Andrzej Stepulak

Expression of glutamate receptor subunits in human cancers

Emerging evidence suggests a role for glutamate and its receptors in the biology of cancer. This study was designed to systematically analyze the expression of ionotropic and metabotropic glutamate receptor subunits in various human cancer cell lines, compare expression levels to those in human brain tissue and, using electrophysiological techniques, explore whether cancer cells respond to glutamate receptor agonists and antagonists. Expression analysis of glutamate receptor subunits NR1-NR3B, GluR1-GluR7, KA1, KA2 and mGluR1-mGluR8 was performed by means of RT-PCR in human rhabdomyosarcoma/medulloblastoma (TE671), neuroblastoma (SK-NA-S), thyroid carcinoma (FTC 238), lung carcinoma (SK-LU-1), astrocytoma (MOGGCCM), multiple myeloma (RPMI 8226), glioma (U87-MG and U343), lung carcinoma (A549), colon adenocarcinoma (HT 29), T cell leukemia cells (Jurkat E6.1), breast carcinoma (T47D) and colon adenocarcinoma (LS180). Analysis revealed that all glutamate receptor subunits were differentially expressed in the tumor cell lines. For the majority of tumors, expression levels of NR2B, GluR4, GluR6 and KA2 were lower compared to human brain tissue. Confocal imaging revealed that selected glutamate receptor subunit proteins were expressed in tumor cells. By means of patch-clamp analysis, it was shown that A549 and TE671 cells depolarized in response to application of glutamate agonists and that this effect was reversed by glutamate receptor antagonists. This study reveals that glutamate receptor subunits are differentially expressed in human tumor cell lines at the mRNA and the protein level, and that their expression is associated with the formation of functional channels. The potential role of glutamate receptor antagonists in cancer therapy is a feasible goal to be explored in clinical trials.

MicroRNAs-Role in Lung Cancer

Regulation of gene expression is essential for normal physiological functions; thus deregulation of gene expression is common in disease conditions. One level of regulation of gene expression is performed by noncoding RNAs, among which microRNAs (miRNA) are the best studied. Abnormal expression of these molecular players can lead to pathogenic processes such as heart disease, immune system abnormalities, and carcinogenesis, to name but a few. Of a length of 18–25 nucleotides miRNAs are involved in binding partial complementary sequences within the 3′-UTR (3′-untranslated region) of the target mRNAs. Depending on the type of neoplastic transformation, miRNAs can act both as oncogenes (oncomirs) or as tumor suppressors. Because of the great importance of miRNAs, most researches focus on either their role as biomarkers or their potential as therapeutic targets. Herein, we present the review of microRNA biology, function, and tumorigenic potential with emphasis on their role in lung cancer.

Whole-tissue biopsy phenotyping of three-dimensional tumours reveals patterns of cancer heterogeneity

Intratumoral heterogeneity is a critical factor when diagnosing and treating patients with cancer. Marked differences in the genetic and epigenetic backgrounds of cancer cells have been revealed by advances in genome sequencing, yet little is known about the phenotypic landscape and the spatial distribution of intratumoral heterogeneity within solid tumours. Here, we show that three-dimensional light-sheet microscopy of cleared solid tumours can identify unique patterns of phenotypic heterogeneity, in the epithelial-to-mesenchymal transition and in angiogenesis, at single-cell resolution in whole formalin-fixed paraffin-embedded (FFPE) biopsy samples. We also show that cleared FFPE samples can be re-embedded in paraffin after examination for future use, and that our tumour-phenotyping pipeline can determine tumour stage and stratify patient prognosis from clinical samples with higher accuracy than current diagnostic methods, thus facilitating the design of more efficient cancer therapies.A method that identifies patterns of tumour heterogeneity in intact biopsy samples using 3D light-sheet microscopy stratifies patients by tumour stage.

Inhibition of mitochondrial 2-oxoglutarate dehydrogenase impairs viability of cancer cells in a cell-specific metabolism-dependent manner

2-Oxoglutarate dehydrogenase (OGDH) of the tricarboxylic acid (TCA) cycle is often implied to be inactive in cancer, but this was not experimentally tested. We addressed the question through specific inhibition of OGDH by succinyl phosphonate (SP). SP action on different cancer cells was investigated using indicators of cellular viability and reactive oxygen species (ROS), metabolic profiling and transcriptomics. Relative sensitivity of various cancer cells to SP changed with increasing SP exposure and could differ in the ATP- and NAD(P)H-based assays. Glioblastoma responses to SP revealed metabolic sub-types increasing or decreasing cellular ATP/NAD(P)H ratio under OGDH inhibition. Cancer cell homeostasis was perturbed also when viability indicators were SP-resistant, e.g. in U87 and N2A cells. The transcriptomics database analysis showed that the SP-sensitive cells, such as A549 and T98G, exhibit the lowest expression of OGDH compared to other TCA cycle enzymes, associated with higher expression of affiliated pathways utilizing 2-oxoglutarate. Metabolic profiling confirmed the dependence of cellular SP reactivity on cell-specific expression of the pathways. Thus, oxidative decarboxylation of 2-oxoglutarate is significant for the interdependent homeostasis of NAD(P)H, ATP, ROS and key metabolites in various cancer cells. Assessment of cell-specific responses to OGDH inhibition is of diagnostic value for anticancer strategies.

Assessment of Interactions between Cisplatin and Two Histone Deacetylase Inhibitors in MCF7, T47D and MDA-MB-231 Human Breast Cancer Cell Lines - An Isobolographic Analysis

Histone deacetylase inhibitors (HDIs) are promising anticancer drugs, which inhibit proliferation of a wide variety of cancer cells including breast carcinoma cells. In the present study, we investigated the influence of valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA, vorinostat), alone or in combination with cisplatin (CDDP) on proliferation, induction of apoptosis and cell cycle progression in MCF7, T47D and MDA-MB-231 human breast carcinoma cell lines. The type of interaction between HDIs and CDDP was determined by an isobolographic analysis. The isobolographic analysis is a very precise and rigorous pharmacodynamic method, to determine the presence of synergism, addition or antagonism between different drugs with using variety of fixed dose ratios. Our experiments show that the combinations of CDDP with SAHA or VPA at a fixed-ratio of 1:1 exerted additive interaction in the viability of MCF7 cells, while in T47D cells there was a tendency to synergy. In contrast, sub-additive (antagonistic) interaction was observed for the combination of CDDP with VPA in MDA-MB-231 “triple-negative” (i.e. estrogen receptor negative, progesterone receptor negative, and HER-2 negative) human breast cancer cells, whereas combination of CDDP with SAHA in the same MDA-MB-231 cell line yielded additive interaction. Additionally, combined HDIs/CDDP treatment resulted in increase in apoptosis and cell cycle arrest in all tested breast cancer cell lines in comparison with a single therapy. In conclusion, the additive interaction of CDDP with SAHA or VPA suggests that HDIs could be combined with CDDP in order to optimize treatment regimen in some human breast cancers.

Xanthohumol inhibits the extracellular signal regulated kinase (ERK) signalling pathway and suppresses cell growth of lung adenocarcinoma cells

Aberrant activation of the Ras/MEK/ERK signaling pathway has been frequently observed in non-small-cell lung carcinoma (NSCLC) and its important role in cancer progression and malignant transformation has been documented. Hence, the ERK1/2 kinase cascade becomes a potential molecular target in cancer treatment. Xanthohumol (XN, a prenylated chalcone derived from hope cones) is known to possess a broad spectrum of chemopreventive and anticancer activities. In our studies, the MTT and BrdU assays revealed that XN demonstrated greater antiproliferative activity against A549 lung adenocarcinoma cells than against the lung adenocarcinoma H1563 cell line. We observed that XN was able to suppress the activities of ERK1/2 and p90RSK kinases, followed by inhibition of phosphorylation and activation of the CREB protein. Additionally, the XN treatment of the cancer cells caused upregulation of key cell cycle regulators p53 and p21 as well as downregulation of cyclin D1. As a result, the cytotoxic effect of XN was attributed to the cell cycle arrest at G1 phase and induction of apoptosis indicated by increased caspase-3 activity. Thus, XN might be a promising anticancer drug candidate against lung carcinomas.

REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering.

Mutagenesis is an important tool to study gene regulation, model disease-causing mutations and for functional characterisation of proteins. Most of the current methods for mutagenesis involve multiple step procedures. One of the most accurate methods for genetically altering DNA is recombineering, which uses bacteria expressing viral recombination proteins. Recently, the use of in vitro seamless assembly systems using purified enzymes for multiple-fragment cloning as well as mutagenesis is gaining ground. Although these in vitro isothermal reactions are useful when cloning multiple fragments, for site-directed mutagenesis it is unnecessary. Moreover, the use of purified enzymes in vitro is not only expensive but also more inaccurate than the high-fidelity recombination inside bacteria. Here we present a single-step method, named REPLACR-mutagenesis (Recombineering of Ends of linearised PLAsmids after PCR), for creating mutations (deletions, substitutions and additions) in plasmids by in vivo recombineering. REPLACR-mutagenesis only involves transformation of PCR products in bacteria expressing Red/ET recombineering proteins. Modifications in a variety of plasmids up to bacterial artificial chromosomes (BACs; 144 kb deletion) have been achieved by this method. The presented method is more robust, involves fewer steps and is cost-efficient.

Matrix metalloproteinases and atherosclerosis

Matrix metalloproteinases (MMPs), a family of proteolytic enzymes that degrade extracellular matrix (ECM), seem to have an important role in pathogenesis of atherosclerosis. Released by inflammatory cells and smooth muscle cells, MMPs regulate the vascular remodeling process. The expression and activity of MMPs is regulated at the level of transcription by a variety of cytokines, proenzyme activation by several cellular and serum proteases, as well as by endogenous and exogenous inhibitors. Overproduction of MMPs could promote arteriosclerosis, leading to atherosclerotic plaque formation, and plaque rupture, resulting in clinical consequences such as myocardial infarction, or critical limb ischemia. Increased plasma levels of some MMPs are now regarded as potential biomarkers of atherosclerosis and cardiovascular risk. The potential significance of MMP inhibitors in atherosclerosis therapy is discussed.

Xanthohumol inhibits cell cycle progression and proliferation of larynx cancer cells in vitro.

Xanthohumol (XN), a prenylflavonoid derived from the hop plant (Humulus lupulus L.) has been found to exhibit a broad spectrum of biological properties, including anti-cancer activity. In this study, the mechanisms involved in anti-cancer activity of XN in human RK33 and RK45 larynx cancer cell lines were investigated. The effect of XN on the viability of larynx cancer and normal cells (human skin fibroblasts HSF and rat oligodendroglia-derived cells, OLN-93) was compared. Additionally, the influence of XN on proliferation, cell cycle progression, induction of apoptosis in larynx cancer cells, as well as the molecular mechanisms underlying in these processes were analyzed. XN promoted the reduction of cell viability in cancer cells, but showed low cytotoxicity to normal cells. The decrease in cell viability in the cancer cells was coupled with induction of apoptosis via two pathways. The mechanisms involved in these effects of XN were associated with cell growth inhibition by induction of cell cycle arrest in the G1 phase, increased p53 and p21/WAF1 expression levels, downregulation of cyclin D1 and Bcl-2, and activation of caspases-9, -8, and -3. Moreover, this compound inhibited phosphorylation of ERK1/2, suggesting a key role of the ERKs pathway in the XN-mediated growth suppressing effects against the studied cells. These results indicate that XN could be used as a potential agent for the treatment of patients with larynx cancer.

How to Train a Cell–Cutting-Edge Molecular Tools

In biological systems, the formation of molecular complexes is the currency for all cellular processes. Traditionally, functional experimentation was targeted to single molecular players in order to understand its effects in a cell or animal phenotype. In the last few years, we have been experiencing rapid progress in the development of ground-breaking molecular biology tools that affect the metabolic, structural, morphological, and (epi)genetic instructions of cells by chemical, optical (optogenetic) and mechanical inputs. Such precise dissection of cellular processes is not only essential for a better understanding of biological systems, but will also allow us to better diagnose and fix common dysfunctions. Here, we present several of these emerging and innovative techniques by providing the reader with elegant examples on how these tools have been implemented in cells, and, in some cases, organisms, to unravel molecular processes in minute detail. We also discuss their advantages and disadvantages with particular focus on their translation to multicellular organisms for in vivo spatiotemporal regulation. We envision that further developments of these tools will not only help solve the processes of life, but will give rise to novel clinical and industrial applications.