Kunitz-type serine protease inhibitor rBmTI-6 as a potential therapeutic lead for lung adenocarcinoma
Gilmara B Lima1, Everton Nencioni1, Fábio Thimoteo1
1Center for Natural and Human Sciences, Federal University of ABC, Brazil.
Abstract:
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.
Insights
A novel Kunitz-type serine protease inhibitor, rBmTI-6, shows significant antitumor activity against lung adenocarcinoma cells. This inhibitor selectively induces apoptosis in cancer cells, offering potential for new protease-targeted cancer therapies.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Lung cancer is a leading cause of cancer mortality globally.
- Targeting tumor-associated proteolytic pathways is crucial for effective cancer therapy.
- The potential of Kunitz-type serine protease inhibitors in cancer treatment remains largely unexplored.
Purpose of the Study:
- To investigate the antitumor activity of rBmTI-6, a recombinant Kunitz-type serine protease inhibitor from Rhipicephalus microplus.
- To evaluate the tumor selectivity and mechanism of action of rBmTI-6 in a human lung adenocarcinoma model.
- To explore the potential of rBmTI-6 as a therapeutic agent for lung cancer.
Main Methods:
- Heterologous expression of rBmTI-6 in Pichia pastoris and purification via affinity chromatography.
- Biochemical characterization including inhibitory activity against trypsin.
- In vitro cellular assays using A549 lung adenocarcinoma cells and MRC-5 human lung fibroblasts.
- Mechanistic studies involving caspase-3 activation, apoptosis assays, and morphological analysis.
- Evaluation of rBmTI-6 activity under serum-free conditions.
Main Results:
- rBmTI-6 demonstrated potent inhibitory activity against trypsin, confirming its functional integrity.
- rBmTI-6 significantly reduced A549 cell viability in a dose-dependent manner.
- The inhibitor exhibited marked selectivity, preserving viability of non-tumorigenic MRC-5 cells.
- rBmTI-6 induced caspase-dependent apoptosis in lung adenocarcinoma cells.
- Cytotoxic activity was enhanced under serum-free conditions, suggesting influence of extracellular interactions.
Conclusions:
- rBmTI-6 possesses significant antitumor activity against lung adenocarcinoma.
- The inhibitor selectively induces apoptosis in lung cancer cells via a caspase-dependent pathway.
- rBmTI-6 represents a promising molecular scaffold for developing novel protease-targeted lung cancer therapies.
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