Effects of combined cannabidiol (CBD) and hops (Humulus lupulus) terpene extract treatment on RAW 264.7 macrophage viability and inflammatory markers.

This study investigates the potential of cannabidiol (CBD), one major cannabinoid of the plant Cannabis sativa, alone and in combination with a terpene-enriched extract from Humulus lupulus ("Hops 1"), on the LPS-response of RAW 264.7 macrophages as an established in vitro model of inflammation. With the present study, we could support earlier findings of the anti-inflammatory potential of CBD, which showed a dose-dependent [0-5 µM] reduction in nitric oxide and tumor necrosis factor-alpha (TNF-α) released by LPS-stimulated RAW 264.7 macrophages. Moreover, we observed an additive anti-inflammatory effect after combined CBD [5 µM] and hops extract [40 µg/mL] treatment. The combination of CBD and Hops 1 showed effects in LPS-stimulated RAW 264.7 cells superior to the single substance treatments and akin to the control hydrocortisone. Furthermore, cellular CBD uptake increased dose-dependently in the presence of terpenes from Hops 1 extract. The anti-inflammatory effect of CBD and its cellular uptake positively correlated with terpene concentration, as indicated by comparison with a hemp extract containing both CBD and terpenes. These findings may contribute to the postulations for the so-called "entourage effect" between cannabinoids and terpenes and support the potential of CBD combined with phytomolecules from a non-cannabinoid source, such as hops, for the treatment of inflammatory diseases.

Comparison of Three Low-Molecular-Weight Fluorescent Probes for Measuring Free Zinc Levels in Cultured Mammary Cells.

Free zinc is a critical regulator in signal transduction and affects many cellular processes relevant to cancer, including proliferation and cell death. Acting as a second messenger, altered free intracellular zinc has fundamental effects on regulating enzymes such as phosphatases and caspases. Therefore, the determination of free intracellular zinc levels is essential to assess its influence on the signaling processes involved in cancer development and progression. In this study, we compare three low-molecular-weight fluorescent probes, ZinPyr-1, TSQ, and FluoZin-3, for measuring free zinc in different mammary cell lines (MCF10A, MCF7, T47D, and MDA-MB-231). In summary, ZinPyr-1 is the most suitable probe for free Zn quantification. It responds well to calibration based on minimal fluorescence in the presence of the chelator TPEN (N,N,N',N'-Tetrakis(2-pyridylmethyl)ethylenediamine) and maximal fluorescence by saturation with ZnSO4, resulting in the detection of free intracellular zinc in breast cancer subtypes ranging from 0.62 nM to 1.25 nM. It also allows for measuring the zinc fluxes resulting from incubation with extracellular zinc, showing differences in the zinc uptake between the non-malignant MCF10A cell line and the other cell lines. Finally, ZinPyr-1 enables the monitoring of sub-cellular distributions by fluorescence microscopy. Altogether, these properties provide a basis for the further exploration of free zinc in order to realize its full potential as a possible biomarker or even therapeutic target in breast cancer.

Systematic Studies on the Antioxidant Capacity and Volatile Compound Profile of Yellow Mealworm Larvae (T. molitor L.) under Different Drying Regimes.

The yellow mealworm (Tenebrio molitor L., Coleoptera: Tenebrionidae) is an edible insect and due to its ubiquitous occurrence and the frequency of consumption, a promising candidate for the cultivation and production on an industrial scale. Moreover, it is the first insect to be approved by EFSA 2021 following the Novel Food Regulation. Industrial production of mealworms necessitates optimized processing techniques, where drying as the first postharvest procedure is of utmost importance for the quality of the final product. The focus of the present study was to analyse the chemical composition, antioxidant capacity, volatile compound profile and colouring of mealworm larvae dried in various regimes (freeze-drying, microwave drying, infrared drying, rack-oven drying and high-frequency drying). Proximate composition and fatty acid profile were similar for all dried larvae. Freeze dried larvae were predominantly marked by lipid oxidation with significantly higher peroxide values, secondary/tertiary oxidation products in the headspace GC-MS profiles and lower antioxidant capacity. High-temperature treatment in the rack oven-and to some extent also infrared or microwave drying-led to mealworm larvae darkening and the appearance of volatile Maillard secondary products such as 2-methylpropanoic acid, 2-/3-methylbutanoic acid and alkylpyrazines. High-frequency drying as a new emerging technology in insect processing was the most cost-effective method with energy costs of solely 0.09 Є/kg T. molitor L. leading to final larval material characterized by both lipid oxidation and nonenzymatic Maillard-browning.

A 2,7-dichlorofluorescein derivative to monitor microcalcifications.

Herein, we report the crystal structure of 2,7-dichlorofluorescein methyl ester (DCF-ME) and its fluorescence response to hydroxyapatite binding. The reported fluorophore is very selective for staining the bone matrix and provides turn-on fluorescence upon hydroxyapatite binding. The reported fluorophore can readily pass the cell membrane of the C2C12 cell line, and it is non-toxic for the cell line. The reported fluorophore DCF-ME may find applications in monitoring bone remodeling and microcalcification as an early diagnosis tool for breast cancer and age-related macular degeneration.

Time- and Zinc-Related Changes in Biomechanical Properties of Human Colorectal Cancer Cells Examined by Atomic Force Microscopy.

Monitoring biomechanics of cells or tissue biopsies employing atomic force microscopy (AFM) offers great potential to identify diagnostic biomarkers for diseases, such as colorectal cancer (CRC). Data on the mechanical properties of CRC cells, however, are still scarce. There is strong evidence that the individual zinc status is related to CRC risk. Thus, this study investigates the impact of differing zinc supply on the mechanical response of the in vitro CRC cell lines HT-29 and HT-29-MTX during their early proliferation (24-96 h) by measuring elastic modulus, relaxation behavior, and adhesion factors using AFM. The differing zinc supply severely altered the proliferation of these cells and markedly affected their mechanical properties. Accordingly, zinc deficiency led to softer cells, quantitatively described by 20-30% lower Young's modulus, which was also reflected by relevant changes in adhesion and rupture event distribution compared to those measured for the respective zinc-adequate cultured cells. These results demonstrate that the nutritional zinc supply severely affects the nanomechanical response of CRC cell lines and highlights the relevance of monitoring the zinc content of cancerous cells or biopsies when studying their biomechanics with AFM in the future.

Dietary zinc enrichment reduces the cadmium burden of mealworm beetle (Tenebrio molitor) larvae.

The industrial production of Tenebrio molitor L. requires optimized rearing and processing conditions to generate insect biomass with high nutritional value in large quantities. One of the problems arising from processing is a tremendous loss in mineral accessibility, affecting, amongst others, the essential trace element Zn. As a feasible strategy this study investigates Zn-enrichment of mealworms during rearing to meet the nutritional requirements for humans and animals. Following feeding ZnSO4-spiked wheat bran substrates late instar mealworm larvae were evaluated for essential micronutrients and human/animal toxic elements. In addition, growth rate and viability were assessed to select optimal conditions for future mass-rearing. Zn-feeding dose-dependently raised the total Zn content, yet the Znlarvae/Znwheat bran ratio decreased inversely related to its concentration, indicating an active Zn homeostasis within the mealworms. The Cu status remained stable, suggesting that, in contrast to mammals, the intestinal Cu absorption in mealworm larvae is not affected by Zn. Zn biofortification led to a moderate Fe and Mn reduction in mealworms, a problem that certainly can be overcome by Fe/Mn co-supplementation during rearing. Most importantly, Zn feeding massively reduced the levels of the human/animal toxicant Cd within the mealworm larvae, a technological novelty of outstanding importance to be implemented in the future production process to ensure the consumer safety of this edible insect species.

Fluorescent Arylphosphonic Acids: Synergic Interactions between Bone and the Fluorescent Core.

Herein, we report the third generation of fluorescent probes (arylphosphonic acids) to target calcifications, particularly hydroxyapatite (HAP). In this study, we use highly conjugated porphyrin-based arylphosphonic acids and their diesters, namely 5,10,15,20-tetrakis[m-(diethoxyphosphoryl)phenyl]porphyrin (m-H8 TPPA-OEt8 ) and 5,10,15,20-tetrakis [m-phenylphosphonic acid]porphyrin (m-H8 TPPA), in comparison with their positional isomers 5,10,15,20-tetrakis[p-(diisopropoxyphosphoryl)phenyl]porphyrin (p-H8 TPPA-iPr8 ) and 5,10,15,20-tetrakis [p-phenylphosphonic acid]porphyrin (p-H8 TPPA), which have phosphonic acid units bonded to sp2 carbon atoms of the fluorescent core. The conjugation of the fluorescent core is thus extended to the (HAP) through sp2 -bonded -PO3 H2 units, which generates increased fluorescence upon HAP binding. The resulting fluorescent probes are highly sensitive towards the HAP in rat bone sections. The designed probes are readily taken up by cells. Due to the lower reported toxicity of (p-H8 TPPA), these probes could find applications in monitoring bone resorption or adsorption, or imaging vascular or soft tissue calcifications for breast cancer diagnosis etc.

Sarcosine is a prostate epigenetic modifier that elicits aberrant methylation patterns through the SAMe-Dnmts axis.

DNA hypermethylation is one of the most common epigenetic modifications in prostate cancer (PCa). Several studies have delineated sarcosine as a PCa oncometabolite that increases the migration of malignant prostate cells while decreasing their doubling time. Here, we show that incubation of prostate cells with sarcosine elicited the upregulation of sarcosine N-demethylation enzymes, sarcosine dehydrogenase and pipecolic acid oxidase. This process was accompanied by a considerable increase in the production of the major methyl-donor S-adenosylmethionine (SAMe), together with an elevation of cellular methylation potential. Global DNA methylation analyses revealed increases in methylated CpG islands in distinct prostate cell lines incubated with sarcosine, but not in cells of nonprostate origin. This phenomenon was further associated with marked upregulation of DNA methyltransferases (Dnmts). Epigenetic changes were recapitulated through blunting of Dnmts using the hypomethylating agent 5-azacytidine, which was able to inhibit sarcosine-induced migration of prostate cells. Moreover, spatial mapping revealed concomitant increases in sarcosine, SAMe and Dnmt1 in histologically confirmed malignant prostate tissue, but not in adjacent or nonmalignant tissue, which is in line with the obtained in vitro data. In summary, we show here for the first time that sarcosine acts as an epigenetic modifier of prostate cells and that this may contribute to its oncometabolic role.

Sulforaphane inhibits damage-induced poly (ADP-ribosyl)ation via direct interaction of its cellular metabolites with PARP-1.

SCOPE: The isothiocyanate sulforaphane, a major breakdown product of the broccoli glucosinolate glucoraphanin, has frequently been proposed to exert anticarcinogenic properties. Potential underlying mechanisms include a zinc release from Kelch-like ECH-associated protein 1 followed by the induction of detoxifying enzymes. This suggests that sulforaphane may also interfere with other zinc-binding proteins, e.g. those essential for DNA repair. Therefore, we explored the impact of sulforaphane on poly (ADP-ribose)polymerase-1 (PARP-1), poly (ADP-ribosyl)ation (PARylation), and DNA single-strand break repair (SSBR) in cell culture. METHODS AND RESULTS: Immunofluorescence analyses showed that sulforaphane diminished H2 O2 -induced PARylation in HeLa S3 cells starting from 15 µM despite increased lesion induction under these conditions. Subcellular experiments quantifying the damage-induced incorporation of (32) P-ADP-ribose by PARP-1 displayed no direct impact of sulforaphane itself, but cellular metabolites, namely the glutathione conjugates of sulforaphane and its interconversion product erucin, reduced PARP-1 activity concentration dependently. Interestingly, this sulforaphane metabolite-induced PARP-1 inhibition was prevented by thiol compounds. PARP-1 is a stimulating factor for DNA SSBR-rate and we further demonstrated that 25 µM sulforaphane also delayed the rejoining of H2 O2 -induced DNA strand breaks, although this might be partly due to increased lesion frequencies. CONCLUSION: Sulforaphane interferes with damage-induced PARylation and SSBR, which implies a sulforaphane-dependent impairment of genomic stability.

MNNG-induced cell death is controlled by interactions between PARP-1, poly(ADP-ribose) glycohydrolase, and XRCC1.

PARP-1 (poly(ADP-ribose) polymerases) modifies proteins with poly(ADP-ribose), which is an important signal for genomic stability. ADP-ribose polymers also mediate cell death and are degraded by poly(ADP-ribose) glycohydrolase (PARG). Here we show that the catalytic domain of PARG interacts with the automodification domain of PARP-1. Furthermore, PARG can directly down-regulate PARP-1 activity. PARG also interacts with XRCC1, a DNA repair factor that is recruited by DNA damage-activated PARP-1. We investigated the role of XRCC1 in cell death after treatment with supralethal doses of the alkylating agent MNNG. Only in XRCC1-proficient cells MNNG induced a considerable accumulation of poly(ADP-ribose). Similarly, extracts of XRCC1-deficient cells produced large ADP-ribose polymers if supplemented with XRCC1. Consequently, MNNG triggered in XRCC1-proficient cells the translocation of the apoptosis inducing factor from mitochondria to the nucleus followed by caspase-independent cell death. In XRCC1-deficient cells, the same MNNG treatment caused non-apoptotic cell death without accumulation of poly(ADP-ribose). Thus, XRCC1 seems to be involved in regulating a poly(ADP-ribose)-mediated apoptotic cell death.

Roles of DNA ligase III and XRCC1 in regulating the switch between short patch and long patch BER.

Damaged DNA bases are repaired by base excision repair (BER), which can proceed via two pathways: short patch and long patch BER. During the latter, a stretch of several nucleotides is replaced by strand displacement DNA synthesis. We recently demonstrated that the ATP concentration may govern the decision between these BER sub-pathways. Employing a reconstituted BER complex containing among others DNA polymerase beta (Pol beta), DNA ligase III (Lig III) and XRCC1, here we show that Lig III and XRCC1 are essential mediators of this regulation. XRCC1 stimulates Pol beta strand displacement activity and releases inhibition of Pol beta by DNA-bound Lig III if ligation is prevented. XRCC1 is thus able to strongly promote strand displacement and long patch BER under conditions of ATP shortage. If sufficient ATP is available, ligation by Lig III prevents strand displacement, leading to short patch BER. Ligation-inactive mutants of Lig III do not prevent strand displacement by Pol beta under the same conditions. Consequently, the preferred use of short patch BER depends on the ligation competence of Lig III. Accordingly, lowering the levels of the XRCC1/Lig III complex in HeLa cells using siRNA decreases ligation capacity but enhances Pol beta-dependent DNA synthesis.

Effect of surface modified liposomes on the aggregation of platelets and tumor cells.

Metastasis is still the most serious reason for the high mortality of cancer patients. It is a complex process in which platelets play a crucial role. Several attempts have been performed to inhibit the metastatic process, some of these using modified liposomes. The aggregation behaviour of human platelets and HT29 colon carcinoma cells in the presence of liposomes with a modified surface has been investigated in the present study. Liposomes (PC/CH/DMPE) were unmodified, sterically stabilized by polyethylene glycol (PEG-DSPE), or equipped with the carbohydrate ligand sialyl Lewis(X) (conjugated to PEG-DMPE or DMPE as anchor) intended to specifically compete with ligands expressed by HT29 cells. We found in vitro that an addition of surface modified liposomes to human platelets in plasma caused an up to 2.9-fold increase in platelet aggregation. In addition, when HT29 tumor cells were mixed with platelets and surface modified liposomes, the number of tumor cells found in aggregates increased significantly from 8.3 % (only tumor cells) to 30.2 %. This result was supported by fluorescence micrographs demonstrating a strong association of platelets and liposomes around the tumor cells. In addition, a clear decrease in number and a change in the distribution of metastases after intravenous injection of HT29 cells in combination with liposomes was observed in vivo. While in control mice metastases in lung, liver and in intestine were prevailing, liposomal treatment resulted in a new localization of metastases in muscles. Taking together, the ability of surface modified liposomes to enhance aggregate formation of platelets and tumor cells has been demonstrated for the first time. The capability of these vesicles to interfere with the metastatic process might have implications for the use of such liposomes for therapeutic applications.

Poly(ADP-ribosylation) and genomic stability.

Poly(ADP-ribose) polymerases (PARPs) catalyze the synthesis of ADP-ribose polymers and attach them to specific target proteins. To date, 6 members of this protein family in humans have been characterized. The best-known PARP, PARP-1, is located within the nucleus and has a major function in DNA repair but also in the execution of cell death pathways. Other PARP enzymes appear to carry out highly specific functions. Most prominently, the tankyrases modify telomere-binding proteins and thereby regulate telomere maintenance. Since only a single enzyme, poly(ADP-ribose) glycohydrolase (PARG), has been identified, which degrades poly(ADP-ribose), it is expected that this protein has important roles in PARP-mediated regulatory processes. This review summarizes recent observations indicating that poly(ADP-ribosylation) represents a major mechanism to regulate genomic stability both when DNA is damaged by exogenous agents and during cell division.

Tannins elevate the level of poly(ADP-ribose) in HeLa cell extracts.

Phenolic phytochemicals such as tannins, which are natural constituents of green tea, red wine, and other plant products, are considered to have cancer-preventive properties. An important endogenous mediator of tumorigenesis is the nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP-1). PARP-1 synthesizes polymers of ADP-ribose (PAR), which, in turn, are degraded by the catabolic enzyme poly(ADP-ribose) glycohydrolase (PARG). In the present study, we investigated the effects of tannins on the level of PAR in HeLa nuclear extracts. The addition of tannins to nuclear extracts led to a 40-fold elevation of PAR-levels. The observed increased PAR-levels resulted from inhibition of the catalytic activity of PARG. Additionally, the human PARG cDNA was cloned and the recombinant enzyme was overexpressed and isolated. Recombinant PARG was immobilized using an affinity column composed of tannins covalently linked to Sepharose beads. Finally, an interaction between immobilized PARG and endogenous PARP-1 from HeLa cell extracts is demonstrated.