Diffractaic acid exhibits thioredoxin reductase 1 inhibition in lung cancer A549 cells.

Lung cancer is the leading cause of cancer-related deaths all over the world. Therefore, it has gained importance in the development of new chemotherapeutic strategies to identify anticancer agents with low side effects, reliable, high anticancer potential, and specific to lung cancer cells. Thioredoxin reductase 1 (TrxR1) is an important therapeutic target for lung cancer treatment because of its overexpression in tumor cells. Here, we aimed to examine the anticancer effect of diffractaic acid, a lichen secondary metabolite, in A549 cells by comparing it with the commercial chemotherapeutic drug carboplatin and also to investigate whether the anticancer effect of diffractaic acid occurs via TrxR1-targeting. The IC50 value of diffractaic acid on A549 cells was determined as 46.37 µg/mL at 48 h, and diffractaic acid had stronger cytotoxicity than carboplatin in A549 cells. qPCR results revealed that diffractaic acid promoted the intrinsic apoptotic pathway through the upregulation of the BAX/BCL2 ratio and P53 gene in A549 cells, which is consistent with the flow cytometry results. Furthermore, migration analysis results indicated that diffractaic acid impressively suppressed the migration of A549 cells. While the enzymatic activity of TrxR1 was inhibited by diffractaic acid in A549 cells, no changes were seen in the quantitative expression levels of gene and protein. These findings provide fundamental data on the anticancer effect of diffractaic acid on A549 cells targeting TrxR1 activity, suggesting that it could be considered a chemotherapeutic agent for lung cancer therapy.

Lichen-Derived Diffractaic Acid Inhibited Dengue Virus Replication in a Cell-Based System.

Dengue is a mosquito-borne flavivirus that causes 21,000 deaths annually. Depsides and depsidones of lichens have previously been reported to be antimicrobials. In this study, our objective was to identify lichen-derived depsides and depsidones as dengue virus inhibitors. The 18 depsides and depsidones of Usnea baileyi, Usnea aciculifera, Parmotrema dilatatum, and Parmotrema tsavoense were tested against dengue virus serotype 2. Two depsides and one depsidone inhibited dengue virus serotype 2 without any apparent cytotoxicity. Diffractaic acid, barbatic acid, and Parmosidone C were three active compounds further characterized for their efficacies (EC50), cytotoxicities (CC50), and selectivity index (SI; CC50/EC50). Their EC50 (SI) values were 2.43 ± 0.19 (20.59), 0.91 ± 0.15 (13.33), and 17.42 ± 3.21 (8.95) µM, respectively. Diffractaic acid showed the highest selectivity index, and similar efficacies were also found in dengue serotypes 1-4, Zika, and chikungunya viruses. Cell-based studies revealed that the target was mainly in the late stage with replication and the formation of infectious particles. This report highlights that a lichen-derived diffractaic acid could become a mosquito-borne antiviral lead as its selectivity indices ranged from 8.07 to 20.59 with a proposed target at viral replication.

Antiproliferative, antimigratory, and apoptotic effects of diffractaic and vulpinic acids as thioredoxin reductase 1 inhibitors on cervical cancer.

Cervical cancer is among the most frequently observed cancer types in females. New therapeutic targets are needed because of the side impacts of existing cancer drugs and the inadequacy of treatment methods. Thioredoxin reductase 1 (TrxR1) is often overexpressed in many cancer cells, and targeting TrxR1 has become an attractive target for cancer therapy. This study investigated the anticancer impacts of diffractaic and vulpinic acids, lichen secondary metabolites, on the cervical cancer HeLa cell line. XTT findings demonstrated showed that diffractaic and vulpinic acids suppressed the proliferation of HeLa cells in a dose- and time-dependent manner and IC50 values were 22.52 µg/ml and 66.53 µg/ml at 48 h, respectively. Each of these lichen metabolites significantly suppressed migration. Diffractaic acid showed an increase in both the BAX/BCL2 ratio by qPCR analysis and the apoptotic cell population via flow cytometry analysis on HeLa cells. Concerning vulpinic acid, although it decreased the BAX/BCL2 ratio in this cells, it increased apoptotic cells according to the flow cytometry analysis results. Diffractaic and vulpinic acids significantly suppressed TrxR1 enzyme activity rather than the gene and protein expression levels in HeLa cells. This research demonstrated for the first time, that targeting TrxR1 with diffractaic and vulpinic acids was an effective therapeutic strategy for treating cervical cancer.

Diffractaic acid exerts anti-cancer effects on hepatocellular carcinoma HepG2 cells by inducing apoptosis and suppressing migration through targeting thioredoxin reductase 1.

Hepatocellular carcinoma (HCC) represents one of the most common malignant tumors worldwide. Due to the limited number of available drugs and their side effects, the development of new chemotherapeutic strategies for HCC treatment has become increasingly important. This study is aimed at investigating whether diffractaic acid (DA), one of the secondary metabolites of lichen, exhibits a potential anticancer effect on HepG2 cells and whether its anticancer effect is mediated by inhibition of thioredoxin reductase 1 (TRXR1), which is a target of chemotherapeutic strategies due to overexpression in tumor cells including HCC. XTT assay results showed that DA exhibited strong cytotoxicity on HepG2 cells with an IC50 value of 78.07 µg/mL at 48 h. Flow cytometric analysis results revealed that DA displayed late apoptotic and necrotic effects on HepG2 cells. Consistent with these findings, real-time PCR results showed that DA did not alter the BAX/BCL2 ratio in HepG2 cells but upregulated the P53 gene. Moreover, the wound healing assay results revealed a strong anti-migratory effect of DA in HepG2 cells. Real-time PCR and Western blot analyses demonstrated that DA increased TRXR1 gene and protein expression levels, whereas enzyme activity studies disclosed that DA inhibited TRXR1. These findings suggest that DA has an anticancer effect on HepG2 cells by targeting the enzymatic inhibition of TRXR1. In conclusion, DA as a TRXR1 inhibitor can be considered an effective chemotherapeutic agent which may be a useful lead compound for the treatment of HCC.

Diffractaic acid, a novel TrxR1 inhibitor, induces cytotoxicity, apoptosis, and antimigration in human breast cancer cells.

Breast cancer represents one of the most frequently encountered cancer types among women worldwide. Thioredoxin reductase 1 (TrxR1) is a therapeutic target for breast cancer therapy due to its overexpression in tumor cells. The current research aims to determine the anticancer effect of diffractaic acid, a lichen acid, in breast cancer, and research whether the anticancer effect of diffractaic acid occurs through TrxR1 targeting. According to the XTT assay results, diffractaic acid induced cytotoxicity in both MCF-7 and MDA-MB-453 cells with IC50 values of 51.32 µg/ml and 87.03 µg/ml, respectively. Flow cytometry and cell migration analyses revealed the apoptotic, necrotic, and antimigratory effects of diffractaic acid. qPCR analysis indicated the upregulation of the BAX/BCL2 ratio and the P53 gene in MCF-7 cells with only the P53 gene in MDA-MB-453 cells. The gene, protein, and enzyme activity of TrxR1 were suppressed in MCF-7 cells, whereas only enzyme activity was suppressed in MDA-MB-453 cells. These findings illustrate the anticancer effect of diffractaic acid on breast cancer targeting TrxR1. In conclusion, these data reveal that diffractaic acid may be considered an effective therapeutic agent for breast cancer treatment.

DFT and QTAIM based investigation on the structure and antioxidant behavior of lichen substances Atranorin, Evernic acid and Diffractaic acid.

In this study, the structural and antioxidant behavior of the three lichen-derived natural compounds such as atranorin (AT), evernic acid (EV) and diffractaic acid (DF) has been investigated in the gas and water phase using both B3LYP and M06-2X functional level of density functional theory (DFT) with two different basis sets 6-31+G (d, p) and 6-311++G (d, p). The intramolecular H-bonds (IHB) strength, aromaticity and noncovalent interactions (NCI) have been computed with the help of the quantum theory of atoms in molecules (QTAIM). This calculation gives major structural characteristics that indirectly influence the antioxidant behavior of the investigated compounds. The spin density (SD) delocalization of the unpaired electron is found to be the main stabilizing factor of neutral and cationic radical species. The main mechanisms, recommended in the literature, for the antioxidant action of polyphenols as radical scavengers such as hydrogen atom transfer (HAT), single electron transfer followed by proton transfer (SET-PT), and sequential proton loss electron transfer (SPLET), were examined. The result shows that the HAT and SPLET mechanism are the most conceivable one for the antioxidant action of this class of compounds in gas and water phase respectively. Preference of SPLET over HAT in water phase is due to the significantly lower value of proton affinity (PA) compared to the bond dissociation enthalpy (BDE) value. This study reveals that O2-H3, O9-H26 and O4-H45 respectively are the most favored site of AT, EV and DF for homolytic as well as heterolytic OH bond breaking.

Molecular modeling and cytotoxicity of diffractaic acid: HP-ß-CD inclusion complex encapsulated in microspheres.

In this pioneer study, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) was used to improve the solubility of the diffractaic acid (DA) via inclusion complex (DA:HP-ß-CD). Subsequently, DA:HP-ß-CD was incorporated into poly-ε-caprolactone (PCL) microspheres (DA:HP-ß-CD-MS). Microspheres containing DA (DA-MS) or DA:HP-ß-CD (DA:HP-ß-CD-MS) were prepared using the multiple W/O/W emulsion-solvent evaporation technique. The phase-solubility diagram of DA in HP-ß-CD (10-50mM) showed an AL type curve with a stability constant K1:1=821M-1. 1H NMR, FTIR, X-ray diffraction and thermal analysis showed changes in the molecular environment of DA in DA:HP-ß-CD. The molecular modeling approach suggests a guest-host complex formation between the carboxylic moiety of both DA and the host (HP-ß-CD). The mean particle size of the microspheres were ∅DA-MS=5.23±1.65µm and ∅DA:HP-ß-CD-MS=4.11±1.39µm, respectively. The zeta potential values of the microspheres were ζDA-MS=-7.85±0.32mV and ζDA:HP-ß-CD-MS=-6.93±0.46mV. Moreover, the encapsulation of DA:HP-ß-CD into microspheres resulted in a more slower release (k2=0.042±0.001; r2=0.996) when compared with DA-MS (k2=0.183±0.005; r2=0.996). The encapsulation of DA or DA:HP-ß-CD into microspheres reduced the cytotoxicity of DA (IC50=43.29µM) against Vero cells (IC50 of DA-MS=108.48µM and IC50 of DA:HP-ß-CD-MS=142.63µM).

The investigation of cytogenetic and oxidative effects of diffractaic acid on human lymphocyte cultures

Diffractaic acid (DA) is a naturally occurring depside derivative found in several lichen species. It has a wide range of important biological effects such as analgesic and antiviral properties, although its cytotoxic, cytogenetic and oxidative effects have not been investigated in human blood tissue yet. Therefore, increasing concentrations (1, 5, 10, 25, 50, 100 and 200 mgL-1) of DA was added into human whole blood cultures. 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyl tetrazolium bromide (MTT) assay was used to assess the cell viability and/or cytotoxicity and genotoxic damage potential of DA using chromosome aberration (CA) and micronucleus (MN) tests were performed. In addition, oxidative alterations were determined by the total antioxidant capacity (TAC) and total oxidant status (TOS) assays. The results revealed that DA reduced cell viability at higher concentrations than 50 mgL-1. The all tested concentrations of DA were non-genotoxic. In vitro treatments with DA led to increases of TAC levels in the cultured blood cells without changing the TOS levels as compared to the control group. Consequently, DA exhibited a significant non-mutagenic and antioxidant potential in vitro.