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Документ Відкритий доступ Combination Treatment with Free Doxorubicin and Inductive Moderate Hyperthermia for Sarcoma Saos-2 Cells(2025) Orel, Valerii E.; Diedkov, Anatolii G.; Ostafiichuk, Vasyl V.; Lyalkin, Sergii A.; Tkachenko, Igor O.; Kolesnyk, Denys L.; Orel, Valerii B.; Dasyukevich, Olga Yo.; Rykhalskyi, Oleksandr Yu.; Movchan, Oleksii V.; Galkin, Alexander Yu.; Prosvietova, Anna B.Background: Osteosarcoma (OS) is the most common primary malignant bone tumor. Doxorubicin (DOX) is extensively used in OS chemotherapy, yet improving patient outcomes remains challenging. This study investigated the effect of free DOX combined with inductive moderate hyperthermia (IMH) on Saos-2 human OS cells. Methods: Cell viability was assessed by trypan blue exclusion. Flow cytometry analyzed apoptosis, necrosis, and reactive oxygen species (ROS) in cells exposed to control (no treatment), IMH (42 MHz frequency, 500 µT magnetic field induction, 564 V/m electric field strength, 15 W output power, and 30 min duration) alone, DOX (0.06 µg/mL) alone, or DOX combined with IMH. The expression of p14ARF tumor suppressor and epidermal growth factor receptor (EGFR) was evaluated by immunocytochemistry. Spatial autocorrelation analysis quantified the heterogeneity of p14ARF and EGFR distributions in acquired images. Results: The half maximal inhibitory concentration (IC50) of DOX in Saos-2 cells had minimal variation between 48 h (0.060 ± 0.01 µg/mL) and 72 h (0.055 ± 0.003 µg/mL). DOX + IMH resulted in a 15% increase in early apoptosis and a 20% elevation in ROS levels compared with DOX alone. Immunocytochemical analysis revealed a 37% increase in p14ARF and a 32% reduction in EGFR expression following combined treatment in comparison to DOX alone. Image analysis showed that DOX + IMH treatment caused the highest Moran’s index values for p14ARF and EGFR, reflecting less heterogeneous spatial distributions (p < 0.05). Conclusions: IMH enhanced DOX-induced cytotoxicity in Saos-2 cells by initiating ROS-mediated apoptosis and reducing heterogeneity of cellular responses.