Kunitz-type serine protease inhibitor rBmTI-6 as a potential therapeutic lead for lung adenocarcinoma.

Lung cancer remains one of the leading causes of cancer-related mortality worldwide, underscoring the urgent need for therapeutic strategies that target tumor-associated proteolytic pathways. Here, we report the previously unrecognized antitumor activity of rBmTI-6, a recombinant Kunitz-type serine protease inhibitor derived from the cattle tick Rhipicephalus microplus, in a human lung adenocarcinoma model. rBmTI-6 was heterologously expressed in Pichia pastoris, purified by affinity chromatography, and biochemically characterized. The recombinant inhibitor displayed potent inhibitory activity against trypsin, confirming its functional integrity as a Kunitz-type protease inhibitor. In cellular assays, rBmTI-6 significantly reduced the viability of A549 lung adenocarcinoma cells in a dose-dependent manner while preserving the viability of non-tumorigenic human lung fibroblasts (MRC-5), demonstrating marked tumor selectivity. Mechanistic analyses revealed activation of caspase-3 and a progressive increase in apoptotic cell populations, indicating that rBmTI-6 primarily induces caspase-dependent apoptosis in tumor cells. Morphological alterations observed by microscopy further supported the activation of programmed cell death pathways. Interestingly, the cytotoxic activity of rBmTI-6 was markedly enhanced under serum-free conditions, suggesting that extracellular protein interactions may influence the effective bioavailability of the inhibitor in vitro. Collectively, these findings identify rBmTI-6 as a Kunitz-type inhibitor capable of selectively inducing apoptotic cell death in lung adenocarcinoma cells and highlight its potential as a molecular scaffold for the development of protease-targeted cancer therapy.