Chemotherapy and zoledronate sensitize solid tumour cells to Vγ9Vδ2 T cell cytotoxicity

Combinations of cellular immune-based therapies with chemotherapy and other antitumour agents may be of significant clinical benefit in the treatment of many forms of cancer. Gamma delta (γδ) T cells are of particular interest for use in such combined therapies due to their potent antitumour cytotoxicity and relative ease of generation in vitro. Here, we demonstrate high levels of cytotoxicity against solid tumour-derived cell lines with combination treatment utilizing Vγ9Vδ2 T cells, chemotherapeutic agents and the bisphosphonate, zoledronate. Pre-treatment with low concentrations of chemotherapeutic agents or zoledronate sensitized tumour cells to rapid killing by Vγ9Vδ2 T cells with levels of cytotoxicity approaching 90%. In addition, zoledronate enhanced the chemotherapy-induced sensitization of tumour cells to Vγ9Vδ2 T cell cytotoxicity resulting in almost 100% lysis of tumour targets in some cases. Vγ9Vδ2 T cell cytotoxicity was mediated by perforin following TCR-dependent and isoprenoid-mediated recognition of tumour cells. Production of IFN-γ by Vγ9Vδ2 T cells was also induced after exposure to sensitized targets. We conclude that administration of Vγ9Vδ2 T cells at suitable intervals after chemotherapy and zoledronate may substantially increase antitumour activities in a range of malignancies.

An integrated approach to the prediction of chemotherapeutic response in patients with breast cancer.

BACKGROUND: A major challenge in oncology is the selection of the most effective chemotherapeutic agents for individual patients, while the administration of ineffective chemotherapy increases mortality and decreases quality of life in cancer patients. This emphasizes the need to evaluate every patient's probability of responding to each chemotherapeutic agent and limiting the agents used to those most likely to be effective. METHODS AND RESULTS: Using gene expression data on the NCI-60 and corresponding drug sensitivity, mRNA and microRNA profiles were developed representing sensitivity to individual chemotherapeutic agents. The mRNA signatures were tested in an independent cohort of 133 breast cancer patients treated with the TFAC (paclitaxel, 5-fluorouracil, adriamycin, and cyclophosphamide) chemotherapy regimen. To further dissect the biology of resistance, we applied signatures of oncogenic pathway activation and performed hierarchical clustering. We then used mRNA signatures of chemotherapy sensitivity to identify alternative therapeutics for patients resistant to TFAC. Profiles from mRNA and microRNA expression data represent distinct biologic mechanisms of resistance to common cytotoxic agents. The individual mRNA signatures were validated in an independent dataset of breast tumors (P = 0.002, NPV = 82%). When the accuracy of the signatures was analyzed based on molecular variables, the predictive ability was found to be greater in basal-like than non basal-like patients (P = 0.03 and P = 0.06). Samples from patients with co-activated Myc and E2F represented the cohort with the lowest percentage (8%) of responders. Using mRNA signatures of sensitivity to other cytotoxic agents, we predict that TFAC non-responders are more likely to be sensitive to docetaxel (P = 0.04), representing a viable alternative therapy. CONCLUSIONS: Our results suggest that the optimal strategy for chemotherapy sensitivity prediction integrates molecular variables such as ER and HER2 status with corresponding microRNA and mRNA expression profiles. Importantly, we also present evidence to support the concept that analysis of molecular variables can present a rational strategy to identifying alternative therapeutic opportunities.

Dual targeting of tumour cells and host endothelial cells by novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines

PURPOSE: Some members of a novel series of pyrrolo-1,5-benzoxazepines (PBOXs) are microtubule-targeting agents capable of inducing apoptosis in a variety of human cancerous cells, hence, they are currently being developed as potential anti-cancer agents. The purpose of this study was to first characterise the activities of a novel PBOX analogue, PBOX-16 and then investigate the anti-angiogenic potential of both PBOX-16 and its prototype PBOX-6.

METHODS: The effects of PBOX-6 and -16 on cancerous cells (chronic myeloid leukaemia K562 cells and ovarian carcinoma A2780 cells) and primary cultured human umbilical vein endothelial cells (HUVECs) were examined by assessing cell proliferation, microtubular organisation, DNA analysis of cell cycle progression and caspase-3/7 activity. Their anti-angiogenic properties were then investigated by examining their ability to interfere with HUVEC differentiation into capillary-like structures and vascular endothelial growth factor (VEGF)-stimulated HUVEC migration.

RESULTS: PBOX-6 and -16 inhibited proliferation of K562, A2780 and HUVEC cells in a concentration-dependent manner. PBOX-16, confirmed as a novel depolymerising agent, was approximately tenfold more potent than PBOX-6. Inhibition of cell proliferation was mediated by G(2)/M arrest followed by varying degrees of apoptosis depending on the cell type; endothelial cells underwent less apoptosis than either of the cancer cell lines. In addition to the antitumourigenic properties, we also describe a novel antiangiogenic function for PBOXs: treatment with PBOXs inhibited the spontaneous differentiation of HUVECs into capillary-like structures when grown on a basement membrane matrix preparation (Matrigel™) and also significantly reduced VEGF-stimulated HUVEC migration.

CONCLUSION: Dual targeting of both the tumour cells and the host endothelial cells by PBOX compounds might enhance the anti-cancer efficacy of these drugs.

Pyrrolo-1,5-benzoxazepines induce apoptosis in chronic myelogenous leukemia (CML) cells by bypassing the apoptotic suppressor bcr-abl

Expression of the transforming oncogene bcr-abl in chronic myelogenous leukemia (CML) cells is reported to confer resistance against apoptosis induced by many chemotherapeutic agents such as etoposide, ara-C, and staurosporine. In the present study some members of a series of novel pyrrolo-1,5-benzoxazepines potently induce apoptosis, as shown by cell shrinkage, chromatin condensation, DNA fragmentation, and poly(ADP-ribose) polymerase (PARP) cleavage, in three CML cell lines, K562, KYO.1, and LAMA 84. Induction of apoptosis by a representative member of this series, PBOX-6, was not accompanied by either the down-regulation of Bcr-Abl or by the attenuation of its protein tyrosine kinase activity up to 24 h after treatment, when approximately 50% of the cells had undergone apoptosis. These results suggest that down-regulation of Bcr-Abl is not part of the upstream apoptotic death program activated by PBOX-6. By characterizing the mechanism in which this novel agent executes apoptosis, this study has revealed that PBOX-6 caused activation of caspase 3-like proteases in only two of the three CML cell lines. In addition, inhibition of caspase 3-like protease activity using the inhibitor z-DEVD-fmk blocked caspase 3-like protease activity but did not prevent the induction of apoptosis, suggesting that caspase 3-like proteases are not essential in the mechanism by which PBOX-6 induces apoptosis in CML cells. In conclusion, this study demonstrates that PBOX-6 can bypass Bcr-Abl-mediated suppression of apoptosis, suggesting an important potential use of these compounds in the treatment of CML.

Benzopyrans from Curvularia sp., an endophytic fungus associated with Ocotea corymbosa (Lauraceae)

An isolate of Curvularia sp. was obtained from the leaves of Ocotea corymbosa, a native plant of the Brazilian Cerrado. The ethyl acetate extract from culture of this fungus afforded two benzopyran derivatives: (2′S)-2-(propan-2′-ol)-5-hydroxy-benzopyran-4-one (2) and 2,3-dihydro-2-methyl-benzopyran-4,5-diol (4); and two known benzopyrans: 2-methyl-5-methoxy-benzopyran-4-one (1) and (2R)-2,3-dihydro-2-methyl-5-methoxy- benzopyran-4-one (3). The structures of 2 and 4 were established on the basis of comprehensive spectroscopic analysis, mainly using 1D and 2D NMR experiments. The benzopyrans 1 and 2 showed weak in vitro antifungal activity against Cladosporium sphaerospermum and C. cladosporioides. Analyses of the biological activities were also carried out on HeLa (human cervix tumor) and CHO (Chinese hamster ovary) cells, aiming to evaluate their potential effects on mammalian cell line proliferation. Results from both cell lines indicated that compound 2 was able to induce cell proliferation: 70% on HeLa cells and 25% on CHO cells. © 2005 Elsevier Ltd. All rights reserved.

Effects of acetylsalicylic acid and acetic acid solutions in VX2 carcinoma cells. In vitro analysis

Purpose: To analyze, in vitro, the effects of acetylsalicylic acid (aspirin) and acetic acid solutions on VX2 carcinoma cells in suspension and to examine the correlation between these effects and neoplastic cell death. Methods: The VX2 tumor cells (107 cells/ml) were incubated in solutions containing differing concentrations (2.5% and 5%) of either acetylsalicylic acid or acetic acid, or in saline solution (controls). Every five minutes, cell viability was tested (using the trypan blue test) and analyzed under light microscopy. Results: Tumor cell viability (in %) decreased progressively and, by 30 minutes, neoplastic cell death had occurred in all solutions. Conclusion: Based on this experimental model and the methodology employed, we conclude that these solutions cause neoplastic cell death in vitro.

If you don’t know, you can’t help: A practical guide for alcohol and other drug screening in psychological services

Alcohol is implicated in over 60 diseases and injuries and accounted for 6.2 per cent of all male deaths globally in 2004 (WHO, 2011). Alcohol and other drug (AOD) abuse causes significant individual, family and social harms at all age levels and across all socioeconomic groups. These may result from intoxication (e.g., overdose, vulnerability to physical injury/trauma or death, consequences of impulsive behaviour, aggression and driving under the influence) and longer-term consequences (e.g., alcohol or drug-related brain injury, cardiovascular and liver diseases, blood borne viruses e.g., Chikritzhs et al., 2003, Roxburgh et al., 2013). Mental health problems may be triggered or exacerbated, and family breakdown, poor self-esteem, legal issues and lack of community engagement may also be evident. Despite the prevalence of substance use disorders and evident consequences for the individual, family and wider community, it would seem that health professionals, including psychologists, are reluctant to ask about substance use.

Quantifying intrinsic specificity: A potential complement to affinity in drug screening

We report here the investigation of a novel description of specificity in protein-ligand binding based on energy landscape theory. We define a new term, intrinsic specificity ratio (ISR), which describes the level of discrimination in binding free energies of the native basin for a protein-ligand complex from the weaker binding states of the same ligand. We discuss the relationship between the intrinsic specificity we defined here and the conventional definition of specificity. In a docking study of molecules with the enzyme COX-2, we demonstrate a statistical correspondence between ISR value and geometrical shapes of the small molecules binding to COX-2. We further observe that the known selective (nonselective) inhibitors of COX-2 have higher (lower) ISR values. We suggest that intrinsic specificity ratio may be a useful new criterion and a complement to affinity in drug screening and in searching for potential drug lead compounds.

Development of an in vitro multicellular tumor spheroid model using microencapsulation and its application in anticancer drug screening and testing

In this study, an in vitro multicellular tumor spheroid model was developed using microencapsulation, and the feasibility of using the microencapsulated. multicellular tumor spheroid (MMTS) to test the effect of chemotherapeutic drugs was investigated. Human MCF-7 breast cancer cells were encapsulated in alginate-poly-L-lysine-alginate (APA) microcapsules, and a single multicellular spheroid 150 mu m in diameter was formed in the microcapsule after 5 days of cultivation. The cell morphology, proliferation, and viability of the MMTS were characterized using phase contrast microscopy, BrdU-Iabeling, MTT stain, calcein AM/ED-2 stain, and H&E stain. It demonstrated that the MMTS was viable and that the proliferating cells were mainly localized to the periphery of the cell spheroid and the apoptotic cells were in the core. The MCF-7 MMTS was treated with mitomycin C (MC) at a concentration of 0.1, 1, or 10 times that of peak plasma concentration (ppc) for up to 72 h. The cytotoxicity was demonstrated. clearly by the reduction in cell spheroid size and the decrease in cell viability. The MMTS was further used to screen the anticancer effect of chemotherapeutic drugs, treated with MC, adriamycin (ADM) and 5-fluorouracil (5-FU) at concentrations of 0.1, 1, and 10 ppc for 24, 48, and 72 h. MCF-7 monolayer culture was used as control. Similar to monolayer culture, the cell viability of MMTS was reduced after treatment with anticancer drugs. However, the inhibition rate of cell viability in MMTS was much lower than that in monolayer culture. The MMTS was more resistant to anticancer drugs than monolayer culture. The inhibition rates of cell viability were 68.1%, 45.1%, and 46.8% in MMTS and 95.1%, 86.8%, and 91.6% in monolayer culture treated with MC, ADM, and 5-FU at 10 ppc for 72 h, respectively. MC showed the strongest cytotoxicity in both MMTS and monolayer, followed by 5-FU and ADM. It demonstrated that the MMTS has the potential to be a rapid and valid in vitro model to screen chemotherapeutic drugs with a feature to mimic in vivo three-dimensional (3-D) cell growth pattern.

Human Vascular Microphysiological System for in vitro Drug Screening.

In vitro human tissue engineered human blood vessels (TEBV) that exhibit vasoactivity can be used to test human toxicity of pharmaceutical drug candidates prior to pre-clinical animal studies. TEBVs with 400-800 μM diameters were made by embedding human neonatal dermal fibroblasts or human bone marrow-derived mesenchymal stem cells in dense collagen gel. TEBVs were mechanically strong enough to allow endothelialization and perfusion at physiological shear stresses within 3 hours after fabrication. After 1 week of perfusion, TEBVs exhibited endothelial release of nitric oxide, phenylephrine-induced vasoconstriction, and acetylcholine-induced vasodilation, all of which were maintained up to 5 weeks in culture. Vasodilation was blocked with the addition of the nitric oxide synthase inhibitor L-N(G)-Nitroarginine methyl ester (L-NAME). TEBVs elicited reversible activation to acute inflammatory stimulation by TNF-α which had a transient effect upon acetylcholine-induced relaxation, and exhibited dose-dependent vasodilation in response to caffeine and theophylline. Treatment of TEBVs with 1 μM lovastatin for three days prior to addition of Tumor necrosis factor - α (TNF-α) blocked the injury response and maintained vasodilation. These results indicate the potential to develop a rapidly-producible, endothelialized TEBV for microphysiological systems capable of producing physiological responses to both pharmaceutical and immunological stimuli.

A comparative study of qualitative immunochemical screening assays for the combined measurement of T-2/HT-2 in cereals and cereal-based products

Many different immunochemical platforms exist for the screening of naturally occurring contaminants in food from the low cost enzyme linked immunosorbent assays (ELISA) to the expensive instruments such as optical biosensors based on the phenomenon of surface plasmon resonance (SPR). The primary aim of this study was to evaluate and compare a number of these platforms to assess their accuracy and precision when applied to naturally contaminated samples containing HT-2/T-2 mycotoxins. Other important factors considered were the speed of analysis, ease of use (sample preparation techniques and use of the equipment) and ultimately the cost implications. The three screening procedures compared included an SPR biosensor assay, a commercially available ELISA and an enzyme-linked immunomagnetic electrochemical array (ELIME array). The qualitative data for all methods demonstrated very good overall agreements with each other, however on comparison with mass spectrometry confirmatory results, the ELISA and SPR assay performed slightly better than the ELIME array, exhibiting an overall agreement of 95.8% compared to 91.7%. Currently, SPR is more costly than the other two platforms and can only be used in the laboratory whereas in theory both the ELISA and ELIME array are portable and can be used in the field, but ultimately this is dependent on the sample preparation techniques employed. Sample preparative techniques varied for all methods evaluated, the ELISA was the most simple to perform followed by that of the SPR method. The ELIME array involved an additional clean-up step thereby increasing both the time and cost of analysis. Therefore in the current format, field use would not be an option for the ELIME array. In relation to speed of analysis, the ELISA outperformed the other methods.

Evaluation of methods of detecting cell reactive oxygen species production for drug screening and cell cycle studies

Intracellular reactive oxygen species (ROS) production is essential to normal cell function. However, excessive ROS production causes oxidative damage and cell death. Many pharmacological compounds exert their effects on cell cycle progression by changing intracellular redox state and in many cases cause oxidative damage leading to drug cytotoxicity. Appropriate measurement of intracellular ROS levels during cell cycle progression is therefore crucial in understanding redox-regulation of cell function and drug toxicity and for the development of new drugs. However, due to the extremely short half-life of ROS, measuring the changes in intracellular ROS levels during a particular phase of cell cycle for drug intervention can be challenging. In this article, we have provided updated information on the rationale, the applications, the advantages and limitations of common methods for screening drug effects on intracellular ROS production linked to cell cycle study. Our aim is to facilitate biomedical scientists and researchers in the pharmaceutical industry in choosing or developing specific experimental regimens to suit their research needs.

Development of screening assays to test novel integrin antagonists in allergic inflammation

Aim of the research: to develop a prototype of homogeneous high-throughput screening (HTS) for identification of novel integrin antagonists for the treatment of ocular allergy and to better understand the mechanisms of action of integrin-mediated levocabastine antiallergic action. Results: This thesis provides evidence that adopting scintillation proximity assay (SPA) levocabastine (IC50=406 mM), but not the first-generation antihistamine chlorpheniramine, displaces [125I]fibronectin (FN) binding to human a4b1 integrin. This result is supported by flow cytometry analysis, where levocabastine antagonizes the binding of a primary antibody to integrin a4 expressed in Jurkat E6.1 cells. Levocabastine, but not chlorpheniramine, binds to a4b1 integrin and prevents eosinophil adhesion to VCAM-1, FN or human umbilical vein endothelial cells (HUVEC) cultured in vitro. Similarly, levocabastine affects aLb2/ICAM-1-mediated adhesion of Jurkat E6.1 cells. Analyzing the supernatant of TNF-a-treated (24h) eosinophilic cells (EoL-1), we report that levocabastine reduces the TNF-a-induced release of the cytokines IL-12p40, IL-8 and VEGF. Finally, in a model of allergic conjunctivitis, levocastine eye drops (0.05%) reduced the clinical aspects of the early and late phase reactions and the conjunctival expression of a4b1 integrin by reducing infiltrated eosinophils. Conclusions: SPA is a highly efficient, amenable to automation and robust binding assay to screen novel integrin antagonists in a HTS setting. We propose that blockade of integrinmediated cell adhesion might be a target of the anti-allergic action of levocabastine and may play a role in preventing eosinophil adhesion and infiltration in allergic conjunctivitis.