Antiproliferative and Antiangiogenic Properties of New VEGFR-2-targeting 2-thioxobenzo[g]quinazoline Derivatives (In Vitro).

A series of 3-ethyl(methyl)-2-thioxo-2,3-dihydrobenzo[g]quinazolines (1-17) were synthesized, characterized, and evaluated in vitro for their antiangiogenesis VEGFR-2-targeting, antiproliferative, and antiapoptotic activities against breast MCF-7 and liver HepG2 cells. Flow cytometry was used to determine cancer-cell cycle distributions, and apoptosis was detected using annexin-V-FITC (V) and propidium iodide (PI) dyes. Fluorescence microscopy, in combination with Hoechst staining was used to detect DNA fragmentation. Most of the tested benzo[g]quinazolines demonstrated promising activity (IC50 = 8.8 ± 0.5-10.9 ± 0.9 µM) and (IC50 = 26.0 ± 2.5-40.4 ± 4.1 µM) against MCF-7 and HepG2, respectively. Doxorubicin was used as a reference drug. Compounds 13-15 showed the highest activity against both cancer cell lines. Differential effects were detected by cell-cycle analysis, indicating similarities in the actions of 13 and 14 against both MCF7 and HepG2, involving the targeting of G1 and S phases, respectively. Compound 15 showed similar indices against both cells, indicating that its cytotoxicity toward the examined cancer cells could be unselective. Interestingly, 14 and 15 showed the highest apoptosis (30.76% and 25.30%, respectively) against MCF-7. The DNA fragmentation results agreed well with the apoptosis detected by flow cytometry. In terms of antiangiogenesis activity, as derived from VEGFR-2 inhibition, 13 and 15 were comparable to sorafenib and effected 1.5- and 1.4-fold inhibition relative to the standard sorafenib. A docking study was conducted to investigate the interaction between the synthesized benzo[g]quinazolines and the ATP-binding site within the catalytic domain of VEGFR-2.

Differential effects of c-myc and ABCB1 silencing on reversing drug resistance in HepG2/Dox cells.

Multidrug resistance (MDR) in various kinds of cancers represents a true obstacle which hinders the successes of most of current available chemotherapies. ATP-binding cassette (ABC) trasporter proteins have been shown to contribute to the majority of MDR in various types of malignancies. c-myc has recently been reported to participate, at least partly, in MDR to some types of cancers. This study aimed to test whether c-myc could play a role, solely or with coordination with other ABCs, in the resistance of HepG2 cells to doxorubicin (Dox). MDR has been induced in wild-type HepG2 and has been verified both on gene and protein levels. Various assays including efflux assays as well as siRNA targeting ABCB1 and c-myc have been employed to explore the role of both candidate molecules in MDR in HepG2. Results obtained, with regard to ABCB1 silencing on HepG2/Dox cells, have shown that ABCB1-deficient cells exhibited a significant reduction in ABCC1 expression as compared to ABCB1-sufficient cells. However, these cells did not show a significant reduction in other tested ABCs (ABCC5 and ABCC10) while c-myc silencing had no significant effect on any of the studied ABCs. Moreover, silencing of ABCB1 on HepG2 significantly increased fluorescent calcein retention in HepG2 cells as compared to the control cells while downregulation of c-myc did not have any effect on fluorescent calcein retention. Altogether, this work clearly demonstrates that c-myc has no role in MDR of HepG2 to Dox which has been shown to be ABCB1-mediated in a mechanism which might involve ABCC1.

In-vitro evaluation of selected Egyptian traditional herbal medicines for treatment of Alzheimer disease.

BACKGROUND: Egyptians recognized the healing power of herbs and used them in their medicinal formulations. Nowadays, "Attarin" drug shops and the public use mainly the Unani medicinal system for treatment of their health problems including improvement of memory and old age related diseases. Numerous medicinal plants have been described in old literature of Arabic traditional medicine for treatment of Alzheimer's disease (AD) (or to strengthen memory). METHODS: In this study, some of these plants were evaluated against three different preliminary bioassays related to AD to explore the possible way of their bio-interaction. Twenty three selected plants were extracted with methanol and screened in vitro against acetylcholinesterase (AChE) and cycloxygenase-1 (COX-1) enzymes. In addition, anti-oxidant activity using DPPH was determined. RESULTS: Of the tested plant extracts; Adhatoda vasica and Peganum harmala showed inhibitory effect on AChE at IC50 294 µg/ml and 68 µg/ml respectively. Moreover, A. vasica interacted reversibly with the enzyme while P. harmala showed irreversible inhibition. Ferula assafoetida (IC50 3.2 µg/ml), Syzygium aromaticum (34.9 µg/ml) and Zingiber officinalis (33.6 µg/ml) showed activity against COX-1 enzyme. Potent radical scavenging activity was demonstrated by three plant extracts Terminalia chebula (EC50 2.2 µg/ml), T. arjuna (3.1 µg/ml) and Emblica officinalis (6.3 µg/ml). CONCLUSION: Interestingly, differential results have been obtained which indicate the variability of the mode of actions for the selected plants. Additionally, the reversible interaction of A. vasica against AChE and the potent activity of F. assafoetida against COX-1 make them effective, new and promising agents for treatment of AD in the future, either as total extracts or their single bioactive constituents.

Bioactive compounds from Matricaria chamomilla: structure identification, in vitro antiproliferative, antimigratory, antiangiogenic, and antiadenoviral activities.

During our exploring the anticancer activity of some medicinal plants and their major metabolites, the aerial parts of the Egyptian Matricaria chamomilla (flowers and stems) were studied. GC-MS analysis of the organic soluble extracts of the flowers and stems fractions revealed the presence of 43 and 45 compounds, respectively. Individual chromatographic purification of the flowers and stems' extracts afforded three major compounds. Structures of these compounds were identified by 1D- and 2D-NMR and HRESI-MS spectroscopic data as bisabolol oxide A (1) and (E)-tonghaosu (2) (as mixture of ratio 2:1) from the flowers extract, meanwhile apigenin-7-ß-d-glucoside (3) from the stems fraction. Biologically, the chamomile extracts announced significant antiproliferative activities exceeded in potency by ∼1.5 fold in case of the stem, recording GI50 13.16 and 17.04 µg/mL against Caco-2 and MCF-7, respectively. Both fractions were approximately equipotent against the migration of the same cell type down to 10 µg/mL together, compounds 1, 2 but not 3, showed considerable growth inhibition of the same cells at GI50 13.36 and 11.83 µg/mL, respectively. Interestingly, they were able to suppress Caco-2 colon cancer cells migration at 5.8 µg/mL and potently inactivate the VEGFR2 angiogenic enzyme (1.5-fold relative to sorafenib. The obtained compounds and corresponding chamomile extracts were evaluated against Adeno-7 virus, revealing that both chamomiles' extracts (flowers and stems) and their corresponding obtained compounds (1-3) were potent in their depletion to the Adeno 7 infectivity titer, however, the flower extract and compounds 1-2 were more effective than those of the stem extract and its end-product (3).

Monascin and monascinol, azaphilonoid pigments from Mortierella polycephala AM1: in silico and in vitro targeting of the angiogenic VEGFR2 kinase.

The fungus, Mortierella polycephala is one of the most productive sources of anticancer bioactive compounds namely those of pigment nature. During our investigation of the produced bioactive metabolites by the terrestrial M. polycephala AM1 isolated from Egyptian poultry feather waste, two main azaphilonoid pigments, monascin (1) and monascinol (2) were obtained as major products; their structures were identified by 1D (1H&13C) and 2D (1H-1H COSY, HMBC) NMR and HRESI-MS spectroscopic data. Biologically, cytotoxic activities of these compounds were broadly studied compared with the fungal extract. To predict the biological target for the presumed antitumor activity, an in silico study was run toward three proteins, topoisomerase IIα, topoisomerase IIß, and VEGFR2 kinase. Monascinol (2) was expected to be moderately active against VEGFR2 kinase without any anticipated inhibition toward topo II isoforms. The in vitro study confirmed the docked investigation consistently and introduced monascinol (2) rather than its counterpart (1) as a potent inhibitor to the tested VEGFR2 kinase. Taxonomically, the fungus was identified using morphological and genetic assessments.

Calamusins J-K: new anti-angiogenic sesquiterpenes from Sarcophyton glaucum.

Chemical investigation of the organic extract of the marine soft coral Sarcophyton glaucum from Red Sea, Egypt, afforded two new hydroazulenes; calamusin J (1) and its hydroperoxide derivative calamusin K (2) in addition to eight known compounds. Structure of compounds 1-2 were confirmed by intensive NMR and mass spectrometry studies. The coral extract and the obtained compounds were examined against a set of diverse microorganisms. The in vitro anti-cancer properties were assessed against colon (Caco-2) and breast (MCF-7) cell lines together with their exerted cytotoxicity on the immortalized normal epithelium (hTERT-RPE1) cell type. The anti-angiogenic power was also highlighted through suppressing MCF-7 cell migration and the significant inactivation of VEGFR2 enzyme. Compounds 1,2 are the most potent angiogenic inhibitors (represented by 1.2- and 1.4-fold enzyme inactivation, respectively) relative to sorafenib. The polyhydroxy sterol; 5α-3ß,6α,11-trihydroxy-24-methyl-9,11-seco-5a-cholest-7-en-9-one (S4) inhibited effectively the growth of Caco-2 and MCF-7 with GI50 of 0.62 and 2.3 µM, respectively.

In vitro chemotherapeutic and antiangiogenic properties of cardenolides from Acokanthera oblongifolia (Hochst.) Codd.

Acovenoside A (Acov-A) and acobioside A (Acob-A) were isolated from Acokanthera oblongifolia. Their anticancer properties were explored regarding, antiproliferative and antiangiogenic activities. The study included screening phase against six cancer cell lines followed by mechanistic investigation against HepG2 cancer cell line. The sulforhodamine-B (SRB) was used to determine their growth inhibitory power. In the other hand, flow cytometry techniques were recorded the cell death type and cell cycle analysis. The clonogenic (colony formation) and wound healing assays, enzyme-linked immunosorbent assay (ELISA) and molecular docking, were performed to evaluate the antiangiogenesis capability. Both compounds were strongly, inhibited four cancer cell lines at GI50 less than 100 nM. The in vitro mechanistic investigation against HepG2 resulted in cell accumulations at G2M phase and induction of apoptosis upon treating cells separately, with 400 nM Acov-A and 200 nM Acob-A. Interestingly, the same concentrations were able to activate caspase-3 by 7.2 and 4.8-fold, respectively. Suppressing the clonogenic capacity of HepG2 cells (20 and 40 nM) and inhibiting the migration of the colon Caco-2 cancer cells were provoke the results of vascular endothelial growth factor receptor2 (VEGFR2) kinase enzyme inactivation. The docked study was highly supportive, to the antiangiogenic approach of both cardenolides. The isolated cardenolides could orchestrate pivotal events in fighting cancer.

Metal-free domino amination-Knoevenagel condensation approach to access new coumarins as potent nanomolar inhibitors of VEGFR-2 and EGFR.

A metal-free, atom-economy and simple work-up domino amination-Knoevenagel condensation approach to construct new coumarin analogous (4a-f and 8a-e) was described. Further, new formyl (5a,d-f) and nitro (9a,d-f) coumarin derivatives were synthesized via C-N coupling reaction of various cyclic secondary amines and 4-chloro-3-(formyl-/nitro)coumarins (1a,c), respectively. The confirmed compounds were screened for their in vitro anti-proliferative activity against KB-3-1, A549 and PC3 human cancer cell lines using resazurin cellular-based assay. Among them, coumarin derivatives 4e and 8e displayed the best anti-cervical cancer potency (KB-3-1) with IC50 values of 15.5 ± 3.54 and 21 ± 4.24 µM, respectively. Also, 4e showed the most promising cytotoxicity toward A549 with IC50 value of 12.94 ± 1.51 µM. As well, 9d presented a more significant impact of potency against PC3 with IC50 7.31 ± 0.48 µM. Moreover, 8d manifested selectivity against PC3 (IC50 = 20.16 ± 0.07 µM), while 8e was selective toward KB-3-1 cell line (IC50 = 21 ± 4.24 µM). Matching with docking profile, the enzymatic assay divulged that 8e is a dual potent single-digit nanomolar inhibitor of VEGFR-2 and EGFR with IC50 values of 24.67 nM and 31.6 nM that were almost equipotent to sorafenib (31.08 nM) and erlotinib (26.79 nM), respectively.

Cardenolides and pentacyclic triterpenes isolated from Acokanthera oblongifolia leaves: their biological activities with molecular docking study.

Pentacyclic triterpenes and cardenolides were isolated from Acokanthera oblongifolia leaves. Their chemical structures were determined based on comprehensive 1D and 2D NMR spectroscopy. Their MIC was determined against 12 microorganisms. Their exerted cytotoxicity on the immortalized normal cells, hTERT-RPE1 was assessed by the sulforhodamine-B assay. The viral inhibitory effects of compounds against Newcastle disease virus (NDV) and H5N1 influenza virus IV were evaluated. Four in vitro antioxidant assays were performed in comparison with BHT and trolox and a weak activity was exhibited. Acovenoside A was with potent against H5N1-IV and NDV with IC50 ≤ 3.2 and ≤ 2.1 µg/ml and SI values of 93.75 and 95.23%, respectively, in comparison to ribavirin. Its CC50 record on Vero cells was > 400 and 200 µg/ml, respectively. Acobioside A was the most active compound against a broad range of microbes while Pseudomonas aeruginosa was the most sensitive. Its MIC (0.07 µg/ml) was 1/100-fold of the recorded CC50 (7.1 µg/ml/72 h) against hTERT-RPE1. The molecular docking of compounds on human DNA topoisomerase I (Top1-DNA) and IV glycoprotein hemagglutinin were studied using MOE program. This study has introduced the cardenolides rather than triterpenoids with the best docking score and binding interaction with the active site of the studied proteins.

Synthesis and biological evaluation of novel 5-chloro-N-(4-sulfamoylbenzyl) salicylamide derivatives as tubulin polymerization inhibitors.

A novel series of sulfonamide derivatives, coupled with a salicylamide scaffold, was designed and synthesized. The structures of the synthesized compounds were established using 1H NMR, 13C NMR and high-resolution mass spectroscopy. The synthesized compounds were tested in vitro against five types of human cell lines. Two were breast adenocarcinoma, including the hormone-dependent MCF-7 and the hormone-independent MDA-MB-231. The others were the colorectal adenocarcinoma Caco-2, the carcinoma HCT-116 and the immortalized retinal-pigmented epithelium, hTERT-RPE1. Nine sulfonamides were able to inhibit the growth of the four tested cancer cells. Compound 33 was the most active against the selected colon cancer (Caco-2 and HCT-116) subtypes, while compound 24 showed the best efficacy against the examined breast cancer (MCF-7 and MDA-MB-231) cells. The selectivity index introduced compounds 24 and 33 as having the best selectivity among the breast and colon subtypes, respectively. In vitro tubulin polymerization experiments and flow cytometric assays showed that compounds 24 and 33 led to cell cycle arrest at the G2/M phase in a dose-dependent manner by effectively inhibiting tubulin polymerization. Furthermore, the results of the molecular docking studies indicate that this class of compounds can bind to the colchicine-binding site of tubulin.

Phylogenetic Analysis and Biological Evaluation of Marine Endophytic Fungi Derived from Red Sea Sponge Hyrtios erectus.

Forty-four endophytic fungal isolates obtained from marine sponge, Hyrtios erectus, were evaluated and screened for their hydrolase activities. Most of the isolates were found to be prolific producers of hydrolytic enzymes. Only 11 isolates exhibited maximum cellular contents of lipids, rhamnolipids, and protein in the fungal isolates under the isolation numbers MERVA5, MERVA22, MERVA25, MERVA29, MERVA32, MERVA34, MERV36, MERVA39, MERVA42, MERVA43, and MERVA44. These isolate extracts exhibit the highest reducing activities against carbohydrate-metabolizing enzymes including α-amylase, α-glucosidase, ß-glucosidase, ß-glucuronidase, and tyrosinase. Consequently, based on morphological and cultural criteria, as well as sequence information and phylogenetic analysis, these isolates could be identified and designated as Penicillium brevicombactum MERVA5, Arthrinium arundinis MERVA22, Diaporthe rudis MERVA25, Aspergillus versicolor MERVA29, Auxarthron alboluteum MERVA32, Dothiorella sarmentorum MERVA34, Lophiostoma sp. MERVA36, Fusarium oxysporum MERVA39, Penicillium chrysogenum MERVA42, Penicillium polonicum MERVA43, and Trichoderma harzianum MERVA44. The endophytic fungal species, D. rudis MERVA25, P. polonicum MERVA43, Lophiostoma sp. MERVA36, A. alboluteum MERVA32, T. harzianum MERVA44, F. oxysporum MERVA39, A. versicolor MERVA29, and P. chrysogenum MERVA42 extracts, showed significant hepatitis C virus (HCV) inhibition. Moreover, D. sarmentorum MERVA34, P. polonicum MERVA43, and T. harzianum MERVA44 extracts have the highest antitumor activity against human hepatocellular carcinoma cells (HepG2).

Phenolic constituents of Pulicaria undulata (L.) C.A. Mey. sub sp. undulata (Asteraceae): Antioxidant protective effects and chemosystematic significances.

One new naturally isoflavone compound, 5,7,2',3',4' penta hydroxyl isoflavone-4'-O-ß-glucopyranoside (1) was isolated from the aqueous methanol extract (AME) of Pulicaria undulata subsp. undulata, together with seven known compounds: kaempferol (2), kaempferol 3-O-ß-glucoside (3), quercetin (4), quercetin 3-O-ß-glucoside (5), quercetin 3-O-ß-galactoside (6), quercetin 3,7-di OCH3 (7), and caffeic acid (8). Their structures were established through chemical (acid hydrolysis) and spectral analysis (UV, NMR, and ESIM). The AME and some isolated compounds were evaluated as protective agents. Free radical scavenging using a microscaled 2,2-diphenyl-1-picrylhydrazyl assay was used to assess the direct antioxidant properties that were evaluated by the ability to protect murine Hepa1c1c7 liver cells against damage induced by the organic peroxide tert-butyl hydroperoxide. The neutral red uptake assay (NRU) was used to record the activity. Results of the 2,2-diphenyl-1-picrylhydrazyl assay recorded differential scavenging properties in ascending order: 5,7,2',3',4' penta hydroxyl isoflavone-4'-O-ß-glucopyranoside>quercetin>quercetin 3-O-galactoside>caffeic acid>quercetin 3,7-di-OCH3>kaempferol with 50% inhibitory concentrations of 3.9 µM, 7.5 µM, 11.4 µM, 12.2 µM, 78.1 µM, and 252.3 µM, respectively. The antioxidative potential reveals the potency of AME, quercetin, and quercetin 3,7-di-OCH3. The latter compound showed full protection at 100 µM (33 µg/mL) against the induced toxicant effect where the 50% effective concentration was calculated as 33.6±1.7 µM (11.1 µg/mL). In addition to quercetin, which was extensively shown previously as a cytoprotective agent, AME was less potent; it was capable of protecting 75% at 100 µg/mL with 50% effective concentration of 92.3±4 µg/mL. Moreover, the isolated flavonoids were found to be significantly chemosystematic markers.