Usnic acid induced changes in biomolecules and their association with apoptosis in squamous carcinoma (A-431) cells: A flow cytometry, FTIR and DLS spectroscopic study

Abstract

Many natural products induce apoptotic cell death in cancer cells, though studies on their interactions with macromolecules are limited. For the first time, this study demonstrated the cytotoxic potential of usnic acid (UA) against squamous carcinoma (A-431) cells and the associated changes in cell surface proteins, lipids and DNA by attenuated total reflection- fourier transform infrared spectroscopy (ATR-FTIR) and dynamic light scattering (DLS) spectroscopic studies. The IC₅₀ for UA was 98.9 µM after treatment of A-431 cells for 48 h, while the IC₅₀ reduced to 39.2 µM after 72 h of incubation time. UA induced oxidative stress in treated cells as confirmed by DCFHDA flow cytometry assay, depletion in reduced glutathione and increase in lipid peroxidation. The oxidative stress resulted in conformation change in amide I, amide II protein bands and DNA as observed by ATR-FTIR in UA treated A-431 cells. Shift in secondary structures of proteins from α helix to β sheets and structural changes in DNA was observed in UA treated A-431 cells. An increase in the band intensity of phospholipids, increased distribution of lipid and change in membrane potential was noted in UA treated cells, which was confirmed by externalization of phosphatidylserine to the outer membrane by annexin V-FITC/PI assay. Increase in mitochondrial membrane potential, cell cycle arrest at G0/G1 phase by flow cytometry and activation of caspase-3/7 dependent proteins confirmed the UA induced apoptosis in treated A-431 cells. FTIR and DLS spectroscopy confirmed the changes in biomolecules after UA treatment, which were associated with apoptosis, as observed by flow cytometry.

Keywords: Apoptosis; FTIR; Natural product; Secondary structure; Zeta potential.