Anticancer Water-Soluble Organoruthenium Complexes: Synthesis and Preclinical Evaluation.

The synthesis, characterisation, and evaluation of the in vitro cytotoxicity of five maleonitriledithiolate-based ruthenium metal complexes bearing various phosphine ligands towards two ovarian cancer cell lines (A2780 and A2780cisR), one non-small-cell lung cancer cell line (H460) and one normal prostate cell line (PNT2) are presented herein. These 18-electron complexes were designed with four water-soluble phosphine ligands to increase the water-solubility character of the corresponding electron-deficient ruthenium complex which showed great in vitro promises, and triphenylphosphine for comparison. The complexes with triphenylphosphine-3,3',3''-trisulfonic acid and triphenylphosphine present similar cytotoxicity compared to the 16-electron precursor, with equal cytotoxicity to both A2780 and A2780cisR. Hints at the mechanism of action suggest an apoptotic pathway based on reactive oxygen species (ROS) production. No toxicity was observed in preliminary in vivo pilot studies for these two complexes in subcutaneous A2780 and A2780cisR xenograft models, with some evidence of tumour growth delay.

Controlled Bioactive Delivery Using Degradable Electroactive Polymers.

Biomaterials capable of precisely controlling the delivery of agrochemicals/biologics/drugs/fragrances have significant markets in the agriscience/healthcare industries. Here, we report the development of degradable electroactive polymers and their application for the controlled delivery of a clinically relevant drug (the anti-inflammatory dexamethasone phosphate, DMP). Electroactive copolymers composed of blocks of polycaprolactone (PCL) and naturally occurring electroactive pyrrole oligomers (e.g., bilirubin, biliverdin, and hemin) were prepared and solution-processed to produce films (optionally doped with DMP). A combination of in silico/in vitro/in vivo studies demonstrated the cytocompatibility of the polymers. The release of DMP in response to the application of an electrical stimulus was observed to be enhanced by ca. 10-30% relative to the passive release from nonstimulated samples in vitro. Such stimuli-responsive biomaterials have the potential for integration devices capable of delivering a variety of molecules for technical/medical applications.

An efficient assay for identification and quantitative evaluation of potential polysialyltransferase inhibitors.

The polysialyltransferases (polySTs) catalyse the polymerisation of polysialic acid, which plays an important role in tumour metastasis. While assays are available to assess polyST enzyme activity, there is no methodology available specifically optimised for identification and quantitative evaluation of potential polyST inhibitors. The development of an HPLC-fluorescence-based enzyme assay described within includes a comprehensive investigation of assay conditions, including evaluation of metal ion composition, enzyme, substrate and acceptor concentrations, temperature, pH, and tolerance to DMSO, followed by validation using known polyST inhibitors. Thorough analysis of each of the assay components provided a set of optimised conditions. Under these optimised conditions, the experimentally observed Ki value for CMP, a competitive polyST inhibitor, was strongly correlated with the predicted Ki value, based on the classical Cheng-Prusoff equation [average fold error (AFE) = 1.043]. These results indicate that this assay can provide medium-throughput analysis for enzyme inhibitors with high accuracy, through determining the corresponding IC50 values with substrate concentration at the KM, without the need to perform extensive kinetic studies for each compound. In conclusion, an in vitro cell-free assay for accurate assessment of polyST inhibition is described. The utility of the assay for routine identification of potential polyST inhibitors is demonstrated, allowing quantitative measurement of inhibition to be achieved, and exemplified through assessment of full competitive inhibition. Given the considerable and growing interest in the polySTs as important anti-metastatic targets in cancer drug discovery, this is a vital tool to enable preclinical identification and evaluation of novel polyST inhibitors.

Tubulin-binding dibenz[c,e]oxepines: Part 2. Structural variation and biological evaluation as tumour vasculature disrupting agents.

5,7-Dihydro-3,9,10,11-tetramethoxybenz[c,e]oxepin-4-ol 1, prepared from a dibenzyl ether precursor via Pd-catalysed intramolecular direct arylation, possesses broad-spectrum in vitro cytotoxicity towards various tumour cell lines, and induces vascular shutdown, necrosis and growth delay in tumour xenografts in mice at sub-toxic doses. The biological properties of 1 and related compounds can be attributed to their ability to inhibit microtubule assembly at the micromolar level, by binding reversibly to the same site of the tubulin αß-heterodimer as colchicine 2 and the allocolchinol, N-acetylcolchinol 4.

Tumor-targeted prodrug ICT2588 demonstrates therapeutic activity against solid tumors and reduced potential for cardiovascular toxicity.

Development of therapeutic strategies for tumor-selective delivery of therapeutics through exploitation of the proteolytic tumor phenotype has significant scope for improvement of cancer treatment. ICT2588 is a peptide-conjugated prodrug of the vascular disrupting agent (VDA) azademethylcolchicine developed to be selectively hydrolyzed by matrix metalloproteinase-14 (MMP-14) within the tumor. In this report, we extend our previous proof-of-concept studies and demonstrate the therapeutic potential of this agent against models of human colorectal, lung, breast, and prostate cancer. In all tumor types, ICT2588 was superior to azademethylcolchicine and was greater or comparable to standard clinically used agents for the respective tumor type. Prodrug activation in clinical human lung tumor homogenates relative to stability in human plasma and liver was observed, supporting clinical translation potential. A major limiting factor to the clinical value of VDAs is their inherent cardiovascular toxicity. No increase in plasma von Willebrand factor (vWF) levels, an indicator of systemic vascular dysfunction and acute cardiovascular toxicity, was detected with ICT2588, thereby supporting the tumor-selective activation and reduced potential of ICT2588 to cause cardiovascular toxicity. Our findings reinforce the improved therapeutic index and tumor-selective approach offered by ICT2588 and this nanotherapeutic approach.

A Proteomic Investigation to Discover Candidate Proteins Involved in Novel Mechanisms of 5-Fluorouracil Resistance in Colorectal Cancer.

One of the main obstacles to therapeutic success in colorectal cancer (CRC) is the development of acquired resistance to treatment with drugs such as 5-fluorouracil (5-FU). Whilst some resistance mechanisms are well known, it is clear from the stasis in therapy success rate that much is still unknown. Here, a proteomics approach is taken towards identification of candidate proteins using 5-FU-resistant sublines of human CRC cell lines generated in house. Using a multiplexed stable isotope labelling with amino acids in cell culture (SILAC) strategy, 5-FU-resistant and equivalently passaged sensitive cell lines were compared to parent cell lines by growing in Heavy medium with 2D liquid chromatography and Orbitrap Fusion™ Tribrid™ Mass Spectrometry analysis. Among 3003 commonly quantified proteins, six (CD44, APP, NAGLU, CORO7, AGR2, PLSCR1) were found up-regulated, and six (VPS45, RBMS2, RIOK1, RAP1GDS1, POLR3D, CD55) down-regulated. A total of 11 of the 12 proteins have a known association with drug resistance mechanisms or role in CRC oncogenesis. Validation through immunodetection techniques confirmed high expression of CD44 and CD63, two known drug resistance mediators with elevated proteomics expression results. The information revealed by the sensitivity of this method warrants it as an important tool for elaborating the complexity of acquired drug resistance in CRC.

NCAM polysialylation during adherence transitions: live cell monitoring using an antibody-mimetic EGFP-endosialidase and the viability dye DRAQ7.

Polysialylation of neural cell adhesion molecule (NCAM) in small-cell lung cancer (SCLC) is thought to regulate NCAM-mediated cell-surface interactions, imparting antiadhesive properties to cells. However, SCLC cells in culture demonstrate anchorage-independent growth and spontaneously generate adherent forms. Here, the ability of polySia-NCAM to influence cell proliferation and adherence is unclear. We analyzed live SCLC cell polySia-NCAM expression by flow cytometry, using the novel combination of a polySia antibody-mimetic eGFP-tagged endosialidase and the viability dye DRAQ7. Enrichment for adherence (<30 population doublings) in SCLC cell lines resolved populations with increased (SHP-77 and COR-L279) or negligible (NCI-H69) polysialylation compared with nonadherent parent populations. Adherent forms retained NCAM expression as confirmed by immunofluorescence and immunoblotting. Initial transition to adherence and loss of polysialylation in NCI-H69 was linked to a reduced proliferation rate with no increase in cell death. This reduced proliferation rate was reiterated in vivo as determined by the growth of noninvasive subcutaneous xenografts in mice. Continued selection for enhanced substrate adherence in NCI-H69 (>150 population doublings) resolved cells with stable re-expression of polySia and increased growth rates both in vitro and in vivo. Endoneuraminidase removal of polySia from re-expressing cells showed that rapid adherence to extracellular matrix components was functionally independent of polySia. PolySia expression was not altered when isolated adherent forms underwent enforced cell-cell contact in three-dimensional culture. Coculture of polySia expression variants modulated overall polySia expression profiles indicating an influence of SCLC microcommunity composition independent of substrate adherence potential. We conclude that an obligatory linkage between substrate adherence potential and polySia expression is rejected for SCLC cells. We suggest that a degree of homeostasis operates to regulate polysialylation within heterogeneous cell populations. The findings suggest a new model for SCLC progression while the application of live cell profiling of polysialylation could be used to assess polySia-NCAM-targeted therapies.

Synthesis and Biological Evaluation of Cyclobutane-Based ß3 Integrin Antagonists: A Novel Approach to Targeting Integrins for Cancer Therapy.

The Arg-Gly-Asp (RGD)-binding family of integrin receptors, and notably the ß3 subfamily, are key to multiple physiological processes involved in tissue development, cancer proliferation, and metastatic dissemination. While there is compelling preclinical evidence that both αvß3 and αIIbß3 are important anticancer targets, most integrin antagonists developed to target the ß3 integrins are highly selective for αvß3 or αIIbß3. We report the design, synthesis, and biological evaluation of a new structural class of ligand-mimetic ß3 integrin antagonist. These new antagonists combine a high activity against αvß3 with a moderate affinity for αIIbß3, providing the first evidence for a new approach to integrin targeting in cancer.

Synthesis and biological evaluation of colchicine C-ring analogues tethered with aliphatic linkers suitable for prodrug derivatisation.

Colchicine was modified at the 10-OCH(3) position of the C-ring by reaction with heterocyclic amines or commercially available amines to afford a library of target colchicinoids in high yields (62-99%). Molecular modeling revealed that the incorporation of the linker groups led to a reduction in entropy and therefore binding affinity when compared with colchicine. Some colchicinoids were shown to be equicytotoxic with colchicine when evaluated in the DLD-1 colon cancer cells and retained activity in resistant A2780AD or HeLa cells with mutant Class III ß-tubulin. Importantly, unlike colchicine, the analogues in this study are amenable for prodrug derivatisation and with potential for tumor-selective delivery.

Semi-Synthetic Analogues of Cryptolepine as a Potential Source of Sustainable Drugs for the Treatment of Malaria, Human African Trypanosomiasis, and Cancer.

The prospect of eradicating malaria continues to be challenging in the face of increasing parasite resistance to antimalarial drugs so that novel antimalarials active against asexual, sexual, and liver-stage malaria parasites are urgently needed. In addition, new antimalarials need to be affordable and available to those most in need and, bearing in mind climate change, should ideally be sustainable. The West African climbing shrub Cryptolepis sanguinolenta is used traditionally for the treatment of malaria; its principal alkaloid, cryptolepine (1), has been shown to have antimalarial properties, and the synthetic analogue 2,7-dibromocryptolepine (2) is of interest as a lead toward new antimalarial agents. Cryptolepine (1) was isolated using a two-step Soxhlet extraction of C. sanguinolenta roots, followed by crystallization (yield 0.8% calculated as a base with respect to the dried roots). Semi-synthetic 7-bromo- (3), 7, 9-dibromo- (4), 7-iodo- (5), and 7, 9-dibromocryptolepine (6) were obtained in excellent yields by reaction of 1 with N-bromo- or N-iodosuccinimide in trifluoroacetic acid as a solvent. All compounds were active against Plasmodia in vitro, but 6 showed the most selective profile with respect to Hep G2 cells: P. falciparum (chloroquine-resistant strain K1), IC50 = 0.25 µM, SI = 113; late stage, gametocytes, IC50 = 2.2 µM, SI = 13; liver stage, P. berghei sporozoites IC50 = 6.13 µM, SI = 4.6. Compounds 3-6 were also active against the emerging zoonotic species P. knowlesi with 5 being the most potent (IC50 = 0.11 µM). In addition, 3-6 potently inhibited T. brucei in vitro at nM concentrations and good selectivity with 6 again being the most selective (IC50 = 59 nM, SI = 478). These compounds were also cytotoxic to wild-type ovarian cancer cells as well as adriamycin-resistant and, except for 5, cisplatin-resistant ovarian cancer cells. In an acute oral toxicity test in mice, 3-6 did not exhibit toxic effects at doses of up to 100 mg/kg/dose × 3 consecutive days. This study demonstrates that C. sanguinolenta may be utilized as a sustainable source of novel compounds that may lead to the development of novel agents for the treatment of malaria, African trypanosomiasis, and cancer.

The microtubule depolymerizing agent CYT997 causes extensive ablation of tumor vasculature in vivo.

The orally active microtubule-disrupting agent (S)-1-ethyl-3-(2-methoxy-4-(5-methyl-4-((1-(pyridin-3-yl)butyl)amino)pyrimidin-2-yl)phenyl)urea (CYT997), reported previously by us (Bioorg Med Chem Lett 19:4639-4642, 2009; Mol Cancer Ther 8:3036-3045, 2009), is potently cytotoxic to a variety of cancer cell lines in vitro and shows antitumor activity in vivo. In addition to its cytotoxic activity, CYT997 possesses antivascular effects on tumor vasculature. To further characterize the vascular disrupting activity of CYT997 in terms of dose and temporal effects, we studied the activity of the compound on endothelial cells in vitro and on tumor blood flow in vivo by using a variety of techniques. In vitro, CYT997 is shown to potently inhibit the proliferation of vascular endothelial growth factor-stimulated human umbilical vein endothelial cells (IC(50) 3.7 ± 1.8 nM) and cause significant morphological changes at 100 nM, including membrane blebbing. Using the method of corrosion casting visualized with scanning electron microscopy, a single dose of CYT997 (7.5 mg/kg i.p.) in a metastatic cancer model was shown to cause destruction of tumor microvasculature in metastatic lesions. Furthermore, repeat dosing of CYT997 at 10 mg/kg and above (intraperitoneally, b.i.d.) was shown to effectively inhibit development of liver metastases. The time and dose dependence of the antivascular effects were studied in a DLD-1 colon adenocarcinoma xenograft model using the fluorescent dye Hoechst 33342. CYT997 demonstrated rapid and dose-dependent vascular shutdown, which persists for more than 24 h after a single oral dose. Together, the data demonstrate that CYT997 possesses potent antivascular activity and support continuing development of this promising compound.

Recent Advances in the Chemistry and Pharmacology of Cryptolepine.

Cryptolepine, the principal constituent of the West African climbing shrub Cryptolepis sanguinolenta, continues to be of interest as a lead to new therapeutic agents, especially for the treatment of protozoal infections and cancer. This contribution reviews the research published in the last decade, highlighting new synthesis routes to cryptolepine and to analogs of this alkaloid, as well as their pharmacology. Studies relating to the use of C. sanguinolenta as an herbal medicine for the treatment of malaria are discussed, as well as the development of analogs of cryptolepine as leads to new agents for the treatment of malaria, trypanosomiasis, and cancer with an emphasis on the pharmacological mechanisms involved. Other potential therapeutic applications include antimicrobial, antidiabetic, and anti-inflammatory activities; the pharmacokinetics and toxicity of cryptolepine are also reviewed.

An assay for quantitative analysis of polysialic acid expression in cancer cells.

Polysialic acid (polySia) is a linear polysaccharide comprised of N-acetylneuraminic acid residues and its over-expression in cancer cells has been correlated with poor clinical prognosis. An assay has been developed for quantitative analysis of cellular polySia expression. This was achieved by extracting and purifying released polySia from glycoproteins by mild acid hydrolysis and optimised organic extraction. The polySia was further hydrolysed into Sia monomers, followed by fluorescent labelling and quantitative analysis. The assay was qualified utilising endoneuraminidase-NF to remove polySia from the surface of C6-ST8SiaII cancer cells (EC50 = 2.13 ng/mL). The result was comparable to that obtained in a polySia-specific cellular ELISA assay. Furthermore, the assay proved suitable for evaluation of changes in polySia expression following treatment with a small molecule inhibitor of polysialylation. Given the importance of polySia in multiple disease states, notably cancer, this is a potentially vital tool with applications in the fields of drug discovery and glycobiology.

Cytochrome P450 isoforms 1A1, 1B1 AND 2W1 as targets for therapeutic intervention in head and neck cancer.

Epidemiological studies have shown that head and neck cancer (HNC) is a complex multistage process that in part involves exposure to a combination of carcinogens and the capacity of certain drug-metabolising enzymes including cytochrome P450 (CYP) to detoxify or activate such carcinogens. In this study, CYP1A1, CYP1B1 and CYP2W1 expression in HNC was correlated with potential as target for duocarmycin prodrug activation and selective therapy. In the HNC cell lines, elevated expression was shown at the gene level for CYP1A1 and CYP1B1 whereas CYP2W1 was hardly detected. However, CYP2W1 was expressed in FaDu and Detroit-562 xenografts and in a cohort of human HNC samples. Functional activity was measured in Fadu and Detroit-562 cells using P450-Glo™ assay. Antiproliferative results of duocarmycin prodrugs ICT2700 and ICT2706 revealed FaDu and Detroit-562 as the most sensitive HNC cell lines. Administration of ICT2700 in vivo using a single dose of ICT2700 (150 mg/kg) showed preferential inhibition of small tumour growth (mean size of 60 mm3) in mice bearing FaDu xenografts. Significantly, our findings suggest a potential targeted therapeutic approach to manage HNCs by exploiting intratumoural CYP expression for metabolic activation of duocarmycin-based prodrugs such as ICT2700.

Preclinical Anticancer Activity of an Electron-Deficient Organoruthenium(II) Complex.

Ruthenium compounds have been shown to be promising alternatives to platinum(II) drugs. However, their clinical success depends on achieving mechanisms of action that overcome Pt-resistance mechanisms. Electron-deficient organoruthenium complexes are an understudied class of compounds that exhibit unusual reactivity in solution and might offer novel anticancer mechanisms of action. Here, we evaluate the in vitro and in vivo anticancer properties of the electron-deficient organoruthenium complex [(p-cymene)Ru(maleonitriledithiolate)]. This compound is found to be highly cytotoxic: 5 to 60 times more potent than cisplatin towards ovarian (A2780 and A2780cisR), colon (HCT116 p53+/+ and HCT116 p53-/-), and non-small cell lung H460 cancer cell lines. It shows no cross-resistance and is equally cytotoxic to both A2780 and A2780cisR cell lines. Furthermore, unlike cisplatin, the remarkable in vitro antiproliferative activity of this compound appears to be p53-independent. In vivo evaluation in the hollow-fibre assay across a panel of cancer cell types and subcutaneous H460 non-small cell lung cancer xenograft model hints at the activity of the complex. Although the impressive in vitro data are not fully corroborated by the in vivo follow-up, this work is the first preclinical study of electron-deficient half-sandwich complexes and highlights their promise as anticancer drug candidates.

Application of small molecule FPR1 antagonists in the treatment of cancers.

The formylpeptide receptor-1 (FPR1) is a member of the chemotactic GPCR-7TM formyl peptide receptor family, whose principle function is in trafficking of various leukocytes into sites of bacterial infection and inflammation. More recently, FPR1 has been shown to be expressed in different types of cancer and in this context, plays a significant role in their expansion, resistance and recurrence. ICT12035 is a selective and potent (30 nM in calcium mobilisation assay) small molecule FPR1 antagonist. Here, we demonstrate the efficacy of ICT12035, in a number of 2D and 3D proliferation and invasion in vitro assays and an in vivo model. Our results demonstrate that targeting FPR1 by a selective small molecule antagonist, such as ICT12035, can provide a new avenue for the treatment of cancers.

Use of the hollow fibre assay for studies of tumor neovasculature.

In vivo preclinical assays are required to screen potential agents that target the tumor vasculature. Here, a hollow fibre-based assay for the quantification of neovasculature in the presence or absence of an agent that potentially targets tumor neovasculature is described. The neovasculature is developed as a consequence of the presence of tumor cells encapsulated in hollow fibres, which are transplanted subcutaneously in the dorsal flanks of mice.

Tumor growth suppression using a combination of taxol-based therapy and GSK3 inhibition in non-small cell lung cancer.

Glycogen synthase kinase-3 (GSK3) is over-expressed and hyperactivated in non-small cell lung carcinoma (NSCLC) and plays a role in ensuring the correct alignment of chromosomes on the metaphase plate during mitosis through regulation of microtubule stability. This makes the enzyme an attractive target for cancer therapy. We examined the effects of a selective cell-permeant GSK3 inhibitor (CHIR99021), used alone or in combination with paclitaxel, using an in vitro cell growth assay, a quantitative chromosome alignment assay, and a tumor xenograft model. CHIR99021 inhibits the growth of human H1975 and H1299 NSCLC cell lines in a synergistic manner with paclitaxel. CHIR99021 and paclitaxel promoted a synergistic defect in chromosomal alignment when compared to each compound administered as monotherapy. Furthermore, we corroborated our in vitro findings in a mouse tumor xenograft model. Our results demonstrate that a GSK3 inhibitor and paclitaxel act synergistically to inhibit the growth of NSCLC cells in vitro and in vivo via a mechanism that may involve converging modes of action on microtubule spindle stability and thus chromosomal alignment during metaphase. Our findings provide novel support for the use of the GSK3 inhibitor, CHIR99021, alongside taxol-based chemotherapy in the treatment of human lung cancer.

Guaianolide sesquiterpenes from Pulicaria crispa (Forssk.) Oliv.

A phytochemical study of the asteraceous herb Pulicaria crispa (Forssk.) Oliv. resulted in the characterisation of three guaianolide sesquiterpenes, 2alpha,4alpha-dihydroxy-7alphaH,8alphaH,10alphaH-guaia-1(5),11(13)-dien-8beta,12-olide (1), 1alpha,2alpha-epoxy-4beta-hydroxy-5alphaH,7alphaH,8alphaH,10alphaH-guaia-11(13)-en-8beta,12-olide (2) and 5,10-epi-2,3-dihydroaromatin (3). The structures were assigned on the basis of extensive 1 and 2D NMR experiments. Compound 3 exhibited weak antimycobacterial activity against Mycobacterium phlei with a minimum inhibitory concentration of 0.52 mM and cytotoxicity (IC50 of 5.8+/-0.2 microM) in a human bladder carcinoma cell line, EJ-138.

Hypoxia modulates CCR7 expression in head and neck cancers.

BACKGROUND: The chemokine receptor CCR7 is expressed on lymphocytes and dendritic cells and is responsible for trafficking of these cells in and out of secondary lymphoid organs. It has recently been shown that CCR7 expression is elevated in a number of cancers, including head and neck cancers, and that its expression correlates to lymph node (LN) metastasis. However, little is known about the factors that can induce CCR7 expression in head and neck cancers. METHOD: We compared the protein expression and functional responses of CCR7 under normoxia and hypoxia in head and neck cancer cell lines OSC-19, FaDu, SCC-4, A-253 and Detroit-562 cultured as monolayers, spheroids, and grown in vivo as xenografts in balb/c mice. In addition, we analysed the correlation between hypoxia marker HIF-1α and CCR7 expression in a tissue microarray comprising 80 clinical samples with various stages and grades of malignant tumour and normal tissue. RESULTS: Under hypoxia, the expression of CCR7 is elevated in both in vitro and in vivo models. Furthermore, in malignant tissue, a correlation is observed between hypoxia marker HIF-1α and CCR7 across all clinical stages. This correlation is also strong in early histological grade of tumours. CONCLUSION: Hypoxia plays a role in the regulation of the expression of CCR7 and it may contribute to the development of a metastatic phenotype in head and neck cancers through this axis.