Cell Line-Specific In Vitro Effects of Boric Acid and Meis1i-2 in Leukemia Cells: Apoptosis, Cell-Cycle Modulation,...

Leukemia, a hematological malignancy resulting from genetic mutations and environmental factors, leads to uncontrolled proliferation and impaired differentiation of hematopoietic cells. Current treatment modalities face limitations such as drug resistance and off-target toxicity, highlighting the need for novel therapeutic strategies. This study evaluates the individual and fixed-dose combined in vitro effects of boric acid (BA) and Myeloid Ecotropic Viral Integration Site 1 Inhibitor-2 (Meis1i-2) in leukemia cell lines, focusing on apoptosis, cell-cycle distribution, and selected transcript-level changes. Three distinct leukemia cell lines (K562, RS4-11, CCRF-CEM) were cultured under standardized conditions and treated with varying concentrations of BA and Meis1i-2 individually and in combination. Apoptotic cell death was evaluated using flow cytometry-based Annexin V-FITC/PI staining, while cell cycle dynamics were assessed via Hoechst 33342 and Pyronin Y staining followed by flow cytometric analysis. Additionally, quantitative RT-PCR (qRT-PCR) was employed to determine the expression levels of apoptosis-related and cell cycle regulatory genes. At the 72-h endpoint, BA and Meis1i-2 produced measurable changes in viability, apoptosis, and cell-cycle distribution in a cell-line- and concentration-dependent manner. The high-dose fixed combination of BA (2000 µM) and Meis1i-2 (10 µM) reduced viability compared with DMSO controls in several assays; however, this condition was above the RS4-11 IC₅₀ values and should be interpreted as a supra-IC₅₀ exploratory condition rather than evidence of superiority over the most active single agent. Annexin V/PI and cell-cycle analyses showed treatment-associated apoptotic changes and redistribution of G1, S, and G2/M phases depending on the cell line. Targeted qRT-PCR indicated transcript-level modulation of selected apoptosis- and cell-cycle-related genes, but these changes were not uniform across all models. These findings reveal that BA and Meis1i-2 exhibit biologically measurable antileukemic effects through apoptosis induction and cell cycle modulation. However, Chou-Talalay analysis in RS4-11 cells yielded CI values greater than 1 at the tested concentrations, suggesting an antagonistic rather than non-synergistic under the tested conditions. Therefore, under the current experimental conditions, the combination demonstrates concentration-specific antileukemic activity rather than pharmacological interaction. Future studies using expanded sub-IC₅₀ dose matrices, protein-level and functional validation, and in vivo models are required.