Brain malignancies: Glioblastoma and brain metastases.

Brain, the major organ of the central nervous system controls and processes most of body activities. Therefore, the most aggressive brain tumor - glioblastoma and metastases from other organs to the brain are lethal leaving the patients with very short time of survival. The brain tissue landscape is very different from any other tissues and the specific microenvironment, comprising stem cells niches and blood-brain barrier, significantly influences the low rate of glioblastoma metastasis out of the brain, but better accommodates brain-invading cancer. In contrast to low frequency (0.5%) of all glioblastoma metastases, 10%-45% of other primary cancers do metastasize to the brain. This review addresses general cellular and molecular pathways that are to some extent similar in both types of metastases, involving circulating tumor cells (CTCs) with cancer stem cells (CSCs) characteristics, and metastatic niches. The invasion is a dynamic process involving reversible epithelial-to-mesenchymal (EMT) cell process, creating a transient gradient state that is inter-connected with epigenetic plasticity of the metastasizing (m)CSCs. These cells can switch between stationary, low proliferating/dormant state to a migratory, mesenchymal-like state. Settling in their respective niches as dormant CSCs in the secondary organ is a common feature in all types of metastases. In glioblastoma metastasis, the malignant mGSC cells express markers of mesenchymal GSC subtype (MES-GSC), such as CD44 and YK-40 and their major obstacle seems to be propagating in the in various organs' microenvironments, different from the niches that home GSCs in the primary glioblastoma. Focusing on one stromal component in the glioblastoma niches, the mesenchymal stem cells (MSCs), we report herein on their differential effects on glioblastoma cells, highly depending on their genetic subtype. On the other hand, in brain metastases, the major hindrance to metastatic progression of mCSCs seem to be crossing the blood-brain-barrier. Novel therapeutic approaches for brain metastases from various cancer types are advancing slowly, and the general trends involve targeting metastatic sub-clones and selective determinants of their niches. The update on the four most common brain metastases from lung, breast, melanoma and colorectal carcinoma is presented.

Cathepsin K cleavage of SDF-1α inhibits its chemotactic activity towards glioblastoma stem-like cells.

Glioblastoma (GBM) is the most aggressive primary brain tumor with poor patient survival that is at least partly caused by malignant and therapy-resistant glioma stem-like cells (GSLCs) that are protected in GSLC niches. Previously, we have shown that the chemo-attractant stromal-derived factor-1α (SDF-1α), its C-X-C receptor type 4 (CXCR4) and the cysteine protease cathepsin K (CatK) are localized in GSLC niches in glioblastoma. Here, we investigated whether SDF-1α is a niche factor that through its interactions with CXCR4 and/or its second receptor CXCR7 on GSLCs facilitates their homing to niches. Furthermore, we aimed to prove that SDF-1α cleavage by CatK inactivates SDF-1α and inhibits the invasion of GSLCs. We performed mass spectrometric analysis of cleavage products of SDF-1α after proteolysis by CatK. We demonstrated that CatK cleaves SDF-1α at 3 sites in the N-terminus, which is the region of SDF-1α that binds to its receptors. Confocal imaging of human GBM tissue sections confirmed co-localization of SDF-1α and CatK in GSLC niches. In accordance, 2D and 3D invasion experiments using CXCR4/CXCR7-expressing GSLCs and GBM cells showed that SDF-1α had chemotactic activity whereas CatK cleavage products of SDF-1α did not. Besides, CXCR4 inhibitor plerixafor inhibited invasion of CXCR4/CXCR7-expressing GSLCs. In conclusion, CatK can cleave and inactivate SDF-1α. This implies that CatK activity facilitates migration of GSLCs out of niches. We propose that activation of CatK may be a promising strategy to prevent homing of GSLCs in niches and thus render these cells sensitive to chemotherapy and radiation.

In Silico Selection Approach to Develop DNA Aptamers for a Stem-like Cell Subpopulation of Non-small Lung Cancer Adenocarcinoma Cell Line A549.

BACKGROUND: Detection of circulating lung cancer cells with cancer-stem like characteristics would represent an improved tool for disease prognosis. However, current antibodies based methods have some disadvantages and therefore cell SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was used to develop DNA aptamers, recognizing cell surface markers of non-small lung carcinoma (NSLC) cells. MATERIALS AND METHODS: The human adenocarcinoma cell line A549 was used for selection in seven cell SELEX cycles. We used human blood leukocytes for negative selection, and lung stem cell protein marker CD90 antibody binding A549 cells for positive selection. RESULTS: The obtained oligonucleotide sequences after the seventh SELEX cycle were subjected to in silico selection analysis based on three independent types of bioinformatics approaches, selecting two closely related aptamer candidates in terms of consensus sequences, structural motifs, binding affinity (Kd) and stability (ΔG). We selected and identified the aptamer A155_18 with very good binding characteristics to A459 cells, selected for CD90 antibody binding. The calculated phylogenetic tree showed that aptamers A155_18 and the known A549 cell aptamer S6 have a close structural relationship. MEME sequence analysis showed that they share two unique motifs, not present in other sequences. CONCLUSIONS: The novel aptamer A155_18 has strong binding affinity for A549 lung carcinoma cell line subpopulation that is expressing stem cell marker CD90, indicating a possible stemness, characteristic for the A459 line, or a subpopulation present within this cell line. This aptamer can be applied as diagnostic tool, identifying NSLC circulating cells.

Complexity of cancer protease biology: Cathepsin K expression and function in cancer progression.

Proteases, including lysosomal cathepsins, are functionally involved in many processes in cancer progression from its initiation to invasion and metastatic spread. Only recently, cathepsin K (CatK), the cysteine protease originally reported as a collagenolytic protease produced by osteoclasts, appeared to be overexpressed as well in various types of cancers. In this review, the physiological functions of CatK are presented and compared to its potential role in pathobiolology of processes associated with tumour growth, invasion and metastasis of cancer cells and their interactions with the tumour microenvironment. CatK activity is either indirectly affecting signalling pathways, or directly degrading extracellular matrix (ECM) proteins, for example in bone metastases. Recently, CatK was also found in glioma, possibly regulating cancer stem-like cell mobilisation and modulating recently found physiological CatK substrates, including chemokines and growth factors. Moreover, CatK may be useful in differential diagnosis and may have prognostic value. Finally, the application of CatK inhibitors, which are already in clinical trials for treatment of osteoporosis, has a potential to attenuate cancer aggressiveness.

Glioblastoma-mesenchymal stem cell communication modulates expression patterns of kinin receptors: Possible involvement of bradykinin in information flow.

The most aggressive subtype of brain tumors is glioma WHO grade IV, the glioblastoma (GBM). The present work aims to elucidate the role of kinin receptors in interactions between GBM cells and mesenchymal stem cells (MSC). The GBM cell line U87-MG was stably transfected to express dsRed protein, single cell cloned, expanded, and cultured with MSC, both in the direct co-cultures (DC) and indirect co-cultures (IC) at equal cell number ratio for 72 h. Up- and down-regulation of matrix metalloproteases (MMP)-9 expression in U87-MG and MSC cells, respectively, in direct co-culture points to possible MSC participation in tumor invasion. MMP9 expression is in line with significantly increased expression of kinin B1 (B1R) and B2 receptor (B2R) in U87-MG cells and their decreased levels in MSC, as confirmed by quantitative assessment using flow cytometric analysis. Similarly, in indirect cultures (IC), lacking the contact between GBM and MSC cells, an increase of B1 and B2 receptor expression was again noted in U87-MG cells, and no significant changes in kinin receptors in MSC was observed. Functionality of kinin-B1 and B2 receptors was evidenced by stimulation of intracellular calcium fluxes by their respective agonists, des-Arg9-bradykinin (DBK) and bradykinin (BK). Moreover, BK showed a feedback control on kinin receptor expression in mono-cultures, direct and indirect co-cultures. The treatment with BK resulted in down-regulation of B1 and B2 receptors in MSC, with simultaneous up-regulation of these receptors in U87-MG cells, suggesting that functions of BK in information flow between these cells is important for tumor progression and invasion. © 2015 International Society for Advancement of Cytometry.

Topoisomerase IIß mediates the resistance of glioblastoma stem cells to replication stress-inducing drugs.

BACKGROUND: Glioblastoma stem cells (GSC) have been extensively recognized as a plausible cause of glioblastoma resistance to therapy and recurrence resulting in high glioblastoma mortality. Abnormalities in the DNA repair pathways might be responsible for the inability of the currently used chemotherapeutics to eliminate the (GSC) subpopulation. METHODS: In this work, we compared the expression of sixty DNA repair related genes between primary glioblastoma cell cultures and the glioblastoma enriched stem cell primary cultures. MTT test was used to analyze the effect of selected drugs and immunofluorescence to evaluate the load of DNA damage. RESULTS: We found several differentially expressed genes and we identified topoisomerase IIß (Top2ß) as the gene with highest up-regulation in GSC. Also among the tested cell lines the expression of Top2ß was the highest in NCH421k cells, a well-characterized glioblastoma cell line with all the stemness characteristics. On the other hand, Top2ß expression markedly decreased upon the induction of differentiation by all trans-retinoic acid. Depletion of Top2ß increased the sensitivity of NCH421k cells to replication stress inducing drugs, such as cisplatin, methyl-methanesulfonate, hydrogen peroxide, and temozolomide. Consistently, we found an increased load of DNA damage and increased Chk1 activation upon Top2ß depletion in NCH421k cells. CONCLUSION: We suggest that Top2ß may represent a new target for gene therapy in glioblastoma. In addition, the other genes that we found to be up-regulated in GSC versus glioblastoma primary cells should be further investigated as glioblastoma theranostics.

Imaging of human glioblastoma cells and their interactions with mesenchymal stem cells in the zebrafish (Danio rerio) embryonic brain.

BACKGROUND: An attractive approach in the study of human cancers is the use of transparent zebrafish (Danio rerio) embryos, which enable the visualization of cancer progression in a living animal. MATERIALS AND METHODS: We implanted mixtures of fluorescently labeled glioblastoma (GBM) cells and bonemarrow-derived mesenchymal stem cells (MSCs) into zebrafish embryos to study the cellular pathways of their invasion and the interactions between these cells in vivo. RESULTS: By developing and applying a carbocyanine-dye-compatible clearing protocol for observation of cells in deep tissues, we showed that U87 and U373 GBM cells rapidly aggregated into tumor masses in the ventricles and midbrain hemispheres of the zebrafish embryo brain, and invaded the central nervous system, often using the ventricular system and the central canal of the spinal cord. However, the GBM cells did not leave the central nervous system. With co-injection of differentially labeled cultured GBM cells and MSCs, the implanted cells formed mixed tumor masses in the brain. We observed tight associations between GBM cells and MSCs, and possible cell-fusion events. GBM cells and MSCs used similar invasion routes in the central nervous system. CONCLUSIONS: This simple model can be used to study the molecular pathways of cellular processes in GBM cell invasion, and their interactions with various types of stromal cells in double or triple cell co-cultures, to design anti-GBM cell therapies that use MSCs as vectors.

Aptamer for imaging and therapeutic targeting of brain tumor glioblastoma.

Aptamers are short single-stranded nucleic acids (RNA or ssDNA), identified by an in vitro selection process, denominated SELEX, from a partially random oligonucleotide library. They bind to a molecular target, a protein or other complex macromolecular structures of interest with high affinity and specificity, comparable to those of antibodies. Recently, aptamer selection protocols were developed for targeting living cells, including tumors. Chemical modifications of the aptamers and modalities of their detection and delivery systems are already available with high selectivity and targeting ability for the desired cancer cell type, making them promising for diagnosis and therapy. Glioblastoma multiformae represents the most malignant and fatal stage of glioma, and is also the most frequent brain tumor. Glioblastoma-specific aptamers were developed by either targeting the whole cell surface or known glioma biomarkers. These aptamers may gain importance for imaging, tumor cell isolation from biopsies and drug delivery. In biomedical imaging techniques, aptamers coupled with radionuclide or fluorescent labels, bioconjugates and nanoparticles offer an advanced, noninvasive manner for defining the glioblastoma tissue border. Though single modality aptamer imaging probes have some limitations, these are overcome by the use of multimodal probes. Due to selectivity and chemical characteristics, aptamers can be coupled to functionalized nanoparticles and loaded with a drug, appeared promising for in vivo targeting of glioblastoma. Finally, aptamers are effective mediators for gene silencing when coupled to small interfering RNA and a viral vector, thus providing a novel tool with enhanced targeting capability in drug delivery, designed for tailored treatment of glioblastoma patients.

Identification of new peptide amides as selective cathepsin L inhibitors: the first step towards selective irreversible inhibitors?

A small library of peptide amides was designed to profile the cathepsin L active site. Within the cathepsin family of cysteine proteases, the first round of selection was on cathepsin L and cathepsin B, and then selected hits were further evaluated for binding to cathepsin K and cathepsin S. Five highly selective sequences with submicromolar affinities towards cathepsin L were identified. An acyloxymethyl ketone warhead was then attached to these sequences. Although these original irreversible inhibitors inactivate cathepsin L, it appears that the nature of the warhead drastically impact the selectivity profile of the resulting covalent inhibitors.

Expansive growth of two glioblastoma stem-like cell lines is mediated by bFGF and not by EGF.

BACKGROUND: Patient-derived glioblastoma (GBM) stem-like cells (GSCs) represent a valuable model for basic and therapeutic research. GSCs are usually propagated in serum-free Neural Basal medium supplemented with bFGF and EGF. Yet, the exact influence of these growth factors on GSCs is still unclear. Recently it was suggested that GBM stem-like cells with amplified EGFR should be cultured in stem cell medium without EGF, as the presence of EGF induced rapid loss of EGFR amplification. However, patient biopsies are usually taken into culture before their genomic profiles are defined. Thus, an important question remains whether GBM cells without EGFR amplification also can be cultured in stem cell medium without EGF. MATERIALS AND METHODS: [corrected] To address this question, we used two heterogeneous glioblastoma GSC lines (NCH421k and NCH644) that lack EGFR amplification. RESULTS: Although both cell lines showed very low EGFR expression under standard growth conditions, bFGF stimulation induced higher expression of EGFR in NCH644. In both cell lines, expression of the stem cell markers nestin and CD133 was higher upon stimulation with bFGF compared to EGF. Importantly, bFGF stimulated the growth of both cell lines, whereas EGF had no effect. We verified that the growth stimulation by bFGF was either mediated by proliferation (NCH421k) or resistance to apoptosis (NCH644). CONCLUSIONS: We demonstrate that GSC cultures without EGFR amplification can be maintained and expanded with bFGF, while the addition of EGF has no significant effect and therefore can be omitted.

The regulation of cysteine cathepsins and cystatins in human gliomas.

Cysteine cathepsins play an important role in shaping the highly infiltrative growth pattern of human gliomas. We have previously demonstrated that the activity of cysteine cathepsins is elevated in invasive glioblastoma (GBM) cells in vitro, in part due to attenuation of their endogenous inhibitors, the cystatins. To investigate this relationship in vivo, we established U87-MG xenografts in non-obese diabetic (NOD)/severe combined immunodeficiency (SCID)-enhanced green fluorescent protein (eGFP) mice. Here, tumor growth correlated with an elevated enzymatic activity of CatB both in the tumor core and at the periphery, whereas CatS and CatL levels were higher at the xenograft edge compared to the core. Reversely, StefB expression was detected in the tumor core, but it was generally absent in the tumor periphery, suggesting that down-regulation of this inhibitor correlates with in vivo invasion. In human GBM samples, all cathepsins were elevated at the tumor periphery compared to brain parenchyma. CatB was also typically associated with angiogenic endothelia and necrotic areas. StefB was mainly detected in the tumor core, whereas CysC and StefA were evenly distributed, reflecting the observations in the xenografts. However, at the mRNA level, no differences in cathepsins and cystatins were observed between the tumor center and the periphery in both human biopsies and xenografts. Interestingly, in human tumors, cathepsin and stefin transcript levels correlated with CD68 and CXCR4 levels, but not with epidermal growth factor receptor (EGFR). Moreover, we reveal for the first time that an elevated StefA mRNA level is a highly significant prognostic factor for patient survival.

A novel photoaffinity-based probe for selective detection of cathepsin L active form.

Detecting the active forms of proteases by using activity-based probes in complex proteomes has become an intensively investigated field of research over the past years because many pathogenic conditions involve alterations in protease activities. The detection of lysosomal cysteine proteases, the cathepsins, has mostly relied on the use of probes that incorporate reactive electrophilic moieties to modify a cysteine in the active site covalently. Here we report the first example of an activity-based probe that targets the cathepsins and incorporates a photoactivatable benzophenone group for covalent labelling. When tested on a set of five cathepsins (B, K, L, S and V), this probe selectively labelled the active site of cathepsin L. Furthermore, when tested on crude cell extracts, the probe specifically detected cathepsin L quantities as low as a few picomoles. This study suggests that photoaffinity labelling is a promising approach for developing highly selective and useful cathepsin L probes. In particular, this probe might allow the detection of small amounts of the secreted active cathepsin L form in the cellular microenvironment in vitro and ex vivo.

Fast assay to predict multipotent mesenchymal stromal cell replicative senescence dynamics.

The major obstacle to the application of mesenchymal stromal cells (MSCs) in regenerative medicine is the expansion of the donor-derived cells in vitro to obtain high cell numbers in the shortest possible time. However, MSCs gradually undergo replicative senescence after a variable number of divisions that reduce their therapeutic efficacy, which needs to be determined before administration. The authors developed a fast and simple evaluation assay testing two senescence inducers, mitoxantrone (Mxt) and trichostatin A (TSA), to predict the onset of spontaneous replicative senescence of adipose-derived mesenchymal stromal cells (ASCs) and have confirmed the correlation between induced senescence and spontaneous replicative senescence in the assay using Mxt. This protocol facilitates the standardization of therapeutic ASCs and MSCs from other origins before application.

Bioactive peptides from venoms against glioma progression.

Venoms are complex mixtures of different molecules and ions. Among them, bioactive peptides have been found to affect cancer hallmarks, such as cell proliferation, cell invasion, cell migration, and can also modulate the immune response of normal and cancer-bearing organisms. In this article, we review the mechanisms of action on these cancer cell features, focusing on bioactive peptides being developed as potential therapeutics for one of the most aggressive and deadly brain tumors, glioblastoma (GB). Novel therapeutic approaches applying bioactive peptides may contribute to multiple targeting of GB and particularly of GB stem cells. Bioactive peptides selectively target cancer cells without harming normal cells. Various molecular targets related to the effects of bioactive peptides on GB have been proposed, including ion channels, integrins, membrane phospholipids and even immunomodulatory treatment of GB. In addition to therapy, some bioactive peptides, such as disintegrins, can also be used for diagnostics or are used as labels for cytotoxic drugs to specifically target cancer cells. Given the limitations described in the last section, successful application in cancer therapy is rather low, as only 3.4% of such peptides have been included in clinical trials and have passed successfully phases I to III. Combined approaches of added bioactive peptides to standard cancer therapies need to be explored using advanced GB in vitro models such as organoids. On the other hand, new methods are also being developed to improve translation from research to practice and provide new hope for GB patients and their families.

The Cytotoxic Effects of Cannabidiol and Cannabigerol on Glioblastoma Stem Cells May Mostly Involve GPR55 and TRPV1 Signalling.

Glioblastoma (GBM) is one of the most aggressive cancers, comprising 60-70% of all gliomas. The large G-protein-coupled receptor family includes cannabinoid receptors CB1, CB2, GPR55, and non-specific ion receptor protein transporters TRPs. First, we found up-regulated CNR1, GPR55, and TRPV1 expression in glioma patient-derived tissue samples and cell lines compared with non-malignant brain samples. CNR1 and GPR55 did not correlate with glioma grade, whereas TRPV1 negatively correlated with grade and positively correlated with longer overall survival. This suggests a tumour-suppressor role of TRPV1. With respect to markers of GBM stem cells, preferred targets of therapy, TRPV1 and GPR55, but not CNR1, strongly correlated with different sets of stemness gene markers: NOTCH, OLIG2, CD9, TRIM28, and TUFM and CD15, SOX2, OCT4, and ID1, respectively. This is in line with the higher expression of TRPV1 and GPR55 genes in GSCs compared with differentiated GBM cells. Second, in a panel of patient-derived GSCs, we found that CBG and CBD exhibited the highest cytotoxicity at a molar ratio of 3:1. We suggest that this mixture should be tested in experimental animals and clinical studies, in which currently used Δ9-tetrahydrocannabinol (THC) is replaced with efficient and non-psychoactive CBG in adjuvant standard-of-care therapy.

Inhibition of cathepsin L lowers the apoptotic threshold of glioblastoma cells by up-regulating p53 and transcription of caspases 3 and 7.

Despite all the progress in cancer treatment, glioblastoma, the most malignant tumor of the central nervous system, remains a terminal disease and new therapeutic approaches are urgently needed. A combination of chemotherapy with modifications that lower the apoptotic threshold of cancer cells could be effective. Cathepsin L inhibition was suggested as one of such modifications but the mechanism of cathepsin L anti-apoptotic activity is largely unknown. In the present study we show that, in U87 glioblastoma cells, cathepsin L is present in the nucleus and regulates the transcription of effector caspases 3 and 7. In cells with low cathepsin L expression, p53 and prohibitin--transcription factors that regulate caspase 7 expression--accumulate in the nuclei. The importance of p53 in this process is highlighted by the fact that in U87 cells with inhibited p53 transcriptional activity or in p53-negative cells U251, cathepsin L inhibition did not influence caspase 7 expression and had minimal effect on the level of apoptosis. Since p53 pathways are often mutated in glioblastoma, the findings of our study need to be considered before using cathepsin L inhibition for glioblastoma therapy and suggest that such adjuvant therapy may be effective only for a subpopulation of p53 wild type glioblastoma patients.

Immunohistochemical Detection of Neural Stem Cells and Glioblastoma Stem Cells in the Subventricular Zone of Glioblastoma Patients.

Glioblastoma usually recurs after therapy consisting of surgery, radiotherapy, and chemotherapy. Recurrence is at least partly caused by glioblastoma stem cells (GSCs) that are maintained in intratumoral hypoxic peri-arteriolar microenvironments, or niches, in a slowly dividing state that renders GSCs resistant to radiotherapy and chemotherapy. Because the subventricular zone (SVZ) is a major niche for neural stem cells (NSCs) in the brain, we investigated whether GSCs are present in the SVZ at distance from the glioblastoma tumor. We characterized the SVZ of brains of seven glioblastoma patients using fluorescence immunohistochemistry and image analysis. NSCs were identified by CD133 and SOX2 but not CD9 expression, whereas GSCs were positive for all three biomarkers. NSCs were present in all seven samples and GSCs in six out of seven samples. The SVZ in all samples were hypoxic and expressed the same relevant chemokines and their receptors as GSC niches in glioblastoma tumors: stromal-derived factor-1α (SDF-1α), C-X-C receptor type 4 (CXCR4), osteopontin, and CD44. In conclusion, in glioblastoma patients, GSCs are present at distance from the glioblastoma tumor in the SVZ. These findings suggest that GSCs in the SVZ niche are protected against radiotherapy and chemotherapy and protected against surgical resection due to their distant localization and thus may contribute to tumor recurrence after therapy.

Cannabigerol Is a Potential Therapeutic Agent in a Novel Combined Therapy for Glioblastoma.

Glioblastoma is the most aggressive cancer among primary brain tumours. As with other cancers, the incidence of glioblastoma is increasing; despite modern therapies, the overall mean survival of patients post-diagnosis averages around 16 months, a figure that has not changed in many years. Cannabigerol (CBG) has only recently been reported to prevent the progression of certain carcinomas and has not yet been studied in glioblastoma. Here, we have compared the cytotoxic, apoptotic, and anti-invasive effects of the purified natural cannabinoid CBG together with CBD and THC on established differentiated glioblastoma tumour cells and glioblastoma stem cells. CBG and THC reduced the viability of both types of cells to a similar extent, whereas combining CBD with CBG was more efficient than with THC. CBD and CBG, both alone and in combination, induced caspase-dependent cell apoptosis, and there was no additive THC effect. Of note, CBG inhibited glioblastoma invasion in a similar manner to CBD and the chemotherapeutic temozolomide. We have demonstrated that THC has little added value in combined-cannabinoid glioblastoma treatment, suggesting that this psychotropic cannabinoid should be replaced with CBG in future clinical studies of glioblastoma therapy.

Differential role of cathepsins B and L in autophagy-associated cell death induced by arsenic trioxide in U87 human glioblastoma cells.

Arsenic trioxide (arsenite) was the first chemotherapeutic drug to be described and is now being rediscovered in cancer treatment, including glioblastoma multiforme. Arsenite toxicity triggers autophagy in cancer cells, although final stages of the process involve executive caspases, suggesting an interplay between autophagic and apoptotic pathways that awaits to be explained at a molecular level. We evaluated the contribution of the lysosomal cathepsins (Cat) L and B, which are upregulated in glioblastomas, in the mechanism of arsenite toxicity in human glioblastoma cells. Arsenite treatment induced autophagosome formation and permeabilization of mitochondria, followed by caspase 3/7-mediated apoptosis. The autophagy inhibitor 3-methyladenine protected from arsenite toxicity, whereas bafilomycin A1 did not. Furthermore, arsenite significantly decreased CatB levels and selectively inhibited its cellular and recombinant protein activity, while not affecting CatL. However, downregulation of CatL greatly enhanced apoptosis by arsenite. Our results show that arsenite toxicity involves a complex interplay between autophagy and apoptosis in human glioblastoma cells and is associated with inhibition of CatB, and that this toxicity is highly exacerbated by simultaneous CatL inhibition. The latter points to a synergy that could be used in clinical treatment to lower the therapeutic dose, thus avoiding the toxic side effects of arsenite in glioblastoma management.

Development of human cell biosensor system for genotoxicity detection based on DNA damage-induced gene expression.

BACKGROUND: Human exposure to genotoxic agents in the environment and everyday life represents a serious health threat. Fast and reliable assessment of genotoxicity of chemicals is of main importance in the fields of new chemicals and drug development as well as in environmental monitoring. The tumor suppressor gene p21, the major downstream target gene of activated p53 which is responsible for cell cycle arrest following DNA damage, has been shown to be specifically up-regulated by genotoxic carcinogens. The aim of our study was to develop a human cell-based biosensor system for simple and fast detection of genotoxic agents. METHODS: Metabolically active HepG2 human hepatoma cells were transfected with plasmid encoding Enhanced Green Fluorescent Protein (EGFP) under the control of the p21 promoter (p21HepG2GFP). DNA damage was induced by genotoxic agents with known mechanisms of action. The increase in fluorescence intensity, due to p21 mediated EGFP expression, was measured with a fluorescence microplate reader. The viability of treated cells was determined by the colorimetric MTS assay. RESULTS: The directly acting alkylating agent methylmethane sulphonate (MMS) showed significant increase in EGFP production after 48 h at 20 µg/mL. The indirectly acting carcinogen benzo(a)pyren (BaP) and the cross-linking agent cisplatin (CisPt) induced a dose- dependent increase in EGFP fluorescence, which was already significant at concentrations 0.13 µg/mL and 0.41 µg/mL, respectively. Vinblastine (VLB), a spindle poison that does not induce direct DNA damage, induced only a small increase in EGFP fluorescence intensity after 24 h at the lowest concentration (0.1 µg/mL), while exposure to higher concentrations was associated with significantly reduced cell viability. CONCLUSIONS: The results of our study demonstrated that this novel assay based on the stably transformed cell line p21HepG2GFP can be used as a fast and simple biosensor system for detection of genetic damage caused by chemical agents.