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Experimental studies on ErbB targeted therapy in malignant melanoma
Linköping University, Department of Clinical and Experimental Medicine, Oncology. Linköping University, Faculty of Health Sciences.
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Malignant melanoma has one of the fastest increasing incidences among the different types of cancer in the Western world. This raise can partly be ascribed to the change in sun habits that has taken place during the last decades, since the major external risk factor for melanoma is exposure to ultraviolet radiation. In the case of patients with early stages of melanoma, the prognosis is usually good and the disease may be cured by surgery alone. However, with conventional anti-cancer treatments, patients diagnosed with unresectable or metastatic melanoma have a very low 5-year survival rate ranging from less than 10 percent to about 20 percent, depending on the location and extent of metastatic spread. Despite the development of novel promising targeted drugs, such as the immunomodulating antibody ipilimumab and the B-raf inhibitor vemurafenib, that have been shown to significantly extend patient survival, there is still an urgent need for new and improved treatment strategies which can further increase the survival of patients with advanced malignant melanoma.

The aim of this thesis was to investigate the anti-tumor effect of two different tyrosine kinase inhibitors (TKIs), gefitinib and canertinib, on human malignant melanoma cell lines with wild-type BRAF and NRAS. We investigated the effect of these two drugs on cell proliferation, survival and on the ErbB1-4 receptor phosphorylation, as well as the downstream signaling molecules Akt, Erk1/2 and Stat3. We also established a melanoma cell line resistant to gefitinib treatment and studied the resistance mechanisms developed by the cells.

The aim of this thesis was to investigate the anti-tumor effect of two different tyrosine kinase inhibitors (TKIs), gefitinib and canertinib, on human malignant melanoma cell lines with wild-type BRAF and NRAS. We investigated the effect of these two drugs on cell proliferation, survival and on the ErbB1-4 receptor phosphorylation, as well as the downstream signaling molecules Akt, Erk1/2 and Stat3. We also established a melanoma cell line resistant to gefitinib treatment and studied the resistance mechanisms developed by the cells.

In conclusion, gefitinib and canertinib display promising anti-tumor effects on ErbB-expressing malignant melanoma and might be used in future studies in combination with conventional chemotherapy or other targeted therapies in the treatment of malignant melanoma patients not harboring BRAF or NRAS mutations.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2012. , 104 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1336
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-85665ISBN: 978-91-7519-775-3 (print)OAI: oai:DiVA.org:liu-85665DiVA: diva2:572437
Public defence
2012-12-13, Eken, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 13:00 (Swedish)
Opponent
Supervisors
Available from: 2012-11-27 Created: 2012-11-27 Last updated: 2013-03-11Bibliographically approved
List of papers
1. ErbB receptor tyrosine kinases contribute to proliferation of malignant melanoma cells: inhibition by gefitinib (ZD1839)
Open this publication in new window or tab >>ErbB receptor tyrosine kinases contribute to proliferation of malignant melanoma cells: inhibition by gefitinib (ZD1839)
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2009 (English)In: Melanoma research, ISSN 0960-8931, E-ISSN 1473-5636, ISSN 0960-8931, Vol. 19, no 3, 156-166 p.Article in journal (Refereed) Published
Abstract [en]

Members of the epidermal growth factor (EGF) family of structurally related tyrosine kinase receptors, known as the ErbB receptors (EGFR/ErbB1/HER1, ErbB2/HER2/neu, ErbB3/HER3 and ErbB4/HER4) and their respective ligands, have been suggested to be involved in the development and progression of malignant melanoma. Here we investigate the effects of the ErbB1 tyrosine kinase inhibitor gefitinib (ZD1839, Iressa) on human malignant melanoma cells (RaH3 and RaH5) in vitro. ZD1839 inhibited proliferation of exponentially growing RaH3 and RaH5 cells in a dose-dependent manner with a half-maximally effective dose of 3.5 and 2.0 mu mol/l, respectively. Cell growth was inhibited at 0.1 mu mol/l ZD1839 in both cell lines. Maximal inhibition was accomplished at 10 mu mol/l ZD1839; however, the effect was not complete as both cell lines showed a continuous slow growth during the treatment period. Flow cytometry analysis of cell-cycle distribution showed that ZD1839 treatment caused accumulation of RaH3 and RaH5 cells in the G, phase. The growth arrest induced by ZD1839 coincided with upregulation of the cyclin-dependent kinase inhibitor p27(KIP1). There was no increase in apoptosis as determined by analysis of plasma phosphatidyl serine redistribution. Western blot analysis revealed that ZD1839 substantially reduced tyrosine phosphorylation of ErbB1 as well as ErbB2 and ErbB3. This was accompanied by a concomitant decrease in Akt-phosphorylation, Erk1/2-phosphorylation, and Stat3-phosphorylation. Our results show that ZD1839 interferes with the growth of human malignant melanoma cells by cytostatic effects. These findings indicate the possible use of ErbB receptor kinase inhibitors as a novel treatment strategy in malignant melanoma.

Keyword
antiproliferative, ErbB receptors, gefitinib, melanoma, tyrosine kinase inhibitor
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-19129 (URN)10.1097/CMR.0b013e32832c6339 (DOI)
Available from: 2009-06-12 Created: 2009-06-12 Last updated: 2017-12-13Bibliographically approved
2. The pan-ErbB receptor tyrosine kinase inhibitor canertinib promotes apoptosis of malignant melanoma in vitro and displays anti-tumor activity in vivo
Open this publication in new window or tab >>The pan-ErbB receptor tyrosine kinase inhibitor canertinib promotes apoptosis of malignant melanoma in vitro and displays anti-tumor activity in vivo
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2011 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 414, no 3, 563-568 p.Article in journal (Refereed) Published
Abstract [en]

The ErbB receptor family has been suggested to constitute a therapeutic target for tumor-specific treatment of malignant melanoma. Here we investigate the effect of the pan-ErbB tyrosine kinase inhibitor canertinib on cell growth and survival in human melanoma cells in vitro and in vivo. Canertinib significantly inhibited growth of cultured melanoma cells, RaH3 and RaH5, in a dose-dependent manner as determined by cell counting. Half-maximum growth inhibitory dose (IC(50)) was approximately 0.8 mu M and by 5 mu M both cell lines were completely growth-arrested within 72 h of treatment. Incubation of exponentially growing RaH3 and RaH5 with 1 mu M canertinib accumulated the cells in the G(1)-phase of the cell cycle within 24 h of treatment without induction of apoptosis as determined by flow cytometry. Immunoblot analysis showed that 1 mu M canertinib inhibited ErbB1-3 receptor phosphorylation with a concomitant decrease of Akt-, Erk1/2- and Stat3 activity in both cell lines. In contrast to the cytostatic effect observed at doses less than= 5 mu M canertinib, higher concentrations induced apoptosis as demonstrated by the Annexin V method and Western blot analysis of PARP cleavage. Furthermore, canertinib significantly inhibited growth of RaH3 and RaH5 melanoma xenografts in nude mice. Pharmacological targeting of the ErbB receptors may prove successful in the treatment of patients with metastatic melanoma.

Place, publisher, year, edition, pages
Elsevier, 2011
Keyword
Malignant melanoma; Tyrosine kinase inhibitor; Canertinib; ErbB-receptor; Apoptosis
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-73740 (URN)10.1016/j.bbrc.2011.09.118 (DOI)000298519500021 ()
Note

On the day of the defence date the status of this article was Manuscript and the title "The pan-ErbB receptor tyrosine kinase inhibitor Canertinib (CI-1033)promotes cell cycle arrest and apoptosis of human malignantmelanoma in vitro".

Available from: 2012-01-12 Created: 2012-01-12 Last updated: 2017-12-08
3. Resistance to gefitinib in malignant melanoma cells is related to increased expression of Met and the insulin receptor and sustained Akt signaling
Open this publication in new window or tab >>Resistance to gefitinib in malignant melanoma cells is related to increased expression of Met and the insulin receptor and sustained Akt signaling
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2012 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: Acquired resistance to cancer therapy, including targeted therapies such as epidermal growth factor receptor (ErbB) tyrosine kinase inhibitors (TKIs), constitutes a major clinical problem in treating patients with malignant disease. Several drug resistance mechanisms for ErbB1 TKIs involving abnormal activation of growth factor receptors or activation of intracellular signaling pathways have been discovered. ErbB TKIs have recently been shown to inhibit growth in melanoma cells. This study was undertaken to develop a gefitinib-resistant melanoma cell line in order to find any resistance mechanism to gefitinib in melanoma cells lacking activating mutation in BRAF or NRAS.

Material and methods: A malignant melanoma cell line (RaH5) was made resistant to the ErbB1 TKI gefitinib by continuous culture with stepwise increasing concentrations of the drug up to 10 μM. The phosphorylation status of 42 different human receptor tyrosine kinases was screened in a protein array in resistant (RaH5ZDR) and wild-type RaH5 cells treated with or without gefitinib. The PI3K, MAPK and Stat3 signaling pathways were studied in an analogous way by Western blot analysis; 2-D gel electrophoresis was performed to determine other potential proteins involved in gefitinib resistance in RaH5 cells. In addition, the effect of the pan-ErbB TKI canertinib on gefitinib-resistant cells was investigated.

Results: Protein array experiments showed that only Met and the insulin receptor (IR) exhibited substantially increased activation in RaH5ZDR cells as compared to their nonresistant counterparts. Interestingly, following gefitinib treatment ErbB2 and ErbB3 receptor signaling in resistant cells were equally well suppressed as in non-resistant cells. However, downstream Akt and Erk1/2 phosphorylation was inhibited to a greater extent in non-resistant RaH5 cells.

Conclusion: Resistance to gefitinib in RaH5 cells appears to be related to an increased expression of Met and IR and linked to a more persistent signaling through Akt and Erk1/2. However, additional studies are required to further elucidate the resistance to gefitinib in our experimental system.

Keyword
Malignant melanoma, gefitinib, canertinib, gefitinib resistance, ErbB, Erk1/2, Akt, Stat3, Met, IR
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-85664 (URN)
Available from: 2012-11-27 Created: 2012-11-27 Last updated: 2012-11-27Bibliographically approved

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Severinsson, Emelie

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