Application of decoy oligodeoxynucleotides strategy for inhibition of cell growth and reduction of metastatic properties in nonresistant and erlotinib-resistant SW480 cell line.

Signal transducer and activator of transcription 3 (STAT3) is a critical regulator for angiogenesis, cell cycle progression, apoptosis, and drug resistance. Resistance toward EGF receptor (EGFR) inhibitors is a significant clinical concern for metastatic colon cancer patients. The present study aimed to evaluate the blocking influences of STAT3 decoy oligodeoxynucleotides (ODNs) on the STAT3 survival signaling pathway in nonresistant and erlotinib-resistant SW480 colon cancer cells. First, STAT3 decoy and scramble ODNs were designed according to STAT3 elements in the promoter region of MYCT1 gene and tested for the interaction of STAT3 protein with designed ODNs via in silico molecular docking study. Then, the efficiency of transfection and subcellular localization of ODNs were assessed using flow cytometry and fluorescence microscopy, respectively. Cell viability, cell cycle, and apoptosis tests, scratch and colony formation assays, and real-time PCR were also used to study the cancerous properties of cells. A considerable decrease in proliferation of colon cancer cells was observed with blockade of STAT3 signaling due to cell cycle arrest and induced apoptosis via downregulation of cyclin D1 and Bcl-XL, respectively. Furthermore, upon transfecting STAT3 decoy ODNs, colony formation potential and migration activity in both SW480 colon cancer cell lines were decreased compared to the control groups. From this study, it could be concluded that STAT3 is critical for cell growth inhibition and metastatic properties reduction of resistant SW480 colon cancer cells; therefore, STAT3 decoy ODNs could be considered as potential therapeutics along with current remedies for treating drug-resistant colon cancer.

Role of Oct4-Sox2 complex decoy oligodeoxynucleotides strategy on reverse epithelial to mesenchymal transition (EMT) induction in HT29-ShE encompassing enriched cancer stem-like cells.

Cancer stem cells are commonly tolerant toward chemotherapy and radiotherapy. Oct4 and Sox2 transcription factors are shown to be overexpressed in various cancers. At the current research, inhibition of Oct4 and Sox2 transcription factors was performed through application of decoy oligodeoxynucleotides (ODNs) strategy via repressing stemness properties in HT29-ShE cells encompassing enriched cancer stem-like cells. Designed Oct4-Sox2 complex decoy ODNs were transfected into HT29-ShE cells with Lipofectamine reagent. At the next step, ODNs efficiency transfection and subcellular localization were determined via flow cytometry and fluorescence microscopy, respectively. Further investigations such as cell proliferation and apoptosis analysis, colonosphere formation, invasion and migration, and real-time PCR assays were also carried out. Obtained results shed light on the fact that the designed complex decoys were effectively transfected into HT29-ShE cells, and they were found to be localized in subcellular compartments. Oct4-Sox2 decoy ODNs led to decreased cell viability, arresting the cell cycle in G0/G1 phases, increasing apoptosis, inhibition of migration/invasion and colonosphere formation ability of HT29-ShE cells in comparison with control and scramble groups. Furthermore, Oct4-Sox2 complex decoy could modulate the MET process via alteration of mRNA expression of downstream genes. It could be concluded that application of Oct4-Sox2 transcription factor decoy strategy in cells with stemness potential could lead to inhibiting the cell growth and triggering differentiation. Therefore, this technique could be applied along with usual remedies (chemotherapy and radiotherapy) as high potential method for treating cancer.

Investigation of specific binding of designed oligodeoxynucleotide decoys to transcription factors in HT29 cell line undergoing epithelial-mesenchymal transition (EMT).

Expression of master transcriptional regulators of stem cells (Oct4 and Sox2) is associated with mediating tumor proliferation and tumor differentiation. The main goal of this study is the investigation of specific binding of designed Oct4-Sox2 transcription factors decoy oligodeoxynucleotides (ODNs) sequence to their nucleus-extracted proteins in HT29-ShE cells containing enriched cancer stem-like cells (SCLCs). First, gene expression of Oct4, Sox2, and E-cadherin revealed the overexpression of Oct4 and Sox2 and downregulation of E-cadherin in HT29-ShE cells compared with HT29 wild-type and HT29-ShC cells. Next, Oct4-Sox2 complex decoy ODNs were designed according to their elements in the promoter region of Sox2 gene. Then, the interactions of Oct4 and Sox2 proteins to designed ODNs were evaluated in silico. Finally, DNA-protein interactions of decoy ODNs and their corresponding proteins were examined by electrophoretic mobility shift assay (EMSA). Analysis of gel shift retardation assay admitted the specific binding of designed ODNs sequence to the nuclear extracted Oct4 and Sox2 proteins. The results will be a promising approach to target cancer stem cells for potential use in differentiation therapy before chemotherapy and radiotherapy of cancers.

Inhibition of transcription factor T-cell factor 3 (TCF3) using the oligodeoxynucleotide strategy increases embryonic stem cell stemness: possible application in regenerative medicine.

The transcription factor T-cell factor 3 (TCF3), one component of the Wnt pathway, is known as a cell-intrinsic inhibitor of many pluripotency genes in embryonic stem cells (ESCs) that influences the balance between pluripotency and differentiation. In this study, the effects of inhibition of TCF3 transcription factor on the stemness of mouse ESCs (mESCs) were investigated using the decoy oligodeoxynucleotides (ODNs) strategy. The TCF3 decoy and its scramble ODNs were designed and synthesized. The interaction specificity of the TCF3 decoy with the TCF3 transcription factor was evaluated by the electrophoretic mobility shift assay. Subcellular localization was carried out using fluorescence and confocal microscopy. Self-renewal and pluripotency of mESCs were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), cell cycle and apoptosis, alkaline phosphatase (ALP), embryoid body (EB) formation, and real-time assays. All experiments were performed in triplicate. The results showed that knockdown of TCF3 by decoy ODNs transfection in mESCs led to an increase in the cell proliferation, ALP enzyme activity, and master regulatory stemness genes and a decrease in the number and diameter of EBs. These results supported TCF3 as a potential target to maintain the pluripotency and self-renewal capacity of mESCs. Knockdown of the TCF3 transcription factor using decoy ODNs can be a promising method to maintain the stemness of stem cells in regenerative medicine and cell therapy researches.