Effects Of17-DMAG On Colon Cancer HT-29Cells And Proteomics Study

BackgroundColon cancer is a common and deadly disease with high rising incidence rate in recent years. The five-year survival rate of patients with advanced colon cancer has not significantly improved in the last few decades, despite major progresses in surgical techniques and chemotherapy. The traditional surgery and chemotherapy have their limits, therefore, it is necessary to find new therapy for colon cancer. Heat shock protein90(HSP90) is a molecular chaperone involved in folding and stabilization of several client proteins regulating the survival of cancer cells. Clinical trials of HSP90inhibitors for some malignant diseases have been performed and have obtained promising results. Among them,17-DMAG was more potent inhibitor of HSP90, which suggests that17-DMAG may become a novel therapeutic agent for advanced colon cancer.ObjectveTo investigate the effects of17-DMAG on colon cancer HT-29cells and proteomics study.Methods1CCK-8assay was performed to evaluate inhibition rates at different concentrations (0,0.1μmol/L,0.25μmol/L,0.5μmol/L,1.0μmol/L,2.5μmol/L,5.0μmol/L) of17-DMAG on HT-29cells in24,48and72h.2Contrast phase microscope was used to observe cellular morphologic changes.317-DMAG induced apoptosis of HT-29cells was delineated by DAPI staining assay.4Annexin V PI double labeling FCM was used to determine cell apoptotic rate. 5Westen blotting analysis was used to determine caspase-3and cleaved caspase-3protein expression.6Intracellular reactive oxygen species (ROS) generation was measured by microplate reader.7Furthermore, protein separation in17-DMAG (1μM) treated group for24h and control group was performed by two dimensional gel electrophoresis(2-DE). After G-250staining, the images of the gels were acquired by the scanner and analyzed by using ImageMaster2D Platinum6.0software to find the differentially expressed protein spots in each group.8The different protein spots were cut out from preparation gels and then digested with trypsin for17h.Through electrospray ionization spectrometry (ESI) detection and the mascot database search, the proteins were identified.Results1After treated with17-DMAG at different concentrations(0,0.1μmol/L,0.25μmol/L,0.5μmol/L,1.0μmol/L,2.5μmol/L,5.0μmol/L) for24h,48h,72h, the proliferation inhibition rate (%) was14.36±0.95,22.17±1.15,28.45±1.16,35.04±1.58,46.85±2.44,57.19±2.06respectively(24h);20.80±1.17,27.55±0.65,33.33±1.23,46.20±4.76,55.45±4.47,61.75±2.72respectively (48h);29.62±2.27,39.19±1.74,44.29±2.00,50.66±2.17,58.84±3.18,70.74±2.65respectively (72h). CCK-8assay demonstrated that the proliferation of HT-29cells were suppressed significantly by17-DMAG in dose and time dependent manner (P﹤0.05).217-DMAG at different concentrations after48h induced the morphologic changes in HT-29cells, including cell shrinkage, membrane blebbing and cell number decrease.3DAPI staining showed that HT-29cells treated with17-DMAG displayed chromatin condensation and nuclear fragmentation which are typical changes of apoptosis. 4AnnexinV-FITC double staining and flow cytometry analysis showed that the early apoptotic rate (%) treated with17-DMAG under different concentrations(0,0.25μmol/L,0.5μmol/L,1.0μmol/L and2.5μmol/L) for24h was1.87±0.34,3.65±0.67,4.44±0.46,6.01±1.8and6.93±1.93respectively and the total apoptotic rate (%) for24hour was2.72±0.57,5.38±0.46,6.88±0.52,10.44±0.32and17.87±4.66respectively. Both the early and total apoptotic rates in17-DMAG treated group for24hour were markedly higher than those in the control group (P=0.000). Moreover, the apoptotic rate with17-DMAG treated group at a concentration of1.0μmol/L for6,12,24or48h was also higher than that in the corresponding control group (P<0.05).5The expression of procaspase-3decreased, while cleaved Caspase-3increased in the presence of17-DMAG at different concentrations for24or48hours.6The HT-29cells generated ROS under different concentrations (0,0.25μmol/L,0.5μmol/L,1μmol/L or2.5μmol/L) of17-DMAG for12h and24h. The level of ROS in the17-DMAG-treated group for12or24hour was dramatically higher than that in the corresponding control group (P<0.05).17-DMAG induced ROS accumulation in HT-29cells in a dose dependent manner.7There were24different expression protein dots between control group and17-DMAG (1μM) treated group for24h. Out of them,2protein dots were stronger while22protein dots were weaker in17-DMAG (1μM) treated group for24h than in control group.8Twenty-four protein spots were detected by ESI. There were16proteins identified out including adenosylmethionine decarboxylase, Lamin-B1, Beta-catenin-like protein1, Src substrate cortactin, Keratin, type Ⅱ cytoskeletal2epidermal, Zinc finger protein326, Prelamin-A/C, Heat shock cognate71kDa protein, Heat shock70kDa protein1A/1B, Keratin, type Ⅱ cytoskeletal8, Keratin, type Ⅰ cytoskeletal10, Heterogeneous nuclear ribonucleoprotein H, Keratin, type Ⅰ cytoskeletal18, Heterogeneous nuclear ribonucleoprotein H3, L-Iactate dehydrogenase B chain and Translationally-controlled tumor protein. Conclusions17-DMAG can markedly inhibit the growth and promote apoptosis of HT-29cells through caspase-3pathway; and this effect of17-DMAG may be associated with an intracellular ROS increase. Furthermore, we found that there were16proteins identified out of24differentially expressed protein spots between17-DMAG (1μM) treated group for24h and control group.