Monitoring ZEO apoptotic potential in 2D and 3D cell cultures and associated spectroscopic evidence on mode of interaction with DNA

Abstract

Recognizing new anticancer compounds to improve Breast cancer treatment seems crucial. Essential oil of Zataria Multiflora (ZEO) is a secondary metabolite with some biological properties, yet underlying cellular and molecular anticancer properties of ZEO is unclear. GC/MS analysis revealed that carvacrol is the major ingredient of the essential oil. ZEO increasingly suppressed viability in MDA-MB-231, MCF-7 and T47D Breast cancer cells while nontoxic to L929 normal cells in monolayer cell cultures (2D), whereas MDA-MB-231 multicellular spheroids (3D) were more resistant to inhibition. ZEO significantly induced cell apoptosis confirmed by fluorescent staining, flow cytometry analysis and DNA fragmentation in MDA-MB-231 2D and 3D cell cultures. ZEO increased ROS generation and subsequent loss of ΔΨm, caspase 3 activation and DNA damage which consequently caused G1 and G2/M cell cycle arrest in a dose- and time-dependent manner in 2D. S phase arrest occurred in cell spheroids therefore ZEO possible DNA interaction with gDNA was investigated and revealed ZEO binds DNA via intercalation. Altogether, these data corroborate anticancer properties of ZEO and suggest that cell culture format (2D monolayer vs. 3D spheroid) plays a critical role in drug response and provides new insights into the mechanisms underlying ZEO cytotoxicity effect on Breast cancer cells.

In-vitro evaluation of apoptotic effect of OEO and thymol in 2D and 3D cell cultures and the study of their interaction mode with DNA

“…After 4 days, for measurement of spheroid size, we took some photos using invert microscope (Ax overt 25, Zeiss, Germany at 5 × and 10 × magnitudes) and then used ImageJ software to measure size and volume of spheroids. Our analysis showed spheroids with diameters ranging from 500–600 μm 13 , 72 .…”

“…The temperature and carrier gas was the same as that of above and ionization source temperature was set on 280 °C. The identity of the oil constituents was assigned by comparison of Retention indices (relative to a homologous series of n-alkanes (C8-C25)) with those reported in the literature and the internal reference mass spectra library 13 .…”

“…Essential Oils (EOs), described as ‘the soul of plants’ are volatile complexes found in the aromatic plants and are used in pharmaceutical, and food industries for their anti-inflammatory, anti-microbial and anti-oxidant properties 9 – 11 . Additionally, anticancer activities of some EOs 12 , 13 have been demonstrated in recent years. Terpenes and their oxygenated derivatives are the main components of EOs 14 .…”

Oxidative DNA damage induced by ROS-modulating agents with the ability to target DNA: A comparison of the biological characteristics of citrus pectin and apple pectin

“…ROS overload also prompts oxidative damage to the cell’s biomacromolecules, which culminates in the cells dysfunction and death. ROS overproduction causes DNA damage via oxidation and DNA strand brakes 61 , 65 . The level of 8-oxo-dG which is the major product of DNA oxidation by reactive oxygen species, increased significantly after CP, and AP treatment in MDA-MB-231 cells.…”

“…In order to generate suitable cell spheroids for screening the compounds, a combination of hanging drop (HD) and liquid overlay (LO) techniques was used as described previously 61 . In brief, 50 µl drops of a cell suspension with the concentration of about 1.0 × 10 5 cells/ml (5000 cells/drop) were deposited on the interior surface of a petri dish lid, and were incubated until the spheroids were formed.…”