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Evaluating of OCT-4 and NANOG was differentially regulated by a new derivative indole in leukemia cell line
Mojgan Noroozi Karimabad1, Mehdi Mahmoodi2, Abdollah Jafarzadeh3
1Molecular Medicine Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran.
Background:
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.
Methodology:
The NB4 cells were cultured in RPMI1640 medium contained NI-3-CD and I3F (15.12-1000μg/mL) for 24, 48 and 72h. 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 real-time-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 72h 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 72h were 225.77, 123.13 and 63.72M respectively while for I3C were 728.05, 407.82 and 277.92M 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.
Insights
A novel indole derivative, NI-3-CD, effectively inhibits cancer stem cell genes NANOG and OCT-4 in NB4 leukemia cells. This compound demonstrates significant anticancer activity and induces apoptosis, suggesting therapeutic potential.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Cancer stem cells (CSCs) play a crucial role in leukemia development and treatment resistance.
- Identifying therapeutic targets that inhibit CSC self-renewal is essential for improving leukemia treatment.
- OCT-4 and NANOG are key genes regulating self-renewal in cancer stem cells.
Purpose of the Study:
- To synthesize and evaluate a new Indole-3-carbaldehyde derivative (NI-3-CD) for its potential to inhibit cancer stem cell gene expression.
- To investigate the effect of NI-3-CD on the self-renewal regulatory factors NANOG and OCT-4 in the NB4 leukemia cell line.
Main Methods:
- NB4 leukemia cells were treated with varying concentrations of NI-3-CD and I3C for 24, 48, and 72 hours.
- Cell viability was assessed using trypan blue staining and MTT assays.
- Apoptosis was quantified by flow cytometry.
- Gene and protein expression levels of NANOG and OCT-4 were determined using real-time PCR and Western blotting, respectively.
Main Results:
- NI-3-CD significantly inhibited NB4 cell viability and demonstrated higher anticancer activity compared to I3C.
- Flow cytometry revealed a significant increase in apoptotic NB4 cells following NI-3-CD treatment.
- Real-time PCR and Western blot analyses confirmed significant downregulation of NANOG and OCT-4 expression in NI-3-CD treated cells.
Conclusions:
- NI-3-CD treatment effectively reduces the expression of stem cell genes NANOG and OCT-4 in NB4 leukemia cells.
- The study suggests that NI-3-CD possesses anticancer properties and may decrease the sphere-forming ability of NB4 cells.
- NI-3-CD represents a promising therapeutic agent for targeting cancer stem cells in leukemia.
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