Alluri Kiran K, Malleswarapu Mahesh, Pyadala Naga Babu
Biochemistry, MNR Medical College and Hospital, Hyderabad, IND.
Life Sciences, University of Hyderabad, Hyderabad, IND.
Cureus. 2025 Aug 18;17(8):e90420. doi: 10.7759/cureus.90420. eCollection 2025 Aug.
Background Physiological zinc levels play a crucial role in regulating cell viability and proliferation. Muscle tissue, a major zinc reservoir, is generally resistant to fluctuations in zinc concentration; however, zinc status can influence muscle cell duplication and survival. Zinc homeostasis is regulated by zinc transporters and signaling pathways such as Notch1 and PI3K/AKT, which control the expression of p21, a key regulator of cell cycle progression and apoptosis. This study aimed to investigate the impact of zinc status on human rhabdomyosarcoma (RD) cells and elucidate the involvement of the metal-regulatory transcription factor 1 (MTF1)-Notch1-PI3K/AKT-p21 axis in zinc depletion-induced cell death. Methodology Zinc depletion was induced in RD cells by treatment with 2.5-15 µM N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), an intracellular zinc chelator. Zinc sufficiency was restored using 25 µM zinc sulfate (ZnSO₄·7H₂O). Cell viability was assessed by the MTT assay, cell cycle progression was evaluated using propidium iodide-based flow cytometry, and protein expression levels (Notch1, pAKT, p21, Bid, Bad, Bax, Caspase-3, MTF1, Znt1, Zip10, and SOCS3) were analyzed by Western blotting. mRNA expression of SOCS3 was quantified to evaluate the effect of zinc depletion on inflammation via the Stat3 pathway. Results Treatment with TPEN led to a dose-dependent reduction in Notch1 and pAKT levels, resulting in decreased p21 expression and increased apoptosis through a caspase-mediated mechanism involving Bid, Bad, Bax, and Caspase-3. Zinc depletion lowered MTF1 levels, thereby affecting the expression of zinc transporters Znt1 and Zip10 and disrupting zinc homeostasis. Propidium iodide cell cycle analysis showed that severe zinc depletion (10 and 15 µM TPEN) caused G1 phase arrest and significantly increased cell death (p < 0.05). An inverse correlation (p < 0.05) was observed between higher TPEN concentrations and p21 levels. Mild zinc depletion (2.5 and 5 µM TPEN) had no significant effect on SOCS3 mRNA levels or cell viability compared to controls, suggesting an adaptive cellular response under mild deficiency. Conclusions Zinc status critically influences the viability of RD cells by modulating MTF1-mediated zinc transporter expression and the Notch1-PI3K/AKT-p21 signaling axis. Severe zinc depletion disrupts zinc homeostasis, downregulates p21, induces G1 cell cycle arrest, and triggers apoptosis through caspase activation, while mild depletion is well tolerated. These findings highlight the importance of zinc homeostasis in skeletal muscle cell survival and provide mechanistic insights into zinc-related muscle pathology.
生理锌水平在调节细胞活力和增殖中起关键作用。肌肉组织是主要的锌储存库，通常对锌浓度波动具有抗性；然而，锌状态可影响肌肉细胞的复制和存活。锌稳态由锌转运体和信号通路如Notch1以及PI3K/AKT调节，这些通路控制细胞周期进程和凋亡的关键调节因子p21的表达。本研究旨在探讨锌状态对人横纹肌肉瘤（RD）细胞的影响，并阐明金属调节转录因子1（MTF1）-Notch1-PI3K/AKT-p21轴在锌缺乏诱导的细胞死亡中的作用。
用细胞内锌螯合剂2.5 - 15 μM N,N,N',N'-四（2 - 吡啶甲基）-1,2 - 乙二胺（TPEN）处理RD细胞以诱导锌缺乏。使用25 μM硫酸锌（ZnSO₄·7H₂O）恢复锌充足状态。通过MTT法评估细胞活力，使用基于碘化丙啶的流式细胞术评估细胞周期进程，并通过蛋白质印迹分析蛋白质表达水平（Notch1、pAKT、p21、Bid、Bad、Bax、Caspase - 3、MTF1、Znt1、Zip10和SOCS3）。对SOCS3的mRNA表达进行定量，以评估锌缺乏通过Stat3途径对炎症的影响。
TPEN处理导致Notch1和pAKT水平呈剂量依赖性降低，导致p21表达减少，并通过涉及Bid、Bad、Bax和Caspase - 3的caspase介导机制增加细胞凋亡。锌缺乏降低了MTF1水平，从而影响锌转运体Znt1和Zip10的表达并破坏锌稳态。碘化丙啶细胞周期分析表明，严重锌缺乏（10和15 μM TPEN）导致G1期阻滞并显著增加细胞死亡（p < 0.05）。观察到较高TPEN浓度与p21水平之间呈负相关（p < 0.05）。与对照组相比，轻度锌缺乏（2.5和5 μM TPEN）对SOCS3 mRNA水平或细胞活力无显著影响，表明在轻度缺乏下细胞有适应性反应。
锌状态通过调节MTF1介导的锌转运体表达和Notch1 - PI3K/AKT - p21信号轴严重影响RD细胞的活力。严重锌缺乏破坏锌稳态，下调p21，诱导G1细胞周期阻滞并通过caspase激活触发凋亡，而轻度缺乏则耐受性良好。这些发现突出了锌稳态在骨骼肌细胞存活中的重要性，并为锌相关的肌肉病理学提供了机制性见解。
