| 1. |
- Zheng, Lin, 1978-, et al.
(författare)
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Autophagy of amyloid beta-protein in differentiated neuroblastoma cells exposed to oxidative stress
- 2006
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Ingår i: Neuroscience Letters. - : Elsevier BV. - 0304-3940 .- 1872-7972. ; 394:3, s. 184-189
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Tidskriftsartikel (refereegranskat)abstract
- Oxidative stress is considered important for the pathogenesis of Alzheimer disease (AD), which is characterized by the formation of senile plaques rich in amyloid beta-protein (Aβ). Aβ cytotoxicity has been found dependent on lysosomes, which are abundant in AD neurons and are shown to partially co-localize with Aβ. To determine whether oxidative stress has any influence on the relationship between lysosomes and Aβ1-42 (the most toxic form of Aβ), we studied the effect of hyperoxia (40% versus 8% ambient oxygen) on the intracellular localization of Aβ1-42 (assessed by immunocytochemistry) in retinoic acid differentiated SH-SY5Y neuroblastoma cells maintained in serum-free OptiMEM medium. In control cells, Aβ1-42 was mainly localized to small non-lysosomal cytoplasmic granules. Only occasionally Aβ1-42 was found in large (over 1 μm) lysosomal-associated membrane protein 2 positive vacuoles, devoid of the early endosomal marker rab5. These large Aβ1-42-containing lysosomes were not detectable in the presence of serum (known to suppress autophagy), while their number increased dramatically (up to 24-fold) after exposure of cells to hyperoxia during 5 days. Activation of autophagy by hyperoxia was confirmed by transmission electron microscopy. Furthermore, an inhibitor of autophagic sequestration 3-methyladenine prevented the accumulation of Aβ1-42-positive lysosomes due to hyperoxia. In parallel experiments, intralysosomal accumulation of Aβ1-40 following oxidative stress has been found as well. The results suggest that Aβ can be autophagocytosed and its accumulation within neuronal lysosomes is enhanced by oxidative stress. © 2005 Elsevier Ireland Ltd. All rights reserved.
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| 2. |
- Zheng, Lin, 1978-, et al.
(författare)
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Oxidative Stress Induces Intralysosomal Accumulation of Alzheimer Amyloid β-Protein in Cultured Neuroblastoma Cells
- 2006
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Ingår i: Annals of the New York Academy of Sciences. - : Wiley. - 0077-8923 .- 1749-6632. ; 1067, s. 248-251
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Tidskriftsartikel (refereegranskat)abstract
- Oxidative stress is considered important for the pathogenesis of Alzheimer's disease (AD), which is characterized by the formation of extracellular senile plaques, mainly composed of amyloid β-protein (Aβ). Aβ also accumulates within AD neurons and is believed to exert cellular toxicity through lysosomal labilization. We report that the exposure of human neuroblastoma cells to hyperoxia (40% vs. 8% ambient oxygen) induced the accumulation of large (over 1 μM) Aβ-containing lysosomes, which were not typical of control cells, showing a distinct localization of Aβ and lysosomal markers. An inhibitor of autophagy, 3-methyladenine, suppressed the effect of hyperoxia. The results suggest a link between the involvement of oxidative stress and lysosomes in AD.
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| 3. |
- D`Herde, Katharina, et al.
(författare)
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Morphological changes in dying cells
- 2003
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Ingår i: Cell proliferation & apoptosis. - Linköping : Linköpings universitet. - 9780203495865 - 0203495861 ; , s. 201-231
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- Cell Proliferation and Apoptosis provides a detailed and novel practical guide to cell proliferation and apoptosis detection methods.
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| 4. |
- Garner, B, et al.
(författare)
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Distribution of ferritin and redox-active transition metals in normal and cataractous human lenses
- 2000
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Ingår i: Experimental Eye Research. - : Elsevier BV. - 0014-4835 .- 1096-0007. ; 71:6, s. 599-607
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Tidskriftsartikel (refereegranskat)abstract
- Previous studies have shown that lenticular levels of Fe and Cu are elevated in age-related cataract. However, it is not known if these metals are present in a state that is permissive for redox reactions that may lead to the formation of free radicals. In addition, there is little data available concerning the concentration and lenticular distribution of ferritin, the major intracellular Fe-sequestering protein, in the lens. The aim of the present work was therefore to determine the distribution of ferritin and the redox-availability of Fe and Cu in healthy and cataractous lenses. Lens ferritin distribution was assessed by ELISA and immunohistochemistry. A modified ELISA detected ferritin in an 'insoluble' lens protein fraction. Ferritin levels were not significantly different in the cortex vs nucleus of healthy lenses. In contrast, ferritin levels in the cataractous lens nuclei appeared to be 70 % lower compared to the cortex. This was at least partially due to the presence of ferritin within an insoluble protein fraction of the homogenized lenses. In normal lenses, ferritin staining was most intense in the epithelium, with diffuse staining observed throughout the cortex and nucleus. The redox-availability of lenticular metals was determined using: (1) autometallography, (2) Ferene-S as a chromogenic Fe chelator, and (3) NO release from nitrosocysteine to probe for redox-active Cu. The antometallography studies showed that the cataractous lenses stained more heavily for redox-active metals in both the nucleus and cortex when compared to age-matched control lenses. Chelatable Fe was detected in homogenized control lenses after incubation with Ferene-S, with almost three-fold higher levels detected in the cataractous lenses on average. The Cu-catalysed liberation of NO from added nitrosocysteine was not demonstrated in any lens sample. When exogenous Cu (50 nM) was added to the lenses, it was rapidly chelated. The cataractous samples were approximately twice as effective at redox-inactivation of added Cu. These studies provide evidence that a chelatable pool of potentially redox-active Fe is present at increased concentrations in human cataractous lenses. In contrast, it seems that lenticular Cu may not be readily available for participation in redox reactions. (C) 2000 Academic Press.
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| 5. |
- Garner, B, et al.
(författare)
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On the cytoprotective role of ferritin in macrophages and its ability to enhance lysosomal stability.
- 1997
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Ingår i: Free radical research. - : Taylor & Francis. - 1071-5762 .- 1029-2470. ; 27, s. 487-500
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Macrophages have a great capacity to take up (e.g. by endocytosis and phagocytosis) exogenous sources of iron which could potentially become cytotoxic, particularly following the intralysosomal formation of low-molecular weight, redox active iron, and under conditions of oxidative stress. Following autophagocytosis of endogenous ferritin/apoferritin, these compounds may serve as chelators of such lysosomal iron and counteract the occurrence of iron-mediated intralysosomal oxidative reactions. Such redox-reactions have been shown to lead to destabilisation of lysosomal membranes and result in leakage of damaging lysosomal contents to the cytosol. In this study we have shown: (i) human monocyte-derived macrophages to accumulate ferritin in response to iron exposure; (ii) iron to destabilise macrophage secondary lysosomes when the cells are exposed to H2O2; and (iii) endocytosed apoferritin to act as a stabiliser of the acidic vacuolar compartment of iron-loaded macrophages. While the endogenous ferritin accumulation which was induced by iron exposure was not sufficient to protect cells from the damaging effects of H2O2, exogenously added apoferritin, as well as the potent iron chelator desferrioxamine, afforded significant protection. It is suggested that intralysosomal formation of haemosiderin, from partially degraded ferritin, is a protective strategy to suppress intralysosomal iron-catalysed redox reactions. However, under conditions of severe macrophage lysosomal iron-overload, induction of ferritin synthesis is not enough to completely prevent the enhanced cytotoxic effects of H2O2.
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| 6. |
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| 7. |
- Jedlinski, Adam, 1978-, et al.
(författare)
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Cetuximab sensitivity of head and neck squamous cell carcinoma xenografts is associated with treatment-induced reduction in EGFR, pEGFR, and pSrc.
- 2017
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Ingår i: Journal of Oral Pathology & Medicine. - : Wiley. - 0904-2512 .- 1600-0714. ; 46:9, s. 717-724
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The aims of this study were to validate in vitro drug sensitivity testing of head and neck squamous cell carcinoma (HNSCC) cell lines in an in vivo xenograft model and to identify treatment-induced changes in the epidermal growth factor receptor (EGFR) signaling pathway that could be used as markers for cetuximab treatment response.MATERIALS AND METHODS: The in vitro and in vivo cetuximab sensitivity of two HNSCC cell lines, UT-SCC-14 and UT-SCC-45, was assessed using a crystal violet assay and xenografts in nude mice, respectively. The expression of EGFR, phosphorylated EGFR (pEGFR), phosphorylated Src (pSrc), and Ki-67 was investigated by immunohistochemistry. To verify these results, the in vitro expression of EGFR and pEGFR was analyzed with ELISA in a panel of 10 HNSCC cell lines.RESULTS: A close correlation was found between in vitro and in vivo cetuximab sensitivity data in the two investigated HNSCC cell lines. In treatment sensitive UT-SCC-14 xenografts, there was a decrease in EGFR, pEGFR, and pSrc upon cetuximab treatment. Interestingly, in insensitive UT-SCC-45 xenografts, an increased expression of these three proteins was found. The change in EGFR and pEGFR expression in vivo was confirmed in cetuximab-sensitive and cetuximab-insensitive HNSCC cell lines using ELISA.CONCLUSION: High sensitivity to cetuximab was strongly associated with a treatment-induced reduction in pEGFR both in vivo and in vitro in a panel of HNSCC cell lines, suggesting that EGFR and pEGFR dynamics could be used as a predictive biomarker for cetuximab treatment response.
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| 8. |
- Matić, Nataša, 1977-
(författare)
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Biomarkers in the tumor microenvironment with impact on treatment response and survival in head and neck cancer
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Head and neck squamous cell cancer (HNSCC) is the sixth most common cancer type worldwide and disease detects at a locally advanced stage in approximately 60% of all patients with HNSCC. Despite the advances in both diagnosis and treatment of cancer, therapy failure with local recurrence and second primary tumors as consequences is still a huge problem. Furthermore, HNSCC is heterogeneous disease and treatment response differs largely within this patient’s group. Owing to such heterogeneity, the individually designed treatment approach, based on biomarkers predicting treatment response is needed. The personalized treatment strategy is a new and promising form of cancer therapy to improve effectiveness of anti-cancer treatment and reduce suffering in patients diagnosed with HNSCC.The main aim of this thesis was to search for new predictive biomarkers with potential to foresee treatment response in HNSCC patients by investigating components and properties of the tumor microenvironment, such as metabolism, hypoxia and cancer-associated fibroblasts (CAF).In paper I we developed an in vitro method for the measurement of intracellular glucose (18F FDG) uptake with gamma spectrometry (GS). Quantity of glucose uptake was associated with treatment (radiation or cetuximab) response, where the radiosensitive cell line and the most cetuximab-sensitive cell lines showed a significant decrease of glucose uptake after treatment. The results were compared with those of a clinical PET/CT scanner and the results in glucose uptake between radiated cells and controls were similar in both methods. Furthermore, we investigated GLUT1 mRNA expression in cell lines after cetuximab treatment and our analysis showed a significant increase of GLUT1 mRNA expression.In paper II, we found a negative impact of hypoxia on radiotherapy response in HNSCC cells and enhanced expression of epithelial–mesenchymal transition (EMT)- and cancer stem cells (CSC)-associated genes during culturing cells in hypoxic conditions. With cDNA microarrays analysis we identified a number of hypoxia regulated genes that were involved in multiple biological functions as well as support growth and proliferation of cancer cells. Furthermore, with use of siRNA silencing, we investigated a possible impact of a panel of hypoxia-responsive genes (HIF-1α, CDH2, CA9, SERPINE1, AREG and EREG) on radiotherapy treatment. Nevertheless, downregulation of these hypoxia regulated genes did not affect the sensitivity to radiotherapy of the investigated HNSCC cell lines.In the following study (paper III) we continued to investigate the most hypoxia-dysregulated genes from previous study (CA9, CASP14, LOX, GLUT3, SERPINE1, AREG, EREG, CCNB1 and KIF14), and their impact on the survival of HNSCC patients treated with radiotherapy. Patients with high KIF14 mRNA expression showed significantly longer overall survival (OS) compared with patients with low KIF14 mRNA expression. Moreover, HNSCC patients with high mRNA expression of KIF14 and low mRNA expression of CA9 (hypoxia marker) showed better OS compared with those with the opposite mRNA expression.In paper IV, we investigated the influence of cancer-associated fibroblasts (CAFs) on tumor cell gene expression profile. Cells were cultured in 2D and 3D models where HNSCC cells and CAFs derived from the same tumor were co-cultured. The microarray analysis revealed a higher number of CAF-regulated genes in tumor cells grown in spheroids compared to tumor cells grown in 2D. Next, the expression pattern of most changed CAF-regulated genes (MMP1, MMP9, POSTN, GREM1, FMOD, COL1A2, GREM1, IVL) was analyzed in normal oral tissue and in pretreatment biopsies from HNSCC patients treated with radiotherapy showing differences in gene expression between HNSCC tumor tissue and normal oral tissue. High mRNA expression of MMP9 and FMOD were found to be significantly associated with better overall survival (OS) in patients treated with radiotherapy.Taken together, we developed a reliable in vitro method for the measurement of intracellular glucose uptake with gamma spectrometry and glucose uptake was associated with treatment response. Furthermore, we found that hypoxia has a negative impact on radiotherapy in HNSCC cells and we identified a panel of hypoxia-dysregulated genes involved in the multiple biological functions in cancer cells, however downregulation of single hypoxia-regulated genes did not affect response to radiotherapy. Further analysis indicated KIF14 mRNA as potential predictor of radiotherapy response but more studies with a larger patient cohort are required. Beside hypoxia, CAFs have an important role in cancer progression. The gene expression profile in HNSCC cell lines was found to be dysregulated by CAF-derived signals in vitro. Additionally, CAF-regulated genes, MMP9 and FMOD are potential candidates as biomarkers of OS in HNSCC patients treated with radiotherapy, however more studies must be undertaken to investigate our hypothesis.
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| 9. |
- Melissaridou, Styliani, et al.
(författare)
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The effect of 2D and 3D cell cultures on treatment response, EMT profile and stem cell features in head and neck cancer 11 Medical and Health Sciences 1112 Oncology and Carcinogenesis
- 2019
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Ingår i: Cancer Cell International. - : Springer Science and Business Media LLC. - 1475-2867. ; 19:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Head and Neck Squamous Cell Carcinoma (HNSCC) tumors are often resistant to therapies. Therefore searching for predictive markers and new targets for treatment in clinically relevant in vitro tumor models is essential. Five HNSCC-derived cell lines were used to assess the effect of 3D culturing compared to 2D monolayers in terms of cell proliferation, response to anti-cancer therapy as well as expression of EMT and CSC genes. Methods: The viability and proliferation capacity of HNSCC cells as well as induction of apoptosis in tumor spheroids cells after treatment was assessed by MTT assay, crystal violet- and TUNEL assay respectively. Expression of EMT and CSC markers was analyzed on mRNA (RT-qPCR) and protein (Western blot) level. Results: We showed that HNSCC cells from different tumors formed spheroids that differed in size and density in regard to EMT-associated protein expression and culturing time. In all spheroids, an up regulation of CDH1, NANOG and SOX2 was observed in comparison to 2D but changes in the expression of EGFR and EMT markers varied among the cell lines. Moreover, most HNSCC cells grown in 3D showed decreased sensitivity to cisplatin and cetuximab (anti-EGFR) treatment. Conclusions: Taken together, our study points at notable differences between these two cellular systems in terms of EMT-associated gene expression profile and drug response. As the 3D cell cultures imitate the in vivo behaviour of neoplastic cells within the tumor, our study suggest that 3D culture model is superior to 2D monolayers in the search for new therapeutic targets.
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| 10. |
- Perskvist, Nasrin, 1952-, et al.
(författare)
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Rab5a GTPase regulates fusion between pathogen-containing phagosomes and cytoplasmic organelles in human neutrophils
- 2002
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Ingår i: Journal of Cell Science. - 0021-9533 .- 1477-9137. ; 115:6, s. 1321-1330
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Tidskriftsartikel (refereegranskat)abstract
- Biogenesis of phagolysosomes proceeds through a sequential series of interactions with endocytic organelles, a process known to be regulated by Rab and SNARE proteins. The molecular mechanisms underlying phagosome maturation in neutrophils are, however, not clearly understood. We investigated fusion between phagosomes containing the intracellular pathogen Mycobacterium tuberculosis versus the extracellular pathogen Staphylococcus aureus (designated MCP for mycobacteria-containing phagosome and SCP for S. aureus-containing phagosome) and cytoplasmic compartments in human neutrophils. Western blot analysis of phagosomes isolated after internalisation revealed that lactoferrin (a constituent of secondary granules) and LAMP-1 were incorporated into both SCP and MCP, whereas hck (marker of azurophil granules) interacted solely with SCP. The subcellular distribution of the proteins Rab5a and syntaxin-4 suggested a role in docking of granules and/or endosomes to the target membrane in the neutrophil. We observed that during phagocytosis, Rab5a in GTP-bound form interacted with syntaxin-4 on the membrane of MCP and were retained for up to 90 minutes, whereas the complex was recruited to the SCP within 5 minutes but was selectively depleted from these vacuoles after 30 minutes of phagocytosis. Downregulation of Rab5a by antisense oligonucleotides efficiently reduced the synthesis of Rab5a, the binding of syntaxin-4 to MCP and SCP and the capacity for fusion exhibited by the pathogen-containing phagosomes, but it had no effect on bacteria internalisation. These data indicate that the difference in granule fusion is correlated with a difference in the association of Rab5a and syntaxin-4 with the phagosomes. Intracellular pathogen-containing phagosomes retain Rab5a and syntaxin-4, whereas extracellular pathogen-containing phagosomes bind briefly to this complex. These results also identified Rab5a as a key regulator of phagolysosome maturation in human neutrophils.
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