Tubulin-interactive stilbene derivatives as anticancer agents

Microtubules are dynamic polymers that occur in eukaryotic cells and play important roles in cell division, motility, transport and signaling. They form during the process of polymerization of α- and β-tubulin dimers. Tubulin is a significant and heavily researched molecular target for anticancer drugs. Combretastatins are natural cis-stilbenes that exhibit cytotoxic properties in cultured cancer cells in vitro. Combretastatin A-4 (3′-hydroxy-3,4,4′, 5-tetramethoxy-cis-stilbene; CA-4) is a potent cytotoxic cis-stilbene that binds to β-tubulin at the colchicine-binding site and inhibits tubulin polymerization. The prodrug CA-4 phosphate is currently in clinical trials as a chemotherapeutic agent for cancer treatment. Numerous series of stilbene analogs have been studied in search of potent cytotoxic agents with the requisite tubulin-interactive properties. Microtubule-interfering agents include numerous CA-4 and transresveratrol analogs and other synthetic stilbene derivatives. Importantly, these agents are active in both tumor cells and immature endothelial cells of tumor blood vessels, where they inhibit the process of angiogenesis. Recently, computer-aided virtual screening was used to select potent tubulin-interactive compounds. This review covers the role of stilbene derivatives as a class of antitumor agents that act by targeting microtubule assembly dynamics. Additionally, we present the results of molecular modeling of their binding to specific sites on the α- and β-tubulin heterodimer. This has enabled the elucidation of the mechanism of stilbene cytotoxicity and is useful in the design of novel agents with improved anti-mitotic activity. Tubulin-interactive agents are believed to have the potential to play a significant role in the fight against cancer.     

Resveratrols in Grape Berry Skins and Leaves in Vitis Germplasm

Background

Resveratrol is an important stilbene that benefits human health. However, it is only distributed in a few species including grape and is very expensive. At present, grape has been an important source resveratrol. However, the details are scarce on resveratrol distribution in different Vitis species or cultivars.

Methodology/Principal Finding

The composition and content of resveratrols were investigated by HPLC for assessing genotypic variation in berry skins and leaves of 75 grape cultivars, belonging to 3 species and 7 interspecific hybrids. Trans-resveratrol, cis-piceid and trans-piceid were detected in berry skins and leaves, but cis-resveratrol was not. Resveratrol content largely varied with genetic background as well as usage. In most cultivars, total resveratrol including the above three compounds was higher in berry skins than leaves. In berry skins of most cultivars and leaves of almost all cultivars, cis-piceid was the most abundant resveratrol; trans-resveratrol and trans-piceid were minor components. Some specific cultivars were found with extremely high levels of trans-resveratrol, cis- piceid, trans-piceid or total resveratrols in berry skins or leaves. In skins and leaves, rootstock cultivars had a higher content of total resveratrols, and the cultivated European type cultivars and their hybrids with V. labrusca had relatively low totals. There were no significant correlations of the amounts of total resveratrols or any individual resveratrol between berry skins and leaves. All 75 cultivars can be divided into four groups based on the composition of resveratrols and their concentration by principal component analysis.

Conclusion

Resveratrol content of grape berries and leaves varied largely with their genetic background and usage. Rootstock cultivars had a higher content of total resveratrols than the other germplasm. Total resveratrols were lower in leaves than berry skins in most cultivars. Cis-piceid was the most abundant resveratrol in most cultivars, and trans-res and trans-pd were minor components.     

Metabolomic analysis of resveratrol-induced effects in the human breast cancer cell lines MCF-7 and MDA-MB-231

Resveratrol is a naturally occurring anticancer compound present in grapes and wine with antiproliferative properties against breast cancer cells and xenografts. Our objective was to investigate the metabolic alterations that characterize the effects of resveratrol in the human breast cancer cell lines MCF-7 and MDA-MB-231 using high-throughput liquid chromatography-based mass spectrometry. In both cell lines, growth inhibition was dose dependent and accompanied by substantial metabolic changes. For all 21 amino acids analyzed levels increased more than 100-fold at a resveratrol dose of 100?μM with far lower concentrations in MDA-MB-231 compared to MCF-7 cells. Among the biogenic amines and modified amino acids (n?=?16) resveratrol increased the synthesis of serotonin, kynurenine, and spermindine in both cell lines up to 61-fold indicating that resveratrol strongly interacts with cellular biogenic amine metabolism. Among the eicosanoids and oxidized polyunsaturated fatty acids (n?=?17) a pronounced increase in arachidonic acid and its metabolite 12S-HETE was observed in MDA-MB-231 and to a lesser extent in MCF-7 cells, indicating release from cell membrane phospholipids upon activation of phospholipase A? and subsequent metabolism by 12-lipoxygenase. In conclusion, metabolomic analysis elucidated several small molecules as markers for the response of breast cancer cells to resveratrol.     

Synthesis,characterization and Akt phosphorylation inhibitory activity of cyclopentanecarboxylate-substituted alkylphosphocholines

Akt is activated in most human cancers and contributes to cell growth, proliferation and cellular survival pathway. Accordingly, it is an attractive target for anticancer therapy. A series of novel alkylphosphocholines, incorporating cyclopentanecarboxylate in the phospholipid head group with trans and cis orientations, were synthesized and evaluated for their Akt phosphorylation inhibitory activities and cytotoxicities against human cancer cell lines, A549, MCF-7 and KATO III. Among the synthesized compounds, 5a, 5b and 6c exhibited potent inhibitory Akt phosphorylation effects with IC₅₀ value of 3.1, 2.0 and 3.0 μM, respectively, and their potencies were better than those of three reference compounds miltefosine, perifosine and edelfosine. All the new compounds, except 5d and 6e, displayed more potent growth inhibition against A549 cells than reference compounds. Specifically, compound 5b exhibited most remarkable cytotoxicities on A549 cells as well as MCF-7 and KATO III cells. Importantly, the cytotoxic effects of these compounds correlated with their Akt phosphorylation inhibitory activities.     

Scavenging of hydroxyl radical by resveratrol and related natural stilbenes after hydrogen peroxide attack on DNA

Resveratrol (3,5,4′-trihydroxystilbene) is of interest due to its role in prevention and therapy of degenerative diseases as cancer and aging. However, depending on its concentration and cell type studied, resveratrol activity appears conflicting. It exerts antioxidant action, as a scavenger of free radicals and as promoter of antioxidant enzyme activity, but resveratrol acts also as a pro-oxidant. Here we present experimental and theoretical studies for resveratrol and two methoxy-derivatives found in plants, pterostilbene and 3,5,4′-trimethoxystilbene. We show that both methoxy-derivatives induce less DNA damage than resveratrol. The protective effects of the three molecules against oxidative DNA damage induced by hydrogen peroxide treatment were analyzed on mammalian cells in vitro. Our data show for the first time that methoxylated derivatives of resveratrol are very efficient in reducing DNA damage: using the same concentration of the three molecules we obtain a relative reduction of 85.5% (pterostilbene), 43.7% (trimethoxystilbene) and 21.1% (resveratrol). Analysis of the crystal structures of pterostilbene and 3,5,4′-trimethoxystilbene, compared to resveratrol, show fewer intermolecular interactions and a lack of planarity, due to packing forces, which is confirmed by density functional theory (DFT) calculations. We also describe the results of DFT calculations (including water solvent effects) in which the three stilbene species scavenge the hydroxyl radical (associated with the H₂O₂ insult).     

Contents of Resveratrol,Piceid, and Their Isomers in Commercially Available Wines Made from Grapes Cultivated in Japan

The presence of resveratrol (3,5,4′-trihydroxystilbene) and its β-glucoside, piceid (resveratrol-3-β-D-glucopyranoside), together with their isomers in wine appears to be one of the beneficial factors conferring a protective effect against cardiovascular disease through red wine ingestion. A total of 42 red and white wines was collected in arears from Hokkaido to Kyushu in Japan. The wines were fractionated with a C₁₈ Sep-pak cartridge, and the active principles were eluted with ethyl acetate. Crude trans- and cis-piceid were extracted from a Chinese medicine, ‘Kojohkon’ (Polygonum cuspidatum), and their retention times and UV absorption were confirmed by HPLC. trans- and cis-Resveratrol, and trans- and cis-piceid were analyzed in a short C₁₈ HPLC column, and cis-resveratrol was quantified from the amount of cis-isomer converted from authentic trans-resveratrol that had been treated by UV irradiation. The content of piceid is shown as the resveratrol equivalent. The average content of total stilbene compounds was 4.37mg/liter in red wines, while only 0.68 mg/liter in white wines. Red wines made from Pinot noir, Merlot, and Zweigeltrebe grapes all had a high resveratrol content.     

Design,synthesis and anticancer activities of stilbene-coumarin hybrid compounds: Identification of novel proapoptotic agents

The naturally occurring coumarins and resveratrol, attract great attention due to their wide range of biological properties, including anticancer, antileukemic, antibacterial and anti-inflammatory activities; moreover, their cancer chemopreventive property have been recently emphasized. A novel class of hybrid compounds, obtained by introducing a substituted trans-vinylbenzene moiety on a coumarin backbone, was synthesized and evaluated for the antitumor profile. A number of derivatives showed a good antiproliferative activity, in some cases higher to that of the reference compound resveratrol. The most promising compounds in this series were 14 and 17, endowed with excellent antiproliferative and proapoptotic activities. The present study suggests that the 7-methoxycoumarin nucleus, together with the 3,5-disubstitution pattern of the trans-vinylbenzene moiety, are likely promising structural features to obtain excellent antitumor compounds endowed with a apoptosis-inducing capability.     

Design,synthesis and biological evaluation of trans 2-(thiophen-2-yl)vinyl heteroaromatic iodides

A modeling approach based on physico-chemical and pharmacokinetic properties, called Volsurf+, was used to design new trans 2-(thiophen-2-yl)vinyl heteroaromatic iodides with antiproliferative activity. The synthesis and in vitro antitumor tests on two cell lines (MCF-7 and LNCap) confirmed Volsurf predicted activity values. An Almond model, derived to have an overall structural insight on the above compounds, supported the validity of Volsurf and provided guidelines for the synthesis of new compounds.     

Synthesis and cytotoxicity studies of steroid-functionalized titanocenes as potential anticancer drugs: sex steroids as potential vectors for titanocenes

Six titanocenyls functionalized with steroidal esters have been synthesized and characterized by infrared, ¹H, and ¹³C NMR spectroscopy and elemental analysis. Among those steroids, dehydroepiandrosterone, trans-androsterone, and androsterone are androgens and pregnenolone is a progesterone precursor. Clionasterol is a natural steroid compound. These steroid-functionalized titanocenyls were tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay for in vitro cytotoxicity for MCF-7 breast cancer and HT-29 colon cancer cells. All complexes exhibited more cytotoxicity than titanocene dichloride. The titanocenyls containing androgen and progesterone derivatives as pendant groups had higher antiproliferative activities than those with cholesterol steroid compounds. Of particular significance is titanocenyl–dehydroepiandrosterone complex, which is 2 orders of magnitude more cytotoxic than titanocene dichloride and also shows much more sensitivity and selectivity for the MCF-7 cell line.     

Synthesis and biological evaluation of benzo[b]furo[3,4-e][1,4]diazepin-1-one derivatives as anti-cancer agents

A new series of novel Podophyllotoxin-like benzo[b]furo[3,4-e][1,4]diazepin-1-ones possessing structural elements of 4-aza-2,3-didehydropodophyllotoxins with central diazepine ring was designed and synthesized as anti-cancer agents. In initial assessment, the cytotoxic activity of the synthesized compounds was evaluated against three cancer cell lines including MCF-7, PC3 and B16-F10 employing the MTT assay. Some of compounds (12h, 13a, 13c and 14b) showed significant cytotoxic activity. So, we investigated the cytotoxicity of compounds 12h, 13a, 13c and 14b, along with podophyllotoxin as the reference drug in different cancer cell lines including A549, A2780, DU145, HeLa, and normal Huvec cell line. Among these four compounds, 13c showed promising antiproliferative activity against all cancer cells stronger than the other compounds and comparable to reference drug podophyllotoxin in some cancer cells. All these four compounds did not show significant cytotoxicity on normal Huvec cell line. The flow cytometry analysis of the MCF-7, PC3 and A2780 human cancer cell lines treated with 13c showed that 13c, induced apoptosis in the MCF-7, PC3 and A2780 human cancer cell lines, which is in good agreement to its cytotoxic activity as well. Compound 13c did not show significant influence on tubulin assembly and exert its cytotoxic effects via induction of apoptosis and has potent and selective cytotoxic effects in cancer cells.     

DNA damage induced by resveratrol and its synthetic analogues in the presence of Cu (II) ions: Mechanism and structure-activity relationship

The prooxidant effect of resveratrol (3,5,4′-trihydroxy-trans-stibene) and its synthetic analogues (ArOH), that is, 3,4,4′-trihydroxy-trans-stibene (3,4,4′-THS), 3,4,5-trihydroxy-trans-stibene (3,4,5-THS), 3,4-dihydroxy-trans-stibene (3,4-DHS), 4,4′-dihydroxy-trans-stibene (4,4′-DHS), 2,4-dihydroxy-trans-stilbene (2,4-DHS), 3,5-dihydroxy-trans-stilbene (3,5-DHS) and 3,5,4′-trimethoxy-trans-stibene (3,5,4′-TMS), on supercoiled pBR322 plasmid DNA strand breakage and calf thymus DNA damage in the presence of Cu (II) ions has been studied. It was found that the compounds bearing ortho-dihydroxyl groups (3,4-DHS, 3,4,4′-THS, and 3,4,5-THS) or bearing 4-hydroxyl groups (2,4-DHS, 4,4′-DHS, and resveratrol) exhibit remarkably higher activity in the DNA damage than the ones bearing no such functionalities. Kinetic analysis by UV-visible spectra demonstrates that the formation of ArOH-Cu (II) complexes, the stabilization of oxidative intermediate derived from ArOH and Cu (II)/Cu (I) redox cycles, might be responsible for the DNA damage. This study also reveals a good correlation between antioxidant and prooxidant activity, as well as cytotoxicity against human leukemia (HL-60 and Jurkat) cell lines. The mechanisms and implications of these observations are discussed.     

Antioxidative Activity of Resveratrol and Its Derivatives Isolated from Seeds of Paeonia lactiflora

Seven stilbenes, a new cis-ε-viniferin and the six known stilbenes, trans-resveratrol, trans-resveratrol-4′-O-β-D-glucopyranoside, trans-ε-viniferin, gnetin H, and suffruticosol A and B, were isolated and identified from seeds of Paeonia lactiflora. The antioxidative activity of these stilbene derivatives was evaluated against the 2-deoxyribose degradation and rat liver microsomal lipid peroxidation induced by the hydroxyl radical generated via a Fenton-type reaction. Among these stilbenes, trans-ε-viniferin and gnetin H significantly inhibited 2-deoxyribose degradation and lipid peroxidation in rat liver microsomes. In addition, cis-ε-viniferin, and suffruticosol A and B also exhibited moderate antioxidative activity. These results suggest that resveratrol dimers and trimers, together with resveratrol from seeds of Paeonia lactiflora may be useful as potential sources of natural antioxidants.     

Selective fraction of Scutellaria baicalensis and its chemopreventive effects on MCF-7 human breast cancer cells

Based on our previous observation, the whole Scutellaria baicalensis extract (SbE) did not show significant breast cancer cell inhibitory effect. In this study, we isolated a baicalin-deprived-fraction (SbF1) of Scutellaria baicalensis, and baicalin-fraction (SbF3), and evaluated their anti-breast cancer properties using MCF-7 cells. The content of four flavonoids in extract/fractions were determined using high performance liquid chromatography. Analytical data showed that in SbF1, the major constituents are baicalein and wogonin, while SbF3 only contains baicalin. The antiproliferative effects of fractions and SbE were assayed using modified trichrome stain method. SbF1 showed significant antiproliferative effect. Treated with 100 μg/ml of SbF1 for 72 h inhibited MCF-7 cell growth by 81.6%, while in the same treatment concentration, SbF3 increased cell growth by 22.6%. SbF1 was recognized as an active fraction of SbE. The effects of four flavonoids in SbE, scutellarin, baicalin, baicalein and wogonin, were determined, and data showed that baicalein and wogonin significantly inhibited MCF-7 cell growth. In contrast, in certain concentrations, scutellarin and baicalin increased cancer cell growth. The effects of SbF1 on cell cycle and apoptosis were assayed using flow cytometry. SbF1 arrested MCF-7 cells in S- and G2/M-phases, and significantly increased induction of cell apoptosis. These combined phytochemical and biological data provide evidence for further chemopreventive studies of the baicalin-deprived SbE on breast cancer.     

Evaluation of cytotoxic activities of snake venoms toward breast (MCF-7) and skin cancer (A-375) cell lines

Snake venoms are mixtures of bioactive proteins and peptides that exhibit diverse biochemical activities. This wide array of pharmacologies associated with snake venoms has made them attractive sources for research into potentially novel therapeutics, and several venom-derived drugs are now in use. In the current study we performed a broad screen of a variety of venoms (61 taxa) from the major venomous snake families (Viperidae, Elapidae and “Colubridae”) in order to examine cytotoxic effects toward MCF-7 breast cancer cells and A-375 melanoma cells. MTT cell viability assays of cancer cells incubated with crude venoms revealed that most venoms showed significant cytotoxicity. We further investigated venom from the Red-bellied Blacksnake (Pseudechis porphyriacus); venom was fractionated by ion exchange fast protein liquid chromatography and several cytotoxic components were isolated. SDS-PAGE and MALDI-TOF mass spectrometry were used to identify the compounds in this venom responsible for the cytotoxic effects. In general, viper venoms were potently cytotoxic, with MCF-7 cells showing greater sensitivity, while elapid and colubrid venoms were much less toxic; notable exceptions included the elapid genera Micrurus, Naja and Pseudechis, which were quite cytotoxic to both cell lines. However, venoms with the most potent cytotoxicity were often not those with low mouse LD₅₀s, including some dangerously venomous viperids and Australian elapids. This study confirmed that many venoms contain cytotoxic compounds, including catalytic PLA₂s, and several venoms also showed significant differential toxicity toward the two cancer cell lines. Our results indicate that several previously uncharacterized venoms could contain promising lead compounds for drug development.     

In vitro cytotoxic potential of extracts from Aristolochia foetida Kunth against MCF-7 and bMECs cell lines

The aim of this study was to evaluate the cytotoxic potential of Aristolochia foetida Kunth. Stems and leaves of A. foetida Kunth (Aristolochiaceae) have never been investigated pharmacologically. Recent studies of species of the Aristolochiaceae family found significant cytotoxic activities. Hexane, dichloromethane, ethyl acetate and methanol extracts were analyzed by ¹H NMR and GC–MS to know the metabolites in each extract. In GC–MS analysis, the main compounds were methyl hexadecanoate (3); hexadecanoic acid (4); 2-butoxyethyl dodecanoate (9); ethyl hexadecanoate (20); methyl octadeca-9,12,15-trienoate (28) and (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid (40). The results showed a significant reduction in cell viability of the MCF-7 (breast cancer) cell line caused by organic extracts in a dose-dependent manner. The cytotoxicity activity of the dichloromethane extract from the stems (DSE) showed IC₅₀ values of 45.9 μg/mL and the dichloromethane extract of the leaves (DLE) showed IC₅₀ values of 47.3 μg/mL. DSE and DLE had the highest cytotoxic potential in an in vitro study against the MCF-7 cell line and non-tumor cells obtained from the bovine mammary epithelial (bMECs). DSE and DLE induced a loss in mitochondrial membrane potential (ΔΨm) and can cause cell death by apoptosis through the intrinsic pathway in the MCF-7 cell line. DSE and DLE are cytotoxic in cancer cells and cause late apoptosis. Higher concentrations of DSE and DLE are required to induce a cytotoxic effect in healthy mammary epithelial cells. This is the first report of the dichloromethane extract of A. foetida Kunth that induces late apoptosis in MCF-7 cancer cells and may be a candidate for pharmacological study against breast cancer.     

Hormetic/Cytotoxic Effects of Nigella sativa Seed Alcoholic and Aqueous Extracts on MCF-7 Breast Cancer Cells Alone or in Combination with Doxorubicin

In this study, we investigate the possible cytotoxic effects of different Nigella sativa seed extracts on human MCF-7 breast cancer cells and screening the effects of a wide range of extracts concentrations and their application as an adjuvant therapy to doxorubicin. The results obtained showed that the cytotoxic solvent dimethyl sulfoxide can be used for permeation assay in concentration range 697.5–0.341 mmol/ml without affecting the viability of MCF-7 cells. N. sativa lipid extract is cytotoxic to MCF-7 cells with LC₅₀ of 2.72 ± 0.232 mg/ml, while its aqueous extract cytotoxicity exhibited when the applied concentration is high as ≈ 50 mg/ml. The results of this study reveal for the first time that low concentrations of aqueous extract of the seed has a hormetic rather than cytotoxic effect. It is also possible to use cell culture medium or bovine serum to dilute the oil extract for the permeation assay. In conclusion, N. sativa aqueous extract should not be used as antitumor compound by its own. The oil is a promising antitumor compound and its cytotoxicity was greatly enhanced with its nanoemulsion formulation. Antitumor activity of doxorubicin was enhanced, as a function of time, when N. sativa extracts were involved as adjunct therapeutic compounds. Adding doxorubicin to the prepared lipid nanoemulsion has a beneficial impact to their bioactivity. These doxorubicin—N. sativa lipid nanoemulsion are promising and potential therapeutic modality.     

Two new flavonol derivatives from the whole plants of Centella asiatica and their cytotoxic activities

Centella asiatica is well known as an important medicinal plant because of its various pharmacological effects. However, most investigations on C. asiatica focused on the pharmacological activity of its extract or asiatic acid. In the present work, we aimed to explore the bioactive compounds of the whole plants of C. asiatica. As a result, seven compounds including two new flavonol derivates named 4′-hydroxyl-7-methoxyl-6-prenyl-3-O-trans-p-coumaroyl-flavonol (1) and (2R,3R,2″S)-3-furanoyl-brosimacutin E (2), and five known compounds (3-7) were isolated. Their chemical structures were elucidated on the basis of spectroscopic analyses. All the isolates were evaluated for in vitro cytotoxic effects on four human cancer cells including A549, HepG2, SGC-7901 and MCF-7. Among them, compounds 1 and 2 exhibited higher cytotoxic activities on HepG2 and SGC-7901 cells compared with 3-7. And compound 2 showed potential cytotoxic activities on HepG2 and SGC-7901 cells with IC₅₀ values of 4.52 and 7.03 μM, respectively.     

Synthesis,cytotoxic evaluation and molecular docking study of 2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-aryl-thiazoles as tubulin polymerization inhibitors

A series of cis-restricted 2-alkylthio-4-(2,3,4-trimethoxyphenyl)-5-aryl-thiazole analogues of combretastatin A-4 were synthesized and investigated for inhibition of cell proliferation against three cancer cell lines, HT-29, MCF-7, and AGS, and a normal mouse fibroblastic cell line, NIH-3T3, using an MTT assay. The biological study showed that 2-(methylthio) substituted compounds showed little cytotoxic activity against the four cell lines. In contrast, the presence of the 2-(benzylthio) group on the thiazole ring resulted in a significant improvement in cytotoxic activity relative to the 2-(methylthio) substituted derivatives. Furthermore, the inhibition of tubulin polymerization by some potent compounds was evaluated. All the compounds studied were moderate tubulin polymerization inhibitors. The flow cytometry analysis confirmed that the synthesized compounds led to cell cycle arrest at the G₂/M phase. Docking simulation was performed to insert these compounds into the crystal structure of tubulin at the colchicine binding site to determine a probable binding model.