Evaluating of OCT-4 and NANOG was differentially regulated by a new derivative indole in leukemia cell line
Evaluating of OCT-4 and NANOG was differentially regulated by a new derivative indole in leukemia cell line
ozra sadat esmaeili,1Mojgan Noroozi Karimabad,2Fariba Rahmani,3Ali Darehkordi,4Gholamhossein Hassanshahi,5,*
1. Department of Clinical Biochemistry, Faculty of Medicine, Rafsanjan University of Medical Sciences, Rafsanjan, Iran 2. Molecular Medicine Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran 3. Department of Chemistry, Faculty of Science, Vali-e-Asr University of Rafsanjan, Rafsanjan, Iran 4. Department of Chemistry, Faculty of Science, Vali-e-Asr University of Rafsanjan, Rafsanjan, Iran 5. Molecular Medicine Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran
Introduction: The potential exists to improve treatment through characterization of tumor stem cells and identification of therapeutic targets Using OCT-4 and NANOG genes. Here we have synthesized and investigated the potential of; New Indole-3-carbaldehyde derivative (NI-3-CD) in inhibiting the expression of self-renewal regulatory factors and cancer stem cell gene in a leukemia cell line NB4.
Methods: The NB4 cells were cultured in RPMI1640 medium contained NI-3-CD and I3F (15.12–1000 μg/
mL) for 24, 48 and 72 h. Inhibition of cell proliferation was assessed by trypan blue staining technique and MTT
assay. The percentage of apoptotic cells was determined by flow cytometry analysis using Annexin V/PI
apoptosis detection kit. The fold changes of NANOG/OCT4 expression against β-actin were determined by realtime-PCR technique. Western blotting analysis was also applied for evaluating the expression of NANOG/OCT4
at protein level. Data were analyzed by student t and repeated measure tests. Differences were considered
significant if (P < 0.01).
Results: There was a significant difference in cell viability, when various concentrations of NI-3- were used for 24, 48 and 72 h in comparison to I3C regarding the cellular viability. Furthermore, the NI-3-CD, had markedly elevated anticancer activity than I3C (IC50 values for novel I3C in 24, 48 and 72 h were 225.77, 123.13 and 63.72 M respectively while for I3C were 728.05, 407.82 and 277.92 M respectively). Flow cytometry results exhibited an obviously significant augmentation in apoptotic NB4 cells. Real Time- PCR analysis indicated that the expression of NANOG/OCT4 was down regulated in compare to untreated control cells and I3C treated cells (P < 0.05). In concert with RT-PCR, western blot analysis showed that the OCT4 expression in NI-3-CD treated cells was also significantly decreased in compare to both untreated control cells and I3C treated cellular populations.
Conclusion: Our results imply that NI-3-CD treatment decreases the sphere-forming ability of NB4 cells. In summary, this study provides valuable information on the presence of stem-cell genes expression in NB4 cells.