Jamal M. Arif

Role of Nigella sativa and a number of its antioxidant constituents towards azoxymethane‐induced genotoxic effects and colon cancer in rats

This study examined the chemopreventive effect of Nigella sativa and some of its antioxidant constituents on a number of colon cancer biomarkers in rats induced with azoxymethane (AOM). Sixty male Sprague‐Dawley rats were randomly assigned into ten subgroups: vehicle (1–5) and experimental (6–10). The rats in each group were fed one of the following diets: basal diet, (200 mg/kg) Nigella sativa, (0.2 mg/kg) selenium, (1.2 mg/kg) all‐trans‐retinol plus (100 mg/kg) dl‐α‐tocopherol and (10 mg/kg) thymoquinone, respectively. Only rats in subgroups 6–10 were injected with AOM (15 mg/kg) once per week for 2 weeks. Both groups were fed their respective diets for 5 weeks. Then they were killed and examined for colonic aberrant crypt foci (ACF). Our result showed that only vitamin supplementation was effective on ACF. Nigella sativa revealed inhibitory effects only on DNA damage (day 34) in the AOM‐treated rat group. Alternatively, selenium, thymoquinone and vitamins inhibited the MDA content in the liver. Although the exact mechanisms involved in the protective role of Nigella sativa against the initiation of colon carcinogenesis are not clearly understood, the results suggest that its inhibitory effects might depend on the combined competitive inhibition of various antioxidant constituents of this plant. Copyright

Novel Marine Compounds: Anticancer or Genotoxic?

In the past several decades, marine organisms have generously gifted to the pharmaceutical industries numerous naturally bioactive compounds with antiviral, antibacterial, antimalarial, anti-inflammatory, antioxidant, and anticancer potentials. But till date only few anticancer drugs (cytarabine, vidarabine) have been commercially developed from marine compounds while several others are currently in different clinical trials. Majority of these compounds were tested in the tumor xenograft models, however, lack of anticancer potential data in the chemical- and/or oncogene-induced pre-initiation animal carcinogenesis models might have cost some of the marine anticancer compounds an early exit from the clinical trials. This review critically discusses importance of preclinical evaluation, failure of human clinical trials with certain potential anticancer agents, the screening tests used, and choice of biomarkers.

Protective Effect of Dietary Tocotrienols against Infection and Inflammation-induced Hyperlipidemia: An In Vivo and In Silico Study

Currently used hypolipidemic drugs, Fluvastatin and Atorvastatin, act via inhibiting the rate‐limiting enzyme 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A (HMG‐CoA) reductase of the mevalonate pathway. The associated severe side‐effects of these statins led us to explore the therapeutic potentials of naturally occurring Tocomin (mixture of dietary α‐, β‐, γ‐ and δ‐tocotrienols). Tocomin (10 mg) was orally administered daily for 10 days before and 12 h after bacterial lipopolysaccharide (200 μg) or 24 h after zymosan (20 mg) or turpentine (0.5 mL) to Syrian hamsters. The data showed that Tocomin significantly reduced the levels of plasma and lipoprotein lipids, cholesterol, apoB, small dense (sd)‐LDL as well as LDL in the hyperlipidemia‐induced hamsters. Further, the mechanism of action of α‐, β‐, γ‐ and δ‐tocotrienols was validated by docking studies with HMG‐CoA reductase enzyme using the Molegro Virtual Docker. The inhibition of HMG‐CoA reductase predicted in terms of MolDockScore and interaction energy suggest the comparative potential in the descending order: Atorvastatin > Fluvastatin ~ δ > γ > β > α. The results favor the daily intake of naturally occurring tocotrienols as dietary supplement in the prevention and treatment of infection/inflammation induced dyslipidemia compared with the hypolipidemic drugs. Copyright

Carcinogen DNA adducts and the risk of colon cancer: case–control study

Abstract Colorectal cancer represents 8.5% of all tumours at the King Faisal Specialist Hospital & Research Centre. Environmental and dietary carcinogens such as polycyclic aromatic hydrocarbons (PAHs) and heterocyclic amines (HCAs) have long been suspected to play a prominent role in colon cancer aetiology. We designed a case–control study to test the hypothesis of whether or not the presence of DNA adducts can play a role in the aetiology of colon cancer. DNA adducts were measured in 24 cancerous and 20 non-cancerous tissue samples of newly diagnosed colon cancer patients by 32P-post-labelling technique. Normal tissue from 19 hospital patients served as controls. The mean levels of adducts per 1010 nucleotides in cancerous and non-cancerous tissue were 151.75±217.27 and 114.81±186.10, respectively; however, only adducts in cancerous tissue were significantly higher than controls (32.78±57.51 per 1010 nucleotides) with p-values of 0.017. No BPDE-DNA adducts were found. No relationship was found between urinary cotinine as a marker of tobacco smoke and 1-hydroxypyrene as an indicator of an individuals internal dose of PAHs and DNA adducts. In a logistic regression model, only adducts in cancerous tissue were associated with the subsequent risk of colon cancer, with an odds ratio of 3.587 (95% confidence interval 0.833–15.448) after adjustment for age and the duration of living in the current region, but of a borderline significance (p=0.086). Although it is difficult to arrive at a definite conclusion from a small dataset, our preliminary results suggest the potential role of DNA adducts in the colon carcinogenesis process. Additional studies with larger sample sizes are needed to confirm our preliminary finding. It is also important to identify the structural characterization of these unknown DNA adducts in order to have a better understanding of whether or not environmental carcinogens play a role in the aetiology of colon cancer.

Antimicrobial, antioxidant, and antimutagenic activities of selected marine natural products and tobacco cembranoids

Multidrug resistance (MDR) in microorganisms is a cause of major concern for clinicians and pharmaceutical industries. Continuous development of new antimicrobial drugs with multiple targets and potentials is expected to efficiently combat MDR in these microorganisms. In a continued exploration of new antimicrobial drug leads, 11 marine natural products, semisynthetic, or related synthetic analogs (1–11) and two tobacco cembranoids (12 and 13) were screened for their antimicrobial, antioxidant, and antimutagenic activities. Eight compounds showed varying levels of both antibacterial and antifungal activities. Compounds such as 17-O-methyllatrunculin-A, verongiaquinol, (1S,2E,4R,6R,7E,11E)-2,7,11-cembratriene-4,6-diol), and manzamine-A showed a broad spectrum of activity, inhibiting six of seven tested bacteria with zone of inhibition diameter from 9 to 30 mm. Four of these active compounds also showed antifungal activity. The findings of the in vitro time-kill assay of the most active compound, verongiaquinol, against Staphylococcus aureus indicated its subinhibitory effect at the level lower than the minimal inhibitory concentration (MIC) values (i.e., 2 and 4 µg/mL). At the MIC (8 µg/mL), bacterial cells were completely killed within 18 hours of incubation. DPPH free radical scavenging activity was demonstrated by five compounds in the range of 89.65–36.19% decolorization. Further, four compounds evaluated for their antimutagenic activity against the directly acting mutagens, methyl methanesulfonate and sodium azide, in Salmonella typhimurium strains, interestingly, showed no sign of mutagenicity. Verongiaquinol and manzamine A, in fact, reduced the mutagenicity by 50–75% at a dose of 5 µg/plate in different test strains. Our study seems to provide some novel antimicrobial leads with strong antioxidant potential and the associated ability of antimutagenicity.

Genotoxic fungicide methyl thiophanate as an oxidative stressor inducing 8-oxo-7,8-dihydro-2′-deoxyguanosine adducts in DNA and mutagenesis.

Dimethyl 4,4′ -(O-phenylene)bis(3-thioallophanate), commonly known as methyl thiophanate (MT), is a systemic fungicide and suspected carcinogen to humans. In this study, the oxidative potential of this category-III acute toxicant has been ascertained based on its capacity of inducing reactive oxygen species (ROS) and promutagenic 8-oxo-7,8-dihydro-2′ -deoxyguanosine (8-oxodG) adducts in DNA. The discernible MT dose-dependent reduction in fluorescence intensity of a cationic dye rhodamine (Rh-123) in human lymphocytes and increased fluorescence intensity of 2′,7′-Dichlorodihydro fluorescein diacetate (DCFH-DA) treated cells signifies decreased mitochondrial membrane potential (Δ Ψ m) due to intracellular ROS generation. The 32P-post-labeling assay demonstrated the MT-induced 8-oxodG adduct formation in calf thymus DNA. Thus, it is concluded that MT, as a potent oxidative stressor, produces ROS leading to mitochondrial dysfunction, oxidative DNA damage and mutagenesis.

Exploitation of in silico potential in prediction, validation and elucidation of mechanism of anti-angiogenesis by novel compounds: comparative correlation between wet lab and in silico data

This study describes in silico validation of the wet lab data from aeroplysinin-1, curcumin and halofuginone using Autodock Tools 4.0. The inhibition patterns of the vascular endothelial cell differentiation and capillary tube formation mediated by anti-angiogenic growth factors from these test compounds were found quite comparable with the in silico results using angiogenic targets (EFGR, bFGF and VEGFR-1). Successful validation of the wet lab results of the selected angiogenic targets by in silico method has led to exploit the hidden potential of in silico tools in preliminary screening of the unknown compounds for anti-angiogenic potential in a cost-effective manner.

Novel Aglycones of Steroidal Glycoalkaloids as Potent Tyrosine Kinase Inhibitors: Role in VEGF and EGF Receptors Targeted Angiogenesis

Receptor tyrosine kinases (RTKs) are crucial targets in the pathway of angiogenesis and the currently used antiangiogenic drugs have limited efficiency with associated toxicity. In our continuous search for new and potent multitarget anti-angiogenic lead compounds, we have conducted in-silico interaction and inhibition studies of the selected aglycones of steroidal glycoalkaloids (GAs) and selected anti-angiogenic drugs, against the EGFR, VEGFR-1, VEGFR-2 using the AutoDock Tools 4.0 and other online bioinformatics softwares. The docking results have been interpreted in terms of binding energies (kcal/mol) and inhibition constant ( M). In our study, aglycones [solanidine (solanid-5-en-3 -ol), solasodine (Solasod-5-en-3 -ol) and tomatidine (5 -Tomatidan-3 -ol)] gave promising and comparable results with the antiangiogenic drugs (Gefitinib, Lapatinib, Axitinib and Motasenib). All the test aglycones expressed significant inhibition against RTKs under study, but tomatidine emerged as a most potent multi-target inhibitor of RTKs. The inhibition constant of tomatidine was observed almost 2-folds less compared to Gefitinib targeting against EGFR and 5-folds less to Axitinib, the standard drug against VEGFR-2. Moreover, these aglycones fulfilled the drug likeliness properties, when analyzed by Lipinski’s Rule of Five. In addition to this, the in-silico toxicity studies of aglycones remarkably showed no mutagenecity and tumorigenecity compared to the standard drugs. Our results for the first time report that these novel aglycones of GAs may be significant lead compounds in the development of new and effective multi-target antiangiogenic drugs with the least or no toxicity. In addition, the wet lab experiments are underway to support these in-silico

ROLE OF INTERMEDIARY BIOMARKERS IN DETERMINING THE ANTICANCER EFFICACY OF MARINE COMPOUNDS

In the present study, two of the probable an umor marine compounds, manzamine A and sarcophine, were screened using benzo[a]pyrene (BP)-derived DNA adduct formation in MCF-7 cells as intermediary biomarker. Briefly, MCF-7 cells were treated with the compounds for 24 h followed by treatment with BP (0.5 μM). After 24h incubation, cellular DNA was isolated and analyzed for BP-derived DNA adducts by 32P-postlabeling technique. Manzamine A and sarcophine increased the BP-DNA adducts by 2 to 4-folds. Further, manzamine A (50 μM) substantially down regulated the expression of p53 while sarcophine (50 μM) slightly induced the level of p21. The residual DNA repair ability was almost completely abolished by manzamine A while sarcophine was ineffective. Based on our preliminary results, these compounds may be classified as potential genotoxic.

Evaluation and Elucidation Studies of Natural Aglycones for Anticancer Potential using Apoptosis-Related Markers: An In silico Study

Exposure to exogenous and endogenous chemicals and subsequent cellular and molecular changes has been linked to enhanced cell proliferation and restricted apoptosis phenomenon. Though in the past decades numerous anticancer drugs inducing programmed cell death in cancer cells by targeting specific apoptotic markers have reached the market, they have been allied with unwanted side effects, ranging from mild to severe toxicity. With further understanding on the functional mechanism of p53 and MDM2 in apoptosis and in our continuous search for new and potent multi-target anticancer lead compounds, we have carried out molecular docking and inhibition studies of the selected aglycones along with selected anticancer leads, against the specific apoptotic and cell cycle markers using AutoDock Tools 4.0 and other computational softwares. The docking results have been analyzed in terms of binding energies (kcal/mol) and inhibition constant (µM). The study clearly proposes our aglycones [solanidine (Solanid-5-en-3β-ol), solasodine (Solasod-5-en-3β-ol), and tomatidine (5α-Tomatidan-3β-ol)] induce apoptosis by inhibiting the p53–MDM2 complex, p21Waf1/Cip1, and Bcl-2 proteins, which were even found comparable with the anticancer drugs nutlin and/or halofuginone. The work further emphasizes that the individual molecular targets such as BAX and Bcl-2 may result in misleading data at any level; however, ratio of responses to BAX and Bcl-2 shall be considered for better clue about a compound to be pro- or anti-apoptotic.