| 1. |
- Holst, Martina, et al.
(författare)
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Comparison of three cytotoxicity tests in the evaluation of the cytotoxicity of a spermine analogue on human breast cancer cell lines
- 2005
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Ingår i: Toxicology in Vitro. - : Elsevier BV. - 1879-3177 .- 0887-2333. ; 19:3, s. 379-387
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Tidskriftsartikel (refereegranskat)abstract
- Using three cytotoxicity assays, we have investigated the effect of the spermine analogue N1,N11-diethylnorspermine (DENSPM)on four human breast cancer cell lines with different known genetic lesions. Cells were seeded in 96 well plates and DENSPM was added 24 h later to give final concentrations from 0.1 to 100 microM. At 24, 48 and 72 h of treatment, the protein content was determined with a modified Lowry assay. Mitochondrial activity was determined with the AlamarBlue and MTT assays. These two assays differ with respect to where in the electron transport chain the reduction of the substrate takes place. Treatment with increasing concentrations of DENSPM resulted in differential responses in the four cell lines. There was a good of agreement between the protein content and the MTT assay showing increased negative effect with increased dose of DENSPM. The AlamarBlue assay on the other hand showed a stimulation of substrate reduction compared to control at DENSPM concentrations that were inhibitory according to the protein content and MTT assay. Thus, the data clearly show that the MTT and AlamarBlue assays are not equivalent. Importantly, the AlamarBlue assay presumably also reflects cytoplasmic reduction of the substrate through DENSPM-induced mechanisms.
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| 2. |
- Holst, Martina, et al.
(författare)
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Differential polyamine analogue effects in four human breast cancer cell lines
- 2006
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Ingår i: Toxicology. - : Elsevier BV. - 0300-483X. ; 223:1-2, s. 71-81
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Tidskriftsartikel (refereegranskat)abstract
- Polyamine analogues have demonstrated anti-tumour activity in a number of solid tumour models. In the present study we compared the cytotoxicities of three polyamine analogues against four breast cancer cell lines. All cell lines are derived from tumours of women with breast cancer and, although we are sampling just a small number of tumours, they represent a spectrum of the genetic plethora of breast cancers. Cytotoxicity, over a dose range from 0.1 to 100 microM, was evaluated with three different cytotoxicity assays performed in 96-well plates. Comparing the effects of the analogues on polyamine pools with data from the cytotoxicity assays indicates that there was not a direct correlation between polyamine pool depletion and cytotoxicity. Flow cytometry was used to investigate analogue-induced cell death as measured by the appearance of a sub-G1 peak. Induction of cell death by the analogues differed in the cell lines, however, cell death when induced was apoptotic, as demonstrated by detection of apoptotic bodies with immunofluorescence microscopy of propidium iodide-stained nuclei. Comparing the flow cytometry-derived data and the data from the cytotoxicity assays reveals that the analogues exert their effects by inhibiting cell growth and/or inducing cell death.
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| 3. |
- Holst, Martina, et al.
(författare)
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Molecular mechanisms underlying N1, N11-diethylnorspermine-induced apoptosis in a human breast cancer cell line.
- 2008
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Ingår i: Anti-Cancer Drugs. - 0959-4973. ; 19:9, s. 871-883
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Tidskriftsartikel (refereegranskat)abstract
- Polyamine analogue treatment results in growth inhibition and sometimes in cell death. Therefore, polyamine analogues are considered in the treatment of cancer; however, the cellular properties that govern sensitivity are not known. The objective of this study was to elucidate molecular mechanisms behind apoptosis induced by the polyamine analogue N, N-diethylnorspermine (DENSPM). Four different breast cancer cell lines were treated with DENSPM. Cell death was evaluated with flow cytometry and a caspase 3 assay. The levels of a number of proapoptotic and antiapoptotic proteins in subcellular compartments were evaluated with western blot. In the most sensitive cell line, DENSPM treatment induced the release of cytochrome c from mitochondria, resulting in activation of caspase 3 but without decreasing the mitochondrial transmembrane potential. However, in the three other cell lines DENSPM treatment did not induce extensive cell death. This is partly explained by the high levels of antiapoptotic proteins Bcl-2 and Bad and low levels of proapoptotic proteins Bax and procaspase 3 in these three cell lines. The results are also partly explained by the degree of activation of the catabolic enzyme spermidine/spermine-N-acetyltransferase and polyamine pool reduction achieved by DENSPM treatment. Our results show that the protein profile of proapoptotic and antiapoptotic proteins may contribute to the outcome to treatment with the polyamine analogue DENSPM. The results also indicate that it should be possible to find molecular markers for sensitivity to DENSPM that could be used in the clinic to predict sensitivity to a polyamine analogue.
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| 4. |
- Holst, Martina, et al.
(författare)
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Novel anti-apoptotic effect of Bcl-2: Prevention of polyamine depletion-induced cell death
- 2008
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Ingår i: Cell Biology International. - : Wiley. - 1095-8355 .- 1065-6995. ; 32, s. 66-74
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Tidskriftsartikel (refereegranskat)abstract
- The spermine analogue N(1),N(11)-diethylnorspermine (DENSPM) efficiently depletes the polyamine pools in the breast cancer cell line L56Br-C1 and induces apoptotic cell death via the mitochondrial pathway. In this study, we have over-expressed the anti-apoptotic protein Bcl-2 in L56Br-C1 cells and investigated the effect of DENSPM treatment. DENSPM-induced cell death was significantly reduced in Bcl-2 over-expressing cells. Bcl-2 over-expression reduced DENSPM-induced release of the pro-apoptotic proteins AIF, cytochrome c, and Smac/DIABLO from the mitochondria. Bcl-2 over-expression reduced the DENSPM-induced activation of caspase-3. Bcl-2 over-expression also prevented DENSPM-induced Bax cleavage and reduction of Bcl-X(L) and survivin levels. The DENSPM-induced activation of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase was reduced by Bcl-2 over-expression, partly preventing polyamine depletion. Thus, Bcl-2 over-expression prevented a number of DENSPM-induced apoptotic effects
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| 5. |
- Holst, Martina
(författare)
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Regulation of Polyamine Analogue-Induced Apoptosis in Human Breast Cancer Cells
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Apoptosis is a defined process for cells to commit suicide through a set of biochemical and morphological cell changes. Activated apoptotic signals converge on the mitochondria, leading to permeabilization of mitochondrial membranes and release of different apoptosis regulatory proteins into the cytosol. With my studies I wanted to investigate the importance of polyamines in the regulation of apoptosis. The homeostasis of polyamines is highly regulated since polyamines are essential for cell proliferation and cell death. A common feature of polyamine analogue treatment is inhibition of cell proliferation, but sometimes cells also die through apoptosis. When several different breast cancer cell lines were treated with polyamine analogues, mitochondrial activity increased indicating a cellular stress response. The stress response may be caused by polyamine analogue-induced polyamine depletion and/or by the presence of high concentration of a polyamine analogue that does not function as a natural polyamine. Polyamine analogue treatment of L56Br-C1 breast cancer cells resulted in a number of responses culminating in mitochondrial apoptotic cell death, as confirmed by the release of a number of apoptosis-related proteins from the mitochondria. Another response to analogue treatment was the activation of the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase (SSAT). Using several different methods we have shown that there is an association of SSAT with the mitochondria implicating that SSAT and polyamine depletion are part of the apoptotic process induced by polyamine analogue treatment. To investigate the role of the antiapoptotic protein Bcl-2 in polyamine analogue-induced apoptosis, a vector carrying the gene for Bcl-2 was introduced into the genome of L56Br-C1 cells. The degree of cell death, polyamine reduction, and SSAT activity decreased in Bcl-2 overexpressing cells compared to control cells. In conclusion, polyamine analogue treatment clearly affects the mitochondria and can induce apoptosis as a response, something which can be prevented by Bcl-2 overexpression.
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| 6. |
- Holst, Martina, et al.
(författare)
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Subcellular distribution of spermidine/spermine N(1)-acetyltransferase
- 2008
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Ingår i: Cell Biology International. - : Wiley. - 1095-8355 .- 1065-6995. ; 32:1, s. 39-47
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Tidskriftsartikel (refereegranskat)abstract
- The subcellular distribution of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) was studied in L56Br-C1 cells treated with 10muM N(1),N(11)-diethylnorspermine (DENSPM) for 24h. Cells were fractioned into three subcellular fractions. A particulate fraction containing the mitochondria was denoted as the mitochondrial fraction. After DENSPM treatment, an increase in SSAT activity was mainly found in the mitochondrial fraction. Western blot analysis showed an increased level of the SSAT protein in the mitochondrial fraction compared to the cytosolic fraction. Immunofluorescence microscopy and immunogold labeling transmission electron microscopy also showed a mitochondrial association of SSAT. Transmission electron microscopy revealed that the endoplasmic reticulum was devoid of ribosomes in DENSPM-treated cells, in contrast to control cells that contained ample ribosomes. An increased SSAT activity in connection with the mitochondria may be part of the mechanism of DENSPM-induced apoptosis.
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| 7. |
- Kvist Reimer, Martina, et al.
(författare)
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Long-term inhibition of dipeptidyl peptidase IV improves glucose tolerance and preserves islet function in mice.
- 2002
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Ingår i: European Journal of Endocrinology. - : Oxford University Press (OUP). - 1479-683X .- 0804-4643. ; 146:5, s. 717-727
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVES: Inhibitors of the glucagon-like peptide-1 (GLP-1)-degrading enzyme, dipeptidyl peptidase IV (DPPIV), are being explored in the treatment of diabetes. We examined the long-term influence of a selective, orally active inhibitor of DPPIV (NVP DPP728), in normal female C57BL/6J mice and such mice rendered glucose-intolerant and insulin-resistant by feeding a high-fat diet. DESIGN: In mice fed a standard diet (11% fat) or a high-fat diet (58% fat), NVP DPP728 (0.12 micromol/g body weight) was administered in the drinking water for an 8 week period. RESULTS: DPPIV inhibition reduced plasma DPPIV activity to 0.01+/-0.03 mU/ml vs 3.26+/-0.19 mU/ml in controls (P<0.001). Glucose tolerance after gastric glucose gavage, as judged by the area under the curve for plasma glucose levels over the 120 min study period, was increased after 8 weeks by NVP DPP728 in mice fed normal diet (P=0.029) and in mice fed a high-fat diet (P=0.036). This was accompanied by increased plasma levels of insulin and intact GLP-1. Glucose-stimulated insulin secretion from islets isolated from NVP DPP728-treated animals after 8 weeks of treatment was increased as compared with islets from control animals at 5.6, 8.3 and 11.1 mmol/l glucose both in mice fed normal diet and in mice fed a high-fat diet (both P<0.05). Islet insulin and glucagon immunocytochemistry revealed that NVP DPP728 did not affect the islet architecture. However, the expression of immunoreactive glucose transporter isoform-2 (GLUT-2) was increased by DPPIV inhibition, and in mice fed a high-fat diet, islet size was reduced after treatment with NVP DPP728 from 16.7+/-2.6 x 10(3) microm(2) in controls to 7.6+/-1.0 x 10(3) microm(2) (P=0.0019). CONCLUSION: Long-term DPPIV inhibition improves glucose tolerance in both normal and glucose-intolerant mice through improved islet function as judged by increased GLUT-2 expression, increased insulin secretion and protection from increased islet size in insulin resistance.
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| 8. |
- Löfdahl, Anna, et al.
(författare)
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5-HT2B receptor antagonists attenuate myofibroblast differentiation and subsequent fibrotic responses in vitro and in vivo
- 2016
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Ingår i: Physiological Reports. - : Wiley. - 2051-817X. ; 4:15
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Tidskriftsartikel (refereegranskat)abstract
- Pulmonary fibrosis is characterized by excessive accumulation of connective tissue, along with activated extracellular matrix (ECM)-producing cells, myofibroblasts. The pathological mechanisms are not well known, however serotonin (5-HT) and 5-HT class 2 (5-HT2) receptors have been associated with fibrosis. The aim of the present study was to investigate the role of 5-HT2B receptors in fibrosis, using small molecular 5-HT2B receptor antagonists EXT5 and EXT9, with slightly different receptor affinity. Myofibroblast differentiation [production of alpha-smooth muscle actin (α-SMA)] and ECM synthesis were quantified in vitro, and the effects of the receptor antagonists were evaluated. Pulmonary fibrosis was also modeled in mice by subcutaneous bleomycin administrations (under light isoflurane anesthesia), and the effects of receptor antagonists on tissue density, collagen-producing cells, myofibroblasts and decorin expression were investigated. In addition, cytokine expression was analyzed in serum. Lung fibroblasts displayed an increased α-SMA (P < 0.05) and total proteoglycan production (P < 0.01) when cultured with TGF-β1 together with 5-HT, which were significantly reduced with both receptor antagonists. Following treatment with EXT5 or EXT9, tissue density, expression of decorin, number of collagen-producing cells, and myofibroblasts were significantly decreased in vivo compared to bleomycin-treated mice. Receptor antagonization also significantly reduced systemic levels of TNF-α and IL-1β, indicating a role in systemic inflammation. In conclusion, 5-HT2B receptor antagonists have potential to prevent myofibroblast differentiation, in vitro and in vivo, with subsequent effect on matrix deposition. The attenuating effects of 5-HT2B receptor antagonists on fibrotic tissue remodeling suggest these receptors as novel targets for the treatment of pulmonary fibrosis.
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| 9. |
- Oredsson, Stina, et al.
(författare)
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Inhibition of cell proliferation and induction of apoptosis by N(1),N(11)-diethylnorspermine-induced polyamine pool reduction
- 2007
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Ingår i: Biochemical Society Transactions. - 0300-5127. ; 35:2, s. 405-409
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Tidskriftsartikel (refereegranskat)abstract
- Reduction of cellular polyamine pools results in inhibition of cell proliferation and sometimes in induction of cell death. Reduction of cellular polyamine pools can be achieved by several strategies involving all the mechanisms of polyamine homoeostasis, i.e. biosynthesis, catabolism and transport across the cell membrane. In the present paper, we concentrate on results achieved using the polyamine analogue DENSPM (N(1),N(11)-diethylnorspermine) on different cell lines. We discuss polyamine levels in DENSPM-treated cells in relation to effects on cell cycle kinetics and induction of apoptosis. To really understand the role of polyamines in cell cycle regulation and apoptosis, we believe it is now time to go through the vast polyamine literature in a meta-analysis-based manner. This short review does not claim to be such a study, but it is our hope to stimulate such studies in the polyamine field. Such work is especially important from the viewpoint of introducing drugs that affect polyamine homoeostasis in the treatment of various diseases such as cancer.
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| 10. |
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