Molecular Medicine Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran.
Molecular Medicine Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran; Department of Clinical Biochemistry, Faculty of Medicine, Rafsanjan University of Medical Sciences, Rafsanjan, Iran.
Immunol Lett. 2017 Oct;190:7-14. doi: 10.1016/j.imlet.2017.06.012. Epub 2017 Jul 8.
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.
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).
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.
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.
通过鉴定肿瘤干细胞和治疗靶点，利用 OCT-4 和 NANOG 基因，有可能改善治疗效果。在这里，我们合成并研究了新型吲哚-3-甲醛衍生物（NI-3-CD）在抑制白血病细胞系 NB4 中自我更新调控因子和癌症干细胞基因表达方面的潜力。
将 NB4 细胞在含有 NI-3-CD 和 I3F（15.12-1000μg/mL）的 RPMI1640 培养基中培养 24、48 和 72h。用台盼蓝染色技术和 MTT 法评估细胞增殖抑制率。通过 Annexin V/PI 凋亡检测试剂盒的流式细胞术分析确定凋亡细胞的百分比。用实时 PCR 技术测定 NANOG/OCT4 表达相对于 β-肌动蛋白的倍数变化。还应用 Western blot 分析评估 NANOG/OCT4 在蛋白水平的表达。数据采用学生 t 检验和重复测量检验进行分析。如果（P<0.01）则认为差异具有统计学意义。
与 I3C 相比，NI-3-CD 在不同浓度下作用 24、48 和 72h 时，细胞活力有显著差异。此外，NI-3-CD 的抗癌活性明显高于 I3C（新型 I3C 在 24、48 和 72h 的 IC50 值分别为 225.77、123.13 和 63.72M，而 I3C 的 IC50 值分别为 728.05、407.82 和 277.92M）。流式细胞术结果显示 NB4 凋亡细胞明显增加。实时 PCR 分析表明，与未处理对照细胞和 I3C 处理细胞相比，NANOG/OCT4 的表达下调（P<0.05）。与 RT-PCR 一致，Western blot 分析显示，NI-3-CD 处理细胞中的 OCT4 表达也明显低于未处理对照细胞和 I3C 处理细胞群体。
我们的结果表明，NI-3-CD 治疗可降低 NB4 细胞的球体形成能力。综上所述，本研究提供了有关 NB4 细胞中存在干细胞基因表达的有价值信息。
