Ternary Deep Eutectic Solvents System of Colchicine, 4-Hydroxyacetophenone, and Protocatechuic Acid and Characterization of Transdermal Enhancement Mechanism.

This study aimed to prepare colchicine (CO), 4-hydroxyacetophenone (HA), and protocatechuic acid (CA) contained in transdermal rubber plasters into a more releasable and acrylate pressure-sensitive adhesive (PSA) to optimize traditional Touguling rubber plasters (TOU) with enhanced transdermal permeability by using deep eutectic solvents (DES) technology. We compared the difference in the release behavior of CO between rubber plaster and PSA, determined the composition of the patch through pharmacodynamic experiments, explored the transdermal behavior of the three components, optimized the patch formula factors, and improved the penetration of CO through the skin. We also focused on elucidating the interactions among the three components of DES and the intricate relationship between DES and the skin. The melting point of DES was determined using DSC, while FTIR, 13C NMR, and ATR-FTIR were used to explore the intricate molecular mechanisms underlying the formation of DES, as well as its enhancement of skin permeability. The results of this investigation confirmed the successful formation of DES, marked by a discernible melting point at 27.33°C. The optimized patch, formulated with a molar ratio of 1:1:1 for CO, HA, and CA, significantly enhanced skin permeability, with the measured skin permeation quantities being 32.26 ± 2.98 µg/cm2, 117.67 ± 7.73 µg/cm2, and 56.79 ± 1.30 µg/cm2 respectively. Remarkably, the optimized patch also demonstrated similar analgesic and anti-inflammatory effects compared to commercial diclofenac diethylamide patches in different pharmacodynamics studies. The formation of DES altered drug compatibility with skin lipids and increased retention, driven by the interaction among the three component molecules through hydrogen bonding, effectively shielding the skin-binding sites and enhancing component permeation. In summary, the study demonstrated that optimized DES patches can concurrently enhance the penetration of CO, HA, and CA, thereby providing a promising approach for the development of DES in transdermal drug delivery systems. The findings also shed light on the molecular mechanisms underlying the transdermal behavior of DES and offer insights for developing more effective traditional Chinese medicine transdermal drug delivery systems.

[Effect of Isodon ternifolius-medicated serum on hepatic stellate cells based on TLR4/NF-κB/NLRP3 signaling pathway].

The present study aimed to investigate the inhibitory effect and mechanism of Isodon terricolous-medicated serum on lipopolysaccharide(LPS)-induced hepatic stellate cell(HSC) activation. LPS-induced HSCs were divided into a blank control group, an LPS model group, a colchicine-medicated serum group, an LPS + blank serum group, an I. terricolous-medicated serum group, a Toll-like receptor 4(TLR4) blocker group, and a TLR4 blocker + I. terricolous-medicated serum group. HSC proliferation was detected by methyl thiazolyl tetrazolium(MTT) assay. Enzyme-linked immunosorbent assay(ELISA) was used to measure type Ⅰ collagen(COL Ⅰ), COL Ⅲ, transforming growth factor-ß1(TGF-ß1), intercellular adhesion molecule-1(ICAM-1), α-smooth muscle actin(α-SMA), vascular cell adhesion molecule-1(VCAM-1), cysteinyl aspartate-specific proteinase-1(caspase-1), and monocyte chemotactic protein-1(MCP-1). Real-time PCR(RT-PCR) was used to detect mRNA expression of TLR4, IκBα, and NOD-like receptor thermal protein domain associated protein 3(NLRP3), nuclear factor-κB(NF-κB) p65, gasdermin D(GSDMD), and apoptosis-associated speck-like protein containing a CARD(ASC) in HSCs. Western blot(WB) was used to detect the protein levels of TLR4, p-IκBα, NF-κB p65, NLRP3, ASC, and GSDMD in HSCs. The results showed that I. terricolous-medicated serum could inhibit the proliferation activity of HSCs and inhibit the secretion of COL Ⅰ, COL Ⅲ, α-SMA, TGF-ß1, caspase-1, MCP-1, VCAM-1, and ICAM-1 in HSCs. Compared with the LPS model group, the I. terricolous-medicated serum group, the colchicine-medicated serum group, and the TLR4 blocker group showed down-regulated expression of p-IκBα, NLRP3, NF-κB p65, GSDMD, and ASC, and up-regulated expression of IκBα. Compared with the TLR4 blocker group, the TLR4 blocker + I. terricolous-medicated serum group showed decreased expression of TLR4, p-IκBα, NLRP3, NF-κB p65, GSDMD, and ASC, and increased expression of IκBα. In conclusion, I. terricolous-medicated serum down-regulates HSC activation by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway.

Anti-inflammatory potential of stevia residue extract against uric acid-associated renal injury in mice.

Abnormal uric acid level result in the development of hyperuricemia and hallmark of various diseases, including renal injury, gout, cardiovascular disorders, and non-alcoholic fatty liver. This study was designed to explore the anti-inflammatory potential of stevia residue extract (STR) against hyperuricemia-associated renal injury in mice. The results revealed that STR at dosages of 150 and 300 mg/kg bw and allopurinol markedly modulated serum uric acid, blood urea nitrogen, and creatinine in hyperuricemic mice. Serum and renal cytokine levels (IL-18, IL-6, IL-1Β, and TNF-α) were also restored by STR treatments. Furthermore, mRNA and immunohistochemistry (IHC) analysis revealed that STR ameliorates UA (uric acid)-associated renal inflammation, fibrosis, and EMT (epithelial-mesenchymal transition) via MMPS (matrix metalloproteinases), inhibiting NF-κB/NLRP3 activation by the AMPK/SIRT1 pathway and modulating the JAK2-STAT3 and Nrf2 signaling pathways. In summary, the present study provided experimental evidence that STR is an ideal candidate for the treatment of hyperuricemia-mediated renal inflammation. PRACTICAL APPLICATIONS: The higher uric acid results in hyperuricemia and gout. The available options for the treatment of hyperuricemia and gout are the use of allopurinol, and colchicine drugs, etc. These drugs possess several undesirable side effect. The polyphenolic compounds are abundantly present in plants, for example, stevia residue extract (STR) exert a positive effect on human health. From this study results, we can recommend that polyphenolic compounds enrich STR could be applied to develop treatment options for the treatment of hyperuricemia and gout.

Phytochemical Screening and Bioactivity Studies of Endophytes Cladosporium sp. Isolated from the Endangered Plant Vateria Indica Using In Silico and In Vitro Analysis.

Vateria indica is persistent tree used in Unani sources for the medication and classified as critically endangered. Thus, endophytes for alternative methods to explore these endangered Plants having rich source pharmaceuticals' active molecules for drug development and production. Endophytes comprises unexplored microbes as a potential source of rich pharmaceutically bioactive compounds attributable to their relationship with the host. In the current study, we have isolated endophyte fungi Cladosporium from the plant Vateria indica and performed phytochemical screening of its ethanolic extract to detect the phytochemicals using thin layer chromatography (TLC), gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), UV-visible spectrophotometry (UV-VIS), and Fourier transform infrared spectroscopy (FTIR). GC-MS analysis revealed the presence of an anticancer compound hydroxymethyl colchicine, antioxidant compound benzoic acid, and antimicrobial 2-(4-chlorophenoxy)-5-nitro in endophyte fungal extract of plant Vateria indica. Moreover, in silico analysis of bioactive compounds identified by GC-MS analysis using the Autodock Vina and SwissADME confirmed excellent anticancer activity methanone, [4-amino-2-[(phenylmethyl) amino]-5-thiazolyl] (4-fluorophenyl)- and hydroxymethyl colchicine against 6VO4 (Bfl-1 protein) as per Lipinski rule. Furthermore, we also demonstrated the excellent antioxidant of endophytic extract compared to plant extract by DPPH and ABTS assay, as well as antimicrobial activity against both Gram (+ ve) and Gram (- ve) bacteria. Moreover, the endophytic extract also showed its antimitotic activity with a mitotic index of 65.32, greater than the plant extract of 32.56 at 10 mg/ml. Thus endophytic fungi Cladosporium species isolated from plant Vateria indica might be used as a potential source for phytochemical anticancer hydroxymethyl colchicine, an antioxidant benzoic acid, and antimicrobial 2-(4-chlorophenoxy)-5-nitro.

Bis-nor-diterpene from Cnidoscolus quercifolius (Euphorbiaceae) induces tubulin depolymerization-mediated apoptosis in BRAF-mutated melanoma cells.

A phytochemical investigation of cytotoxic extract and fractions of Cnidoscolus quercifolius Pohl led to isolation of five terpenoids, including three lupane-type triterpenes (1-3) and two bis-nor-diterpenes (4-5). Compounds 4 (phyllacanthone) and 5 (favelanone) are commonly found in this species and have unique chemical structure. Although their cytotoxic activity against cancer cells has been previously reported, the anticancer potential of these molecules remains poorly explored. In this paper, the antimelanoma potential of phyllacanthone (PHY) was described for the first time. Cell viability assay showed a promising cytotoxic activity (IC50 = 40.9 µM) against chemoresistant human melanoma cells expressing the BRAF oncogenic mutation (A2058 cell line). After 72 h of treatment, PHY inhibited cell migration and induced apoptosis and cell cycle arrest (p < 0.05). Immunofluorescence assay showed that the pro-apoptotic effect of PHY is probably associated with tubulin depolymerization, resulting in cytoskeleton disruption of melanoma cells. Molecular docking investigation confirmed this hypothesis given that satisfactory interaction between PHY and tubulin was observed, particularly at the colchicine binding site. These results suggest PHY from C. quercifolius could be potential leader for the design of new antimelanoma drugs.

Discovery and engineering of colchicine alkaloid biosynthesis.

Few complete pathways have been established for the biosynthesis of medicinal compounds from plants. Accordingly, many plant-derived therapeutics are isolated directly from medicinal plants or plant cell culture1. A lead example is colchicine, a US Food and Drug Administration (FDA)-approved treatment for inflammatory disorders that is sourced from Colchicum and Gloriosa species2-5. Here we use a combination of transcriptomics, metabolic logic and pathway reconstitution to elucidate a near-complete biosynthetic pathway to colchicine without prior knowledge of biosynthetic genes, a sequenced genome or genetic tools in the native host. We uncovered eight genes from Gloriosa superba for the biosynthesis of N-formyldemecolcine, a colchicine precursor that contains the characteristic tropolone ring and pharmacophore of colchicine6. Notably, we identified a non-canonical cytochrome P450 that catalyses the remarkable ring expansion reaction that is required to produce the distinct carbon scaffold of colchicine. We further used the newly identified genes to engineer a biosynthetic pathway (comprising 16 enzymes in total) to N-formyldemecolcine in Nicotiana benthamiana starting from the amino acids phenylalanine and tyrosine. This study establishes a metabolic route to tropolone-containing colchicine alkaloids and provides insights into the unique chemistry that plants use to generate complex, bioactive metabolites from simple amino acids.

Structure-Based Pharmacophore Design and Virtual Screening for Novel Tubulin Inhibitors with Potential Anticancer Activity.

Tubulin inhibitors have been considered as potential drugs for cancer therapy. However, their drug resistance and serious side-effects are the main reasons for clinical treatment failure. Therefore, there is still an urgent need to develop effective therapeutic drugs. Herein, a structure-based pharmacophore model was developed based on the co-crystallized structures of the tubulin with a high resolution. The model including one hydrogen-bond acceptor feature, two aromatic features, and one hydrophobic feature was further validated using the Gunner-Henry score method. Virtual screening was performed by an integrated protocol that combines drug-likeness analysis, pharmacophore mapping, and molecular docking approaches. Finally, five hits were selected for biological evaluation. The results indicated that all these hits at the concentration of 40 µM showed an inhibition of more than 50% against five human tumor cells (MCF-7, U87MG, HCT-116, MDA-MB-231, and HepG2). Particularly, hit 1 effectively inhibited the proliferation of these tumor cells, with inhibition rates of more than 80%. The results of tubulin polymerization and colchicine-site competition assays suggested that hit 1 significantly inhibited tubulin polymerization by binding to the colchicine site. Thus, hit 1 could be used as a potential chemotherapeutic agent for cancer treatment. This work also demonstrated the potential of our screening protocol to identify biologically active compounds.

Saikosaponin b2 enhances the hepatotargeting effect of anticancer drugs through inhibition of multidrug resistance-associated drug transporters.

AIMS: Vinegar-baked Radix Bupleuri (VBRB) potentiates the activity of anticancer drugs in the liver by increasing their hepatic distribution. However, this phenomenon may be associated with drug transporters. We investigated the effect of saikosaponin b2 (SSb2; the main component of VBRB) on the activity and expression of different drug transporters in both normal cells and those that overexpress the transporter. MAIN METHODS: The activities of transporters were analyzed by concentration of their cellular substrates. Concentrations of colchicine (substrate of Pgp and MRP1) and cisplatin (substrate of OCT2 and MRP2) were determined by high-performance liquid chromatography (HPLC). The concentration of rhodamine B was determined by flow cytometry. The expression of transporter gene and protein were determined by qRT-PCR and Western blotting analysis. KEY FINDINGS: SSb2 increased colchicine efflux in HEK293 cells by primarily increasing Mrp1 activity, independent of gene and protein expression. SSb2 enhanced Mrp2 function and increased cisplatin efflux in BRL3A cells by upregulating Mrp2 gene expression, with a marginal effect on Pgp in normal cells. SSb2 increased OCT2 activity in OCT2-HEK293 cells by increasing the expression of OCT2 protein and mRNA; however, SSb2 inhibited MRP2 activity in MRP2-HEK293 cells by decreasing MRP2 protein expression, and decreased Pgp and MRP1 activity in Pgp- and MRP1-HEK293 cells. SIGNIFICANCE: SSb2 might potentially be the key active component of VBRB that enhances the hepatotargeting of anticancer drugs through the inhibition of multidrug resistance-associated drug transporters (Pgp, MRP1, and MRP2) in an environment-dependent manner.

Design, Synthesis, and Biological Evaluation of 6-Substituted Thieno[3,2- d]pyrimidine Analogues as Dual Epidermal Growth Factor Receptor Kinase and Microtubule Inhibitors.

The clinical evidence for the success of tyrosine kinase inhibitors in combination with microtubule-targeting agents prompted us to design and develop single agents that possess both epidermal growth factor receptor (EGFR) kinase and tubulin polymerization inhibitory properties. A series of 6-aryl/heteroaryl-4-(3',4',5'-trimethoxyanilino)thieno[3,2- d]pyrimidine derivatives were discovered as novel dual tubulin polymerization and EGFR kinase inhibitors. The 4-(3',4',5'-trimethoxyanilino)-6-( p-tolyl)thieno[3,2- d]pyrimidine derivative 6g was the most potent compound of the series as an antiproliferative agent, with half-maximal inhibitory concentration (IC50) values in the single- or double-digit nanomolar range. Compound 6g bound to tubulin in the colchicine site and inhibited tubulin assembly with an IC50 value of 0.71 µM, and 6g inhibited EGFR activity with an IC50 value of 30 nM. Our data suggested that the excellent in vitro and in vivo profile of 6g may be derived from its dual inhibition of tubulin polymerization and EGFR kinase.

Indirubin, a bis-indole alkaloid binds to tubulin and exhibits antimitotic activity against HeLa cells in synergism with vinblastine.

Indirubin, a bis-indole alkaloid used in traditional Chinese medicine has shown remarkable anticancer activity against chronic myelocytic leukemia. The present work was aimed to decipher the underlying molecular mechanisms responsible for its anticancer attributes. Our findings suggest that indirubin inhibited the proliferation of HeLa cells with an IC50 of 40 µM and induced a mitotic block. At concentrations higher than its IC50, indirubin exerted a moderate depolymerizing effect on the interphase microtubular network and spindle microtubules in HeLa cells. Studies with goat brain tubulin indicated that indirubin bound to tubulin at a single site with a dissociation constant of 26 ±â€¯3 µM and inhibited the in vitro polymerization of tubulin into microtubules in the presence of glutamate as well as microtubule-associated proteins. Molecular docking analysis and molecular dynamics simulation studies indicate that indirubin stably binds to tubulin at the interface of the α-ß tubulin heterodimer. Further, indirubin stabilized the binding of colchicine on tubulin and promoted the cysteine residue modification by 5,5'-dithiobis-2-nitrobenzoic acid, indicating towards alteration of tubulin conformation upon binding. In addition, we found that indirubin synergistically enhanced the anti-mitotic and anti-proliferative activity of vinblastine, a known microtubule-targeted agent. Collectively our studies indicate that perturbation of microtubule polymerization dynamics could be one of the possible mechanisms behind the anti-cancer activities of indirubin.

Biotic elicitation of ginsenoside metabolism of mutant adventitious root culture in Panax ginseng.

Biotic elicitation is an important biotechnological strategy for triggering the accumulation of secondary metabolites in adventitious root cultures. These biotic elicitors can be obtained from safe, economically important strains of bacteria found in the rhizosphere and fermented foods. Here, we assayed the effects of filtered cultures of five nitrogen-fixing bacteria and four types of fermentation bacteria on mutant adventitious Panax ginseng root cultures induced in a previous study by colchicine treatment. The biomass, pH, and electrical conductivity (EC) of the culture medium were altered at 5 days after treatment with bacteria. The saponin content was highest in root cultures treated with Mesorhizobium amorphae (GS3037), with a concentration of 105.58 mg g-1 dry weight saponin present in these cultures versus 74.48 mg g-1 dry weight in untreated root cultures. The accumulation of the ginsenosides Rb2 and Rb3 dramatically increased (19.4- and 4.4-fold, and 18.8- and 4.8-fold) 5 days after treatment with M. amorphae (GS3037) and Mesorhizobium amorphae (GS336), respectively. Compound K production increased 1.7-fold after treatment with M. amorphae (GS3037) compared with untreated root cultures. These results suggest that treating mutant adventitious root cultures with biotic elicitors represents an effective strategy for increasing ginsenoside production in Panax ginseng.

Omega 3 fatty acid-enriched nanoemulsion of thiocolchicoside for transdermal delivery: formulation, characterization and absorption studies.

Thiocolchicoside (TCC) is an effective therapeutic agent against the orthopaedic, traumatic and rheumatologic disorders but it suffer from the drawback of poor bioavailability due to extensive first pass metabolism and low permeability via the oral route. The aim of the present study was to evaluate the potential of nanoemulsion (NE) for bioavailability enhancement of TCC through the transdermal route. The NEs were developed using Linseed: sefsol in 1:1 ratio as the oil phase, span 80, Transcutol P and distilled water as surfactant, co-surfactant and aqueous phase. Furthermore, selected formulations were subjected to physical stability and consequently evaluated for in vitro permeation using porcine skin. The optimized formulation had small average globule diameter of 117 nm with polydispersity index of 0.285. The globules were spherical in shape as observed by transmission electron microscopy. The in vitro skin permeation profile of optimized NE was compared with aqueous solution of TCC. Significant increase in permeability parameters were observed in NEs formulation (p < 0.05) as compared to aqueous solution of TCC. The steady-state flux (Jss) and permeability coefficient (Kp) for optimized NE formulation (C1) were found to be 30.63 ± 4.18 µg/cm(2)/h and 15.21 × 10(-3) ± 2.81cm(2)/h, respectively. The results of enhanced permeation through transdermal route suggest that water-in-oil NEs which are compatible with the lipophilic sebum environment of the hair follicle facilitate the transport of TCC, and such transport might be predominantly transfollicular in nature. Overall, these results suggested that water-in-oil NEs are good carriers for transdermal delivery of TCC.

Antimitotic and antivascular activity of heteroaroyl-2-hydroxy-3,4,5-trimethoxybenzenes.

This study reports the synthesis of a series of heteroaroyl-2-hydroxy-3,4,5-trimethoxybenzenes, which are potent antitubulin agents. Compound 13, (2-hydroxy-3,4,5-trimethoxyphenyl)-(6-methoxy-1H-indol-3-yl)-methanone exhibits marked antiproliferative activity against KB and MKN45 cells with IC50 values of 8.8 and 10.5 nM, respectively, binds strongly to the colchicine binding site and leads to inhibition of tubulin polymerization. It also behaves as a vascular disrupting agent which suppresses the formation of capillaries. The C2-OH group in the A-ring of this compound not only retains the biological activity but has valuable physicochemical properties.

The complete chloroplast genome of colchicine plants (Colchicum autumnale L. and Gloriosa superba L.) and its application for identifying the genus.

MAIN CONCLUSION: The complete chloroplast genome of two colchicine medicinal plants is reported for the first time. Deletion of ycf 15 gene occurred only in Colchicum but not in Gloriosa and suggests this as a potential marker for delineating the two species. Colchicum autumnale L. and Gloriosa superba L. are well-known sources of colchicine, a type of alkaloid and an ancient anti-inflammatory drug used to prevent gout. Accordingly, this alkaloid has been used as a chemical marker for identifying the expanded Colchicaceae family. In the present study, we report the complete chloroplast genome (cpDNA) sequence of two colchicine medicinal plants (G. superba and C. autumnale) that belong to the tribe Colchiceae of the Colchicaceae family. In C. autumnale, the circular double-stranded cpDNA sequence of 156,462 bp consists of two inverted repeat (IR) regions of 27,741 bp each, a large single-copy region (LSC) of 84,246 bp, and a small single-copy region (SSC) of 16,734 bp. The cpDNA sequence of G. superba is longer than that of C. autumnale (157,924 bp), which consists of two IRs (28,063 bp), an SSC (16,786 bp), and an LSC (85,012 bp). Significant structural differences between them were observed in the ycf15 gene. ycf15 gene was absent from C. autumnale cpDNA and affected the length of the chloroplast genome between the species. Furthermore, this gene loss event was specific to the expanded genus of Colchicum sensu Vinnersten and Manning. Therefore, this gene may be an effective and powerful molecular marker for identifying the Colchicum genus within the family.

Combined Molecular Docking, 3D-QSAR, and Pharmacophore Model: Design of Novel Tubulin Polymerization Inhibitors by Binding to Colchicine-binding Site.

Interference with dynamic equilibrium of microtubule-tubulin has proven to be a useful tactics in the clinic. Based on investigation into the structure-activity relationship (SAR) studies of tubulin polymerization inhibitors obtained from several worldwide groups, we attempted to design 691 compounds covering several main heterocyclic scaffolds as novel colchicine-site inhibitors (CSIs). Evaluated by a series of combination of commonly used computer methods such as molecular docking, 3D-QSAR, and pharmacophore model, we can obtain the ultimate 16 target compounds derived from five important basic scaffolds in the field of medicinal chemistry. Among these compounds, compound A-132 with in silico moderate activity was synthesized, and subsequently validated for preliminary inhibition of tubulin polymerization by immunofluorescence assay. In additional, the work of synthesis and validation of biological activity for other 15 various structure compounds will be completed in our laboratory. This study not only developed a hierarchical strategy to screen novel tubulin inhibitors effectively, but also widened the spectrum of chemical structures of canonical CSIs.

Improved growth and colchicine concentration in Gloriosa superba on mycorrhizal inoculation supplemented with phosphorus-fertilizer.

BACKGROUND: Gloriosa superba produces an array of alkaloids including colchicine, a compound of interest in the treatment of various diseases. The tuber of Gloriosa superba is a rich source of colchicine which has shown anti-gout, anti-inflammatory, and anti-tumor activity. However, this promising compound remains expensive and Gloriosa superba is such a good source in global scale. Increase in yield of naturally occurring colchicine is an important area of investigation. MATERIALS AND METHODS: The effects of inoculation by four arbuscular mycorrhizal (AM), fungi, Glomus mossae, Glomus fasciculatum, Gigaspora margarita and Gigaspora gilmorei either alone or supplemented with P-fertilizer, on colchicine concentration in Gloriosa superba were studied. The concentration of colchicine was determined by high-performance thin layer chromatography. RESULTS: The four fungi significantly increased concentration of colchicine in the herb. Although there was significant increase in concentration of colchicine in non-mycorrhizal P-fertilized plants as compared to control, the extent of the increase was less compared to mycorrhizal plants grown with or without P-fertilization. This suggests that the increase in colchicine concentration may not be entirely attributed to enhanced P-nutrition and improved growth. Among the four AM fungi Glomus mossae was found to be best. The total colchicine content of plant (mg / plant) was significantly high in plants inoculated with Glomus mossae and 25 mg kg(-1)phosphorus fertilizer (348.9 mg /plant) while the control contain least colchicine (177.87 mg / plant). CONCLUSION: The study suggests a potential role of AM fungi in improving the concentration of colchicine in Gloriosa superba tuber.

Novel colchicine-site binders with a cyclohexanedione scaffold identified through a ligand-based virtual screening approach.

Vascular disrupting agents (VDAs) constitute an innovative anticancer therapy that targets the tumor endothelium, leading to tumor necrosis. Our approach for the identification of new VDAs has relied on a ligand 3-D shape similarity virtual screening (VS) approach using the ROCS program as the VS tool and as query colchicine and TN-16, which both bind the α,ß-tubulin dimer. One of the hits identified, using TN-16 as query, has been explored by the synthesis of its structural analogues, leading to 2-(1-((2-methoxyphenyl)amino)ethylidene)-5-phenylcyclohexane-1,3-dione (compound 16c) with an IC50 = 0.09 ± 0.01 µM in HMEC-1 and BAEC, being 100-fold more potent than the initial hit. Compound 16c caused cell cycle arrest in the G2/M phase and interacted with the colchicine-binding site in tubulin, as confirmed by a competition assay with N,N'-ethylenebis(iodoacetamide) and by fluorescence spectroscopy. Moreover, 16c destroyed an established endothelial tubular network at 1 µM and inhibited the migration and invasion of human breast carcinoma cells at 0.4 µM. In conclusion, our approach has led to a new chemotype of promising antiproliferative compounds with antimitotic and potential VDA properties.

Enhancement of artemisinin content in tetraploid Artemisia annua plants by modulating the expression of genes in artemisinin biosynthetic pathway.

Tetraploid Artemisia annua plants were successfully inducted by using colchicine, and their ploidy was confirmed by flow cytometry. Higher stomatal length but lower frequency in tetraploids were revealed and could be considered as indicators of polyploidy. The average level of artemisinin in tetraploids was increased from 39% to 56% than that of the diploids during vegetation period, as detected by high-performance liquid chromatography-evaporative light scattering detector. Gene expressions of 10 key enzymes related to artemisinin biosynthetic pathway in different ploidy level were analyzed by semiquantitative polymerase chain reaction and significant upregulation of FPS, HMGR, and artemisinin metabolite-specific Aldh1 genes were revealed in tetraploids. Slight increased expression of ADS was also detected. Our results suggest that higher artemisinin content in tetraploid A. annua may result from the upregulated expression of some key enzyme genes related to artemisinin biosynthetic pathway.

Microtubule-binding natural products for cancer therapy.

Natural products, especially microtubule-binding natural products, play important roles in the war against cancer. From the clinical use of vinblastine in 1961, paclitaxel in 1992, to ixabepilone in 2007, microtubule-binding natural products have continually contributed to the development of cancer therapy. The present review summarizes the development of representative microtubule-binding natural products including agents binding to the colchicine-binding site, the VINCA alkaloid-binding site, the taxane-binding site and other binding sites. Future directions for the development of new anticancer microtubule-binding natural products are discussed. Finding new formulations, new targets and new sources of microtubule-binding natural products may enable more members of this kind of agent to be introduced into the clinic for cancer therapy.

Hydropathic analysis and biological evaluation of stilbene derivatives as colchicine site microtubule inhibitors with anti-leukemic activity.

The crucial role of the microtubule in cell division has identified tubulin as a target for the development of therapeutics for cancer; in particular, tubulin is a target for antineoplastic agents that act by interfering with the dynamic stability of microtubules. A molecular modeling study was carried out to accurately represent the complex structure and the binding mode of a new class of stilbene-based tubulin inhibitors that bind at the alphabeta-tubulin colchicine site. Computational docking along with HINT (Hydropathic INTeractions) score analysis fitted these inhibitors into the colchicine site and revealed detailed structure-activity information useful for inhibitor design. Quantitative analysis of the results was in good agreement with the in vitro antiproliferative activity of these derivatives (ranging from 3 nM to 100 muM) such that calculated and measured free energies of binding correlate with an r(2) of 0.89 (standard error +/- 0.85 kcal mol(-1)). This correlation suggests that the activity of unknown compounds may be predicted.