| 1. |
- Chatzakos, Vicky, 1977-, et al.
(författare)
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Bacillus Calmette-Guérin treatment specifically induces SK1 protein expression inurothelial bladder-cancer cells
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Bladder instillation with Bacillus Calmétte-Guerin (BCG) is an established treatmentmodality for superficial high-risk urinary bladder-cancer and carcinoma in situ (CIS), but theanti-tumor mechanisms following BCG-instillations remain largely unknown. In this study weexamined the effects of BCG-treatment on SK1 protein expression in the murine bladdercancer cell line MBT2. To simulate in vivo BCG-instillations, where the immune systemplays a vital role for successful BCG-treatment, we also stimulated MBT2 cells withsupernatant from BCG-treated (SupBCG) or un-treated Raw 264.7 macrophages. BCGtreatment as well as SupBCG treatment induced SK1 protein expression. Treatment with thesphingosine-kinase 1 (SK1) inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole (SK1-II) induced reduced viability in a dose dependent manner. However, combined treatment withBCG diminished this viability reduction suggesting a protective effect of BCG, but not ofSup-BCG, from SK1-II induced toxicity. Apoptosis was detected, as PARP-cleavage, in cellstreated with SupBCG whereas BCG-only or supernatant from untreated macrophages did notinduce apoptosis. A substantial increase in ceramides C18, C18:1 and C20:1 was observedafter BCG-treatment. These ceramide subspecies, as well as ceramides C14, C16, C20, C22and C22:1, were also induced by 20 or 30M of SK1-II, but the induction was abrogated byco-treatement with BCG. Following treatment with Sup-BCG, the levels of all the abovementioned ceramide subspecies were increased. Levels of ceramides C14, C16, C18, C18:1,C20, C20:1, C22 and C22:1 were more than two times higher in response to combinedtreatment with Sup-BCG and 10M of SK1-II as compared to treatment with Sup-BCG orSK1-II alone. Taken together these data suggest the sphingolipid metabolism as an importantpathway in the response to BCG-therapy.
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| 2. |
- Chatzakos, Vicky, 1977-, et al.
(författare)
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Inhibition of sphingosine kinase 1 enhances cytotoxicity, ceramide levels and ROS formation in liver cancer cells treated with selenite
- 2012
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Ingår i: Biochemical Pharmacology. - : Elsevier BV. - 0006-2952 .- 1356-1839 .- 1873-2968. ; 84:5, s. 712-721
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Tidskriftsartikel (refereegranskat)abstract
- High doses of selenite have been shown to induce cell death in acute myeloid leukemia and lung cancercells. In this study, we combined selenite treatment with modulators of sphingolipid metabolism in thehepatocellular carcinoma cell line Huh7. Treatment with 20 mM of selenite reduced the viability of Huh7cells by half and increased the levels of long chain C14-, C16-, C18- and C18:1- ceramides by two-fold.Inhibition of neutral sphingomyelinase with 3-O-methylsphingosine significantly reduced the cytotoxiceffect of selenite. In line with this result, selenite caused a 2.5-fold increase in the activity of neutralsphingomyelinase. The sphingosine kinase 1 (SK1) inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophe-nyl)thiazole (SK1-II) sensitized the cells to the cytotoxic effects of selenite. Preincubation with 10 mM ofSK1-II prior to treatment with 10 mM of selenite caused induction of apoptosis and gave rise to a 2.5-foldincrease in C14-, C16-, C18- and C18:1- ceramides. Instead, 50 mM of SK1-II combined with 10 mM ofselenite caused accumulation of cells in G1/S phases, but less apoptosis and accumulation of ceramides.The formation of reactive oxygen species (ROS) after treatment with 10 mM of selenite was maximallyenhanced by 1 mM of SK1-II. Moreover, combined treatment with SK1-II and 10 mM of selenitesynergistically reduced the number of viable Huh7 cells, while the non-tumorigenic hepatocyte cell lineMIHA remained unaffected by the same treatment. These results raise the possibility that a combinationof selenite and SK1 inhibitors could be used to treat liver cancer cells, that are regarded as drug resistant,at doses that are non-toxic to normal liver cells.
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| 3. |
- Chatzakos, Vicky, 1977-
(författare)
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Studies of bioactive lipids in cancer
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Lipids are a broad class of molecules that, besides being a major form of energy storage and components of cell membranes, act as bioactive signalling molecules. N-acyl taurines are structurally related to endocannabinoids that are known to exert antiproliferative actions in a variety of cancer cells. We have evaluated the cytotoxicity of N-oleoyl taurine and N-arachidonoyl taurine and found N-acyl taurines to reduce proliferation of prostate cancer cells.The sphingolipids ceramide and sphingosine act as tumour suppressors. We found selenite treatment to cause reduced viability, induction of neutral sphingomyelinase activity and accumulation of ceramide in the liver cancer cells. Inhibition of sphingosine kinase 1 (SK1) sensitized the cells to selenite treatment with regards to ceramide accumulation, arrest of cells in the G1/S phases of the cell cycle and formation of reactive oxygen species. Whereas combined selenite treatment and SK1 inhibition synergistically reduced the number of viable cells, the non-tumorigenic hepatocyte cell line, remained unaffected.Furthermore, we studied the involvement of sphingolipid metabolism in bladder cancer cells treated with Bacillus Calmette-Guérin (BCG), and found BCG to induce formation of nitric oxide and upregulation of nitric oxide synthase 2 as well as SK1 protein levels. Additionally, pharmacological inhibition of SK1 enhanced the viability reduction, ceramide accumulation and induction of apoptosis observed following BCG treatment.In conclusion, our findings have shown that N-acyl taurines exert antiproliferative effects on prostate cancer cells. Furthermore, sphingolipids were shown to be involved in cytotoxic treatment with selenite and BCG in liver cancer and bladder cancer cells respectively, and inhibition of SK1 increased the cytotoxicity. Our findings raise the possibility that modulation of ceramide-metabolizing enzymes could be used to enhance the effects of selenite and BCG in these cancer cell types.
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| 4. |
- Flygare, Jenny
(författare)
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Expression and regulation of Rad51 in human cells
- 2000
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Double strand breaks (DSBs) can be caused by exogenous DNA damaging agents or by endogenous processes, and may lead to chromosomal breakage and rearrangement resulting in apoptosis or tumorigenesis. The repair of DSBs in higher eukaryotes is to a large extent accomplished by homologous recombination (HR), a process in which DNA from a complementary strand is used as a template for repair synthesis. This thesis focuses on the expression and regulation of human Rad51 (HsRad51), the key component of a protein complex involved in HR. 1) The expression of the HsRad51 gene during the cell cycle was studied in mitogen stimulated primary human peripheral blood lymphocytes (PBLs). A peak in the HsRad51 mRNA and protein expression was observed in S/G2. Studies with inhibitors of DNA, RNA and protein synthesis indicated transcriptional regulation of the gene and coregulation of the expression with the S-phase. Treatment of PBLs with high doses of genotoxic agents resulted in decreased HsRad51 expression. The protein was shown to be cleaved, producing a 33 kD fragment, during drug- and receptor mediated apoptosis. The time-course of the cleavage coincided with inter-nucleosomal DNA fragmentation and with cleavage of human poly (ADP-ribose) polymerase. The absence of cleavage in MCF-7 cells showed that caspase-3 is essential for HsRad51 cleavage in vivo. Recombinant caspase-3 cleaved in vitro translated HsRad51 and endogenous HsRad51 in Jurkat cell extracts. The cleavage site was mapped to AQVD274¯G. This site is located in a region of the HsRad51 protein sequence that has been shown to be important for the interaction with Rad52. Abrogation of the interaction may lead to decreased activity of HsRad51. Inducible, stable transfectants were generated in order to study the effects of acute HsRad51 overexpression. Induction of HsRad51 protein expression resulted in a dose-dependent decrease in plating efficiency and growth rate. An accumulation of HsRad.51 overexpressing cells in the G2 phase of the cell cycle was observed following release from synchronization. The fraction of apoptotic cells increased with the time of overexpression. Using an array hybridization technique, increased mRNA levels of a set of genes involved in cell cycle regulation, survival and apoptosis was observed in the HsRad51 overexpressors. A selection of cells resistant to the observed negative effects of acute HsRad51 overexpression on end-points related to cell proliferation could conceivably be of importance for tumour progression. In summary, the results show that HsRad51 expression is coregulated with the cell cycle, confirming a role of the protein in HR between sister chromatids. The cleavage of HsRad51 by caspase-3 indicates that HsRad51 belongs to a group of repair proteins which are specifically cleaved during the execution phase of apoptosis. The observed detrimental effects of acute HsRad51 overexpression may be of importance in carcinogenesis.
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| 5. |
- Mattebo, Alexander, et al.
(författare)
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Yippee like 4 (Ypel4) is essential for normal mouse red blood cell membrane integrity
- 2021
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 11:1
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Tidskriftsartikel (refereegranskat)abstract
- The YPEL family genes are highly conserved across a diverse range of eukaryotic organisms and thus potentially involved in essential cellular processes. Ypel4, one of five YPEL family gene orthologs in mouse and human, is highly and specifically expressed in late terminal erythroid differentiation (TED). In this study, we investigated the role of Ypel4 in murine erythropoiesis, providing for the first time an in-depth description of a Ypel4-null phenotype in vivo. We demonstrated that the Ypel4-null mice displayed a secondary polycythemia with macro- and reticulocytosis. While lack of Ypel4 did not affect steady-state TED in the bone marrow or spleen, the anemia-recovering capacity of Ypel4-null cells was diminished. Furthermore, Ypel4-null red blood cells (RBC) were cleared from the circulation at an increased rate, demonstrating an intrinsic defect of RBCs. Scanning electron micrographs revealed an ovalocytic morphology of Ypel4-null RBCs and functional testing confirmed reduced deformability. Even though Band 3 protein levels were shown to be reduced in Ypel4-null RBC membranes, we could not find support for a physical interaction between YPEL4 and the Band 3 protein. In conclusion, our findings provide crucial insights into the role of Ypel4 in preserving normal red cell membrane integrity.
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| 6. |
- Stranneheim, Henrik, et al.
(författare)
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A comparison between protein profiles of B cell subpopulations and mantle cell lymphoma cells
- 2009
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Ingår i: Proteome Science. - : Springer Science and Business Media LLC. - 1477-5956. ; 7, s. 43-
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Tidskriftsartikel (refereegranskat)abstract
- Background: B-cell lymphomas are thought to reflect different stages of B-cell maturation. Based on cytogenetics and molecular markers, mantle cell lymphoma (MCL) is presumed to derive predominantly from naive, pre-germinal centre (pre-GC) B lymphocytes. The aim of this study was to develop a method to investigate the similarity between MCL cells and different B-cell compartments on a protein expression level. Methods: Subpopulations of B cells representing the germinal centre (GC), the pre-GC mantle zone and the post-GC marginal zone were isolated from tonsils using automated magnetic cell sorting (AutoMACS) of cells based on their expression of CD27 and IgD. Protein profiling of the B cell subsets, of cell lines representing different lymphomas and of primary MCL samples was performed using top-down proteomics profiling by surface-enhanced laser detection/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Results: Quantitative MS data of significant protein peaks (p-value < 0.05) separating the three B-cell subpopulations were generated. Together, hierarchical clustering and principal component analysis (PCA) showed that the primary MCL samples clustered together with the pre- and post-GC subpopulations. Both primary MCL cells and MCL cell lines were clearly separated from the B cells representing the GC compartment. Conclusion: AutoMACS sorting generates sufficient purity to enable a comparison between protein profiles of B cell subpopulations and malignant B lymphocytes applying SELDI-TOF-MS. Further validation with an increased number of patient samples and identification of differentially expressed proteins would enable a search for possible treatment targets that are expressed during the early development of MCL.
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| 7. |
- Thiel, Tomas, et al.
(författare)
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Secondary stimulation from Bacillus Calmette-Guerin induced macrophages induce nitric oxide independent cell-death in bladder cancer cells
- 2014
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Ingår i: Cancer Letters. - : Elsevier BV. - 0304-3835 .- 1872-7980. ; 348:1-2, s. 119-125
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Tidskriftsartikel (refereegranskat)abstract
- The anti-tumour mechanisms following Bacillus Calmette-Guerin (BCG) treatment of bladder-cancer remain largely unknown. Previous studies have shown involvement of nitric-oxide (NO) formation in the BCG-mediated effect. We analyzed the effects of macrophage secreted factors (MSFs) from BCG-stimulated RAW264.7 cells on the bladder-cancer cell line MBT2. Direct treatment with BCG did not induce NO in MBT2-cells whereas supernatant from BCG-stimulated macrophages increased NOS2 mRNA and protein expression, NO concentrations and cell-death. Blocking NO-synthesis with the NOS-inhibitor L-NAME did not affect levels of cell-death suggesting cytotoxic pathways involving other signalling molecules than NO. Several such candidate genes were identified in a microarray.
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| 8. |
- Thiel, Tomas, et al.
(författare)
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Secondary stimulation from Bacillus Calmette-Guérin induced macrophages upregulatesNOS2 protein in bladder cancer cells
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Treatment with Bacillus Calmette-Guérin (BCG) bladder instillations is an established treatment modality for superficial urinary bladder cancer and carcinoma in situ (CIS), but the anti-tumor mechanisms following BCG-instillations remain largely unknown. Previous data show increased nitric oxide (NO) concentrations in the urinary bladder from patients treated with BCG suggesting that NO-formation may be involved in the BCG-mediated effect. Using immunohistochemistry we have previously shown nitric oxide synthase 2 (NOS2/iNOS) protein expression in both immune cells and in urothelial cells in bladder cancer patient biopsies. In this study we analysed the influence of macrophage- (RAW 264.7) secreted factors on NO production by stimulating urothelial carcinoma cells (MBT2) with supernatant from BCG-treated macrophages as well as supernatant from untreated macrophages. Using real-time PCR, western blot and chemiluminescence, we found no effect of BCG when added straight to the culture medium of urothelial carcinoma cells. However, when 40% of the culture medium of the bladder cancer cells was substituted with supernatant from BCGstimulated macrophages, we found increased NOS2 mRNA and protein expression as well as increased levels of NO. In addition we found increased cell death only in bladder cancer cells stimulated with supernatant from BCG-treated macrophages, as visualized by cell cycle analysis and PARP cleavage. These results suggest that simultaneous targeting of the microenvironment and subsequent stimulation of adaptive responses can improve conventional BCG-therapy.
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| 9. |
- Vestin Fredriksson, Malin, et al.
(författare)
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Clinical Manifestations and Symptoms of Maxillary Sinusitis of Odontogenic Origin Demonstrated by Cone Beam Computed Tomography
- 2019
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Ingår i: Journal of General Practice. - : OMICS Publishing Group. - 2329-9126. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Objectives: To compare the symptomatology of patients with maxillary sinusitis of dental origin (MSDO) with sinusitis due to upper respiratory tract infection (URTI) with a special focus on time to correct diagnosis. To define the accuracy of cone beam computed tomography (CBCT) in detecting the dental origin of the sinusitis.Methods: Retrospective review of the otolaryngology specialist care, primary health care and dental health care medical records of patients with maxillary sinusitis who has been referred for radiology. All patients were examined by CBCT, which has a better resolution regarding bony structures than low-dose computed tomography. To the best of our knowledge there is no previous study on this topic based on CBCT as diagnostic method.Results: Sixty-one patients were included in the study; of these, 25 had MSDO and 36 had URTI sinusitis. The MSDO patients more frequently reported foul odour and foul taste than patients with URTI sinusitis. The URTI sinusitis patients more frequently reported symptoms such as facial pain, facial congestion and cold-related symptoms. Both the time from the onset of symptoms to the first medical visit and the subsequent time to a correct diagnosis were significantly longer in the MSDO group. The accuracy of CBCT for detecting dental pathology as the underlying cause of sinusitis was 97%.Conclusion: The present study verifies that maxillary sinusitis of dental origin differs from viral-induced rhinosinusitis concerning symptomatology and clinical findings. Certain findings and symptoms could serve as valuable indicators of an underlying dental pathology, because although MSDO is well known, the present study shows that these patients are often misdiagnosed and the correct diagnose and treatment is often delayed several months. Consequently, better assessment is important. The suspicion of MSDO should be raised for patients with unilateral sinusitis presenting little pain, foul odour or foul taste and a long time course. These patients should be referred for radiology, preferably CBCT, to rule out odontogenic cause. CBCT is easy to perform for sinusitis examinations and has advances to common CT, especially regarding detection of pathology in bony structures as the periapical area. Because of this CBCT is a reliable tool in order to detect maxillary sinusitis of dental origin.
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