The siRNA-mediated downregulation of N-Ras sensitizes human melanoma cells to apoptosis induced by selective BRAF inhibitors
The clinical benefit of selective BRAF inhibitor therapies is limited by the emergence of drug resistance. Here, we investigated the molecular basis underlying the acquired resistance to a BRAF inhibitor by comparing the signaling pathways in the parental A375P cells and the resistant subline (A375P...
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| Veröffentlicht in: | Molecular and cellular biochemistry 2014-07, Vol.392 (1-2), p.239-247 |
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| creator | Ahn, Jun-Ho Lee, Michael |
| description | The clinical benefit of selective BRAF inhibitor therapies is limited by the emergence of drug resistance. Here, we investigated the molecular basis underlying the acquired resistance to a BRAF inhibitor by comparing the signaling pathways in the parental A375P cells and the resistant subline (A375P/Mdr). We demonstrate that MAPK re-activation does not contribute to the mechanism of resistance to UAI-201 of A375P/Mdr cells. The relative quantitative analysis using the 2
−ΔΔCt
method revealed that the BRAF inhibitor resistance observed in A375P/Mdr cells is not mediated through the overexpression of MDR proteins. In particular, we found that the expression of N-Ras was upregulated in BRAF inhibitor-resistant A375P/Mdr cells compared with A375P cells. In fact, siRNA-mediated N-Ras knockdown partially conferred UAI-201 sensitivity to A375P/Mdr cells, implying that N-Ras upregulation confers acquired resistance to BRAF inhibition. Notably, the flow cytometric analysis of the N-Ras-knockdown A375P/Mdr cells revealed that UAI-201 causes a significant accumulation of cells in the
G
0
/
G
1
phase with a concomitant decrease in the number of cells in the
S
and
G
2
/
M
phases. However, platelet-derived growth factor receptor
β
(PDGFR
β
) knockdown failed to sensitize A375P/Mdr cells to growth suppression by UAI-201, although a remarkable increase in PDGFRβ was observed in the A375P cells after UAI-201 treatment. Taken together, our results suggest that N-Ras is worth targeting to improve the therapeutic outcome of melanomas with acquired resistance to BRAF inhibitors. |
| doi_str_mv | 10.1007/s11010-014-2034-2 |
| format | Article |
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−ΔΔCt
method revealed that the BRAF inhibitor resistance observed in A375P/Mdr cells is not mediated through the overexpression of MDR proteins. In particular, we found that the expression of N-Ras was upregulated in BRAF inhibitor-resistant A375P/Mdr cells compared with A375P cells. In fact, siRNA-mediated N-Ras knockdown partially conferred UAI-201 sensitivity to A375P/Mdr cells, implying that N-Ras upregulation confers acquired resistance to BRAF inhibition. Notably, the flow cytometric analysis of the N-Ras-knockdown A375P/Mdr cells revealed that UAI-201 causes a significant accumulation of cells in the
G
0
/
G
1
phase with a concomitant decrease in the number of cells in the
S
and
G
2
/
M
phases. However, platelet-derived growth factor receptor
β
(PDGFR
β
) knockdown failed to sensitize A375P/Mdr cells to growth suppression by UAI-201, although a remarkable increase in PDGFRβ was observed in the A375P cells after UAI-201 treatment. Taken together, our results suggest that N-Ras is worth targeting to improve the therapeutic outcome of melanomas with acquired resistance to BRAF inhibitors.</description><identifier>ISSN: 0300-8177</identifier><identifier>EISSN: 1573-4919</identifier><identifier>DOI: 10.1007/s11010-014-2034-2</identifier><identifier>PMID: 24671490</identifier><language>eng</language><publisher>Boston: Springer US</publisher><subject>Analysis ; Apoptosis ; Apoptosis - genetics ; Base Sequence ; Biochemistry ; Biomedical and Life Sciences ; Cardiology ; Cell Line, Tumor ; Cellular biology ; DNA Primers ; Down-Regulation ; Drug resistance ; Gene Knockdown Techniques ; Genes, ras ; Humans ; Life Sciences ; Medical Biochemistry ; Melanoma ; Melanoma - genetics ; Melanoma - pathology ; Molecular biology ; Oncology ; Platelet-derived growth factor ; Proto-Oncogene Proteins B-raf - antagonists & inhibitors ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; RNA, Small Interfering - genetics</subject><ispartof>Molecular and cellular biochemistry, 2014-07, Vol.392 (1-2), p.239-247</ispartof><rights>Springer Science+Business Media New York 2014</rights><rights>COPYRIGHT 2014 Springer</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c542t-3b636bb4dfd65467c1d082ab9488b603787a033c968d62f133b3a72ab7eefeca3</citedby><cites>FETCH-LOGICAL-c542t-3b636bb4dfd65467c1d082ab9488b603787a033c968d62f133b3a72ab7eefeca3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://link.springer.com/content/pdf/10.1007/s11010-014-2034-2$$EPDF$$P50$$Gspringer$$H</linktopdf><linktohtml>$$Uhttps://link.springer.com/10.1007/s11010-014-2034-2$$EHTML$$P50$$Gspringer$$H</linktohtml><link.rule.ids>314,777,781,27905,27906,41469,42538,51300</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24671490$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Ahn, Jun-Ho</creatorcontrib><creatorcontrib>Lee, Michael</creatorcontrib><title>The siRNA-mediated downregulation of N-Ras sensitizes human melanoma cells to apoptosis induced by selective BRAF inhibitors</title><title>Molecular and cellular biochemistry</title><addtitle>Mol Cell Biochem</addtitle><addtitle>Mol Cell Biochem</addtitle><description>The clinical benefit of selective BRAF inhibitor therapies is limited by the emergence of drug resistance. Here, we investigated the molecular basis underlying the acquired resistance to a BRAF inhibitor by comparing the signaling pathways in the parental A375P cells and the resistant subline (A375P/Mdr). We demonstrate that MAPK re-activation does not contribute to the mechanism of resistance to UAI-201 of A375P/Mdr cells. The relative quantitative analysis using the 2
−ΔΔCt
method revealed that the BRAF inhibitor resistance observed in A375P/Mdr cells is not mediated through the overexpression of MDR proteins. In particular, we found that the expression of N-Ras was upregulated in BRAF inhibitor-resistant A375P/Mdr cells compared with A375P cells. In fact, siRNA-mediated N-Ras knockdown partially conferred UAI-201 sensitivity to A375P/Mdr cells, implying that N-Ras upregulation confers acquired resistance to BRAF inhibition. Notably, the flow cytometric analysis of the N-Ras-knockdown A375P/Mdr cells revealed that UAI-201 causes a significant accumulation of cells in the
G
0
/
G
1
phase with a concomitant decrease in the number of cells in the
S
and
G
2
/
M
phases. However, platelet-derived growth factor receptor
β
(PDGFR
β
) knockdown failed to sensitize A375P/Mdr cells to growth suppression by UAI-201, although a remarkable increase in PDGFRβ was observed in the A375P cells after UAI-201 treatment. Taken together, our results suggest that N-Ras is worth targeting to improve the therapeutic outcome of melanomas with acquired resistance to BRAF inhibitors.</description><subject>Analysis</subject><subject>Apoptosis</subject><subject>Apoptosis - genetics</subject><subject>Base Sequence</subject><subject>Biochemistry</subject><subject>Biomedical and Life Sciences</subject><subject>Cardiology</subject><subject>Cell Line, Tumor</subject><subject>Cellular biology</subject><subject>DNA Primers</subject><subject>Down-Regulation</subject><subject>Drug resistance</subject><subject>Gene Knockdown Techniques</subject><subject>Genes, ras</subject><subject>Humans</subject><subject>Life Sciences</subject><subject>Medical Biochemistry</subject><subject>Melanoma</subject><subject>Melanoma - genetics</subject><subject>Melanoma - pathology</subject><subject>Molecular biology</subject><subject>Oncology</subject><subject>Platelet-derived growth factor</subject><subject>Proto-Oncogene Proteins B-raf - antagonists & inhibitors</subject><subject>Real-Time Polymerase Chain Reaction</subject><subject>Reverse Transcriptase Polymerase Chain Reaction</subject><subject>RNA, Small Interfering - 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genetics</topic><topic>Base Sequence</topic><topic>Biochemistry</topic><topic>Biomedical and Life Sciences</topic><topic>Cardiology</topic><topic>Cell Line, Tumor</topic><topic>Cellular biology</topic><topic>DNA Primers</topic><topic>Down-Regulation</topic><topic>Drug resistance</topic><topic>Gene Knockdown Techniques</topic><topic>Genes, ras</topic><topic>Humans</topic><topic>Life Sciences</topic><topic>Medical Biochemistry</topic><topic>Melanoma</topic><topic>Melanoma - genetics</topic><topic>Melanoma - pathology</topic><topic>Molecular biology</topic><topic>Oncology</topic><topic>Platelet-derived growth factor</topic><topic>Proto-Oncogene Proteins B-raf - antagonists & inhibitors</topic><topic>Real-Time Polymerase Chain Reaction</topic><topic>Reverse Transcriptase Polymerase Chain Reaction</topic><topic>RNA, Small Interfering - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Ahn, Jun-Ho</creatorcontrib><creatorcontrib>Lee, Michael</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Immunology Abstracts</collection><collection>Industrial and Applied Microbiology Abstracts (Microbiology A)</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Oncogenes and Growth Factors Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection (ProQuest)</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Science Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central Basic</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Molecular and cellular biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Ahn, Jun-Ho</au><au>Lee, Michael</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>The siRNA-mediated downregulation of N-Ras sensitizes human melanoma cells to apoptosis induced by selective BRAF inhibitors</atitle><jtitle>Molecular and cellular biochemistry</jtitle><stitle>Mol Cell Biochem</stitle><addtitle>Mol Cell Biochem</addtitle><date>2014-07-01</date><risdate>2014</risdate><volume>392</volume><issue>1-2</issue><spage>239</spage><epage>247</epage><pages>239-247</pages><issn>0300-8177</issn><eissn>1573-4919</eissn><abstract>The clinical benefit of selective BRAF inhibitor therapies is limited by the emergence of drug resistance. Here, we investigated the molecular basis underlying the acquired resistance to a BRAF inhibitor by comparing the signaling pathways in the parental A375P cells and the resistant subline (A375P/Mdr). We demonstrate that MAPK re-activation does not contribute to the mechanism of resistance to UAI-201 of A375P/Mdr cells. The relative quantitative analysis using the 2
−ΔΔCt
method revealed that the BRAF inhibitor resistance observed in A375P/Mdr cells is not mediated through the overexpression of MDR proteins. In particular, we found that the expression of N-Ras was upregulated in BRAF inhibitor-resistant A375P/Mdr cells compared with A375P cells. In fact, siRNA-mediated N-Ras knockdown partially conferred UAI-201 sensitivity to A375P/Mdr cells, implying that N-Ras upregulation confers acquired resistance to BRAF inhibition. Notably, the flow cytometric analysis of the N-Ras-knockdown A375P/Mdr cells revealed that UAI-201 causes a significant accumulation of cells in the
G
0
/
G
1
phase with a concomitant decrease in the number of cells in the
S
and
G
2
/
M
phases. However, platelet-derived growth factor receptor
β
(PDGFR
β
) knockdown failed to sensitize A375P/Mdr cells to growth suppression by UAI-201, although a remarkable increase in PDGFRβ was observed in the A375P cells after UAI-201 treatment. Taken together, our results suggest that N-Ras is worth targeting to improve the therapeutic outcome of melanomas with acquired resistance to BRAF inhibitors.</abstract><cop>Boston</cop><pub>Springer US</pub><pmid>24671490</pmid><doi>10.1007/s11010-014-2034-2</doi><tpages>9</tpages></addata></record> |
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| ispartof | Molecular and cellular biochemistry, 2014-07, Vol.392 (1-2), p.239-247 |
| issn | 0300-8177 1573-4919 |
| language | eng |
| recordid | cdi_proquest_miscellaneous_1566856894 |
| source | MEDLINE; Springer Nature - Complete Springer Journals |
| subjects | Analysis Apoptosis Apoptosis - genetics Base Sequence Biochemistry Biomedical and Life Sciences Cardiology Cell Line, Tumor Cellular biology DNA Primers Down-Regulation Drug resistance Gene Knockdown Techniques Genes, ras Humans Life Sciences Medical Biochemistry Melanoma Melanoma - genetics Melanoma - pathology Molecular biology Oncology Platelet-derived growth factor Proto-Oncogene Proteins B-raf - antagonists & inhibitors Real-Time Polymerase Chain Reaction Reverse Transcriptase Polymerase Chain Reaction RNA, Small Interfering - genetics |
| title | The siRNA-mediated downregulation of N-Ras sensitizes human melanoma cells to apoptosis induced by selective BRAF inhibitors |
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