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- Aftab, Obaid, 1984-, et al.
(författare)
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Label-free detection and dynamic monitoring of drug-induced intracellular vesicle formation enabled using a 2-dimensional matched filter
- 2014
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8627 .- 1554-8635. ; 10:1, s. 57-69
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Tidskriftsartikel (refereegranskat)abstract
- Analysis of vesicle formation and degradation is a central issue in autophagy research and microscopy imaging is revolutionizing the study of such dynamic events inside living cells. A limiting factor is the need for labeling techniques that are labor intensive, expensive, and not always completely reliable. To enable label-free analyses we introduced a generic computational algorithm, the label-free vesicle detector (LFVD), which relies on a matched filter designed to identify circular vesicles within cells using only phase-contrast microscopy images. First, the usefulness of the LFVD is illustrated by presenting successful detections of autophagy modulating drugs found by analyzing the human colorectal carcinoma cell line HCT116 exposed to each substance among 1266 pharmacologically active compounds. Some top hits were characterized with respect to their activity as autophagy modulators using independent in vitro labeling of acidic organelles, detection of LC3-II protein, and analysis of the autophagic flux. Selected detection results for 2 additional cell lines (DLD1 and RKO) demonstrate the generality of the method. In a second experiment, label-free monitoring of dose-dependent vesicle formation kinetics is demonstrated by recorded detection of vesicles over time at different drug concentrations. In conclusion, label-free detection and dynamic monitoring of vesicle formation during autophagy is enabled using the LFVD approach introduced.
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| 2. |
- Hernlund, Emma, et al.
(författare)
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The phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 is effective in inhibiting regrowth of tumour cells after cytotoxic therapy
- 2012
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Ingår i: European Journal of Cancer. - : Elsevier BV. - 0959-8049 .- 1879-0852. ; 48:3, s. 396-406
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Tidskriftsartikel (refereegranskat)abstract
- PURPOSE:Regrowth of tumour cells between cycles of chemotherapy is a significant clinical problem. Treatment strategies where antiproliferative agents are used to inhibit tumour regrowth between chemotherapy cycles are attractive, but such strategies are difficult to test using conventional monolayer culture systems.METHODS:We used the in vitro tumour spheroid model to study regrowth of 3-D colon carcinoma tissue after cytotoxic therapy. Colon carcinoma cells with wild-type or mutant phosphatidyl inositol 3-kinase catalytic subunit (PI3KCA) or KRAS alleles were allowed to form multicellular spheroids and the effects of different pharmacological compounds were studied after sectioning and staining for relevant markers of cell proliferation and apoptosis.RESULTS:Studies using colon cancer cells with gene disruptions suggested that the phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathway was essential for proliferation in 3-D culture. The dual PI3K-mTOR inhibitor NVP-BEZ235, currently in clinical trials, was found to inhibit phosphorylation of the mTOR target 4EBP1 in 3-D cultured cells. The ability of NVP-BEZ235 to inhibit tumour cell proliferation and to induce apoptosis was markedly more pronounced in 3-D cultures compared to monolayer cultures. It was subsequently found that NVP-BEZ235 was effective in inhibiting regrowth of 3-D cultured cells after treatment with two cytotoxic inhibitors of the ubiquitin-proteasome system (UPS), methyl-13-hydroxy-15-oxokaurenoate (MHOK) and bortezomib (Velcade®).CONCLUSIONS:The dual PI3K-mTOR inhibitor NVP-BEZ235 was found to reduce cell proliferation and to induce apoptosis in 3-D cultured colon carcinoma cells, NVP-BEZ235 is a promising candidate for use in sequential treatment modalities together with cytotoxic drugs to reduce the cell mass of solid tumours.
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| 3. |
- Kolosenko, Iryna, et al.
(författare)
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Cell crowding induces interferon regulatory factor 9, which confers resistance to chemotherapeutic drugs
- 2015
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Ingår i: International Journal of Cancer. - : Wiley. - 0020-7136 .- 1097-0215. ; 136:4, s. E51-E61
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Tidskriftsartikel (refereegranskat)abstract
- The mechanism of multicellular drug resistance, defined as the reduced efficacy of chemotherapeutic drugs in solid tumors is incompletely understood. Here we report that colon carcinoma cells cultured as 3D microtissues (spheroids) display dramatic increases in the expression of a subset of type I interferon-(IFN)-stimulated genes (ISGs). A similar gene signature was associated previously with resistance to radiation and chemotherapy, prompting us to examine the underlying biological mechanisms. Analysis of spheroids formed by different tumor cell lines and studies using knock-down of gene expression showed that cell crowding leads to the induction of IFN regulatory factor-9 (IRF9) which together with STAT2 and independently of IFNs, is necessary for ISG upregulation. Increased expression of IRF9 alone was sufficient to induce the ISG subset in monolayer cells and to confer increased resistance to clinically used cytotoxic drugs. Our data reveal a novel mechanism of regulation of a subset of ISGs, leading to drug resistance in solid tumors. What's new? Drug resistance remains a major challenge in the management of cancer patients. Using a 3D model of tumor cells the authors identify cell crowding and the interferon response as important mediators of drug resistance. They demonstrate that interferon regulatory factor 9 (IRF9) and a panel of interferon-stimulated genes are induced by cell crowding in this model. These results link unexpected new molecular mechanisms with the therapy resistance of solid tumors.
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| 6. |
- Zhang, Xiaonan, et al.
(författare)
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Induction of mitochondrial dysfunction as a strategy for targeting tumour cells in metabolically compromised microenvironments
- 2014
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 5, s. 3295-
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Tidskriftsartikel (refereegranskat)abstract
- Abnormal vascularization of solid tumours results in the development of microenvironments deprived of oxygen and nutrients that harbour slowly growing and metabolically stressed cells. Such cells display enhanced resistance to standard chemotherapeutic agents and repopulate tumours after therapy. Here we identify the small molecule VLX600 as a drug that is preferentially active against quiescent cells in colon cancer 3-D microtissues. The anticancer activity is associated with reduced mitochondrial respiration, leading to bioenergetic catastrophe and tumour cell death. VLX600 shows enhanced cytotoxic activity under conditions of nutrient starvation. Importantly, VLX600 displays tumour growth inhibition in vivo. Our findings suggest that tumour cells in metabolically compromised microenvironments have a limited ability to respond to decreased mitochondrial function, and suggest a strategy for targeting the quiescent populations of tumour cells for improved cancer treatment.
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