Project description:Gemcitabine (GEM) is a key drug for treating PDAC, and it is commonly used for adjuvant chemotherapy. Although the majority of PDAC is sensitive to GEM at first, GEM cannot control PDAC for very long, suggesting that PDAC develops resistance to GEM after prolonged exposure. No reliable predictors of susceptibility to gemcitabine chemotherapy exist in pancreatic ductal adenocarcinoma. MicroRNAs (miR) are epigenetic gene regulators with tumorsuppressive or oncogenic roles in various carcinomas. This study assesses gemcitabine resistant PDAC for its specific miR expression pattern. Gemcitabine resistant variants of Panc1, a human pancreatic adenocarcinoma cell line, were established. MicroRNA screening was investigated by microarray.
Project description:Pancreatic ductal adenocarcinoma (PDAC) often presents at late clinical stages, and most patients are managed solely through palliative chemotherapy. With no approved treatment modalities for patients who progress on broad-spectrum chemotherapy, we set to identify druggable targets to prevent or reverse resistance to the first line anti-neoplastic Gemcitabine. In our first experiment, we used the well-established Panc1 cell line as an in vitro model of PDAC. Panc1 cells were incubated with a tolerable dose of Gemcitabine in vitro, and examined alterations in gene expression via single cell RNA sequencing. In our subsequent studies, we incubated Panc1 cells with increasing doses of Gemcitabine for several passages, until viable in approximately 10x the known IC50 value. These cells were designated Panc1-GR. Based on our observations in the prior experiment, Panc1-GR cells were compared to those treated with wither the Calmodulin inhibitor W-7, Calcium chelator BAPTA-AM, or the calcium channel blocker Amlodipine. Through these efforts, we hope to better understand the mechanisms of Gemcitabine resistance in PDAC, as well as introduce new therapeutic strategies to reverse drug resistant phenotypes in the clinic.
Project description:microRNA expression profiles show altered patterns in cell lines with acquired resistance to the BRAF inhibitor compared to the parental cell lines, and common miRNAs regulated in the resistant variants
Project description:This experiment describes the differential microRNA expression between parental gemcitabine-sensitive BxPC-3 cells and their resistant subclones, Bx-GEM. To select for the resistant subclones, parental BxPC-3 cells were treated with increasing concentrations of gemcitabine (10, 25, 50, 100 and 200 nM) for more than one year. Cells resistant at each stage of drug dosing were re-cultured in the subsequent dose, and their resistance confirmed via cell viability assays. Subclones resistant to 200 nM gemcitabine, in additon to the parental cells were used for microRNA profiling by array. Total RNA was extracted from the cells using the miRNeasy Mini Kit (Qiagen). Fluorescently-labeled miRNA were prepared according to Agilent protocol miRNA Complete Labeling and Hyb Kit. Labeled miRNA sample were hybridized for at least 20 hr at 55C on Agilent human miRNA Microarray Release 19.0, 8x60k. Gene Expression Microarrays were scanned using the Agilent Scanner G2505C.
Project description:We examined the microarray-based expression profiles of the cells to identify genes associated with gemcitabine resistance Using a set of parental and gemcitabine resistant cell line, MKN28 and RMKN28, we studied mRNA transcription levels by microarray analysis to see if there are any of the key molecules for development of acquisition of the GEM resistance.
Project description:We examined the microarray-based expression profiles of the cells to identify genes associated with gemcitabine resistance Using three set of parental and resistant cell lines, PK-9 and RPK-9, PK-1 and RPK-1, and PK-59 and RPK-59, we studied mRNA transcription levels by microarray analysis to see if there are any of the key molecules for development of acquisition of the GEM resistance.
Project description:Gemcitabine has been a first-line therapeutic agent for pancreatic ductal adenocarcinoma (PDAC) pancreatic cancer; however, acquisition of resistance to gemcitabine remains a major challenge. We analyzed miRNAs expression profiles by array-based miRNAs analysis between gemcitabine–resistant (PANC-1/GEM) and parental PANC-1 cells.
Project description:We reported the transcriptional effects of gemcitabine, genistein and the combined gemcitaibe/gensitein treatments on two pancreatic cancer cell lines MiaPaCa2 and PANC1. The overall purpose of the study to understand the underlying mechanism of genistein chemoenhancing function.
