Suppressing the metastatic properties of the breast cancer cells using STAT3 decoy oligodeoxynucleotides: A promising approach for eradication of cancer cells by differentiation therapy

Abstract

Due to the presence of cancer stem cells (CSCs), breast cancer often relapsed after conventional therapies. Strategies that induce differentiation of CSCs will be helpful in eradication of tumor cells, so we designed an oligodeoxynucleotide (ODNs) for targeting of signal transducer and activator of transcription 3 (STAT3) transcription factor which is involved in stemness, and constitutively activated in triple‐negative breast cancer. Molecular docking and electrophoretic mobility shift assay analysis showed that decoy ODN bound specifically to the DNA binding site of STAT3 protein. The prevalent uptake of Cy3‐labeled ODNs is in the cytoplasm and the nucleus of MDA‐MB‐231 treated cells. STAT3 decoy ODNs treatment showed cell growth inhibition by decreasing cell viability (17%), increasing the percentage of arrested cells in G0/G1 phases (18%), and triggering apoptosis (29%). Migration and invasion potential decreased from 10.77 to 6.76 µm/hr, by wound closure rate, and migrated/invaded percentage by 26.4% and 15.4% in the transwell assays, respectively. CD44 protein expression level on the cell surface also decreased, while CD24 increased. Mammosphere formation efficiency reduced in terms of tumorsphere size by 47%, while the required time increased. Cells morphology was changed, and lipid droplets were accumulated in the cytoplasm compared to the control and scrambled groups, in all assays (repeated triplicate). Furthermore, the gene expression of all downstream targets significantly decreased owing to suppressing the STAT3 transcription factor. Overall, the results confirmed the antitumor effects of STAT3 decoy in MDA‐MB‐231 cells. Thus, it seems that STAT3 decoy ODNs might be considered as an auxiliary tool for breast cancer eradicating by the differentiation therapy approach.

Evaluation of STAT3 decoy oligodeoxynucleotides' synergistic effects on radiation and/or chemotherapy in metastatic breast cancer cell line

“…Twenty‐two‐mer phosphorothioated double‐stranded STAT3 decoy and SCR ODNs (scramble) were designed based on the STAT3 core binding site in the human Bcl‐xL gene (Rahmati, Johari, Kadivar, Rismani, & Mortazavi, 2020). For tracking inside the cells, decoy ODNs were synthetically Cy3‐labeled at the 3′ terminus.…”

“…ODNs were transfected into the cells with Lipofectamine 2000 at optimized concentration of Lipofectamine and ODNs. Cellular transfection assay and their analysis were performed in accordance with a previous report by Rahmati, Johari et al (2020).…”

“…Cell cycle analysis was carried out, as previously reported by Rahmati, Johari et al (2020). To determine XI, MTX, and XI‐MTX exposure effects on cell cycle profile and apoptosis after STAT3 decoy treatment, 7 × 10 4 MDA‐MB‐231 cells were seeded in 12‐well plates (SPL Life Sciences, South Korea).…”

“…Apoptosis analysis was carried out, as previously reported by Rahmati, Johari et al (2020). Apoptosis assay was performed on all cell groups (similar to cell cycle assay plan) by staining pellets with 100 µl Annexin V binding buffer (Invitrogen) containing Annexin V‐FITC (Biolegend) and propidium iodide (Sigma‐Aldrich), according to the manufacturer's instructions.…”

“…The migration and invasion assays were established, as previously described by Bigdelou et al (2020) and Rahmati, Johari et al (2020). Briefly, transfected cells with decoy ODNs, SCR ODNs, and Lipofectamine (test and controls, respectively) were exposed to X‐rays (2 Gy; FD) and MTX (20 nM) and cultured overnight.…”