[Show abstract][Hide abstract]ABSTRACT: Glycogen synthase kinase 3β (GSK3β) is a serine/threonine protein kinase involved in human cancers including glioblastoma. We have previously demonstrated that GSK3β inhibition enhances temozolomide effect in glioma cells. In this report, we investigated the molecular mechanisms of sensitization of glioblastoma cells to temozolomide by GSK3β inhibition, focusing on O(6)-methylguanine DNA methyltransferase (MGMT) gene silencing. Glioblastoma tissues from patients treated with the GSK3β-inhibiting drugs were subjected to immunohistochemistry and methylation-specific polymerase chain reaction (MSP) assay. Human glioblastoma cell lines T98G, U138, U251 and U87 were treated with a small-molecule GSK3β inhibitor, AR-A014418 or GSK3β-specific siRNA. The combined effect of temozolomide and AR-A014418 on cell proliferation was determined by AlamarBlue assay and an isobologram method. MGMT promoter methylation was estimated by MSP and MethyLight assay. MGMT gene expression was evaluated by real-time quantitative reverse transcriptase-polymerase chain reaction. c-Myc and DNA (cytosine-5)-methyltransferase 3A (DNMT3A) binding to the MGMT promoter was estimated by chromatin immunoprecipitation assay. GSK3β inhibition decreased phosphorylation of glycogen synthase and reduced MGMT expression, and increased MGMT promoter methylation in clinical tumors. In glioblastoma cell lines, GSK3β inhibition decreased cell viability, enhanced temozolomide effect, and downregulated MGMT expression with relevant changes in the methylation levels of the MGMT promoter. Here, we showed for the first time that c-Myc binds to the MGMT promoter with consequent recruitment of DNMT3A, regulating the levels of MGMT promoter methylation. The results of this study suggest that GSK3β inhibition enhances temozolomide effect by silencing MGMT expression via c-Myc-mediated promoter methylation..
[Show abstract][Hide abstract]ABSTRACT: Aberrant DNA methylation is a commonly observed epigenetic change in lung cancer. Folate has been suggested to play a role in the homeostasis of DNA methylation and has also been implicated in cancer chemotherapy. We investigated a possible role for folate in DNA methylation by measuring folate concentrations in tumors and adjacent normal tissues from 72 non-small cell lung cancer (NSCLC) patients. These were compared to DNA methylation levels and to clinicopathological features. Folate concentrations were determined as the sum of 5,10-methylenetetrahydrofolate and tetrahydrofolate. The MethyLight assay was used to quantitate methylation in promoter regions of P16(CDKN2A), APC, CDH13, RARB, RASSF1, RUNX3, and MYOD1. Methylation of LINE-1 repeats was used as a surrogate for global methylation. Folate levels in tumors correlated positively with LINE-1, CDH13, and RUNX3 methylation. Folate concentrations and methylation of LINE-1, RASSF1, and RUNX3 were significantly higher in adenocarcinoma compared to squamous cell carcinoma (SCC). Two sets of array-based data retrieved from the Gene Expression Omnibus consistently showed that expression of FOLR1, a folate transport enzyme, and GGH, an enzyme that prevents folate retention, were higher and lower, respectively, in adenocarcinomas compared to SCC. This was independently validated by quantitative RT-PCR in 26 adenocarcinomas and 13 SCC. Our results suggest that folate metabolism plays a role in aberrant DNA methylation in NSCLC. The histological subtype differences in folate concentration and DNA methylation observed here were associated with distinct expression patterns for folate metabolizing enzymes. These findings may have clinical applications for histology-directed chemotherapy with fluoropyrimidine and anti-folates in NSCLC. (Cancer Sci 2009l; 100: 2325–2330)
[Show abstract][Hide abstract]ABSTRACT: Effects of gemciatbine and AR-A014418, alone or in combination, against pancreatic cancer cells. Inhibitory effects of gemcitabine, AR-A014418 and combinations of the two agents at different doses were examined on the survival of pancreatic cancer cells. PANC-1 (A) and MIA PaCa-2 (B) cells were treated with escalating doses of either gemcitabine, AR-A014418 or both agents in combination at the doses indicated. Relative (%) cell survival ratios for each cell line were examined by WST-8 assay at 48 hrs after treatment with the respective agent. IC50 of gemcitabine in the absence (+ DMSO) or presence of AR-A014418 (+ AR) at the indicated doses was determined and is shown in Table S4.
[Show abstract][Hide abstract]ABSTRACT: The major obstacles to treatment of pancreatic cancer are the highly invasive capacity and resistance to chemo- and radiotherapy. Glycogen synthase kinase 3β (GSK3β) regulates multiple cellular pathways and is implicated in various diseases including cancer. Here we investigate a pathological role for GSK3β in the invasive and treatment resistant phenotype of pancreatic cancer.
Pancreatic cancer cells were examined for GSK3β expression, phosphorylation and activity using Western blotting and in vitro kinase assay. The effects of GSK3β inhibition on cancer cell survival, proliferation, invasive ability and susceptibility to gemcitabine and radiation were examined following treatment with a pharmacological inhibitor or by RNA interference. Effects of GSK3β inhibition on cancer cell xenografts were also examined.
Pancreatic cancer cells showed higher expression and activity of GSK3β than non-neoplastic cells, which were associated with changes in its differential phosphorylation. Inhibition of GSK3β significantly reduced the proliferation and survival of cancer cells, sensitized them to gemcitabine and ionizing radiation, and attenuated their migration and invasion. These effects were associated with decreases in cyclin D1 expression and Rb phosphorylation. Inhibition of GSK3β also altered the subcellular localization of Rac1 and F-actin and the cellular microarchitecture, including lamellipodia. Coincident with these changes were the reduced secretion of matrix metalloproteinase-2 (MMP-2) and decreased phosphorylation of focal adhesion kinase (FAK). The effects of GSK3β inhibition on tumor invasion, susceptibility to gemcitabine, MMP-2 expression and FAK phosphorylation were observed in tumor xenografts.
The targeting of GSK3β represents an effective strategy to overcome the dual challenges of invasiveness and treatment resistance in pancreatic cancer.
[Show abstract][Hide abstract]ABSTRACT: Effect of GSK3β inhibitor on pancreatic cancer cell migration. The time course for cell migration was minitored by monolayer-based wound healing assay for MIA PaCa-2 and BxPC-3 cells in the presence of DMSO or AR-A014418 (AR 5 µM, AR 10 µM). The relative widths of wounds were measured and expressed as a percentage of the initial gap at time zero. Values are means ± SD of three separate experiments. *p<0.05, statistically significant difference between cells treated with DMSO or AR-A014418.
[Show abstract][Hide abstract]ABSTRACT: Effects of GSK3β inhibition on expression and phosphorylation of the proteins in pancreatic cancer cells. The levels of expression and phosphorylation of the indicated proteins were examined by Western blotting in pancreatic cancer cells after treatment with the respective agents. (A, B) Expression of GS and β-catenin and their phosphorylation (p-GSS641, p-β-catenin S33/37/T41) were examined and compared between the same pancreatic cancer cells treated with DMSO (DM) or 10 µM AR-A014418 (AR) for 6 hrs. (C) Expression of E-cadherin, N-cadherin and vimentin in pancreatic cancer cells treated with DMSO (DM) or 10 µM AR-A014418 (AR) for 6 hrs. (D) Expression of E-cadherin, N-cadherin, vimentin and GSK3α and GSK3β in pancreatic cancer cells transfected with non-specific siRNA (NS) or GSK3β-specific (S) siRNA (10 nM each). (A–D) The amount of protein extract in each sample was monitored by expression of β-actin.
[Show abstract][Hide abstract]ABSTRACT: Background
Long interspersed nucleotide element 1 (LINE-1) hypomethylation is suggested to play a role in the progression of colorectal cancer (CRC). To assess intra-patient heterogeneity of LINE-1 methylation in CRC and to understand its biological relevance in invasion and metastasis, we evaluated the LINE-1 methylation at multiple tumor sites. In addition, the influence of stromal cell content on the measurement of LINE-1 methylation in tumor tissue was analyzed.
Formalin-fixed paraffin-embedded primary tumor tissue was obtained from 48 CRC patients. Matched adjacent normal colon tissue, lymph node metastases and distant metastases were obtained from 12, 18 and 7 of these patients, respectively. Three different areas were microdissected from each primary tumor and included the tumor center and invasive front. Normal mucosal and stromal cells were also microdissected for comparison with the tumor cells. The microdissected samples were compared in LINE-1 methylation level measured by multicolor MethyLight assay. The assay results were also compared between microdissected and macrodissected tissue samples.
LINE-1 methylation within primary tumors showed no significant intra-tumoral heterogeneity, with the tumor center and invasive front showing identical methylation levels. Moreover, no difference in LINE-1 methylation was observed between the primary tumor and lymph node and distant metastases from the same patient. Tumor cells showed significantly less LINE-1 methylation compared to adjacent stromal and normal mucosal epithelial cells. Consequently, LINE-1 methylation was significantly lower in microdissected samples compared to macrodissected samples. A trend for less LINE-1 methylation was also observed in more advanced stages of CRC.
LINE-1 methylation shows little intra-patient tumor heterogeneity, indicating the suitability of its use for molecular diagnosis in CRC. The methylation is relatively stable during CRC progression, leading us to propose a new concept for the association between LINE-1 methylation and disease stage.
[Show abstract][Hide abstract]ABSTRACT: Inter-ethnic differences in drug handling and frequencies of pharmacogenetic variants are increasingly being characterized. In this study, we systematically assessed the feasibility of inferring ethnic trends in chemotherapy outcomes from inter-ethnic differences in pharmacogenetic variant frequencies. Frequencies of 51 variants and chemotherapy outcomes of East Asian and Caucasian colorectal cancer patients on standard chemotherapy regimens were summarized by meta-analyses, and variant frequencies were validated by MassARRAY analysis. Inferences of relative chemotherapy outcomes were made by considering minor allele function and population differences in their frequency. Significant population differences in genotype distributions were observed for 13/23 (60%) and 27/35 (77%) variants in the meta-analyses and validation series, respectively. Across chemotherapy regimens, East Asians had lower rates of grade 3/4 toxicity for diarrhea and stomatitis/mucositis than Caucasians, which was correctly inferred from 13/18 (72%, P=0.018) informative genetic variants. With appropriate variant selection, inferring relative population toxicity rates from population genotype differences may be relevant.The Pharmacogenomics Journal advance online publication, 26 June 2012; doi:10.1038/tpj.2012.26.