LY-3000328

Ferroptosis is a newly discovered iron-dependent programmed cell death characterized by excess lipid peroxidation. It is emerging as a promising target for tumor therapies. In the present study, we first identify Cathepsin S (CTSS) as a novel ferroptosis regulator. CTSS is upregulated in ferroptosis-resistant hepatocellular carcinoma (HCC) cells, and suppression of CTSS sensitizes HCC cells to ferroptosis. Mechanistically, ferroptosis stress induces CTSS maturation and promotes the autophagy-lysosomal degradation of Kelch-like ECH-associated protein 1 (KEAP1). This process blocks KEAP1-dependent, ubiquitination-mediated degradation of nuclear factor E2-related factor 2 (NRF). Consequently, the accumulated NRF2 translocates from the cytoplasm to the nucleus and drives the transcription of anti-ferroptosis genes. In vivo study reveals that CTSS depletion, achieved through either shRNA or the specific inhibitor LY3000328, in combination with a ferroptosis inducer, inhibits HCC tumor growth in orthotopic xenograft mouse models. In conclusion, the above data suggest that CTSS can potentiate ferroptosis in HCC cells and may be a therapeutic target to overcome ferroptosis resistance in HCC patients.

Airway epithelial dysfunction constitutes a pivotal contributor to the pathogenesis of acute lung injury (ALI). Pannexin 1 (Panx1), a plasma membrane channel activated during epithelial injury, not only helps dampen inflammation but also plays a key role in epithelial repair. Cathepsin S (CTSS) is implicated in ALI pathophysiology. This study investigates the mechanistic link between CTSS and airway epithelial Panx1 dysregulation in ALI pathogenesis. Lipopolysaccharide (LPS) was instilled into the airways of BALB/c mice, followed by intraperitoneal injection of the CTSS inhibitor LY3000328. The effects of recombinant mouse CTSS (rCTSS) were tested in vivo, and Akt inhibition was used to explore the possible mechanism. In vitro, LPS-treated BEAS-2B cells were co-cultured with or without CTSS or Akt inhibitors. LPS exposure significantly increased pulmonary expression of CTSS. Treatment with LY3000328 alleviated the LPS-induced neutrophil accumulation, alveolar permeability and edema. Additionally, we observed decreased expression of Panx1 in the airway epithelium of LPS-exposed mice, accompanied by increased serine phosphorylation of Akt (p-Akt); inhibition of CTSS restored Panx1 and suppressed the p-Akt. Treatment with rCTSS directly downregulated Panx1 expression and induced Akt phosphorylation in the lung, which could be reversed by pharmacological inhibitor of Akt. In BEAS-2B cells, LPS increased CTSS and p-Akt expression alongside decreased levels of junction proteins (E-cadherin and occludin) and Panx1. Blockade of the CTSS/Akt axis restored the disruption of E-cadherin, occludin, and Panx1. Taken together, our data demonstrated that CTSS regulates the dysregulation of airway epithelial Panx1 through the Akt signaling pathway in an LPS-induced ALI model.