Reversal of doxorubicin resistance in lung cancer cells by neferine is explained by nuclear factor erythroid-derived 2-like 2 mediated lung resistance protein down regulation.

Aim: Development of multi drug resistance and dose limiting cardiotoxicity are hindering the use of Doxorubicin (Dox) in clinical settings. Augmented dox efflux induced by lung resistance protein (LRP) over expression has been related to multi drug resistance phenotype in various cancers. An alkaloid from lotus, Neferine (Nef) shows both anticancer and cardioprotective effects. Here, we have investigated the interconnection between nuclear factor erythroid-derived 2-like 2 (NRF2) and LRP in Dox resistance and how Nef can overcome Dox resistance in lung cancer cells by altering this signaling. Methods: Anti-proliferative and apoptotic-inducing effects of Nef and Dox combination in Parental and Dox resistant lung cancer cells were determined in monolayers and 3D spheroids. Intracellular Dox was analyzed using flow cytometry, siRNA knockdown and western blot analysis were used to elucidate NRF2-LRP crosstalk mechanism. Results: We observed that the Dox resistant lung cancer cells expressed higher levels of LRP, reduced glutathione (GSH) and NRF2. Combination of Dox and Nef induced apoptosis, leads to reactive oxygen species (ROS) generation, GSH depletion and reduction in LRP levels contributing to higher intracellular and intranuclear Dox accumulation. The use of N-acetylcysteine and knockdown studies confirmed an important role of ROS and NRF2 in LRP down regulation. Presence of NRF2 binding sites in LRP is support of direct interaction between LRP and NRF2. Conclusion: Nef sensitizes lung cancer cells to Dox by increasing intracellular and/or intra nuclear Dox accumulation via LRP down regulation. This is mediated by redox regulating NRF2. This decoded crosstalk mechanism reinforces the role of NRF2 and LRP in Dox resistance and as an important anticancer target.

Three dimensional in vitro models of cancer: Bioprinting multilineage glioblastoma models.

Three dimensional (3D) bioprinting of multiple cell types within optimised extracellular matrices has the potential to more closely model the 3D environment of human physiology and disease than current alternatives. In this study, we used a multi-nozzle extrusion bioprinter to establish models of glioblastoma made up of cancer and stromal cells printed within matrices comprised of alginate modified with RGDS cell adhesion peptides, hyaluronic acid and collagen-1. Methods were developed using U87MG glioblastoma cells and MM6 monocyte/macrophages, whilst more disease relevant constructs contained glioblastoma stem cells (GSCs), co-printed with glioma associated stromal cells (GASCs) and microglia. Printing parameters were optimised to promote cell-cell interaction, avoiding the 'caging in' of cells due to overly dense cross-linking. Such printing had a negligible effect on cell viability, and cells retained robust metabolic activity and proliferation. Alginate gels allowed the rapid recovery of printed cell protein and RNA, and fluorescent reporters provided analysis of protein kinase activation at the single cell level within printed constructs. GSCs showed more resistance to chemotherapeutic drugs in 3D printed tumour constructs compared to 2D monolayer cultures, reflecting the clinical situation. In summary, a novel 3D bioprinting strategy is developed which allows control over the spatial organisation of tumour constructs for pre-clinical drug sensitivity testing and studies of the tumour microenvironment.

Narrowband Organic Light-Emitting Diodes for Fluorescence Microscopy and Calcium Imaging.

Fluorescence imaging is an indispensable tool in biology, with applications ranging from single-cell to whole-animal studies and with live mapping of neuronal activity currently receiving particular attention. To enable fluorescence imaging at cellular scale in freely moving animals, miniaturized microscopes and lensless imagers are developed that can be implanted in a minimally invasive fashion; but the rigidity, size, and potential toxicity of the involved light sources remain a challenge. Here, narrowband organic light-emitting diodes (OLEDs) are developed and used for fluorescence imaging of live cells and for mapping of neuronal activity in Drosophila melanogaster via genetically encoded Ca2+ indicators. In order to avoid spectral overlap with fluorescence from the sample, distributed Bragg reflectors are integrated onto the OLEDs to block their long-wavelength emission tail, which enables an image contrast comparable to conventional, much bulkier mercury light sources. As OLEDs can be fabricated on mechanically flexible substrates and structured into arrays of cell-sized pixels, this work opens a new pathway for the development of implantable light sources that enable functional imaging and sensing in freely moving animals.

Matrix metalloproteinase (MMP)-7 in Barrett's esophagus and esophageal adenocarcinoma: expression, metabolism, and functional significance.

Matrix metalloproteinase (MMP)-7, unlike many MMPs, is typically expressed in epithelial cells. It has been linked to epithelial responses to infection, injury, and tissue remodeling including the progression of a number of cancers. We have now examined how MMP-7 expression changes in the progression to esophageal adenocarcinoma (EAC), and have studied mechanisms regulating its expression and its functional significance. Immunohistochemistry revealed that MMP-7 was weakly expressed in normal squamous epithelium adjacent to EAC but was abundant in epithelial cells in both preneoplastic lesions of Barrett's esophagus and EAC particularly at the invasive front. In the stroma, putative myofibroblasts expressing MMP-7 were abundant at the invasive front but were scarce or absent in adjacent tissue. Western blot and ELISA revealed high constitutive secretion of proMMP-7 in an EAC cell line (OE33) that was inhibited by the phosphatidylinositol (PI) 3-kinase inhibitor LY294002 but not by inhibitors of protein kinase C, or MAP kinase activation. There was detectable proMMP-7 in cultured esophageal myofibroblasts but it was undetectable in media. Possible metabolism of MMP-7 by myofibroblasts studied by proteomic analysis indicated degradation via extensive endopeptidase, followed by amino- and carboxpeptidase, cleavages. Myofibroblasts exhibited increased migration and invasion in response to conditioned media from OE33 cells that was reduced by MMP-7 knockdown and immunoneutralization. Thus, MMP-7 expression increases at the invasive front in EAC which may be partly attributable to activation of PI 3-kinase. Secreted MMP-7 may modify the tumor microenvironment by stimulating stromal cell migration and invasion.

Cell cycle dependent expression of the CCK2 receptor by gastrointestinal myofibroblasts: putative role in determining cell migration.

The well-known action of the gastric hormone gastrin in stimulating gastric acid secretion is mediated by activation of cholecystokinin-2 receptors (CCK2R). The latter are expressed by a variety of cell types suggesting that gastrin is implicated in multiple functions. During wound healing in the stomach CCK2R may be expressed by myofibroblasts. We have now characterized CCK2R expression in cultured myofibroblasts. Immunocytochemistry showed that a relatively small proportion (1-6%) of myofibroblasts expressed the receptor regardless of the region of the gut from which they were derived, or whether from cancer or control tissue. Activation of CCK2R by human heptadecapeptide gastrin (hG17) increased intracellular calcium concentrations in a small subset of myofibroblasts indicating the presence of a functional receptor. Unexpectedly, we found over 80% of cells expressing CCK2R were also labeled with 5-ethynyl-2'-deoxyuridine (EdU) which is incorporated into DNA during S-phase of the cell cycle. hG17 did not stimulate EdU incorporation but increased migration of both EdU-labeled and unlabelled myofibroblasts; the migratory response was inhibited by a CCK2R antagonist and by an inhibitor of IGF receptor tyrosine kinase; hG17 also increased IGF-2 transcript abundance. The data suggest myofibroblasts express CCK2R in a restricted period of the cell cycle during S-phase, and that gastrin accelerates migration of these cells; it also stimulates migration of adjacent cells probably through paracrine release of IGF. Together with previous findings, the results raise the prospect that gastrin controls the position of dividing myofibroblasts which may be relevant in wound healing and cancer progression in the gastrointestinal tract.

Network pharmacology of cancer: From understanding of complex interactomes to the design of multi-target specific therapeutics from nature.

Despite massive investments in drug research and development, the significant decline in the number of new drugs approved or translated to clinical use raises the question, whether single targeted drug discovery is the right approach. To combat complex systemic diseases that harbour robust biological networks such as cancer, single target intervention is proved to be ineffective. In such cases, network pharmacology approaches are highly useful, because they differ from conventional drug discovery by addressing the ability of drugs to target numerous proteins or networks involved in a disease. Pleiotropic natural products are one of the promising strategies due to their multi-targeting and due to lower side effects. In this review, we discuss the application of network pharmacology for cancer drug discovery. We provide an overview of the current state of knowledge on network pharmacology, focus on different technical approaches and implications for cancer therapy (e.g. polypharmacology and synthetic lethality), and illustrate the therapeutic potential with selected examples green tea polyphenolics, Eleutherococcus senticosus, Rhodiola rosea, and Schisandra chinensis). Finally, we present future perspectives on their plausible applications for diagnosis and therapy of cancer.

Distinct miRNA profiles in normal and gastric cancer myofibroblasts and significance in Wnt signaling.

Stromal cells influence epithelial function in both health and disease. Myofibroblasts are abundant stromal cells that influence the cellular microenvironment by release of extracellular matrix (ECM) proteins, growth factors, proteases, cytokines, and chemokines. Cancer-associated myofibroblasts (CAMs) differ from adjacent tissue (ATMs) and normal tissue myofibroblasts (NTMs), but the basis of this is incompletely understood. We report now the differential expression of miRNAs in gastric cancer CAMs. MicroRNA arrays identified differences in the miRNA profile in gastric and esophageal NTMs and in CAMs from stomach compared with NTMs. miR-181d was upregulated in gastric CAMs. Analysis of differentially regulated miRNAs indicated an involvement in Wnt signaling. Examination of a microarray data set then identified Wnt5a as the only consistently upregulated Wnt ligand in gastric CAMs. Wnt5a stimulated miR-181d expression, and knockdown of miR-181d inhibited Wnt5a stimulation of CAM proliferation and migration. Analysis of miR-181d targets suggested a role in chemotaxis. Conditioned medium from CAMs stimulated gastric cancer cell (AGS) migration more than that from ATMs, and miR-181d knockdown reduced the effect of CAM-CM on AGS cell migration but had no effect on AGS cell responses to ATM conditioned media. The data suggest that dysregulation of miRNA expression in gastric CAMs, secondary to Wnt5a signaling, accounts at least in part for the effect of CAMs in promoting cancer cell migration.

Release of TGFßig-h3 by gastric myofibroblasts slows tumor growth and is decreased with cancer progression.

Tumor progression has been linked to changes in the stromal environment. Myofibroblasts are stromal cells that are often increased in tumors but their contribution to cancer progression is not well understood. Here, we show that the secretomes of myofibroblasts derived from gastric cancers [cancer-associated myofibroblasts (CAMs)] differ in a functionally significant manner from those derived from adjacent tissue [adjacent tissue myofibroblasts (ATMs)]. CAMs showed increased rates of migration and proliferation compared with ATMs or normal tissue myofibroblasts (NTMs). Moreover, conditioned medium (CM) from CAMs significantly stimulated migration, invasion and proliferation of gastric cancer cells compared with CM from ATMs or NTMs. Proteomic analysis of myofibroblast secretomes revealed decreased abundance of the extracellular matrix (ECM) adaptor protein like transforming growth factor-ß-induced gene-h3 (TGFßig-h3) in CAMs, which was correlated with lymph node involvement and shorter survival. TGFßig-h3 inhibited IGF-II-stimulated migration and proliferation of both cancer cells and myofibroblasts, and suppressed IGF-II activation of p42/44 MAPkinase; TGFßig-h3 knockdown increased IGF-II- and CM-stimulated migration. Furthermore, administration of TGFßig-h3 inhibited myofibroblast-stimulated growth of gastric cancer xenografts. We conclude that stromal cells exert inhibitory as well as stimulatory effects on tumor cells; TGFßig-h3 is a stromal inhibitory factor that is decreased with progression of gastric cancers.