Chair of Medical Biochemistry, Jagiellonian University Medical College, ul. Kopernika 7, 31-034 Cracow, Poland.
Department of Cardiovascular Surgery and Transplantology, Institute of Cardiology, Jagiellonian University, John Paul II Hospital, ul. Pra˛dnicka 80, 31-202 Cracow, Poland.
Int J Mol Sci. 2024 Nov 15;25(22):12278. doi: 10.3390/ijms252212278.
The balance between apoptosis and autophagy plays a key role in cancer biology and treatment strategies. The aim of this study was to assess the effect of the mTOR kinase inhibitor everolimus and chloroquine on the regulation of proliferation, caspase-3 activation, and apoptosis in melanoma cells. We studied the activity of caspase-3 and the levels of caspase-3 and -9 using the Western blot technique. Cellular apoptosis was examined using a DNA fragmentation assay, and changes in the cell nucleus and cytoskeleton were examined using fluorescence microscopy DAPI, OA/IP. We also studied the rearrangement of lipid structures using fluorescent dyes: Nile Red and Nile Blue. A low nanomolar concentration of the mTOR kinase inhibitor everolimus in combination with chloroquine activated the apoptosis process and decreased cell proliferation. These changes were accompanied by an obvious change in cell morphology and rearrangement of lipid structures. Alterations in lipid redistribution accompanying the process of apoptosis and autophagy are among the first to occur in the cell and can be easily monitored in in vitro studies. The combination of mTOR inhibitors and chloroquine represents a promising area of research in cancer therapy. It has the potential to enhance treatment efficacy through complementary mechanisms.
细胞凋亡和自噬之间的平衡在癌症生物学和治疗策略中起着关键作用。本研究旨在评估 mTOR 激酶抑制剂依维莫司和氯喹对黑色素瘤细胞增殖、caspase-3 激活和细胞凋亡的调控作用。我们使用 Western blot 技术研究了 caspase-3 的活性以及 caspase-3 和 caspase-9 的水平。通过 DNA 片段化测定法检测细胞凋亡，并通过荧光显微镜 DAPI、OA/IP 观察细胞核和细胞骨架的变化。我们还使用荧光染料 Nile Red 和 Nile Blue 研究了脂质结构的重排。低纳摩尔浓度的 mTOR 激酶抑制剂依维莫司与氯喹联合使用可激活细胞凋亡过程并降低细胞增殖。这些变化伴随着细胞形态的明显变化和脂质结构的重排。伴随细胞凋亡和自噬过程的脂质重新分布改变是细胞内最早发生的改变之一，在体外研究中很容易监测到。mTOR 抑制剂和氯喹的联合使用代表了癌症治疗研究的一个有前途的领域。它具有通过互补机制增强治疗效果的潜力。
