine, DAC) and ALL remains unclear. Here, we examine the combined effects of DAC and anti-leukemic agents such as clofarabine (CLO) and etoposide (ETO) on the ALL cell line CCRF-CEM.

In vitro drug sensitivity was measured using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. We cultured CCRF-CEM cells for 72 hours with or without DAC, and then removed DAC (when present) prior to culturing CCRF-CEM cells for 48 hours with ETO or CLO, or without chemotherapeutic drugs. After culturing for 48 hours, we removed the chemotherapeutic drugs and measured in vitro drug sensitivity using MTT assay. The MTT assay was performed in triplicate. We then evaluated the inhibitory concentration at 50% (IC50). IC50 for ETO, ETO+DAC, CLO, and CLO+DAC was 3.36, 0.625, 4.96, and 1.92, respectively.

The combination Index (CI) was produced with Calcusyn® software, which uses the methodology of Chou and Talalay to perform formal synergy analyses. A CI < 1 indicated a synergistic effect. The CI was 0.026 for ETO+DAC and 0.431 for CLO+DAC.

We assayed with Annexin-V, PI staining, and caspase-3/7 to detect apoptosis. We observed apoptosis rates of 31.6%, 53.3%, 31.2%, and 52.6% for ETO, ETO+DAC, CLO, and CLO+DAC, respectively. We observed greater caspase-3/7 activity with DAC+CLO and DAC+ETO than with CLO and ETO.

Using real-time reverse transcription polymerase chain reaction (RT-qPCR) in CCRF-CEM cells, we examined mRNA expression levels for the pro-apoptotic genes BAK, BID, BAX, BAD, BIM, PUMA, ATM, TP53, and NOXA, as well as those for the anti-apoptotic genes BCL2, BCL2L1, and XIAP. The expression level of each target gene was calculated by normalizing it to the housekeeping gene GAPDH. The RT-qPCR was performed in triplicate. We used Student’s t test to compare the data. We observed DAC increased mRNA expression levels of BAX and NOXA, but decreased those for BAK, BID, PUMA, BCL2L1, ATM, TP53, and XIAP.

We then analyzed the methylation status of pro- and anti-apoptotic genes after 48 hours incubation with or without DAC. Methylation status of BAK, NOXA, BCL2L1 and XIAP incubation with DAC was 1.3%, 3.3%, 2.5% and 72.9%, respectively. Methylation status of BAK, NOXA, BCL2L1 and XIAP incubation without DAC was 1.9%, 3.6%, 0.7% and 92.3%, respectively. There was no significant difference.

Our results showed that DAC synergistically enhances CLO and ETO cytotoxicity, and this cytotoxic effect depends on caspase-3/7 activity. We examined mRNA expression levels of pro- and anti-apoptotic genes. We hypothesized that DAC would increase mRNA expression levels of most pro-apoptotic genes, and decrease mRNA levels of most anti-apoptotic genes. We found that DAC decreased some pro-apoptotic genes, such as BAK, BID, PUMA, ATM, and TP53, which disproves our hypothesis. Our present findings are similar to those of Shin et al., who reported that DAC decreased BID mRNA expression levels. However, they provided no explanation for this activity. Our results show that DAC did not demethylate the CpG of BAK, NOXA, BCL2L1, or XIAP. Thus, DAC must demethylate the CpG of other genes. Nevertheless, many genes are involved in apoptosis, and it remains unclear which genes are demethylated by DAC.

Sakaguchi:Yakult Honsha Company: Research Funding; Japan Leukemia Research Fund: Research Funding; Japan Society for the Promotion of Science: Research Funding; Sanofi: Research Funding; Teijin Pharma: Research Funding.