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Conference Paper: Blockade of Raf/MEK/ERK pathway by Raf265 inhibits tumor growth in colorectal cancer

TitleBlockade of Raf/MEK/ERK pathway by Raf265 inhibits tumor growth in colorectal cancer
Authors
Issue Date2010
PublisherAmerican Association for Cancer Research. The Journal's web site is located at http://www.aacrmeetingabstracts.org/
Citation
The 101st Annual Meeting of the American Association for Cancer Research (AACR 2010), San Francisco, CA., 17-21 April 2010. In AACR Meeting Abstracts, 2010 How to Cite?
AbstractDeregulation of the Raf/MEK/ERK signalling pathway is commonly observed in colorectal cancer (CRC). Since this signalling pathway plays a central role in controlling cell proliferation, apoptosis and differentiation; therefore, a number of therapeutics targeting on the Raf/MEK/ERK pathway has been established recently. Raf265 is an orally bioavailable small molecule which is a potent inhibitor of wild-type and mutant (e.g. V600E) B-raf kinases. The study of Raf265 has entered Phase I clinical trial in subjects with locally advanced or metastatic melanoma. However, its therapeutic efficacy in CRC has not been established. The objective of this study is to examine the functional effects of Raf265 in CRC cells in vitro and in vivo. CRC cell lines of different B-raf status were used for this study. Cell proliferation upon Raf265 treatment (0-50 μM) was determined using MTT cell proliferation assay. Cell cycle distribution and cell apoptosis upon Raf265 treatment (0, 1, 5, and 10 μM) were assessed by flow cytometry. Phosphorylation of molecules including MEK and ERK 1/2, and eIF4E, and expression of Mcl-1 and cyclin D1 was analyzed using Western blot. For in vivo animal studies, subcutaneous tumors were established by subcutaneous injections of 1x106 cells into nude or SCID mice, and tumor growth was monitored. Mice were sacrificed week 16 or when tumor sizes exceeded 30% of their body weight. Raf265 significantly inhibited cell proliferation in a dose-dependent manner with IC50 at 0.83 to 5.54 μM. Increased annexin V positive cells were observed with escalating dose of Raf265, which is indicative of induction of apoptosis in CRC cells. Dose-dependent increase in G1 and decrease in S phase population (cell cycle arrest at G1 phase) was also observed after treatment with Raf265. This was accompanied by the reduction of phosphor-MEK and phosphor-ERK 1/2. Down-regulation of Mcl-1 and cyclin D1, which regulate apoptosis and cell proliferation, respectively, was also observed. Intraperitoneal injections of Raf265 four times weekly demonstrated significant anti-tumor activity in established tumors of xenograft models. Immunohistochemistry demonstrated a close association between inhibition of tumor growth and inhibition of ERK 1/2 phosphorylation in the xenograft tumors, consistent with inhibition of the RAF/MEK/ERK pathway. Additional analyses of microvessel density in the same tumour sections using antimurine CD31 antibodies demonstrated significant inhibition of neovascularization in xenograft models. Furthermore, repeated sequential use of Raf265 and chemotherapy with 5-Fu and/or cisplatin demonstrated synergistic effect of inhibition of tumor growth compared with Raf265 or chemotherapy alone. These pre-clinical data demonstrate robust anti-tumour activity of Raf265 in CRC, providing the basis for exploiting its potential use as a therapeutic for Raf-driven CRC tumors.
DescriptionPoster Session 21 - Anticancer Drugs Targeting Cell Cycle and Proliferation: abstract no. 2508
Persistent Identifierhttp://hdl.handle.net/10722/137921
ISSN

 

DC FieldValueLanguage
dc.contributor.authorChow, AKMen_US
dc.contributor.authorChu, ACYen_US
dc.contributor.authorPoon, RTPen_US
dc.contributor.authorWong, BCYen_US
dc.contributor.authorPang, RWCen_US
dc.date.accessioned2011-08-26T14:36:57Z-
dc.date.available2011-08-26T14:36:57Z-
dc.date.issued2010en_US
dc.identifier.citationThe 101st Annual Meeting of the American Association for Cancer Research (AACR 2010), San Francisco, CA., 17-21 April 2010. In AACR Meeting Abstracts, 2010en_US
dc.identifier.issn1948-3279-
dc.identifier.urihttp://hdl.handle.net/10722/137921-
dc.descriptionPoster Session 21 - Anticancer Drugs Targeting Cell Cycle and Proliferation: abstract no. 2508-
dc.description.abstractDeregulation of the Raf/MEK/ERK signalling pathway is commonly observed in colorectal cancer (CRC). Since this signalling pathway plays a central role in controlling cell proliferation, apoptosis and differentiation; therefore, a number of therapeutics targeting on the Raf/MEK/ERK pathway has been established recently. Raf265 is an orally bioavailable small molecule which is a potent inhibitor of wild-type and mutant (e.g. V600E) B-raf kinases. The study of Raf265 has entered Phase I clinical trial in subjects with locally advanced or metastatic melanoma. However, its therapeutic efficacy in CRC has not been established. The objective of this study is to examine the functional effects of Raf265 in CRC cells in vitro and in vivo. CRC cell lines of different B-raf status were used for this study. Cell proliferation upon Raf265 treatment (0-50 μM) was determined using MTT cell proliferation assay. Cell cycle distribution and cell apoptosis upon Raf265 treatment (0, 1, 5, and 10 μM) were assessed by flow cytometry. Phosphorylation of molecules including MEK and ERK 1/2, and eIF4E, and expression of Mcl-1 and cyclin D1 was analyzed using Western blot. For in vivo animal studies, subcutaneous tumors were established by subcutaneous injections of 1x106 cells into nude or SCID mice, and tumor growth was monitored. Mice were sacrificed week 16 or when tumor sizes exceeded 30% of their body weight. Raf265 significantly inhibited cell proliferation in a dose-dependent manner with IC50 at 0.83 to 5.54 μM. Increased annexin V positive cells were observed with escalating dose of Raf265, which is indicative of induction of apoptosis in CRC cells. Dose-dependent increase in G1 and decrease in S phase population (cell cycle arrest at G1 phase) was also observed after treatment with Raf265. This was accompanied by the reduction of phosphor-MEK and phosphor-ERK 1/2. Down-regulation of Mcl-1 and cyclin D1, which regulate apoptosis and cell proliferation, respectively, was also observed. Intraperitoneal injections of Raf265 four times weekly demonstrated significant anti-tumor activity in established tumors of xenograft models. Immunohistochemistry demonstrated a close association between inhibition of tumor growth and inhibition of ERK 1/2 phosphorylation in the xenograft tumors, consistent with inhibition of the RAF/MEK/ERK pathway. Additional analyses of microvessel density in the same tumour sections using antimurine CD31 antibodies demonstrated significant inhibition of neovascularization in xenograft models. Furthermore, repeated sequential use of Raf265 and chemotherapy with 5-Fu and/or cisplatin demonstrated synergistic effect of inhibition of tumor growth compared with Raf265 or chemotherapy alone. These pre-clinical data demonstrate robust anti-tumour activity of Raf265 in CRC, providing the basis for exploiting its potential use as a therapeutic for Raf-driven CRC tumors.-
dc.languageengen_US
dc.publisherAmerican Association for Cancer Research. The Journal's web site is located at http://www.aacrmeetingabstracts.org/-
dc.relation.ispartofAACR Meeting Abstractsen_US
dc.titleBlockade of Raf/MEK/ERK pathway by Raf265 inhibits tumor growth in colorectal canceren_US
dc.typeConference_Paperen_US
dc.identifier.emailChow, AKM: chowakm@hku.hken_US
dc.identifier.emailChu, ACY: bcccy@hku.hken_US
dc.identifier.emailPoon, RTP: poontp@hku.hken_US
dc.identifier.emailWong, BCY: bcywong@hku.hken_US
dc.identifier.emailPang, RWC: robertap@hku.hken_US
dc.identifier.authorityChu, ACY=rp00505en_US
dc.identifier.authorityPoon, RTP=rp00446en_US
dc.identifier.authorityWong, BCY=rp00429en_US
dc.identifier.authorityPang, RWC=rp00274en_US
dc.description.naturelink_to_OA_fulltext-
dc.identifier.hkuros191900en_US
dc.identifier.hkuros173432-
dc.publisher.placeUnited States-
dc.description.otherThe 101st Annual Meeting of the American Association for Cancer Research (AACR 2010), San Francisco, CA., 17-21 April 2010. In AACR Meeting Abstracts, 2010-

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