Nanoscale insight into biochemical changes in cervical cancer cells exposed to adaptogenic drug.

This paper describes for the first time the application of atomic force microscopy-based infrared spectroscopy (AFM-IR) to evaluate cellular response to adaptogen, based on an in vitro model of cervical cancer. HeLa cervical cells were exposed to different concentrations of withaferin A, a very promising anti-cancer adaptogenic substance. AFM-IR approach was used to image single cells post-adaptogen treatment and to track subtle biochemical changes in cells at the nanoscale level. Partial least squares (PLS) regression was applied to build predictive models that allowed for the identification of spectral markers of adaptogen-induced alterations Spectroscopic studies were enriched with fluorescence staining to determine whether the adaptogen affects cell morphology. The results showed that with the increase in the concentration of adaptogen, changes in the cell nucleus and the actin cytoskeleton become more and more significant. It has been demonstrated that the AFM-IR technique can successfully study the cellular response to the anti-cancer agent at the single-cell level with nanoscale spatial resolution. On the basis of the promising findings presented in this paper, it is possible to conclude that withaferin A has great potential in inhibiting the proliferation of cervical cancer cells in a dose-dependent manner. It has been found that both the increase in the concentration of withaferin A and the increase in incubation time with the adaptogen resulted in a decrease in the intensity of the bands assigned to nucleic acids. This may be due to DNA condensation, internuclear cleavage, or degradation during apoptosis. The findings also suggest changes in the secondary structure of proteins that may be a consequence of disruption of the actin cytoskeleton, progressive apoptosis, or significant biochemical changes. Furthermore, noticeable changes were also observed in the bands originating from lipids vibrations, and an increased share of the band near 2920 cm, considered an important marker of apoptosis, was noted. The metabolism of carbohydrates in cells also changes under the influence of the adaptogen. AFM-IR provides nanoscale insight into the structural and morphological properties of cells after drug treatment and is an indisputable milestone in the development of new anti-cancer approaches.