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Induction of apoptosis by intracellular potassium ion depletion: using the fluorescent dye PBFI in a 96-well plate method in cultured lung cancer cells.
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry. Klinisk kemi.
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry. Klinisk kemi.
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry.
Umeå University, Faculty of Medicine, Department of Radiation Sciences, Oncology. Onkologi.
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2006 (English)In: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 20, no 6, 986-994 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2006. Vol. 20, no 6, 986-994 p.
Keyword [en]
Apoptosis, K+-depletion, mesothelioma, PBFI-AM, small-cell lung cancer
Identifiers
URN: urn:nbn:se:umu:diva-13588DOI: doi:10.1016/J.tiv.2005.12.013PubMedID: 16483738OAI: oai:DiVA.org:umu-13588DiVA: diva2:153259
Available from: 2008-01-11 Created: 2008-01-11 Last updated: 2011-08-31Bibliographically approved
In thesis
1. Manipulation of potassium ion fluxes to induce apoptosis in lung cancer cells
Open this publication in new window or tab >>Manipulation of potassium ion fluxes to induce apoptosis in lung cancer cells
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Apoptosis is a special form of cell death that if non-functional may lead to diseases such as cancer. A reduction of the intracellular potassium ion (K+) content is necessary for activating enzymes important for the execution of apoptosis. Pharmacological modulation of K+ fluxes to reduce intracellular K+ in cancer cells might therefore force the cells into apoptosis and decrease tumour cell mass.

Human malignant pleural mesothelioma (MPM) is a form of cancer often caused by asbestos exposure. Although asbestos has been banned in the Western World, the incidence of MPM is expected to increase. Cisplatin is the first-line chemotherapy for MPM, but acquired resistance to the drug is a clinical problem.

This thesis is mainly based on work with the human malignant pleural mesothelioma cell line (P31 wt) and a cisplatin-resistant sub-line (P31 res). The aim was to first characterize K+ fluxes in P31 wt and P31 res cells, and then manipulate them in order to reduce intracellular K+ and induce apoptosis with K+ manipulation alone or in combination with cisplatin.

Characterization of K+ fluxes in P31 wt cells showed that: 1) ouabain, a digitalis-like drug, and specific blocker of the Na+, K+, ATPase pump, effectively inhibited K+ uptake, 2) bumetanide, a diuretic, and an inhibitor of the Na+, K+, 2Cl-¬-cotransporter, had a transient effect on K+ uptake, and 3) the antifungal drug amphotericin B stimulated K+ efflux.

In order to determine intracellular K+ content, the potassium-binding fluorescent probe PBFI-AM was used in a 96-well plate assay. After a 3-h incubation with ouabain, with or without bumetanide, combined with amphotericin B, the intracellular K+ content was reduced in P31 wt cells but not in P31 res cells.

Ouabain induced apoptosis in both P31 wt and P31 res cells. P31 res cells were sensitized to cisplatin by ouabain, since 10 mg/L cisplatin in combination with ouabain induced about the same percentage of apoptotic cells as 40 mg/L cisplatin. Apoptosis was executed via caspase-3 activation in both P31 wt and P31 res cells. Amphotericin B enhanced ouabain-induced apoptosis in P31 wt cells via caspase-9 activation, with increased caspase-3 activation and DNA fragmentation as consequences. Ouabain-induced apoptosis in P31 res cells was executed via increased expression of pro-apoptotic Bak. The combination of cisplatin with ouabain and amphotericin B was stressful to both P31 wt and P31 res cells, since SAPK/JNK a known factor in stress-induced apoptosis was activated.

In conclusion, K+ flux manipulation with clinical used drugs can induce apoptosis per se and also enhance cisplatin-induced apoptosis in P31 wt and P31 res cells.

Place, publisher, year, edition, pages
Umeå: Medicinsk biovetenskap, 2007. 33 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1086
Keyword
Apoptosis, Cisplatin, Intracellular K+ content, Mesothelioma, Na+ K+ ATPase pump
National Category
Other Clinical Medicine
Identifiers
urn:nbn:se:umu:diva-1014 (URN)978-91-7264-264-5 (ISBN)
Public defence
2007-03-16, Betula, 6M, Umeå universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2007-02-16 Created: 2007-02-16 Last updated: 2009-05-13Bibliographically approved
2. Cisplatin-resistance and cell death in malignant pleural mesothelioma cells
Open this publication in new window or tab >>Cisplatin-resistance and cell death in malignant pleural mesothelioma cells
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Malignant pleural mesothelioma (MPM) is an aggressive, treatment-resistant tumour. Cisplatin (cis-diamminedichloroplatinum (II)) is the best single-agent chemotherapy for MPM, but platinum-based combination therapies give the best overall response rates. However, cisplatin use is limited by resistance and severe side effects. This thesis has increased the knowledge concerning cisplatin-induced cell death in MPM by describing a novel potential therapeutic target, and three novel phenotypes of cisplatin-resistance in a human MPM cell line (P31) and its cisplatin-resistant sub-line (P31res1.2).

The novel potential therapeutic target, and one of the novel phenotypes, was cisplatin-resistant pro-apoptotic BH3-only proteins. In the P31 cells, cisplatin transiently increased pro-apoptotic BH3-only proteins during 6 h of exposure. This response was almost completely abrogated in the P31res1.2 cells. De-regulated caspase activity and activation was the second novel phenotype identified. The P31res1.2 cells had earlier, possibly mitochondria-independent, caspase-3 activation, increased basal caspase-3 activity and increased basal cleavage of caspase-8 and -9. Despite these differences, 6-h equitoxic cisplatin exposures rendered 50-60% of the cells apoptotic in both cell lines. The third novel phenotype was abrogated Na+K+2Cl--cotransporter (NKCC1) activity. Although NKCC1 activity was dispensable for cisplatin-induced apoptosis, balanced potassium transport activity was essential for P31 cell survival. Finally, the survival signalling protein Protein Kinase B (PKB or Akt) isoforms α and γ were constitutively activated in a PI3K-independent manner in P31 cells. In the P31res1.2 cells, PKBα and γ activities were increased, and there was PI3K-dependent activation of PKBβ. However, this increase in PKB isoform activity was not strongly associated to the cisplatin-resistance of the P31res1.2 cells.

Place, publisher, year, edition, pages
Umeå: Medicinsk biovetenskap, 2008. 104 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1192
Keyword
apoptosis, BH3-only proteins, caspase, cell morphology, potassium transport, protein kinase B (PKB/Akt), signalling pathways, time
National Category
Other Clinical Medicine
Identifiers
urn:nbn:se:umu:diva-1680 (URN)978-91-7264-560-8 (ISBN)
Public defence
2008-08-29, E04, 6E, Norrlands Universitetssjukhus, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2008-05-28 Created: 2008-05-28 Last updated: 2010-01-18Bibliographically approved

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Andersson, BrittaJanson, VeronicaBehnam Motlagh, ParvizHenriksson, RogerGrankvist, Kjell

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