Effect of 5-Aza-2'-deoxycytidine on T-cell acute lymphoblastic leukemia cell biological behaviors and PTEN expression

Abstract

Objective: We currently face a sharp increase of T-cell acute lymphoblastic leukemia (T-ALL) incidence and a challenge of unmasking its complex etiology. The deoxycytidine analog 5-Aza-2'-deoxycytidine (5-Aza-dC) is currently the most common nucleoside methyltransferase inhibitor. The objective of this study was to clarify the role of 5-Aza-dC in T-ALL cell biological behaviors and phosphatase and tensin homolog deleted on chromosome ten (PTEN) expression.

Material and methods: T-ALL cell lines were divided into the experimental group with 5-Aza-dC solution treatment, and the control group without treatment. PTEN methylation was detected using methylation-specific polymerase chain reaction (MS-PCR). Following the measurement of cell proliferation, viability, apoptosis, invasion, migration, etc., quantitative reverse transcription-polymerase chain reaction (PCR) was conducted to detect PTEN, DNA methyl-transferases (DNMT1), DNMT3a, MBD2, and MeCP2 expressions; Western blot to detect PTEN, PI3K, AKT, and mTOR protein expressions. In addition, rescue experiments to inhibit and restore the expression of PTEN in different groups were performed for further identification of the results in the former parts.

Results: MS-PCR results showed that in Jurkat cells, the target band was amplified using methylated primers for the PTEN gene promoter region; moreover, at 10 μmol/L of 5-Aza-dC for 24 h, PTEN methylation was completely removed without any un-methylated band observed. The experimental group had significantly lower cell proliferation and viability rates, higher apoptosis rates, decreased cell proportion in S phase, reduced invasion and migration; increased PTEN expression, decreased DNMT1, DNMT3a, MBD2, and MeCP2 mRNA expressions; and decreased PI3K, AKT, and mTOR protein expressions than those in the control group (all P < 0.05). Furthermore, according to the rescue experiment, silenced PTEN expression weakened the beneficial roles of 5-Aza-dC treatment, and resulted in significantly higher cell proliferation and viability rates, lower apoptosis rates, increased cell proportion in S phase, increased cell invasion and migration; decreased PTEN expression, elevated DNMT1, DNMT3a, MBD2, and MeCP2 mRNA expressions, and higher PI3K, AKT, and mTOR protein expressions (all P < 0.05). While restored PTEN expression enhanced functions of 5-Aza-dC treatment, leading to obviously lower cell proliferation and viability rates, higher apoptosis rates, increased cell proportion in G1 phase, and reduced cell invasion and migration; as well as increased PTEN expression, decreased DNMT1, DNMT3a, MBD2, and MeCP2 mRNA expressions, and lower PI3K, AKT, and mTOR protein expressions (all P < 0.05).

Conclusion: Demethylation treatment with 5-Aza-dC can inhibit T-ALL cell malignant biological behaviors and enhance the sensitivity to chemotherapy agents possibly, which may be related to the inhibited expressions of DNMT1, DNMT3a, MBD2, and MeCP2, and restored expression activity of PTEN to negatively regulate the PI3K/AKT signal transduction. Our silencing and restoration of PTEN expressions further support our findings, highlighting that demethylation with 5-Aza-dC to restore the anti-tumor activity of the tumor suppressor gene PTEN may be a promising therapeutic option for treating T-ALL.

Keywords: 5-Aza-2’-deoxycytidine; Demethylation; PTEN; T-cell acute lymphoblastic leukemia; Tumor cell biological behaviors.