Systematic Protein Level Regulation via Degradation Machinery Induced by Genotoxic Drugs

In this study we monitored protein dynamics in response to cisplatin, 5-fluorouracil, and irinotecan with different concentrations and administration modes using “reverse-phase” protein arrays (RPPAs) in order to gain comprehensive insight into the protein dynamics induced by genotoxic drugs. Among 666 protein time-courses, 38% exhibited an increasing trend, 32% exhibited a steady decrease, and 30% fluctuated within 24 h after drug exposure. We analyzed almost 12,000 time-course pairs of protein levels based on the geometrical similarity by correlation distance (<i>dCor</i>). Twenty-two percent of the pairs showed <i>dCor</i> > 0.8, which indicates that each protein of the pair had similar dynamics. These trends were disrupted by a proteasome inhibitor, MG132, suggesting that the protein degradation system was activated in response to the drugs. Among the pairs with high <i>dCor</i>, the average <i>dCor</i> of pairs with apoptosis-related protein was significantly higher than those without, indicating that regulation of protein levels was induced by the drugs. These results suggest that the levels of numerous functionally distinct proteins may be regulated by common degradation machinery induced by genotoxic drugs

Hierarchical clustering of three different matrices and results of immunohistochemical examinations of candidate markers.

<p>(A) Based on a chemosensitivity assay of a cancer cell line panel, the A (activity) × C (cells)  =  AC matrix was created. (B) Quantitative protein expression data of each cell line determined by “reverse-phase” lysate microarray generates the C×P (protein)  =  CP matrix. (C) A heatmap with hierarchical clustering representation of the AP matrix, which is generated from AC and CP matrices. (D) Immunohistochemical stainings of candidate markers for 5-FU treatment.</p

Time-to-relapse (TTR) rates on the basis of NF-κB and JNK protein expression in gastric and colon carcinomas.

<p>Time-to-relapse (TTR) rates on the basis of NF-κB and JNK protein expression in gastric and colon carcinomas.</p

Induction of biomarkers by 5-FU treatment.

<p>(A) Baseline protein expression of NF-κB and JNK in five gastric cancer cell lines. Tublin was used as a loading control. (B) Induction of candidate biomarkers in response to 5-FU treatment in different concentrations in MKN45 and KE39. (C) Examination of protein localization by fluorescent immunocytochemistry using MKN45. (D) Increased inhibitory growth effect by anticancer agents in gastric cancer cell lines after transfection of siRNA for NF-κB p65 and JNK transcripts. Control samples are the corresponding cell lines transfected with the indicated siRNAs without anticancer agents. Abbreviations are: CIS, cisplatinum; DTX, docetaxel; and PXL, paclitaxel; and 5FU, 5-fluorouracil. *<i>p</i><0.05, Student <i>t</i>-test.</p