Identification of Differentially Expressed Genes in Head and Neck Cancer Cells Treated with BAI

Abstract

A novel cyclin-dependent kinase (CDK) inhibitor, 2-[1,1‘- biphenyl]-4-yl-N-[5-(1,1-dioxo-1λ^(6)-isothiazolidin -2-yl)-1H-indazol-3-yl] acetamide (BAI), inhibits the activity of CDK and the growth of many tumor cells. In this study, I investigated anti-tumor effects of BAI and identified differentially expressed gene (DEG) in head and neck cancer cells induced by BAI. BAI treatment for 24 h arrested AMC-HN6 cells at the G2/M phase of the cell cycle. However, it inhibited the growth of AMC-HN4 cells and increased the proportion of cells in the sub-G1 phase. BAI treatment for 48 h resulted in a markedly increased proportion of cells in the sub-G1 phase, cleavages of procaspase-3 and PLC-g1 and release of cytochrome c in both cell lines. By annealing control primer-based gene fishing assay, 10 DEGs were identified in AMC-HN6 cells treated with BAI, including DNA methyltransferase 1 associated protein 1, translocation protein 1, glyceraldehyde-3-phosphate dehydrogenase, replication initiator 1, nitric oxide synthase interacting protein, platelet-activating factor acetylhydrolase, YTH domain family member 2, glutamine synthetase, mitochondrial protein 18, and RNA binding motif protein 5. These DEGs were further analyzed by RT-PCR. Significant decreases in the mRNA levels of DEGs were observed, except GAPDH. Some of the DEGs encode proteins with functions related to key cellular processes. These results indicate that BAI may induce cell death as well as inhibit the function of CDK. The DEGs identified in this study may provide insights into the molecular mechanisms of BAI-mediated cell damage. 새로운 cyclin-dependent kinase (CDK) inhibitor인 2-[1,1‘-biphenyl] -4-yl-N-[5-(1,1-dioxo-1λ^(6)-isothiazolidin-2-yl)-1H-indazol-3-yl] acetamide (BAI)는 CDK 활성과 종양세포 증식을 억제할 수 있다. 이 연구에서는 두경부 종양세포에 대한 BAI의 항종양효과와 BAI 처치에 의해 유발되는 differentially expressed gene (DEG)을 규명하고자 하였다. BAI를 24시간 처치한 경우 AMC-HN6 cells의 세포주기가 G2/M phase에 중지되었으나, AMC-HN4 cell에 대해서는 BAI는 세포 증식을 억제시키고 sub-G1 phase의 세포 수 축적을 증가시켰다. BAI를 48 시간 처치할 경우 두 세포주 모두에서 sub-G1 phase 세포 수 축적과 procaspase-3와 PLC-g1 분절화 및 미토콘드리아 cytochrome c 유리가 증가하였다. BAI를 처치한 AMC-HN6 cell을 대상으로 annealing control primer를 이용한 gene fishing assay를 실시한 결과 10개의 DEGs 즉 DNA methyltransferase1 associated protein 1, translocation protein 1, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), replication initiator 1, nitric oxide synthase interacting protein, platelet- activating factor acetylhydrolase, YTH domain family member 2, glutamine synthetase, mitochondrial protein 18 및 RNA binding motif protein 5가 동정되었다. RT-PCR을 통해 DEG 발현성을 재 확인한 결과 GAPDH를 제외한 나머지 DEGs 모두 BAI 처치에 의해 발현이 저하되었으며, 확인된 DEG 중 일부는 중요한 세포 활동과 연관된 기능을 보이는 것으로 나타났다. 이상의 결과는 BAI는 CDK inhibitor로는 물론 세포 사멸 유도제로도 작용할 수 있음을 나타내며, 이 연구에서 규명된 DEG들은 향후 BAI에 의한 세포손상 분자기전을 규명하는데 중요한 자료로 활용될 수 있다고 생각된다.