HT-29 Colon Carcinoma Research Articles

Green synthesis of NiO nanoparticles using Moringa oleifera extract and their biomedical applications: Cytotoxicity effect of nanoparticles against HT-29 cancer cells

Green protocols for the synthesis of nickel oxide nanoparticles using Moringa oleifera plant extract has been reported in the present study as they are cost effective and ecofriendly, moreover this paper records that the nickel oxide (NiO) nanoparticles prepared from green method shows better cytotoxicity and antibacterial activity. The NiO nanoparticles were characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), High resolution transmission electron microscopy (HRTEM), Energy dispersive X-ray analysis (EDX), and Photoluminescence spectroscopy (PL). The formation of a pure nickel oxide phase was confirmed by XRD and FTIR. The synthesized NiO nanoparticles was single crystalline having face centered cubic phase and has two intense photoluminescence emissions at 305.46nm and 410nm. The formation of nano- and micro-structures was confirmed by HRTEM. The in-vitro cytotoxicity and cell viability of human cancer cell HT-29 (Colon Carcinoma cell lines) and antibacterial studies against various bacterial strains were studied with various concentrations of nickel oxide nanoparticles prepared from Moringa oleifera plant extract. MTT assay measurements on cell viability and morphological studies proved that the synthesized NiO nanoparticles posses cytotoxic activity against human cancer cells and the various zones of inhibition (mm), obtained revealed the effective antibacterial activity of NiO nanoparticles against various Gram positive and Gram negative bacterial pathogens.

Halogenated naphthochalcones and structurally related naphthopyrazolines with antitumor activity

Three 3-(3-halo-4,5-dimethoxyphenyl)-1-(2-naphthyl)prop-2-en-1-ones 1 and three structurally related 2-pyrazolines 2 were prepared and assessed in vitro for anticancer activity. The chalcones 1 were antiproliferative with low double-digit micromolar IC50 values against six tumor cell lines whereas the pyrazolines 2 showed low single-digit micromolar IC50 values against this panel. The pyrazolines inhibited ATP-binding cassette efflux transporters of types P-gp and BCRP while the chalcones inhibited selectively BCRP. All test compounds induced an accumulation of HT-29 colon carcinoma cells in the G2/M phase of the cell cycle and they interfered with the microtubule and F-actin dynamics, but only the chalcones induced apoptosis in 518A2 melanoma cells after 24h.

Identification of a novel human deoxynivalenol metabolite enhancing proliferation of intestinal and urinary bladder cells.

The mycotoxin deoxynivalenol (DON) is an abundant contaminant of cereal based food and a severe issue for global food safety. We report the discovery of DON-3-sulfate as a novel human metabolite and potential new biomarker of DON exposure. The conjugate was detectable in 70% of urine samples obtained from pregnant women in Croatia. For the measurement of urinary metabolites, a highly sensitive and selective LC-MS/MS method was developed and validated. The method was also used to investigate samples from a duplicate diet survey for studying the toxicokinetics of DON-3-sulfate. To get a preliminary insight into the biological relevance of the newly discovered DON-sulfates, in vitroexperiments were performed. In contrast to DON, sulfate conjugates lacked potency to suppress protein translation. However, surprisingly we found that DON-sulfates enhanced proliferation of human HT-29 colon carcinoma cells, primary human colon epithelial cells (HCEC-1CT) and, to some extent, also T24 bladder cancer cells. A proliferative stimulus, especially in tumorigenic cells raises concern on the potential impact of DON-sulfates on consumer health. Thus, a further characterization of their toxicological relevance should be of high priority.

Effects of histidin-2-ylidene vs. imidazol-2-ylidene ligands on the anticancer and antivascular activity of complexes of ruthenium, iridium, platinum, and gold

Couples of N-heterocyclic carbene complexes of ruthenium, iridium, platinum, and gold, each differing only in the carbene ligand being either 1,3-dimethylimidazol-2-ylidene (IM) or 1,3-dimethyl-N-boc-O-methylhistidin-2-ylidene (HIS), were assessed for their antiproliferative effect on seven cancer cell lines, their interaction with DNA, their cell cycle interference, and their vascular disrupting properties. In MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays only the platinum complexes were cytotoxic at single-digit micromolar IC50 concentrations with the (HIS)Pt complex being on average twice as active as the (IM)Pt complex. The former was highly efficacious against cisplatin-resistant HT-29 colon carcinoma cells where the latter had no effect. Both Pt complexes were accumulated by cancer cells and bound to double-helical DNA equally well. Only the (HIS)Pt complex modified the electrophoretic mobility of circular DNA in vitro due to the HIS ligand causing greater morphological changes to the DNA. Both platinum complexes induced accumulation of 518A2 melanoma cells in G2/M and S phase of the cell cycle. A disruption of blood vessels in the chorioallantoic membrane of fertilized chicken eggs was observed for both platinum complexes and the (IM)gold complex. The (HIS)platinum complex was as active as cisplatin in tumor xenografted mice while being tolerated better. We found that the HIS ligand may augment the cytotoxicity of certain antitumoral metal fragments in two ways: by acting as a transmembrane carrier increasing the cellular accumulation of the complex, and by initiating a pronounced distortion and unwinding of DNA. We identified a new (HIS)platinum complex which was highly cytotoxic against cancer cells including cisplatin-resistant ones.

Hexokinase 2 in colorectal cancer: a potent prognostic factor associated with glycolysis, proliferation and migration.

It is well known that proliferating carcinoma cells preferentially use aerobic glycolysis rather than oxidative phosphorylation for energy production. Hexokinase 2 (HK2) plays a pivotal role in the glycolytic pathway. Previous studies have demonstrated that HK2 activity is markedly increased in various malignant neoplasms, but the clinical and biological significance of HK2 remain largely unclear in the colorectal carcinoma. We performed immunohistochemistry for HK2 in 195 colorectal carcinoma tissues. We also used HCT8 and HT29 colon carcinoma cells in in vitro studies. HK2 immunoreactivity was detected in 100 out of 195 (51%) colorectal carcinoma tissues, and the immunohistochemical HK2 status was significantly associated with tumor size, depth of invasion, liver metastasis and TNM stage in these cases. Moreover, the HK2 status was significantly associated with increased incidence of recurrence and overall mortality of the patients, and multivariate analyses demonstrated that HK2 status was an independent prognostic factor for both disease-free and overall survival. Subsequent in vitro experiments revealed that both HCT8 and HT29 colon carcinoma cells transfected with specific siRNA for HK2 significantly decreased the lactate production, proliferation activity and migration property. These results suggest that HK2 plays important roles in the glycolytic, proliferation and migration properties of colorectal carcinoma and, therefore, HK2 status is a potent worse prognostic factor in colorectal cancer patients.

Synthesis, Cytotoxic Evaluation, Docking and QSAR Study of N-(4-Oxo- 2-(4-((5-Aryl-1,3,4-Thiadiazol-2-yl)Amino)Phenyl)Thiazolidin-3-yl) Benzamides as Antitubulin Agents.

In the present study an efficient strategy for the synthesis of thiazole and thiadiazole derivatives was developed and clubbed together both of the substituted nucleus to form the analogues of combretastatin A-4 (tubulin polymerization inhibitors.). Synthesis was started by the reaction of substituted benzoic acid with thionyl chloride followed by the reaction with hydrazine, p-chloro benzaldehyde and thioglycolic acid to form substituted thiazole derivatives. On the other side hydrazides were reacted with ammonium thiocyanate and strong acid to form substituted thiadiazole compounds. Finally thiazole and thiadiazole compounds were clubbed with the help of dioxan and triethylamine. All novel derivatives (TH01-TH40) were screened for their cytotoxicity activity using MTT assay against three cancer cell lines viz. A-549 (lung carcinoma), HT-29 (colon carcinoma), HeLa (cervix carcinoma). Compounds TH08 exhibited highest activity, due to the presence of trimethoxy substitution on phenyl ring. In QSAR study these results were correlated with physicochemical parameters and the correlation of XlogP, kaapa2, Quadrupole1 with cytotoxic activity on A-549 (lung carcinoma) was found highest (r2: 0.941; F: 99.103; Se: 0.0006). In docking study binding of active molecule (TH08) was found very well with α, β tubulin (PDB: 1SA0) protein.

Labile Pd-sulphur and Pt-sulphur bonds in organometallic palladium and platinum complexes [(COD)M(alkyl)(S-ligand)]n+—A speciation study

Reaction of various sulphur ligands L (SEt−, SPh−, SC6F4H-4−, SEt2, StBu2, SnBu2, DMSO, DPSO) with the precursors [(COD)M(R)Cl] (COD=1,5-cyclooctadiene, M=Pd or Pt; R=methyl (Me) or benzyl (Bn); DMSO=dimethyl sulfoxide; DPSO=diphenyl sulfoxide) allowed isolation and characterisation of mononuclear neutral (n=0) or cationic (n=1) complexes [(COD)Pt(R)(L)]n+. Reaction of l-cysteine (HCys) with [(COD)Pt(Me)Cl] under similar conditions gave the binuclear cationic complex in [{(COD)Pt(Me)}2(μ-Cys)]Cl. Detailed NMR spectroscopy and single crystal X-ray diffraction in the case of [(COD)Pt(Me)(SEt2)][SbF6] and [(COD)Pt(Me)(DMSO)][SbF6] reveal markedly labilised Pt–S bonds as a consequence of the highly covalent Pt–C bonds of the R coligands in these organometallic species. Cationic charge (n=1) seems to lower the Pt–S bond strength further. Consequently, most of these complexes are not stable long-term in aqueous DMF (N,N-dimethylformamide) solutions. This made the evaluation of their antiproliferative properties towards HT-29 colon carcinoma and MCF-7 breast adenocarcinoma cell lines impossible. Only the two complexes [(COD)Pt(R)(SC6F4H-4)] with R=Me or SC6F4H-4 coligands could be tested with the R=Me complex showing promising activity (in the range of cisplatin), while the R=SC6F4H-4 derivative is largely inactive, as were the phosphane complexes [(dppe)Pt(SC6F4H-4)2] (dppe=1,2-bis(diphenylphosphino)ethane), cis-[(PPh3)2Pt(SC6F4H-4)2] and cis-[(PPh3)2PtCl2] which were tested for comparison. In turn, our findings might pave the way to new Pt anti-cancer drugs with largely reduced unwanted depletion of incorporated drugs and reduced side-effects from binding to S-containing biomolecules.

Ellagitannin-rich cloudberry inhibits hepatocyte growth factor induced cell migration and phosphatidylinositol 3-kinase/AKT activation in colon carcinoma cells and tumors in Min mice.

Berries have been found to inhibit colon carcinogenesis in animal models, and thus represent a potential source of compounds for prevention and treatment of colorectal cancer. The mechanistic basis for their effects is not well understood. We used human colon carcinoma cells and Min mice to investigate the effects of ellagitannin-rich cloudberry (Rubus chamaemorus) extract on cancer cell migration and underlying cell signaling. Intrinsic and hepatocyte growth factor (HGF) -induced cell motility in human HT29 and HCA7 colon carcinoma cells was assessed carrying out cell scattering and scratch wound healing assays using time-lapse microscopy. Activation of Met, AKT, and ERK in cell lines and tumors of cloudberry-fed Min mice were determined using immunoprecipitation, Western blot and immunohistochemical analyses. Cloudberry extract significantly inhibited particularly HGF-induced cancer cell migration in both cell lines. Cloudberry extract inhibited the Met receptor tyrosine phosphorylation by HGF and strongly suppressed HGF-induced AKT and ERK activation in both HT29 and HCA7 cells. Consistently, cloudberry feeding (10% w/w freeze-dried berries in diet for 10 weeks) reduced the level of active AKT and prevented phosphoMet localization at the edges in tumors of Min mice. These results indicate that cloudberry reduces tumor growth and cancer cell motility by inhibiting Met signaling and consequent activation of phosphatidylinositol 3-kinase/AKT in vitro and in tumors in vivo. As the Met receptor is recognized to be a major target in cancer treatment, our results suggest that dietary phytochemicals may have therapeutic value in reducing cancer progression and metastasis.

YSL-12, a novel microtubule-destabilizing agent, exerts potent anti-tumor activity against colon cancer in vitro and in vivo.

Microtubules play a central role in various fundamental cell functions and thus become an attractive target for cancer therapy. A novel compound YSL-12 is a combretastatin A-4 (CA-4) analogue with more stability. We investigated its anti-tumor activity and mechanisms in vitro and in vivo for the first time. Cytotoxicity was evaluated by MTT method. In vitro microtubule polymerization assay was performed using a fluorescence-based method by multifunction fluorescence microplate reader. Intracellular microtubule network was detected by immunofluorescence method. Cell cycle analysis and apoptosis were measured by flow cytometry. Metabolic stability was recorded by liquid chromatography-ultraviolet detection and liquid chromatography-mass spectrometry. In vivo anti-tumor activity was assessed using HT-29 colon carcinoma xenografts established in BALB/c nude mice. YSL-12 displayed nanomolar-level cytotoxicity against various human cancer cell lines. A high selectivity toward normal cells and potent activity toward drug-resistant cells were also observed. YSL-12 was identified as tubulin polymerization inhibitor evidenced by effectively inhibits tubulin polymerization and heavily disrupted microtubule networks in living HT-29 cells. YSL-12 displayed potent disruption effect of pre-established tumor vasculature in vitro. In addition, YSL-12 treatment also caused cell cycle arrest in the G2/M phase and induced cell apoptosis in a dose-dependent manner. In vitro metabolic stability study revealed YSL-12 displayed considerable better stability than CA-4 in liver microsomes. In vivo, YSL-12 delayed tumor growth with 69.4% growth inhibition. YSL-12 is a promising microtubule inhibitor that has great potential for the treatment of colon carcinoma in vitro and in vivo and worth being a candidate for further development of cancer therapy.

Combretastatin A-4 derived 5-(1-methyl-4-phenyl-imidazol-5-yl)indoles with superior cytotoxic and anti-vascular effects on chemoresistant cancer cells and tumors

5-(1-Methyl-4-phenyl-imidazol-5-yl)indoles 5 were prepared and tested as analogs of the natural vascular-disrupting agent combretastatin A-4 (CA-4). The 3-bromo-4,5-dimethoxyphenyl derivative 5c was far more active than CA-4 with low nanomolar IC50 concentrations against multidrug-resistant KB-V1/Vbl cervix and MCF-7/Topo mamma carcinoma cells, and also against CA-4-resistant HT-29 colon carcinoma cells. While not interfering markedly with the polymerization of tubulin in vitro, indole 5c completely disrupted the microtubule cytoskeleton of cancer cells at low concentrations. It also destroyed real blood vessels, both in the chorioallantoic membrane (CAM) of fertilized chicken eggs and within tumor xenografts in mice, without harming embryo or mouse, respectively. Indole 5c was less toxic than CA-4 to endothelial cells, fibroblasts, and cardiomyocytes. In highly vascularized xenograft tumors 5c induced distinct discolorations and histological features typical of vascular-disrupting agents, such as disrupted vessel structures, hemorrhages, and extensive necrosis. In a first preliminary therapy trial, indole 5c retarded the growth of resistant xenograft tumors in mice. © 2016 Elsevier Science Ltd. All rights reserved.

Iberis amara Extract Induces Intracellular Formation of Reactive Oxygen Species and Inhibits Colon Cancer.

Massively increasing global incidences of colorectal cancer require efficient treatment and prevention strategies. Here, we report unexpected anticancerogenic effects of hydroethanolic Iberis amara extract (IAE), which is known as a widely used phytomedical product for treating gastrointestinal complaints. IAE significantly inhibited the proliferation of HT-29 and T84 colon carcinoma cells with an inhibitory concentration (IC50) of 6 and 9 μg/ml, respectively, and further generated inhibitory effects in PC-3 prostate and MCF7 breast cancer cells. Inhibition of proliferation in HT-29 cells was associated with a G2/M phase cell cycle arrest including reduced expression of various regulatory marker proteins. Notably, in HT-29 cells IAE further induced apoptosis by intracellular formation of reactive oxygen species (ROS). Consistent with predictions derived from our in vitro experiments, bidaily oral gavage of 50 mg/kg of IAE over 4 weeks resulted in significant inhibition of tumor growth in a mouse HT-29 tumor xenograft model. Taken together, Iberis amara extracts could become useful alternatives for preventing and treating the progression of colon cancer.

Facile preparation of multifunctional superparamagnetic PHBV microspheres containing SPIONs for biomedical applications.

The promising potential of magnetic polymer microspheres in various biomedical applications has been frequently reported. However, the surface hydrophilicity of superparamagnetic iron oxide nanoparticles (SPIONs) usually leads to poor or even failed encapsulation of SPIONs in hydrophobic polymer microspheres using the emulsion method. In this study, the stability of SPIONs in poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) solution was significantly increased after surface modification with lauric acid. As a result, magnetic PHBV microspheres with high encapsulation efficiencies (71.0–87.4%) were prepared using emulsion-solvent extraction/evaporation method. Magnetic resonance imaging (MRI) showed significant contrast for the magnetic PHBV microspheres. The toxicity of these magnetic PHBV microspheres towards human T-lymphoma suspension cells and adherent colon carcinoma HT-29 cells was investigated using flow cytometry, and they were shown to be non-toxic in a broad concentration range. A model drug, tetracycline hydrochloride, was used to demonstrate the drug delivery capability and to investigate the drug release behavior of the magnetic PHBV microspheres. The drug was successfully loaded into the microspheres using lauric acid-coated SPIONs as drug carrier, and was released from the microspheres in a diffusion controlled manner. The developed magnetic PHBV microspheres are promising candidates for biomedical applications such as targeted drug delivery and MRI.

Inhibition of histone deacetylases by trans-cinnamic acid and its antitumor effect against colon cancer xenografts in athymic mice.

Previous studies have shown that trans-cinnamic acid (tCA) has a broad spectrum of biological activities, and exhibits antioxidant, anti-inflammatory and anticancer properties. In addition, tCA and a variety of its analogs have been detected as gut microbe-derived metabolites exerting various biological effects in the colon. The aim of this study was to assess the antitumor activity of tCA in vitro and in vivo, in particular its therapeutic efficacy against colon cancer xenografts in athymic mice. Furthermore, it aimed to examine the effects of tCA on histone deacetylases (HDACs) and to identify the underlying molecular mechanisms. Using an MTT assay, tCA was observed to inhibit the proliferation of several cancer cell lines, and the half maximal inhibitory concentration (IC50) in HT29 colon carcinoma cells was ~1 mM. Western blot analysis demonstrated that tCA upregulated the expression of acetyl-H3 and acetyl-H4 proteins, which was consistent with the effects of the HDAC inhibitor, trichostatin A (TSA). Furthermore, expression of Bcl-2 (a marker of cell proliferation) was reduced, and apoptosis was induced. Apoptosis was shown by the activation of cleavage of poly ADP ribose polymerase and the increased expression of Bax. Apoptosis was also confirmed using APC Annexin V and SYTOX Green Nucleic Acid Stain. In addition, the tCA-induced inhibition of the expression of HDAC markers and activation of apoptosis in tumor tissues were further confirmed by immunohistochemistry. Intragastric administration of tCA at doses of 1.0 and 1.5 mmol/kg body weight suppressed the growth of HT29 human colon carcinoma xenografts in athymic mice at well-tolerated doses. No toxic changes were found in the heart, lung, liver, kidney, colon or bone marrow following histopathological examination. This study indicated that tCA is effective against colon cancer xenograft in nude mice. The antitumor mechanism of tCA was mediated, at least in part, by inhibition of HDACs in cancer cells. As an endogenous microbial metabolite predominantly produced in the colon, tCA is an agent of interest for further evaluation.

64Cu-ATSM therapy targets regions with activated DNA repair and enrichment of CD133+ cells in an HT-29 tumor model: Sensitization with a nucleic acid antimetabolite

64Cu-diacetyl-bis (N4-methylthiosemicarbazone) (64Cu-ATSM) is a potential theranostic agent targeting the over-reduced state under hypoxia within tumors. Recent clinical Cu-ATSM positron emission tomography studies have revealed a correlation between uptake and poor prognosis; however, the reason is unclear. Here, using a human colon carcinoma HT-29 model, we demonstrated that the intratumoral 64Cu-ATSM high-uptake regions exhibited malignant characteristics, such as upregulated DNA repair and elevated %CD133+ cancer stem-like cells. Based on this evidence, we developed a strategy to enhance the efficacy of 64Cu-ATSM internal radiotherapy (IRT) by inhibiting DNA repair with a nucleic acid (NA) antimetabolite. The results of the analyses showed upregulation of pathways related to DNA repair along with NA incorporation (bromodeoxyuridine uptake) and elevation of %CD133+ cells in 64Cu-ATSM high-uptake regions. In an in vivo64Cu-ATSM treatment study, co-administration of an NA antimetabolite and 64Cu-ATSM synergistically inhibited tumor growth, with little toxicity, and effectively reduced %CD133+ cells. 64Cu-ATSM therapy targeted malignant tumor regions with activated DNA repair and high concentrations of CD133+ cells in the HT-29 model. NA antimetabolite co-administration can be an effective approach to enhance the therapeutic effect of 64Cu-ATSM IRT.

Rubiaceae-Type Cyclopeptides from Galianthe thalictroides.

Two Rubiaceae-type cyclopeptides, 6-O-methylbouvardin (1) and the new cyclopeptide 5β-hydroxy-RA-III (2), were isolated from the roots of Galianthe thalictroides. Employing the sulforhodamine B assay, compounds 1 and 2 were tested in vitro against three cancer cell lines--786-0 (kidney carcinoma), PC-3 (prostate carcinoma), and HT-29 (colon carcinoma)--and showed GI50 values ranging from 0.06 to 1.80 μg mL(-1). This is the first report on the isolation of Rubiaceae-type cyclopeptides from a genus other than Rubia or Bouvardia.

The effects of formulation and serum albumin on the in vitro photodynamic activity of zinc(II) phthalocyanines substituted with sulfonated quinolineoxy groups

Three zinc(II) phthalocyanines substituted with sulfonated quinolineoxy groups, including the mono-α-substituted 1, tetra-α-substituted 3, and tetra-β-substituted 4 were synthesized and characterized. These compounds (in deprotonated form) were water-soluble and could be formulated with Cremophor EL or phosphate buffered saline. When formulated with phosphate buffered saline, the serum albumin exerted an important effect on the aggregation tendency and spectroscopic properties of these compounds in biological media. In the case of phosphate buffered saline formulation, the photocytotoxicity followed the order 3 > 4 >> 1 for both human colon carcinoma HT29 and human hepatocarcinoma HepG2 cell lines as a result of their different aggregation tendency. Interestingly, when formulated with Cremophor EL, 1 showed the highest photocytotoxicity not only due to its highest cellular uptake but also due to the reduced aggregation. In both of these formulated systems, the tetra-α-substituted compound 3 exhibited a higher potency than the tetra-β-substituted analogue 4.

Dual fluorescent N-(2-hydroxypropyl) methacrylamide-based conjugates for passive tumor targeting with reduction-sensitive drug release: Proof of the concept, tumor accumulation, and biodistribution

In the field of drug delivery, many different carrier systems have been described to date, including nanoparticles, micelles, liposomes, and water-soluble polymer conjugates, where the active compound could be either incorporated non-covalently or linked to its carrier by a degradable chemical bond. In this study, we synthesized, characterized, and investigated the in vivo fate of N-(2-hydroxypropyl)methacrylamide-based polymer conjugates with a drug model bound via a disulfide bond, which is frequently cited in the literature as being completely stable in the bloodstream but readily cleaved after cell internalization. The concept was based on a “dual” labeling of N-(2-hydroxypropyl)methacrylamide copolymers with two different fluorescent dyes, where the first dye was linked via a disulfide bond, thus representing a model drug, while the second dye was attached as an amide and served as a label for the polymer carrier. Two conjugates, differing in their molecular weights (30 and 104 kDa), were examined using a multispectral optical imaging technique in athymic nude mice inoculated with HT-29 and DLD-1 human colon carcinoma xenografts. Additionally, necropsied organs and tumors were examined ex vivo to obtain more detailed information about polymer and model drug biodistribution. In vivo results confirmed preferential tumor accumulation for both conjugates. Moreover, different fluorescence patterns for the polymer and drug model were observed in both mice and necropsied tumors, indicating tumor-specific “drug” release.

Cooperative TRAIL production mediates IFNα/Smac mimetic-induced cell death in TNFα-resistant solid cancer cells.

Smac mimetics antagonize IAP proteins, which are highly expressed in several cancers. Recent reports indicate that Smac mimetics trigger a broad cytokine response and synergize with immune modulators to induce cell death. Here, we identify a differential requirement of TRAIL or TNFα as mediators of IFNα/Smac mimetic-induced cell death depending on the cellular context. Subtoxic concentrations of Smac mimetics cooperate with IFNα to induce cell death in various solid tumor cell lines in a highly synergistic manner as determined by combination index. Mechanistic studies show that IFNα/BV6 cotreatment promotes the formation of a caspase-8-activating complex together with the adaptor protein FADD and RIP1. Assembly of this RIP1/FADD/caspase-8 complex represents a critical event, since RIP1 silencing inhibits IFNα/BV6-induced cell death. Strikingly, pharmacological inhibition of paracrine/autocrine TNFα signaling by the TNFα scavenger Enbrel rescues HT-29 colon carcinoma cells, but not A172 glioblastoma cells from IFNα/BV6-induced cell death. By comparison, A172 cells are significantly protected against IFNα/BV6 treatment by blockage of TRAIL signaling through genetic silencing of TRAIL or its cognate receptor TRAIL receptor 2 (DR5). Despite this differential requirement of TNFα and TRAIL signaling, mRNA and protein expression is increased by IFNα/BV6 cotreatment in both cell lines. Interestingly, A172 cells turn out to be resistant to exogenously added recombinant TNFα even in the presence of BV6, whereas they display a high sensitivity towards TRAIL/BV6. In contrast, BV6 efficiently sensitizes HT-29 cells to TNFα while TRAIL only had limited efficacy. This demonstrates that a differential sensitivity towards TRAIL or TNFα determines the dependency on either death receptor ligand for IFNα/Smac mimetic-induced cell death. Thus, by concomitant stimulation of both death receptor systems IFNα/Smac mimetic combination treatment is an effective strategy to induce cell death in TNFα- or TRAIL-responsive cancers.

Synthesis of Some Polysubstituted Nicotinonitriles and Derived Pyrido[2,3-d]pyrimidines asIn VitroCytotoxic and Antimicrobial Candidates

The synthesis of polysubstituted pyridines, in addition to some derived pyrido[2,3-d]pyrimidine ring systems supported with chemotherapeutically active functionalities, is described. They were evaluated for theirin vitrocytotoxic effects against three different human tumor cell lines (human colon carcinoma HT29, hepatocellular carcinoma Hep-G2, and Caucasian breast adenocarcinoma MCF7). Nine compounds displayed variable cytotoxic potential, among which alkylthio analogs33,34, and37emerged as the most active members, being almost twice as active as doxorubicin against the colon carcinoma HT29 cell line. In addition, the same three analogs showed a clear differential cytotoxic profile as they exhibited a marginal inhibitory effect on the growth of the normal nontransformed human foreskin fibroblast Hs27 cell line. Meanwhile, nineteen compounds were able to exhibit significant antibacterial activity against both Gram-positive and Gram-negative bacteria, together with moderate antifungal activities. The pyrido[2,3-d]pyrimidine-2(1H)-thione30together with its alkylthio derivatives33and34stemmed as the most active antimicrobial members being equipotent to ampicillin againstS. aureus,E. coli,andP. aeruginosa,together with a noticeable antifungal activity againstC. albicans.Compounds33and34could be considered as a promising template for possible dual antimicrobial-anticancer candidates.

Bevacizumab-Induced Inhibition of Angiogenesis Promotes a More Homogeneous Intratumoral Distribution of Paclitaxel, Improving the Antitumor Response

The antitumor activity of angiogenesis inhibitors is reinforced in combination with chemotherapy. It is debated whether this potentiation is related to a better drug delivery to the tumor due to the antiangiogenic effects on tumor vessel phenotype and functionality. We addressed this question by combining bevacizumab with paclitaxel on A2780-1A9 ovarian carcinoma and HT-29 colon carcinoma transplanted ectopically in the subcutis of nude mice and on A2780-1A9 and IGROV1 ovarian carcinoma transplanted orthotopically in the bursa of the mouse ovary. Paclitaxel concentrations together with its distribution by MALDI mass spectrometry imaging (MALDI MSI) were measured to determine the drug in different areas of the tumor, which was immunostained to depict vessel morphology and tumor proliferation. Bevacizumab modified the vessel bed, assessed by CD31 staining and dynamic contrast enhanced MRI (DCE-MRI), and potentiated the antitumor activity of paclitaxel in all the models. Although tumor paclitaxel concentrations were lower after bevacizumab, the drug distributed more homogeneously, particularly in vascularized, non-necrotic areas, and was cleared more slowly than controls. This happened specifically in tumor tissue, as there was no change in paclitaxel pharmacokinetics or drug distribution in normal tissues. In addition, the drug concentration and distribution were not influenced by the site of tumor growth, as A2780-1A9 and IGROV1 growing in the ovary gave results similar to the tumor growing subcutaneously. We suggest that the changes in the tumor microenvironment architecture induced by bevacizumab, together with the better distribution of paclitaxel, may explain the significant antitumor potentiation by the combination.