Zinc Transporters and MTF1-Notch1-P21 Signalling Axis in TPEN-Induced Cell Death in Human Skeletal Muscle (Rhabdomyosarcoma) Cells

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.