Time- and Dose-dependent Intracellular Proteome Alterations by Cobra Venom Cytotoxin: System Toxicology and Mechanistic Perspectives

Naja sumatrana venom cytotoxin (CTX) is a three-finger toxin which has been reported to exhibit caspase-dependent and mitochondrial-mediated apoptosis without transitioning into necrosis in human breast cancer MCF-7 cells. It has also been found to cause membrane permeabilization, leading to secretory phenotype changes. This study investigates the time- and dose-dependent alterations of the intracellular proteome by the cytotoxin from system toxicology and mechanistic perspectives. The cytotoxicity of CTX was determined at 8 and 24 h. MCF-7 cells were treated with IC₂₀, IC₅₀, and IC₈₀ of CTX at 8 h and 24 h. Label-free quantitative (LFQ) proteomics was performed to determine the intracellular proteome changes caused by CTX. Bioinformatic analyses were then carried out to identify the significantly distinguished proteins and pathway enrichment for protein–protein interaction networks. The LFQ proteomics revealed distinctive protein families significantly distinguished at 8 and 24 h. At 8 h, CTX targeted the cell cycle checkpoints and estrogen signalling, Notch, WNT/ β-catenin, and Rho GTPase pathways. On the other hand, at 24 h, proteins related to oxidative stress and MAPK-mediated apoptosis pathways were detected. There was also a concentration-dependent substantial elevation in different protein families associated with chromatin organization, DNA damage checkpoints, and DNA fragmentation during apoptosis. The protein–protein interaction (PPI) networks demonstrated several potential clusters related to toxin internalization, which could lead to intracellular accumulation that triggers cytotoxicity. Nevertheless, the exposure to CTX did not have notable effects on cellular metabolism. In conclusion, CTX induced significant time- and dose-dependent alterations of the intracellular proteome, revealing the involvement of apoptosis-related pathways and the suppression of proliferation in MCF-7. Integrative proteomics and system toxicology uncovered the mechanistic insights of CTX’s progressive actions in causing cell death.