Oncogenic BRAF inhibitor UAI-201 induces cell cycle arrest and autophagy in BRAF mutant glioma cells.
OBJECTIVE
An activating mutation of BRAF (BRAF-V600E) has been reported in a subset of malignant brain tumors. Thus, the aim of the present study was to identify the antiproliferative effect of the new oncogenic B-Raf targeting drug UAI-201 on 6 types of glioma cell lines with differing B-Raf mutational status.


METHODS
The IC50 values of UAI-201 were determined using crystal violet assays in six glioma cell lines. Real-time RT-PCR was performed to assess the functional role of multidrug resistance proteins in response to UAI-201. The effects of UAI-201 on six glioma cells were further examined by immunoblotting analysis, cell cycle analysis, flow cytometric apoptotic assay and autophagy assay. To identify the role of autophagy in UAI-201-induced growth inhibition, Atg5 and Beclin 1 were knocked down by RNA interference.


RESULTS
Real-time RT-PCR analysis showed a poor correlation between UAI-201 activity and the expression level of multidrug resistance proteins. The growth inhibitory effects of UAI-201 correlated with the BRAF-V600E genotype of the glioma cell lines. BRAF blockade with UAI-201 resulted in dose-dependent inhibition of MEK/ERK phosphorylations and increased G0/G1 arrest in glioma cells with BRAF-V600E. Interestingly, UAI-201 preferentially induced autophagy in BRAF-V600E cells, but not in BRAF-WT cells. More notably, autophagy inhibition through siRNA-mediated Beclin 1 knockdown partially attenuated the growth inhibition induced by UAI-201 in BRAF-V600E cells.


CONCLUSIONS
The pro-death autophagic processes could be one of the underlying mechanisms for the sensitization of BRAF-V600E glioma cells toward UAI-201.
Division of Life Sciences, College of Natural Sciences, Incheon National University, Incheon 406-772, Republic of Korea.
Jun-Ho
JH
Ahn
School of Biomedical Engineering and Sciences, Virginia Polytechnic Institute and State University (Virginia Tech), Blacksburg, VA 24061, USA.
Yong Woo
YW
Lee
Division of Life Sciences, College of Natural Sciences, Incheon National University, Incheon 406-772, Republic of Korea; YOUAI Co., Ltd., Suwon-Si, Gyeonggi-Do 443-766, Republic of Korea.
Soon Kil
SK
Ahn
Division of Life Sciences, College of Natural Sciences, Incheon National University, Incheon 406-772, Republic of Korea. Electronic address: mikelee@incheon.ac.kr.
Michael
M
Lee
D000970:Antineoplastic Agents / Q000737:chemistry*; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001343:Autophagy; D000071187:Autophagy-Related Protein 5; D000071186:Beclin-1; D001932:Brain Neoplasms / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle; D059447:Cell Cycle Checkpoints; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004252:DNA Mutational Analysis; D018432:Drug Resistance, Multiple; D005910:Glioma / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020128:Inhibitory Concentration 50; D008565:Membrane Proteins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009154:Mutation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics / Q000378:metabolism*; D011687:Purines / Q000737:chemistry*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D013449:Sulfonamides / Q000737:chemistry*
C502778:ATG5 protein, human; D000970:Antineoplastic Agents; D051017:Apoptosis Regulatory Proteins; D000071187:Autophagy-Related Protein 5; C491997:BECN1 protein, human; D000071186:Beclin-1; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; C570548:N-((3-(3-9H-purin-6-yl)pyridin-2-ylamino)-2,4-difluorophenyl)furan-3-sulfonamide; D011687:Purines; D034741:RNA, Small Interfering; D013449:Sulfonamides; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf
Autophagy; BRAF inhibitor; Cell cycle arrest; Glioma; UAI-201