Compounds derived from Humulus lupulus inhibit SARS-CoV-2 papain-like protease and virus replication.

BACKGROUND: Selected natural compounds exhibit very good antiviral properties. Especially, the medicinal plant Humulus lupulus (hop) contains several secondary plant metabolites some of which have previously shown antiviral activities. Among them, the prenylated chalcone xanthohumol (XN) demonstrated to be a potent inhibitor of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro). HYPOTHESIS/PURPOSE: Following the finding that xanthohumol (XN) is a potent inhibitor of SARS-CoV-2 Mpro, the effect of XN and its major derivatives isoxanthohumol (IXN), 6-prenylnaringenin (6-PN), and 8-prenylnaringenin (8-PN) from hops on SARS-CoV-2 papain-like protease (PLpro) were investigated. STUDY DESIGN: The modulatory effect of the hop compounds on PLpro were studied first in silico and then in vitro. In addition, the actual effect of hop compounds on the replication of SARS-CoV-2 in host cells was investigated. METHODS: In silico docking analysis was used to predict the binding affinity of hop compounds to the active site of PLpro. A recombinant PLpro was cloned, purified, characterized, and analyzed by small-angle X-ray scattering (SAXS), deISGylation assays, and kinetic analyses. Antiviral activity of hop compounds was assessed using the fluorescently labeled wildtype SARS-CoV-2 (icSARS-CoV-2-mNG) in Caco-2 host cells. RESULTS: Our in silico docking suggests that the purified hop compounds bind to the active site of SARS-CoV-2 PLpro blocking the access of its natural substrates. The hop-derived compounds inhibit SARS-CoV-2 PLpro with half maximal inhibitory concentration (IC50) values in the range of 59-162 µM. Furthermore, we demonstrate that XN and 6-PN, in particular, impede viral replication with IC50 values of 3.3 µM and 7.3 µM, respectively. CONCLUSION: In addition to the already known inhibition of Mpro by XN, our results show, for the first time, that hop-derived compounds target also SARS-CoV-2 PLpro which is a promising therapeutic target as it contributes to both viral replication and modulation of the immune system. These findings support the possibility to develop new hop-derived antiviral drugs targeting human coronaviruses.

High-Dose Ascorbate in Combination with Anti-PD1 Checkpoint Inhibition as Treatment Option for Malignant Melanoma.

Ascorbate acts as a prooxidant when administered parenterally at high supraphysiological doses, which results in the generation of hydrogen peroxide in dependence on oxygen. Most cancer cells are susceptible to the emerging reactive oxygen species (ROS). Accordingly, we evaluated high-dose ascorbate for the treatment of the B16F10 melanoma model. To investigate the effects of ascorbate on the B16F10 cell line in vitro, viability, cellular impedance, and ROS production were analyzed. In vivo, C57BL/6NCrl mice were subcutaneously injected into the right flank with B16F10 cells and tumor-bearing mice were treated intraperitoneally with ascorbate (3 g/kg bodyweight), immunotherapy (anti-programmed cell death protein 1 (PD1) antibody J43; 2 mg/kg bodyweight), or both treatments combined. The efficacy and toxicity were analyzed by measuring the respective tumor sizes and mouse weights accompanied by histological analysis of the protein levels of proliferating cell nuclear antigen (Pcna), glucose transporter 1 (Glut-1), and CD3. Treatment of B16F10 melanoma-carrying mice with high-dose ascorbate yielded plasma levels in the pharmacologically effective range, and ascorbate showed efficacy as a monotherapy and when combined with PD1 inhibition. Our data suggest the applicability of ascorbate as an additional therapeutic agent that can be safely combined with immunotherapy and has the potential to potentiate anti-PD1-based immune checkpoint blockades.

Therapeutic Efficacy of Pharmacological Ascorbate on Braf Inhibitor Resistant Melanoma Cells In Vitro and In Vivo.

High-dose ascorbate paradoxically acts as a pro-oxidant causing the formation of hydrogen peroxide in an oxygen dependent manner. Tumor cells (in particular melanoma cells) show an increased vulnerability to ascorbate induced reactive oxygen species (ROS). Therefore, high-dose ascorbate is a promising pharmacological approach to treating refractory melanomas, e.g., with secondary resistance to targeted BRAF inhibitor therapy. BRAF mutated melanoma cells were treated with ascorbate alone or in combination with the BRAF inhibitor vemurafenib. Viability, cell cycle, ROS production, and the protein levels of phospho-ERK1/2, GLUT-1 and HIF-1α were analyzed. To investigate the treatment in vivo, C57BL/6NCrl mice were subcutaneously injected with D4M.3A (BrafV600E) melanoma cells and treated with intraperitoneal injections of ascorbate with or without vemurafenib. BRAF mutated melanoma cell lines either sensitive or resistant to vemurafenib were susceptible to the induction of cell death by pharmacological ascorbate. Treatment of BrafV600E melanoma bearing mice with ascorbate resulted in plasma levels in the pharmacologically active range and significantly improved the therapeutic effect of vemurafenib. We conclude that intravenous high-dose ascorbate will be beneficial for melanoma patients by interfering with the tumor's energy metabolism and can be safely combined with standard melanoma therapies such as BRAF inhibitors without pharmacological interference.

Ex Vivo Evaluation of the Sepsis Triple Therapy High-Dose Vitamin C in Combination with Vitamin B1 and Hydrocortisone in a Human Peripheral Blood Mononuclear Cells (PBMCs) Model.

Sepsis is an extremely complex clinical syndrome, usually involving an excessive inflammatory response including an overshooting cytokine release that damages tissue and organs of the patient. Due to the severity of this condition, it is estimated that over 11 million people die from sepsis each year. Despite intensive research in the field, there is still no specific therapy for sepsis. Many sepsis patients show a marked deficiency of vitamin C. 9 out of 10 sepsis patients have a hypovitaminosis C, and every third patient even shows a clinical deficiency in the scurvy range. In addition, low vitamin C levels of intensive care sepsis patients correlate with a higher need for vasopressors, higher Sequential Organ Failure Assessment (SOFA) scores, and increased mortality. Based on this observation and the conducted clinical trials using vitamin C as sepsis therapy in intensive care patients, the aim of the present ex vivo study was to evaluate the effects of high-dose vitamin C alone and in a triple combination supplemented with vitamin B1 (thiamine) and hydrocortisone on the lipopolysaccharide (LPS)-induced cytokine response in peripheral blood mononuclear cells (PBMCs) from healthy human donors. We found that all corticosteroid combinations strongly reduced the cytokine response on RNA- and protein levels, while high-dose vitamin C alone significantly diminished the PBMC mediated secretion of the cytokines interleukin (IL)-10, IL-23, and monocyte chemo-attractant protein (MCP-1), which mediate the inflammatory response. However, vitamin C showed no enhancing effect on the secretion of further cytokines studied. This data provides important insights into the possible immunomodulatory function of vitamin C in an ex vivo setting of human PBMCs and the modulation of their cytokine profile in the context of sepsis. Since vitamin C is a vital micronutrient, the restoration of physiologically adequate concentrations should be integrated into routine sepsis therapy, and the therapeutic effects of supraphysiological concentrations of vitamin C in sepsis patients should be further investigated in clinical trials.

Nutritional immunology: function of natural killer cells and their modulation by resveratrol for cancer prevention and treatment.

Natural killer (NK) cells as part of the innate immune system represent the first line of defence against (virus-) infected and malignantly transformed cells. The emerging field of nutritional immunology focuses on compounds featuring immune-modulating activities in particular on NK cells, which e.g. can be exploited for cancer prevention and treatment. The plant-based nutrition resveratrol is a ternary hydroxylated stilbene, which is present in many foods and beverages, respectively. In humans it comprises a large variety of distinct biological activities. Interestingly, resveratrol strongly modulates the immune response including the activity of NK cells. This review will give an overview on NK cell functions and summarize the resveratrol-mediated modulation thereof.

Prenylated chalcones and flavonoids for the prevention and treatment of cancer.

Prenylated chalcones and flavonoids gained increasing attention not only in nutrition but also in cancer prevention because of their biological and molecular activities in humans, which have been extensively investigated in vitro or in preclinical studies. These naturally occurring compounds exhibit antioxidant effects, modulate metabolism of carcinogens by inhibition of distinct phase 1 metabolic enzymes and activation of phase 2 detoxifying enzymes, and display antiinflammatory properties. In particular, their potential to prevent proliferation of tumor cells is noteworthy. Some representatives of this subclass of secondary plant compounds exert pronounced anti-tumor-initiating capacities and directly inhibit growth of cancer cells, whereas their toxic effects on healthy tissues are remarkably low. These promising pharmacologic characteristics are countered by low ingestion, low bioavailability, and little knowledge of their metabolism. This review focuses on the great potential of these plant- and nutrient-derived compounds for cancer prevention and therapy. Provided here is a comprehensive summary of the current knowledge and inherent modes of action, focusing on the prenylated chalcones xanthohumol, desmethylxanthohumol, and xanthogalenol, as well as the prenylated flavonoids isoxanthohumol, 6-prenylnaringenin, 8-prenylnaringenin, 6-geranylnaringenin, 8-geranylnaringenin, and pomiferin.

Molecular mechanisms of pharmacological doses of ascorbate on cancer cells.

Intravenous application of high-dose ascorbate (vitamin C) has been used in complementary medicine since the 1970s to treat cancer patients. In recent years it became evident that high-dose ascorbate in the millimolar range bears selective cytotoxic effects on cancer cells in vitro and in vivo. This anticancer effect is dose dependent, catalyzed by serum components and mediated by reactive oxygen species and ascorbyl radicals, making ascorbate a pro-oxidative pro-drug that catalyzes hydrogen peroxide production in tissues instead of acting as a radical scavenger. It further depends on HIF-1 signaling and oxygen pressure, and shows a strong epigenetic signature (alteration of DNA-methylation and induction of tumor-suppressing microRNAs in cancer cells). The detailed understanding of ascorbate-induced antiproliferative molecular mechanisms warrants in-depth preclinical evaluation in cancer-bearing animal models for the optimization of an efficacious therapy regimen (e.g., combination with hyperbaric oxygen or O2-sensitizers) that subsequently need to be evaluated in clinical trials.

Anti-proliferative activity of hop-derived prenylflavonoids against human cancer cell lines.

Flavonoids form a substantial group of secondary plant metabolites that display several health-promoting effects. Therefore, prenylflavonoids, a subclass of flavonoids, have attracted increasing attention. Here, we investigated the possible anti-cancer potential of 6-prenylnaringenin (6-PN) and 8-prenylnaringenin (8-PN), two prenylflavonoids present in hops and beer and demonstrate an unexpectedly pronounced, dose-dependent reduction of cellular proliferation of human PC-3 prostate cancer and UO.31 renal carcinoma cells upon treatment. Based on these findings 6-PN and 8-PN are currently further clinically evaluated in detail.

Epigenetic impacts of ascorbate on human metastatic melanoma cells.

In recent years, increasing evidence has emerged demonstrating that high-dose ascorbate bears cytotoxic effects on cancer cells in vitro and in vivo, making ascorbate a pro-oxidative drug that catalyzes hydrogen peroxide production in tissues instead of acting as a radical scavenger. This anticancer effect of ascorbate is hypoxia-inducible factor-1α- and O2-dependent. However, whether the intracellular mechanisms governing this effect are modulated by epigenetic phenomena remains unknown. We treated human melanoma cells with physiological (200 µM) or pharmacological (8 mM) ascorbate for 1 h to record the impact on DNA methyltransferase (DNMT)-activity, histone deacetylases (HDACs), and microRNA (miRNA) expression after 12 h. The results were analyzed with the MIRUMIR online tool that estimates the power of miRNA to serve as potential biomarkers to predict survival of cancer patients. FACS cell-cycle analyses showed that 8 mM ascorbate shifted BLM melanoma cells toward the sub-G1 fraction starting at 12 h after an initial primary G2/M arrest, indicative for secondary apoptosis induction. In pharmacological doses, ascorbate inhibited the DNMT activity in nuclear extracts of MeWo and BLM melanoma cells, but did not inhibit human HDAC enzymes of classes I, II, and IV. The expression of 151 miRNAs was altered 12 h after ascorbate treatment of BLM cells in physiological or pharmacological doses. Pharmacological doses up-regulated 32 miRNAs (≥4-fold) mainly involved in tumor suppression and drug resistance in our preliminary miRNA screening array. The most prominently up-regulated miRNAs correlated with a significantly increased overall survival of breast cancer or nasopharyngeal carcinoma patients of the MIRUMIR database with high expression of the respective miRNA. Our results suggest a possible epigenetic signature of pharmacological doses of ascorbate in human melanoma cells and support further pre-clinical and possibly even clinical evaluation of ascorbate for melanoma therapy.

The ROS-induced cytotoxicity of ascorbate is attenuated by hypoxia and HIF-1alpha in the NCI60 cancer cell lines.

Intravenous application of high-dose ascorbate is used in complementary palliative medicine to treat cancer patients. Pharmacological doses of ascorbate in the mM range induce cytotoxicity in cancer cells mediated by reactive oxygen species (ROS), namely hydrogen peroxide and ascorbyl radicals. However, little is known about intrinsic or extrinsic factors modulating this ascorbate-mediated cytotoxicity. Under normoxia and hypoxia, ascorbate IC50 values were determined on the NCI60 cancer cells. The cell cycle, the influence of cobalt chloride-induced hypoxia-inducible factor-1α (HIF-1α) and the glucose transporter 1 (GLUT-1) expression (a pro-survival HIF-1α-downstream-target) were analysed after ascorbate exposure under normoxic and hypoxic conditions. The amount of ascorbyl radicals increased with rising serum concentrations. Hypoxia (0.1% O2 ) globally increased the IC50 of ascorbate in the 60 cancer cell lines from 4.5 ± 3.6 mM to 10.1 ± 5.9 mM (2.2-fold increase, P < 0.001, Mann-Whitney t-test), thus inducing cellular resistance towards ascorbate. This ascorbate resistance depended on HIF-1α-signalling, but did not correlate with cell line-specific expression of the ascorbate transporter GLUT-1. However, under normoxic and hypoxic conditions, ascorbate treatment at the individual IC50 reduced the expression of GLUT-1 in the cancer cells. Our data show a ROS-induced, HIF-1α- and O2 -dependent cytotoxicity of ascorbate on 60 different cancer cells. This suggests that for clinical application, cancer patients should additionally be oxygenized to increase the cytotoxic efficacy of ascorbate.