Metabolomics analysis and biological investigation of three Malvaceae plants.

INTRODUCTION: Metabolomics is a fast growing technology that has effectively contributed to many plant-related sciences and drug discovery. OBJECTIVE: To use the non-targeted metabolomics approach to investigate the chemical profiles of three Malvaceae plants, namely Hibiscus mutabilis L. (Changing rose), H. schizopetalus (Dyer) Hook.f. (Coral Hibiscus), and Malvaviscus arboreus Cav. (Sleeping Hibiscus), along with evaluating their antioxidant and anti-infective potential. METHODOLOGY: Metabolic profiling was carried out using liquid chromatography coupled with high-resolution electrospray ionisation mass spectrometry (LC-HR-ESI-MS) for dereplication purposes. The chemical composition of the studied plants was further compared by principal component analysis (PCA). The antioxidant and anti-infective properties of their different extracts were correlated to their phytochemical profiles by orthogonal partial least square discriminant analysis (OPLS-DA). RESULTS: A variety of structurally different metabolites, mostly phenolics, were characterized. Comparing the distribution pattern of these tentatively identified metabolites among the studied plant species/fractions revealed the chemical uniqueness of the dichloromethane fraction of M. arboreus. Some extracts and fractions of these plants demonstrated noteworthy antioxidant and antitrypanosomal potential; the latter was partly attributed to their anti-protease activities. The active principles of these plants were pinpointed before any laborious isolation steps, to avoid the redundant isolation of previously known compounds. CONCLUSION: This study highlighted the use of the established procedure in exploring the metabolomes of these species, which could be helpful for chemotaxonomic and authentication purposes, and might expand the basis for their future phytochemical analysis. Coupling the observed biological potential with LC-MS data has also accelerated the tracing of their bioactive principles.

Metabolomic profiling and biological investigation of the marine sponge-derived bacterium Rhodococcus sp. UA13.

INTRODUCTION: Marine sponge-associated actinomycetes are potent sources of bioactive natural products of pharmaceutical significance. They also contributed to the discovery of several clinically relevant antimicrobials. OBJECTIVE: To apply the non-targeted metabolomics approach in chemical profiling of the sponge-derived bacterium Rhodococcus sp. UA13, formerly recovered from the Red Sea sponge Callyspongia aff. Implexa, along with testing for the anti-infective potential of its different fractions. METHODOLOGY: Metabolomic analysis of the crude extract was carried out using liquid chromatography with high resolution electrospray ionisation mass spectrometry (LC-HR-ESI-MS) for dereplication purposes. Besides, the three major fractions (ethyl acetate, methanol, and n-butanol) obtained by chromatographic fractionation of the crude extract were evaluated for their anti-infective properties. RESULTS: A variety of metabolites, mostly peptides, were characterised herein for the first time from the genus Rhodococcus. Among the tested samples, the n-butanol fraction showed potent inhibitory activities against Staphylococcus aureus, Candida albicans, and Trypanosoma brucei brucei with IC50 values of 9.3, 6.7, and 8.7 µg/mL, respectively, whereas only the ethyl acetate fraction was active against Chlamydia trachomatis (IC50  = 18.9 µg/mL). In contrast, both fractions did not exert anti-infective actions against Enterococcus faecalis and Leishmania major, whereas the methanol fraction was totally inactive against all the tested organisms. CONCLUSION: This study showed the helpfulness of the established procedure in metabolic profiling of marine actinomycetes using liquid chromatography mass spectrometry (LC-MS) data, which aids in reducing the complex isolation steps during their chemical characterisation. The anti-infective spectrum of their metabolites is also interestingly relevant to future drug development.

IR and IGF-1R expression affects insulin induced proliferation and DNA damage.

Diabetes mellitus type 2 is in its prediagnostic and early phase characterized by hyperinsulinemia. Previously, we pointed out hyperinsulinemia as a potential link between diabetes mellitus and the increased cancer risk that is associated with this disease through its induction of oxidative stress and DNA damage. In the present study, we address the relationship between the induction of proliferation and genomic damage in vitro in cell lines with different expression of the insulin and the IGF-1 receptors after treating the cells with insulin and the insulin analog glargine. Contribution of the IGF-1 receptor was further examined by application of the IGF-1R inhibitor ((5R,5aS,8aR,9R)-9-hydroxy-5,8,8a,9-tetrahydro-5-(3,4,5-trimethoxyphenyl)-furo[3_,4_:6,7]-naphtho[2,3-d]-1,3-dioxol-6(5aH)-one) (PPP). Insulin as well as insulin glargine stimulated cell proliferation in IGF-receptor-dominated MCF-7 cells and not in insulin receptor-dominated BT-474 cells and PPP attenuated this effect. Both insulins induced DNA damage which was reduced by PPP in MCF-7 cells only. Overall, we showed in this study that high levels of insulin and insulin glargine can enhance cell proliferation in cells which highly express IGF-1 receptor and induce DNA damage in cells with high and also in those with low IGF-1 receptor levels.

Metformin Protects Kidney Cells From Insulin-Mediated Genotoxicity In Vitro and in Male Zucker Diabetic Fatty Rats.

Hyperinsulinemia is thought to enhance cancer risk. A possible mechanism is induction of oxidative stress and DNA damage by insulin, Here, the effect of a combination of metformin with insulin was investigated in vitro and in vivo. The rationales for this were the reported antioxidative properties of metformin and the aim to gain further insights into the mechanisms responsible for protecting the genome from insulin-mediated oxidative stress and damage. The comet assay, a micronucleus frequency test, and a mammalian gene mutation assay were used to evaluate the DNA damage produced by insulin alone or in combination with metformin. For analysis of antioxidant activity, oxidative stress, and mitochondrial disturbances, the cell-free ferric reducing antioxidant power assay, the superoxide-sensitive dye dihydroethidium, and the mitochondrial membrane potential-sensitive dye 5,5',6,6'tetrachloro-1,1',3,3'-tetraethylbenzimidazol-carbocyanine iodide were applied. Accumulation of p53 and pAKT were analyzed. As an in vivo model, hyperinsulinemic Zucker diabetic fatty rats, additionally exposed to insulin during a hyperinsulinemic-euglycemic clamp, were treated with metformin. In the rat kidney samples, dihydroethidium staining, p53 and pAKT analysis, and quantification of the oxidized DNA base 8-oxo-7,8-dihydro-2'-deoxyguanosine were performed. Metformin did not show intrinsic antioxidant activity in the cell-free assay, but protected cultured cells from insulin-mediated oxidative stress, DNA damage, and mutation. Treatment of the rats with metformin protected their kidneys from oxidative stress and genomic damage induced by hyperinsulinemia. Metformin may protect patients from genomic damage induced by elevated insulin levels. This may support efforts to reduce the elevated cancer risk that is associated with hyperinsulinemia.

Two new antioxidant actinosporin analogues from the calcium alginate beads culture of sponge-associated Actinokineospora sp. strain EG49.

Marine sponge-associated actinomycetes represent an exciting new resource for the identification of new and novel natural products . Previously, we have reported the isolation and structural elucidation of actinosporins A (1) and B (2) from Actinokineospora sp. strain EG49 isolated from the marine sponge Spheciospongia vagabunda. Herein, by employing different fermentation conditions on the same microorganism, we report on the isolation and antioxidant activity of structurally related metabolites, actinosporins C (3) and D (4). The antioxidant potential of actinosporins C and D was demonstrated using the ferric reducing antioxidant power (FRAP) assay. Additionally, at 1.25 µM, actinosporins C and D showed a significant antioxidant and protective capacity from the genomic damage induced by hydrogen peroxide in the human promyelocytic (HL-60) cell line.

Signaling steps in the induction of genomic damage by insulin in colon and kidney cells.

Diabetes mellitus (DM), a disease with almost 350 million people affected worldwide, will be the seventh leading cause of death by 2030. Diabetic patients develop various types of complications, among them an increased rate of malignancies. Studies reported the strong correlation between DM and several cancer types, of which colon and kidney cancers are the most common. Hyperinsulinemia, the high insulin blood level characteristic of early diabetes type 2, was identified as a risk factor for cancer development. In previous studies, we showed that an elevated insulin level can induce oxidative stress, resulting in DNA damage in colon cells in vitro and in kidney cells in vitro and in vivo. In the present study, we elucidate the signaling pathway of insulin-mediated genotoxicity, which is effective through oxidative stress induction in colon and kidney. The signaling mechanism is starting by phosphorylation of the insulin and insulin-like growth factor-1 receptors, followed by activation of phosphatidylinositide 3-kinase (PI3K), which in turn activates AKT. Subsequently, mitochondria and nicotinamide adenine dinucleotide phosphate oxidase (NADPH) isoforms (Nox1 and Nox4 in colon and kidney, respectively) are activated for reactive oxygen species (ROS) production, and the resulting excess ROS can attack the DNA, causing DNA oxidation. We conclude that hyperinsulinemia represents an important risk factor for cancer initiation or progression as well as a target for cancer prevention in diabetic patients.

Insulin mediated DNA damage in mammalian colon cells and human lymphocytes in vitro.

Hyperinsulinemia, the medical term for elevated insulin blood levels, is characteristic for several diseases such as diabetes mellitus, obesity and polycystic ovarian syndrome. Studies reported a direct relationship between hyperinsulinemia and cancer risk, especially for colon cancer. In the present work we investigated for the first time the ability of pathophysiological concentrations of insulin to induce DNA damage in colon cells and human peripheral lymphocytes through the overproduction of reactive oxygen species (ROS). Human colon adenocarcinoma cells (HT29) showed significant elevation of DNA damage using comet assay, FPG modified comet assay and micronucleus frequency analysis upon treatment with 5nM insulin in standard protocols. Extension of the treatment to 6 days lowered the concentration needed to reach significance to 0.5nM. Insulin enhanced the cellular ROS production as examined by the oxidation of the dyes 2',7'-dichlorodihydrofluorescein diacetate (H2DCF-DA) and dihydroethidium (DHE). The radical scavenger tempol protected the cells. To investigate the sources of ROS upon insulin stimulation, apocynin and VAS2870 as NADPH oxidase inhibitors and rotenone as mitochondrial inhibitor were applied in combination with insulin, all of them leading to a reduction of the genomic damage. Studies in another human colon cancer cell line, Caco-2, rat primary colon cells and peripheral human lymphocytes treated in vitro all confirmed the effect of insulin on cellular chromatin. We conclude that pathophysiological levels of insulin can cause DNA damage, which may contribute to the induction or progression of colon cancer. Antioxidants as well as mitochondrial and NADPH oxidase inhibitors may have a cancer preventive potential under certain conditions.

Insulin-mediated oxidative stress and DNA damage in LLC-PK1 pig kidney cell line, female rat primary kidney cells, and male ZDF rat kidneys in vivo.

Hyperinsulinemia, a condition with excessively high insulin blood levels, is related to an increased cancer incidence. Diabetes mellitus is the most common of several diseases accompanied by hyperinsulinemia. Because an elevated kidney cancer risk was reported for diabetic patients, we investigated the induction of genomic damage by insulin in LLC-PK1 pig kidney cells, rat primary kidney cells, and ZDF rat kidneys. Insulin at a concentration of 5nM caused a significant increase in DNA damage in vitro. This was associated with the formation of reactive oxygen species (ROS). In the presence of antioxidants, blockers of the insulin, and IGF-I receptors, and a phosphatidylinositol 3-kinase inhibitor, the insulin-mediated DNA damage was reduced. Phosphorylation of protein kinase B (PKB or AKT) was increased and p53 accumulated. Inhibition of the mitochondrial and nicotinamide adenine dinucleotide phosphatase oxidase-related ROS production reduced the insulin-mediated damage. In primary rat cells, insulin also induced genomic damage. In kidneys from healthy, lean ZDF rats, which were infused with insulin to yield normal or high blood insulin levels, while keeping blood glucose levels constant, the amounts of ROS and the tumor protein (p53) were elevated in the high-insulin group compared with the control level group. ROS and p53 were also elevated in diabetic obese ZDF rats. Overall, insulin-induced oxidative stress resulted in genomic damage. If the same mechanisms are active in patients, hyperinsulinemia might cause genomic damage through the induction of ROS contributing to the increased cancer risk, against which the use of antioxidants and/or ROS production inhibitors might exert protective effects.

Antioxidant and anti-protease activities of diazepinomicin from the sponge-associated Micromonospora strain RV115.

Diazepinomicin is a dibenzodiazepine alkaloid with an unusual structure among the known microbial metabolites discovered so far. Diazepinomicin was isolated from the marine sponge-associated strain Micromonospora sp. RV115 and was identified by spectroscopic analysis and by comparison to literature data. In addition to its interesting preclinical broad-spectrum antitumor potential, we report here new antioxidant and anti-protease activities for this compound. Using the ferric reducing antioxidant power (FRAP) assay, a strong antioxidant potential of diazepinomicin was demonstrated. Moreover, diazepinomicin showed a significant antioxidant and protective capacity from genomic damage induced by the reactive oxygen species hydrogen peroxide in human kidney (HK-2) and human promyelocytic (HL-60) cell lines. Additionally, diazepinomicin inhibited the proteases rhodesain and cathepsin L at an IC50 of 70-90 µM. It also showed antiparasitic activity against trypomastigote forms of Trypanosoma brucei with an IC50 of 13.5 µM. These results showed unprecedented antioxidant and anti-protease activities of diazepinomicin, thus further highlighting its potential as a future drug candidate.