Alteration in miRNA gene expression pattern in acute promyelocytic leukemia cell induced by arsenic trioxide: a possible mechanism to explain arsenic multi-target action

Abstract

MicroRNAs (miRNAs) are involved in cancer pathogenesis, apoptosis, and cell growth, and these miRNAs are thought to be functional as oncogenes and/or tumor suppressors in the gene regulatory networks. We studied the potential contribution of miRNAs in acute promyelocytic leukemia (APL) cell NB4 during the apoptosis induction by arsenic trioxide (ATO). The apoptotic effects of ATO on the NB4 cell line at a pharmacological dose (2 μM) was verified using cell growth and viability assays, MTT assay, BrdU cell proliferation assay, flow cytometric analysis, and caspase-3 activity assay. miRNAs from untreated and 2 μM ATO-treated NB4 cell line were extracted, purified, and converted to complementary DNAs. Differential expressions of 88 cancer-related miRNAs were analyzed by real-time reverse transcription PCR using miRNA PCR cancer-array system. After normalizing to the average Ct value of three housekeeping genes in the array (U6, SNORD47, and SNORD48), the fold change of miRNAs was calculated in the ATO-treated cells as compared to untreated. Among the 88 cancer-focused miRNAs, 51 miRNAs were found to be differentially expressed more than 2-fold after ATO treatment. Of these, 48 miRNAs were upregulated up to 21.65-fold changes, while three miRNAs were downregulated up to 5.19-fold changes. By screening the literature, a majority of these upregulated miRNAs were found to have tumor and/or metastatic suppressors' functions associated with cell cycle arrest and apoptosis, as well as inhibition of angiogenesis, invasion, and metastasis. Our results demonstrate that ATO, at the relevant concentration, modulate a substantial number of cancer-related miRNAs in APL cell line; most of these are known to function as a tumor and/or metastatic suppressors and have confirmed targets involved in cell cycle arrest and apoptosis. The results of this study support the hypothesis that miRNAs may play a mediatory role in eliciting the multi-target and pleiotropic action of ATO.

Arsenic-Associated Changes to the Epigenome: What Are the Functional Consequences?

“…Levels of selected miRNAs also analyzed using real-time qPCR.Malignant transformation associated with reduction of miR-200 family members; downregulation of miR-200b shown to be essential for malignant transformation.Wang et al, 2011 [76] In vivo exposure to arsenite (chick embryos); in vitro exposure to arsenite (human umbilical cord vein cell line)100 nM arsenite (NaAsO 2 ) injected in yolk sac of chick embryos at HH stages 6, 9, and 12; embryos harvested at HH stage 18. EA.hy926 cells exposed to 100 nM arsenite (NaAsO 2 ) for 72 hWhite Leghorn chick (Bovan strain) whole embryos; human umbilical cord vein cell line EA.hy926Total RNA analyzed for miRNA levels using several methods including miRNA PCR arrays and real-time qPCR and Northern blots of selected targets.miR-9 and miR-181b implicated as potential mediators of the toxic developmental effects of arsenic, specifically abnormal angiogenesis.Cui et al, 2012 [75] In vitro exposure to ATO (APL cell line)Therapeutic dose of 2 μM ATO (As 2 O 3 ) for 24 hhuman APL cell line NB4Total RNA analyzed for levels of 88 cancer-associated human miRNAs using a miRNA PCR array.Exposure modulated 88 cancer-associated miRNAs, including those predicted to bind mRNAs of genes involved in cell cycle and apoptosis.Ghaffari et al, 2012 [72] In vitro exposure to arsenite (immortalized human embryonic lung fibroblasts)1.0 μM arsenite (NaAsO 2 ) for ~15 weeks, resulting in acquisition of malignant characteristicsImmortalized human embryonic lung fibroblast cell line HELFTotal RNA analyzed for miR-21 levels using real-time qPCR.Identified ROS-sensitive pathways as inducers of miR-21. MiR-21 upregulation implicated as important event in the acquisition of malignant characteristics.Ling et al, 2012 [78•]

APL: acute myelocytic leukemia

ATO: arsenic trioxide

HH: Hamburger-Hamilton

qPCR: quantitative polymerase chain reaction

ROS: reactive oxygen species

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“…Some of these studies have focused on miRNAs as mediators of the apoptotic effects of arsenic trioxide (ATO) [72, 73], a chemotherapeutic agent used in the treatment of relapsed or refractory acute promyelocytic leukemia [74]. Other studies have indicated miRNAs may play roles in the diverse toxic effects of iAs.…”

Deficiency of SUMO-specific protease 1 induces arsenic trioxide-mediated apoptosis by regulating XBP1 activity in human acute promyelocytic leukemia

“…As 2 O 3 has been successfully used in the treatment of APL (7,35,36), and therapeutic doses of As 2 O 3 could effectively induce complete molecular remission in vivo and trigger the apoptotic death of APL cells (37). Thus, we sought to investigate if the knockdown of SENP1 affects As 2 O 3 -induced apoptosis in NB4 cells.…”

Arsenic Trioxide and Resveratrol Show Synergistic Anti-Leukemia Activity and Neutralized Cardiotoxicity

“…2009104). NRLVMs were pretreated with Rev (5 µM) or Gen (50 µM) for 1 h and then co-incubated with As 2 O 3 (5 µM) [22] for another 24 h. The procedure was the same for NB4 cells, except that a concentration of 2 µM As 2 O 3 was used [23].…”