ROS-Induced DNA-Damage and Autophagy in Oral Squamous Cell Carcinoma by Usnea barbata Oil Extract—An In Vitro Study

Abstract

Oxidative stress is associated with aging, cancers, and numerous metabolic and chronic disorders, and phenolic compounds are well known for their health-promoting role due to their free-radical scavenging activity. These phytochemicals could also exhibit pro-oxidant effects. Due to its bioactive phenolic secondary metabolites, Usnea barbata (L.) Weber ex. F.H. Wigg (U. barbata) displays anticancer and antioxidant activities and has been used as a phytomedicine for thousands of years. The present work aims to analyze the properties of U. barbata extract in canola oil (UBO). The UBO cytotoxicity on oral squamous cell carcinoma (OSCC) CLS-354 cell line and blood cell cultures was explored through complex flow cytometry analyses regarding apoptosis, reactive oxygen species (ROS) levels, the enzymatic activity of caspase 3/7, cell cycle, nuclear shrinkage (NS), autophagy (A), and synthesis of deoxyribonucleic acid (DNA). All these studies were concomitantly performed on canola oil (CNO) to evidence the interaction of lichen metabolites with the constituents of this green solvent used for extraction. The obtained data evidenced that UBO inhibited CLS-354 oral cancer cell proliferation through ROS generation (316.67 × 104), determining higher levels of nuclear shrinkage (40.12%), cell cycle arrest in G0/G1 (92.51%; G0 is the differentiation phase, while during G1 phase occurs preparation for cell division), DNA fragmentation (2.97%), and autophagy (62.98%) than in blood cells. At a substantially higher ROS level in blood cells (5250.00 × 104), the processes that lead to cell death—NS (30.05%), cell cycle arrest in G0/G1 (86.30%), DNA fragmentation (0.72%), and autophagy (39.37%)—are considerably lower than in CLS-354 oral cancer cells. Our work reveals the ROS-mediated anticancer potential of UBO through DNA damage and autophagy. Moreover, the present study suggests that UBO pharmacological potential could result from the synergism between lichen secondary metabolites and canola oil phytoconstituents.

Cancer Metabolism: The Role of ROS in DNA Damage and Induction of Apoptosis in Cancer Cells

“…Subsequent research studies have found that NCX4040 can also induce a TNF-dependent pathway, further increasing oxidative stress through NF-κB-mediated immune responses, leading to increased apoptosis (mediated by NCX4040) [ 33 ]. In addition, Popovici et al revealed that Usnea barbata extract in canola oil showed anticancer potential through ROS generation leading to DNA damage and autophagy [ 34 ]. Therefore, there is a relatively low or high threshold for ROS accumulation to promote or inhibit cancer.…”

Cytotoxicity Analysis and In Silico Studies of Three Plant Extracts with Potential Application in Treatment of Endothelial Dysfunction

“…The separation and introduction into the experimental wells were performed under a stereomicroscope. Microscopic preparations were made (magnification of 100× and 400×) to analyze the details, evaluate the developmental stages and highlight the effects on the larvae; the Visiocam 2 imaging system [ 8 , 9 ] stock solutions were prepared in distilled water (15,900 µg/mL for CE, 17,700 µg/mL for GE, and 16,100 µg/mL for SE), and serial dilutions produced samples. The assay was carried out in two stages, with evaluation in triplicate for each concentration.…”

Antioxidant and antidiabetic activity and phytoconstituents of lichen extracts with temperate and polar distribution

“…As for the DPPH analysis, the three extracts evidenced an IC 50 value much higher than the gallic acid standard, suggesting a medium-low inhibition capacity of the study concentrations used; however, the extract of O. frigida continued to show better antioxidant These results correlate positively with the reports of extracts and compounds isolated from other lichen species that show highly effective phenolic concentration and antioxidant activity through Frontiers in Pharmacology frontiersin.org colorimetric techniques (Luo et al, 2009;Mitrović et al, 2011;Jha et al, 2017;Studzińska-Sroka et al, 2021a;Elečko et al, 2022) and electrochemical, computational, and genetic studies (Kalra et al, 2023;Yañez et al, 2023). Likewise, the variability in the antioxidant properties of lichens is mediated by the geographical, altitudinal, and/or microhabitat conditions in which they develop working concentrations, metabolite isolation efficiency, types of solvent, and forms of extraction and exploration of the mechanisms of action (Ranković et al, 2012;White et al, 2014;Studzińska-Sroka et al, 2021b;Li et al, 2022;Popovici et al, 2022;Sánchez et al, 2022). On the other hand, the evident antioxidant capacity of lichenic species has supported their use for the evaluation of neuroprotective effects in in vitro and in vivo models, yielding positive results regarding cell viability, protection against induced oxidative stress, decrease in reactive oxygen species, improvement of mitochondrial function, and suppression of signaling pathways that induce inflammatory response in astrocytes (Fernández-Moriano et al, 2015;Fernández-Moriano et al, 2016;Lee et al, 2021;Ureña-Vacas et al, 2022b).…”