Relationship between the Expression of Matrix Metalloproteinases and Their Tissue Inhibitors in Patients with Brain Tumors.

Matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) play critical roles in regulating processes associated with malignant behavior. These endopeptidases selectively degrade components of the extracellular matrix (ECM), growth factors, and their receptors, contributing to cancer cell invasiveness and migratory characteristics by disrupting the basal membrane. However, the expression profile and role of various matrix metalloproteinases remain unclear, and only a few studies have focused on differences between diagnoses of brain tumors. Using quantitative real-time PCR analysis, we identified the expression pattern of ECM modulators (n = 10) in biopsies from glioblastoma (GBM; n = 20), astrocytoma (AST; n = 9), and meningioma (MNG; n = 19) patients. We found eight deregulated genes in the glioblastoma group compared to the benign meningioma group, with only MMP9 (FC = 2.55; p = 0.09) and TIMP4 (7.28; p < 0.0001) upregulated in an aggressive form. The most substantial positive change in fold regulation for all tumors was detected in matrix metalloproteinase 2 (MNG = 30.9, AST = 4.28, and GBM = 4.12). Notably, we observed an influence of TIMP1, demonstrating a positive correlation with MMP8, MMP9, and MMP10 in tumor samples. Subsequently, we examined the protein levels of the investigated MMPs (n = 7) and TIMPs (n = 3) via immunodetection. We confirmed elevated levels of MMPs and TIMPs in GBM patients compared to meningiomas and astrocytomas. Even when correlating glioblastomas versus astrocytomas, we showed a significantly increased level of MMP1, MMP3, MMP13, and TIMP1. The identified metalloproteases may play a key role in the process of gliomagenesis and may represent potential targets for personalized therapy. However, as we have not confirmed the relationship between mRNA expression and protein levels in individual samples, it is therefore natural that the regulation of metalloproteases will be subject to several factors.

Involvement of Proteasomal and Endoplasmic Reticulum Stress in Neurodegeneration After Global Brain Ischemia.

A brief period of transient global brain ischemia leads to selective ischemic neurodegeneration associated with death of hippocampal CA1 pyramidal neurons days after reperfusion. The mechanism of such selective and delayed neurodegeneration is still uncertain. Our work aimed to study the involvement of proteasomal and endoplasmic reticulum (ER) stress in ischemic neurodegeneration. We have performed laser scanning confocal microscopy analysis of brain slices from control and experimental animals that underwent global brain ischemia for 15 min and varying times of reperfusion. We have focused on ubiquitin, PUMA, a proapoptotic protein of the Bcl-2 family overexpressed in response to both proteasomal and ER stress, and p53, which controls expression of PUMA. We have also examined the expression of HRD1, an E3 ubiquitin ligase that was shown to be overexpressed after ER stress. We have also examined potential crosstalk between proteasomal and ER stress using cellular models of both proteasomal and ER stress. We demonstrate that global brain ischemia is associated with an appearance of distinct immunoreactivity of ubiquitin, PUMA and p53 in pyramidal neurons of the CA1 layer of the hippocampus 72 h after ischemic insults. Such changes correlate with a delay and selectivity of ischemic neurodegeneration. Immunoreactivity of HRD1 observed in all investigated regions of rat brain was transiently absent in both CA1 and CA3 pyramidal neurones 24 h after ischemia in the hippocampus, which does not correlate with a delay and selectivity of ischemic neurodegeneration. We do not document significant crosstalk between proteasomal and ER stress. Our results favour dysfunction of the ubiquitin proteasome system and consequent p53-induced expression of PUMA as the main mechanisms responsible for selective and delayed degeneration of pyramidal neurons of the hippocampal CA1 layer in response to global brain ischemia.

Immunodetection of Pyruvate Carboxylase Expression in Human Astrocytomas, Glioblastomas, Oligodendrogliomas, and Meningiomas.

Pyruvate carboxylase (PC) is an enzyme catalyzing the carboxylation of pyruvate to oxaloacetate. The enzymatic generation of oxaloacetate, an intermediate of the Krebs cycle, could provide the cancer cells with the additional anaplerotic capacity and promote their anabolic metabolism. Recent studies revealed that several types of cancer cells express PC. The gained anaplerotic capability of cells mediated by PC correlates with their expedited growth, higher aggressiveness, and increased metastatic potential. By immunohistochemical staining and immunoblotting analysis, we investigated PC expression among samples of different types of human brain tumors. Our results show that PC is expressed by the cells in glioblastoma, astrocytoma, oligodendroglioma, and meningioma tumors. The presence of PC in these tumors suppose that PC could support the anabolic metabolism of their cellular constituents by its anaplerotic capability.

Different Approaches for the Profiling of Cancer Pathway-Related Genes in Glioblastoma Cells.

Deregulation of signalling pathways that regulate cell growth, survival, metabolism, and migration can frequently lead to the progression of cancer. Brain tumours are a large group of malignancies characterised by inter- and intratumoral heterogeneity, with glioblastoma (GBM) being the most aggressive and fatal. The present study aimed to characterise the expression of cancer pathway-related genes (n = 84) in glial tumour cell lines (A172, SW1088, and T98G). The transcriptomic data obtained by the qRT-PCR method were compared to different control groups, and the most appropriate control for subsequent interpretation of the obtained results was chosen. We analysed three widely used control groups (non-glioma cells) in glioblastoma research: Human Dermal Fibroblasts (HDFa), Normal Human Astrocytes (NHA), and commercially available mRNAs extracted from healthy human brain tissues (hRNA). The gene expression profiles of individual glioblastoma cell lines may vary due to the selection of a different control group to correlate with. Moreover, we present the original multicriterial decision making (MCDM) for the possible characterization of gene expression profiles. We observed deregulation of 75 genes out of 78 tested in the A172 cell line, while T98G and SW1088 cells exhibited changes in 72 genes. By comparing the delta cycle threshold value of the tumour groups to the mean value of the three controls, only changes in the expression of 26 genes belonging to the following pathways were identified: angiogenesis FGF2; apoptosis APAF1, CFLAR, XIAP; cellular senescence BM1, ETS2, IGFBP5, IGFBP7, SOD1, TBX2; DNA damage and repair ERCC5, PPP1R15A; epithelial to mesenchymal transition SNAI3, SOX10; hypoxia ADM, ARNT, LDHA; metabolism ATP5A1, COX5A, CPT2, PFKL, UQCRFS1; telomeres and telomerase PINX1, TINF2, TNKS, and TNKS2. We identified a human astrocyte cell line and normal human brain tissue as the appropriate control group for an in vitro model, despite the small sample size. A different method of assessing gene expression levels produced the same disparities, highlighting the need for caution when interpreting the accuracy of tumorigenesis markers.

The effect of temozolomide on apoptosis-related gene expression changes in glioblastoma cells.

BACKGROUND: Glioblastoma (GB) is the most common and biologically the most aggressive primary brain tumor of the central nervous system (CNS) in adults. Standard treatment for newly diagnosed GB consists of surgical resection, radiotherapy, and chemotherapy with temozolomide (TMZ). Despite numbers of studies, a resistance to chemotherapy is the major obstacle to successful GB treatment. OBJECTIVES: The aim of our study was to detect the sensitivity of glioblastoma T98G cells to TMZ treatment and subsequently to determine the expression changes of apoptosis-associated genes in glioblastoma cells. MATERIAL AND METHODS: The human glioblastoma cell line (T98G) was treated with specified concentrations of TMZ during different time periods. Their viability was measured by colorimetric MTT assay and the activation of the apoptotic pathway was determined by measuring the caspase 3/7 activity. Commercial pre-designed microfluidic array was used to quantify expression of human apoptosis-associated genes. RESULTS: The untreated control of T98G cell line against human brain total RNA standards reported significant changes in several apoptotic genes expression levels. We identified also a deregulation in geneexpression levels between the TMZ treated and untreated T98G cells associated with apoptotic pathways. After 48 hours of exposure of T98G cells to TMZ, we observed a significant deregulation ofseven genes: BBC3, BCL2L1, RIPK1, CASP3, BIRC2, CARD6 and DAPK1. These results can contribute to the importance of apoptosis in glioblastoma cells metabolism and effect of TMZ treatment. CONCLUSIONS: Identification of apoptotic gene panel in T98G cell line could help to improve understanding of brain tumor cells metabolism. Recognizing of the pro-apoptotic and anti-apoptotic genes expression changes could contribute to clarify the sensitivity to TMZ therapy and molecular base in healthy and tumor cells (Tab. 1, Fig. 2, Ref. 48).

Endoplasmic reticulum stress induces mitochondrial dysfunction but not mitochondrial unfolded protein response in SH-SY5Y cells.

In the present study we have shown that treatment of SH-SY5Y cells with either thapsigargin or tunicamycin is associated with a significant decrease in ROUTINE and ATP-coupled mitochondrial respiration as well as a decrease in spare and maximal respiratory capacity. We have also shown that treating cells with either thapsigargin or tunicamycin is associated with significant changes in mitochondrial membrane potential (ΔΨm) generation, which is mainly associated with the reversal of the succinyl-CoA ligase reaction and a decreased activity of complex II. Despite the induction of endoplasmic reticulum (ER) specific unfolded protein response (UPR), as documented by increased expression of HRD1, ER stress did not induce mitochondrial UPR since the expression of both mitochondrial protease LONP1 and mitochondrial chaperone HSP60 was not significantly altered. Inhibition of IRE1α ribonuclease with STF-083010 did not protect the SH-SY5Y cells from ER stress-induced mitochondrial dysfunction. STF-083010 itself had significant impact on both mitochondrial respiration and generation of ΔΨm, which has mainly been associated with the uncoupling of respiratory chain from ATP synthesis.

Associations between matrix metalloproteinase, tissue inhibitor of metalloproteinase and collagen expression levels in the adjacent rectal tissue of colorectal carcinoma patients.

As the commonest type of cancer in Europe and the third most common type of cancer worldwide, colorectal carcinoma (CRC) poses a challenge for numerous scientific studies. At present, the cause of this disease is remains to be elucidated, but early diagnosis is only one solution to prevent serious health complications. As a structural scaffold, the extracellular matrix (ECM) is in direct contact with tumour cells and significantly interferes with tumour progression. During the process of tumorigenesis, the ECM undergoes structural changes in which collagens serve an important role. Their life cycle is regulated by proteolytic enzymes called matrix metalloproteinases (MMPs), which are controlled by tissue inhibitors of metalloproteinases (TIMPs). The present study analysed the gene expression of MMPs (MMP1-2-8-10-13), TIMPs (TIMP1-2-4) and collagens (COL1A1 and COL3A1) and the correlation with biochemical parameters in the adjacent rectal tissue (ART) of patients with CRC. The patients who underwent standard neoadjuvant pre-therapy showed increased concentrations of collagen in the normal ART. The mRNA levels of COL3A1, TIMP1 and TIMP2 were significantly higher in the ART of CRC patients (with or without pre-therapy) when compared with the control group. This finding suggested that TIMPs served an important role in the regulation of MMPs and in the modification of collagen content in the ECM. Despite the small data set, the present study provided insights into the transcriptomic relationships between the individual genes that are an integral part of the ECM.

Both thapsigargin- and tunicamycin-induced endoplasmic reticulum stress increases expression of Hrd1 in IRE1-dependent fashion.

OBJECTIVES: We have investigated the impact of endoplasmic reticulum (ER) stress, which is often implicated in neurodegenerative diseases, on the expression of Hrd1, an E3 ubiquitin ligase that plays a central role in the process of ER-associated degradation (ERAD). METHODS: SH-SY5Y neuroblastoma cells, a frequently used model for studying neurotoxicity in dopaminergic neurons and the mechanisms of neurodegeneration associated with Parkinson's disease, and parental SK-N-SH cells were studied. RESULTS: We demonstrate that ER stress, induced by thapsigargin or tunicamycin, correlates with the increased expression of Hrd1 in both SH-SY5Y and SK-N-SH cells. Inhibition of PERK does not significantly suppress the thapsigargin- or tunicamycin-induced expression of Hrd1. Nevertheless, PERK inhibition has a positive effect on the survival of SH-SY5Y cells treated with thapsigargin but not on those treated with tunicamycin. Inhibition of IRE1 associated with the inhibition of XBP1 splicing does not affect the survival of SH-SY5Y cells treated with either thapsigargin or tunicamycin but results in the complete suppression of both the thapsigargin- and tunicamycin-induced expression of Hrd1. DISCUSSION: Thus, the ER-stress-induced expression of Hrd1 in SH-SY5Y depends on Hrd1 transcription activation, which is a consequence of IRE1 but not of PERK activation.

Cobalt chloride affects the death of SH-SY5Y cells induced by inhibition of ubiquitin proteasome system. Role of heat shock protein 70 and caspase 3.

The aim our study was to investigate protective effect of cobalt chloride (CoCl2) in the model of proteasome stress of neuroblastoma SH-SY5Y cells induced by bortezomib, an inhibitor of 26S proteasome. We have focused our interests on Hsp70 and activation of caspase 3. Finally, we have compared the effect of CoCl2 with an effect of the pre-treatment of the cells with 17-AAG, an inhibitor of Hsp90 that is capable to induce expression of Hsp70, or with IOX2, an inhibitor of isoform 2 of prolyl hydroxylase that increases stability of hypoxia inducible factor 1α (HIF1α). Pre-treatment of SH-SY5Y cells for 24 h with CoCl2, at concentrations of 150 or 250 µmol/l, and with 17-AAG at concentration 1 µmol/l but not with IOX2 at concentration 100 µmol/l, was associated with significantly increased expression of Hsp70. We have shown that pre-treatment of SH-SY5Y cells with CoCl2 but not with 17-AAG or IOX2 was associated with significant delay of the cell death induced by proteasome stress. CoCl2-mediated effect was consistent with inhibition of bortezomib-induced caspase 3 activation in the cells pre-treated with CoCl2. Despite established neuroprotective properties of Hsp70 our results do not provide strong evidence that the effect of CoCl2 could be mainly attributed to the ability of CoCl2 to induce expression of Hsp70 and other mechanisms have to be considered.

Proteasome Stress Triggers Death of SH-SY5Y and T98G Cells via Different Cellular Mechanisms.

Overload or dysfunction of ubiquitin-proteasome system (UPS) is implicated in mechanisms of neurodegeneration associated with neurodegenerative diseases, e.g. Parkinson and Alzheimer disease, and ischemia-reperfusion injury. The aim of this study was to investigate the possible association between viability of neuroblastoma SH-SY5Y and glioblastoma T98G cells treated with bortezomib, inhibitor of 26S proteasome, and accumulation of ubiquitin-conjugated proteins with respect to direct cytotoxicity of aggregates of ubiquitin-conjugated proteins. Bortezomib-induced death of SH-SY5Y cells was documented after 24 h of treatment while death of T98G cells was delayed up to 48 h. Already after 4 h of treatment of both SH-SY5Y and T98G cells with bortezomib, increased levels of both ubiquitin-conjugated proteins with molecular mass more than 150 kDa and Hsp70 were observed whereas Hsp90 was elevated in T98G cells and decreased in SH-SY5Y cells. With respect to the cell death mechanism, we have documented bortezomib-induced activation of caspase 3 in SH-SY5Y cells that was probably a result of increased expression of pro-apoptotic proteins, PUMA and Noxa. In T98G cells, bortezomib-induced expression of caspase 4, documented after 24 h of treatment, with further activation of caspase 3, observed after 48 h of treatment. The delay in activation of caspase 3 correlated well with the delay of death of T98G cells. Our results do not support the possibility about direct cytotoxicity of aggregates of ubiquitin-conjugated proteins. They are more consistent with a view that proteasome inhibition is associated with both transcription-dependent and -independent changes in expression of pro-apoptotic proteins and consequent cell death initiation associated with caspase 3 activation.