A High-Molecular-Weight Fraction of Planarian Mucus Triggers UPR-Linked Cell Death Pathway in Human Bronchioalveolar Carcinoma Cell Line NCI-H358

Natural products remain a major source of anticancer agents, yet freshwater organisms are largely unexplored. Building on our previous evidence that planarian mucus exerts cyto- static and cytotoxic effects on cancer cells, we investigated the involvement of endoplasmic reticulum stress and unfolded protein response (UPR) pathways. Mucus-induced cytotoxi- city is ROS-dependent and associated with depletion of intracellular reduced glutathione (GSH), not through inhibition of the System Xc− transporter but potentially associated with upregulation of CHAC1, a glutathione-degrading enzyme. Mucus fractionation based on molecular weight identified the high-molecular-weight crude fraction as the one con- taining the bioactive entity, reproducing the effects of whole mucus. Treatment with this fraction early activates the PERK–ATF4 branch of the UPR, which could be responsible for driving CHAC1 induction. Moreover, ATF4 enhances DDIT3 expression, and activates a compensatory NRF2-dependent antioxidant response. At a later stage mucus also activates the IRE1α–XBP1 axis, with no ATF6 involvement, indicating selective UPR engagement in response to oxidative and lipid stress. Overall, our data are consistent with a potential PERK–ATF4–CHAC1–GSH self-sustaining axis promoting oxidative stress that culminates in cell death, supporting the potential of planarian mucus as a source of pleiotropic bioac- tive compounds, although the molecular identity of the active component(s) remains still unresolved.