The Anti-Proliferative Lichen-Compound Protolichesterinic Acid Inhibits Oxidative Phosphorylation and Is Processed via the Mercapturic Pathway in Cancer Cells.

The lichen compound protolichesterinic acid (PA) has an anti-proliferative effect against several cancer cell lines of different origin. This effect cannot be explained by the known inhibitory activity of PA against 5- and 12-lipoxygenases. The aim was therefore to search for mechanisms for the anti-proliferative activity of PA. Two cancer cell lines of different origin, both sensitive to anti-proliferative effects of PA, were selected for this study, T-47D from breast cancer and AsPC-1 from pancreatic cancer. Morphological changes were assessed by transmission electron microscopy, HPLC coupled with TOF spectrometry was used for metabolomics, mitochondrial function was measured using the Agilent Seahorse XFp Real-time ATP assay and glucose/lactate levels by radiometry. Levels of glutathione, NADP/NADPH and reactive oxygen species [ROS] were measured by luminescence. Following exposure to PA both cell lines showed structural changes in mitochondria that were in line with a measured reduction in oxidative phosphorylation and increased glycolysis. These changes were more marked in T-47D, which had poorer mitochondrial function at baseline. PA was processed and expelled from the cells via the mercapturic pathway, which consumes glutathione. Nevertheless, glutathione levels were increased after 24 hours of exposure to PA, implying enhanced synthesis. Redox balance was not much affected and ROS levels were not increased. We conclude that PA is metabolically processed and expelled from cells, leading indirectly to increased glutathione levels with minimal effects on redox balance. The most marked effect was on mitochondrial structure and metabolic function implying that effects of PA may depend on mitochondrial fitness.

Effects of anti-proliferative lichen metabolite, protolichesterinic acid on fatty acid synthase, cell signalling and drug response in breast cancer cells.

BACKGROUND: The lichen compound (+)-protolichesterinic acid (+)-PA, isolated from Iceland moss, has anti-proliferative effects on several cancer cell lines. The chemical structure of (+)-PA is similar to a known fatty acid synthase (FASN) inhibitor C75. AIMS: To test whether the anti-proliferative activity of (+)-PA is associated with effects on FASN and HER2 (human epidermal growth factor receptor 2) and major signalling pathways. Synergism between (+)-PA and lapatinib, a HER2 active drug, was also evaluated. MATERIALS AND METHODS: Pure compound was isolated by preparative high-performance liquid chromatography (HPLC) and purity of (+)-PA analyzed by analytical HPLC. Cell viability was assessed using Crystal violet staining. FASN and HER2 expression was estimated by immunofluorescence. The Meso Scale Discovery (MSD)(®) assay was used to measure activation of ERK1/2 and AKT. Synergism was estimated by the CalcuSyn software. RESULTS: Treatment with (+)-PA increased FASN expression in SK-BR-3 cells, which overexpress FASN and HER2, implying a compensatory response to inhibition of FASN activity. HER2 expression was decreased suggesting secondary downregulation. ERK1/2 and AKT signalling pathways were inhibited, probably due to reduced levels of HER2. No effects were observed in T-47D cells. Synergism between (+)-PA and lapatinib was observed in the SK-BR-3 cells. CONCLUSION: Results suggest that the primary effect of (+)-PA is inhibition of FASN activity. Synergistic effects with lapatinib were seen only in SK-BR-3 cells, and not T-47D cells, further supporting the notion that (+)-PA acts by inhibiting FASN with secondary effects on HER2 expression and signalling. (+)-PA could therefore be a suitable agent for further testing, alone or in combination treatment against HER2-overexpressing breast cancer.

Synergistic cytotoxic effect of the microtubule inhibitor marchantin A from Marchantia polymorpha and the Aurora kinase inhibitor MLN8237 on breast cancer cells in vitro.

Macrocyclic bisbibenzyls are a class of characteristic compounds, exclusively produced by liverworts. They are attracting increasing attention due to their wide range of biological activities, including antibacterial, antifungal, and antioxidative properties as well as cytotoxicity. Marchantin A is a cyclic bisbibenzyl that has previously been isolated from Marchantia polymorpha and other liverwort species and has been shown to exert cytotoxic effects. In the present study we found that the Icelandic M. polymorpha species produces marchantin A and through an in vitro cell growth inhibition assay, marchantin A was shown to induce a reduction in cell viability of breast cancer cell lines A256 (IC50 = 5.5 µM), MCF7 (IC50 = 11.5 µM), and T47D (IC50 = 15.3 µM). The effect was considerably increased in all cell lines in a synergistic manner when the Aurora-A kinase inhibitor MLN8237 was added simultaneously. Fluorescence microscopy confirmed the antimicrotubular effect of marchantin A, and cell cycle analysis indicated enhanced cell division failure when combining this mitotic-spindle inhibitor with the checkpoint modulator.

Cellular mechanisms of the anticancer effects of the lichen compound usnic acid.

The lichen compound usnic acid is used for its antimicrobial activities in cosmetic products and is also a component of slimming agents. Its effect against cancer cells was first noted over 30 years ago. In this study possible mechanisms of this effect were investigated using two human cell lines, the breast cancer cell line T-47D and the pancreatic cancer cell line Capan-2. Pure (+)-usnic acid from CLADONIA ARBUSCULA and (-)-usnic acid from ALECTORIA OCHROLEUCA were shown to be equally effective inhibitors of DNA synthesis, with IC (50) 4.2 microg/mL and 4.0 microg/mL for (+) and (-)-usnic acid against T-47D, and 5.3 microg/mL and 5.0 microg/mL against Capan-2, respectively. Flow cytometric analysis confirmed the inhibited entry into the S-phase and showed reduction in cell size. Classical apoptosis, as assessed by TUNEL staining, was not observed. Necrosis, measured by LDH release, was seen only in Capan-2 after exposure for 48 hours. Staining with the mitochondrial dye JC-1 demonstrated dose-dependent loss of mitochondrial membrane potential following treatment with usnic acid in both cell lines. In conclusion, usnic acid had a marked inhibitory effect on growth and proliferation of two different human cancer cell lines and led to loss of mitochondrial membrane potential. Cell survival was little affected; late necrosis was seen in one of the cell lines. No difference was noted between the two enantiomers.

The cytotoxic effect of two chemotypes of essential oils from the fruits of Angelica archangelica L.

BACKGROUND: The aim of this work was to study the constituents and cytotoxicity of the essential oils from the fruits of Angelica archangelica growing in Iceland. MATERIALS AND METHODS: Three samples of essential oils were prepared by steam distillation. Their composition was established with GC/MS. The effects of the oils were examined in PANC-1 human pancreas cancer cells and Crl mouse breast cancer cells in concentrations ranging from 10-400 microg/ml, measuring the reduction of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl)-2- (4-sulphophenyl) -2H-tetrazolium (MTS) by mitochondrial enzymes. RESULTS: Two types of essential oils were found, differing mainly in the absence or presence of beta-phellandrene. The ED50 of the oils ranged from 48.6 microg/ml to 108.3 microg/ml for PANC-1 and 48.0 microg/ml to 91.8 microg/ml for Crl cells. CONCLUSION: The cytotoxic activity of the essential oils was independent of the quantity of their main components.

Antitumour activity of Angelica archangelica leaf extract.

BACKGROUND: The purpose of this study was to examine the effect of a leaf extract from A. archangelica on the growth of Crl mouse breast cancer cells in vitro and in vivo. MATERIALS AND METHODS: The antiproliferative activity of the extract was measured by 3H-thymidine uptake in the Crl cells in vitro. Twenty mice were injected with the Crl cells, and 11 of them were fed A. archangelica leaf extract, and the progress of the tumours was followed. RESULTS: The leaf extract was mildly antiproliferative on the Crl cells with an EC50 of 87.6 microg/ml The antitumour activity of the extract was expressed in the mice by marked reduction in tumour growth. In the experimental animals, 9 out of 11 mice developed no or very small tumours, whereas control animals, not receiving the extract, developed significantly larger tumours (p<0.01), as estimated by Mann-Whitney U-test. The antitumour activity of the leaf extract could not be explained by the antiproliferative activity of furanocoumarins present in the extract. CONCLUSION: The results demonstrate the antiproliferative activity in vitro and antitumour activity in vivo of a leaf extract from A. archangelica

Anti-proliferative effects of lichen-derived lipoxygenase inhibitors on twelve human cancer cell lines of different tissue origin in vitro.

Lipoxygenases (LOXs) have been implicated in carcinogenesis in various cancer types. In the current study, three structurally different lichen metabolites, protolichesterinic acid (1), lobaric acid (2) and baeomycesic acid (3) were tested for anti-proliferative effects against 12 different human cancer cell lines. All compounds have known in vitro 5-LOX inhibitory activity, and 1 and 2 also inhibit 12-LOX. The activity of the lichen metabolites was compared to that of a specific 5-LOX inhibitor, zileuton (4). The following cancer cell lines were tested: Capan-1, Capan-2 and PANC-1 (all from pancreas), T47-D (breast), PC-3 (prostate), NCI-H1417 (small cell lung), NIH:OVCAR-3 (ovary), AGS (stomach), WiDr (colorectal), HL-60, K-562 and JURKAT (acute promyelocytic, erythro- and T-cell leukemia, respectively). Compound 1 showed the greatest inhibitory effect against all cell lines, with EC50 ranging from 2.4-18.1 microg mL(-1) (7.4-55.8 microM), followed by 2, with EC50 of 15.2 - 65.5 microg mL(-1) (33.2-143.6 microM). The effects of 3 and 4 were of similar orders of magnitude, with EC50 of 28.7 - >80 microg mL(-1) (76.8 - > 213.9 microM) and 12.9 - > 80 microg mL(-1) (50.4 - > 313.7 microM). The dual 5- and 12-LOX inhibitors 1 and to some extent 2 thus exert significant anti-proliferative effects against a variety of human cancer cell lines, while the selective 5-LOX inhibitors 3 and 4 are considerably less active.

Antiproliferative effect of Angelica archangelica fruits.

The aim of this work was to study the antiproliferative effect of a tincture from fruits of Angelica archangelica and the active components using the human pancreas cancer cell line PANC-1 as a model. Significant dose-dependent antiproliferative activity was observed in the tincture with an EC50 value of 28.6 microg/ml. Strong antiproliferative activity resulted from the two most abundant furanocoumarins in the tincture, imperatorin and xanthotoxin. The contribution of terpenes to this activity was insignificant. Imperatorin and xanthotoxin proved to be highly antiproliferative, with EC50 values of 2.7 microg/ml and 3.7 microg/ml, respectively, equivalent to 10 and 17 microM. The results indicate that furanocoumarins account for most of the antiproliferative activity of the tincture.