Project description:Gemcitabine resistance is currently the main problem of chemotherapy for advanced pancreatic cancer patients. The resistance is thought to be caused by altered drug metabolism or reduced apoptosis of cancer cells. However, the underlying mechanism of Gemcitabine resistance in pancreatic cancer remains unclear. In this study, we established Gemcitabine resistant PANC-1 (PANC-1-GR) cell lines and compared the circular RNAs (circRNAs) profiles between PANC-1 cells and PANC-1-GR cells by RNA sequencing. Differentially expressed circRNAs were demonstrated using scatter plot and cluster heatmap analysis. Gene ontology and pathway analysis were performed to systemically map the genes which are functionally associated to those differentially expressed circRNAs identified from our data. The expression of the differentially expressed circRNAs picked up by RNAseq in PANC-1-GR cells was further validated by qRT-PCR and two circRNAs were eventually identified as the most distinct targets. Consistently, by analyzing plasma samples form pancreatic ductal adenocarcinoma (PDAC) patients, the two circRNAs showed more significant expression in the Gemcitabine non-responsive patients than the responsive ones. In addition, we found that silencing of the two circRNAs could restore the sensitivity of PANC-1-GR cells to Gemcitabine treatment, while over-expression of them could increase the resistance of normal PANC-1 and MIA PACA-2 cells, suggesting that they might serve as drug targets for Gemcitabine resistance. Furthermore, the miRNA interaction networks were also explored based on the correlation analysis of the target microRNAs of these two circRNAs. In conclusion, we successfully established new PANC-1-GR cells, systemically characterized the circRNA and miRNA profiles, and identified two circRNAs as novel biomarkers and potential therapeutic targets for Gemcitabine non-responsive PDAC patients.
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. Overall design: The three PDAC cells including PANC-1, PANC-1 GEM 100 and PANC-1 GEM1500 were harvested and RNA were isolated. Two independent experiments were performed for each group.
Project description:Gemcitabine resistance is currently the main problem of chemotherapy for advanced pancreatic cancer patients. The resistance is thought to be caused by altered drug metabolism or reduced apoptosis of cancer cells. However, the underlying mechanism of Gemcitabine resistance in pancreatic cancer remains unclear. In this study, we established Gemcitabine resistant PANC-1 (PANC-1-GR) cell lines and compared the circular RNAs (circRNAs) profiles between PANC-1 cells and PANC-1-GR cells by RNA sequencing. Overall design: Examing 2 conditions, each with 3 replicates
Project description:BACKGROUND:TM4SF1 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and affects the development of this cancer. Also, multidrug resistance (MDR) is generally associated with tumor chemoresistance in pancreatic cancer. However, the correlation between TM4SF1 and MDR remains unknown. This research aims to investigate the effect of TM4SF1 on gemcitabine resistance in PDAC and explore the possible molecular mechanism between TM4SF1 and MDR. METHODS:The expression of TM4SF1 was evaluated in pancreatic cancer cell lines and human pancreatic duct epithelial (HPDE) cell lines by quantitative RT-PCR. TM4SF1 siRNA transfection was carried out using Hiperfect transfection reagent to knock down TM4SF1. The transcripts were analyzed by quantitative RT-PCR, RT-PCR and western blotting for further study. The cell proliferation and apoptosis were obtained to investigate the sensitivity to gemcitabine of pancreatic cancer cells after silencing TM4SF1 in vitro. We demonstrated that cell signaling of TM4SF1 mediated chemoresistance in cancer cells by assessing the expression of multidrug resistance (MDR) genes using quantitative RT-PCR. In vivo, we used orthotopic pancreatic tumor models to investigate the effect of proliferation after silencing TM4SF1 by a lentivirus-mediated shRNA in MIA PaCa-2 cell lines. RESULTS:The mRNA expression of TM4SF1 was higher in seven pancreatic cancer cell lines than in HPDE cell lines. In three gemcitabine-sensitive cell lines (L3.6pl, BxPC-3, SU86.86), the expression of TM4SF1 was lower than that in four gemcitabine-resistant cell lines (MIA PaCa-2, PANC-1, Hs766T, AsPC-1). We evaluated that TM4SF1 was a putative target for gemcitabine resistance in pancreatic cancer cells. Using AsPC-1, MIA PaCa-2 and PANC-1, we investigated that TM4SF1 silencing affected cell proliferation and increased the percentages of cell apoptosis mediated by treatment with gemcitabine compared with cells which were treated with negative control. This resistance was associated with the expression of multidrug resistance genes including ABCB1 and ABCC1. In vivo, silencing of TM4SF1 in MIA PaCa-2 cell lines increased the effectiveness of gemcitabine-based treatment in orthotopic pancreatic tumor models evaluated using noninvasive bioluminescent imaging. CONCLUSION:These findings suggest that TM4SF1 is a surface membrane antigen that is highly expressed in pancreatic cancer cells and increases the chemoresistance to gemcitabine. Thus, TM4SF1 may be a promising target to overcome the chemoresistance of pancreatic cancer.
Project description:Background: Chemotherapy remains a primary treatment method for advanced pancreatic cancer. However, chemotherapy resistance can influence the therapeutic effect of pancreatic cancer. The resistance mechanism of chemotherapeutic agents such as gemcitabine, which is an agent typically used to treat pancreatic cancer, is complicated and can be influenced by genes and the environment. Oridonin is a tetracyclic diterpenoid compound extracted from the traditional Chinese herb Rabdosia labtea. Oridonin may overcome drug resistance in pancreatic cancer, but researching pancreatic cancer drug resistance of chemotherapy by oridonin is not completely understood. Purpose: The present study aimed to assess the impact of oridonin on multidrug resistance proteins, apoptosis-associated proteins and energy metabolism in gemcitabine-resistant PANC-1 (PANC-1/Gem) pancreatic cancer cells. Methods: Gemcitabine resistance in PANC-1/Gem cells was induced using a concentration gradient of gemcitabine. Cell Counting Kit-8 assays were used to detect the impact of gemcitabine and oridonin on the proliferation of PANC-1 and PANC-1/Gem cells. Western blot analysis and immunofluorescence were used to detect the expression of multidrug resistance proteins, apoptosis-associated proteins and low-density lipoprotein receptor protein 1 (LRP1) proteins in PANC-1/Gem cells. The effects of gemcitabine and oridonin on PANC-1/Gem cells apoptosis were detected using flow cytometry. Animal xenograft tumor assays were used to detect the effect of gemcitabine and oridonin on pancreatic cancer in vivo. Furthermore, the ATP Assay kit was used to determine the effects of gemcitabine and oridonin on ATP levels in PANC-1/Gem cells. Immunofluorescence assays were used to detect the effects of gemcitabine and oridonin on the expression of low-density lipoprotein receptor protein 1 (LRP1) in PANC-1/Gem cells. In addition, LRP1 expression was knocked down in PANC-1/Gem cells via lentiviral vector-mediated RNA silencing. Clone formation assays and Western blot analysis were used to detect the effect of LRP1 knockdown on the proliferation of PANC-1/Gem cells. Results: The present results demonstrate that oridonin overcomes PANC-1/Gem cells gemcitabine reistance by regulating GST pi and LRP1/ERK/JNK signaling. Conclusion: In conclusion, the present study indicated that oridonin could overcome gemcitabine resistance in PANC-1/Gem cells by regulating GST pi and LRP1/ ERK/JNK signaling, inducing cell apoptosis. Therefore, oridonin with gemcitabine may be a promising preoperative treatment for patients who suffer from pancreatic cancer.
Project description: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 chemoresistant PDAC for its specific miR expression pattern. Gemcitabine-resistant variants of two mutant p53 human pancreatic adenocarcinoma cell lines were established. MicroRNA screening was investigated by microarray. Gemcitabine-resistant PANC-1 (PANC-1-GR) and MIA-PaCa-2 (MIA-PaCa-2-GR) cell clones were produced by exposing the parental cells to repeated pulsatile gemcitabine treatment over 3 days with constant sublethal concentrations followed by recovery-periods with agent-free medium until the cells recovered exponentially. Parental PANC-1 cells were treated with 0.4µM gemcitabine cycles for approximately 9 months. Parental MIA-PaCa-2 cells were exposed to 0.06µM gemcitabine cycles for approximately 12 months. Affymetrix GeneChip miRNA microarrays (Affymetrix UK Ltd., High Wycombe, UK) were performed in parental and chemoresistant PANC-1 and MIA-PaCa-2 cells after 29 chemotherapy cycles using the manufacturers´ protocols. The samples were prepared from 1µg of total-RNA in accordance with the Affymetrix FlashTag Biotin HSR RNA Labeling Kit. The targets were hybridized overnight to Affymetrix GeneChip miRNA arrays. Following hybridization, the arrays were washed and stained using the Affymetrix GeneChip Fluidics Station 450 and scanned using the Affymetrix GeneChip Scanner 3000 7G. Microarray data quality was checked as recommended by the manufacturer and by the quality metrics in the Partek Genomics Suite software (Partek Inc., St. Louis, MO).
Project description:One of the main obstacles for cancer therapies is to deliver medicines effectively to target sites. Since stroma cells are developed around tumors, chemotherapeutic agents have to go through stroma cells in order to reach tumors. As a method to improve drug delivery to the tumor site, a prodrug approach for gemcitabine was adopted. Amino acid and dipeptide monoester prodrugs of gemcitabine were synthesized and their chemical stability in buffers, resistance to thymidine phosphorylase and cytidine deaminase, antiproliferative activity, and uptake/permeability in HFF cells as a surrogate to stroma cells were determined and compared to their parent drug, gemcitabine. The activation of all gemcitabine prodrugs was faster in pancreatic cell homogenates than their hydrolysis in buffer, suggesting enzymatic action. All prodrugs exhibited great stability in HFF cell homogenate, enhanced resistance to glycosidic bond metabolism by thymidine phosphorylase, and deamination by cytidine deaminase compared to their parent drug. All gemcitabine prodrugs exhibited higher uptake in HFF cells and better permeability across HFF monolayers than gemcitabine, suggesting a better delivery to tumor sites. Cell antiproliferative assays in Panc-1 and Capan-2 pancreatic ductal cell lines indicated that the gemcitabine prodrugs were more potent than their parent drug gemcitabine. The transport and enzymatic profiles of gemcitabine prodrugs suggest their potential for delayed enzymatic bioconversion and enhanced resistance to metabolic enzymes, as well as for enhanced drug delivery to tumor sites, and cytotoxic activity in cancer cells. These attributes would facilitate the prolonged systemic circulation and improved therapeutic efficacy of gemcitabine prodrugs.
Project description:Pancreatic cancer is susceptible to gemcitabine resistance, and patients receive less benefit from gemcitabine chemotherapy. Previous studies report that gambogic acid possesses antineoplastic properties; however, to our knowledge, there have been no specific studies on its effects in pancreatic cancer. Therefore, the purpose of this study was to explore whether increases the sensitivity of pancreatic cancer to gemcitabine, and determine the synergistic effects of gambogic acid and gemcitabine against pancreatic cancer.The effects of gambogic acid on cell viability, the cell cycle, and apoptosis were assessed using 4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) and flow cytometry in pancreatic cancer cell lines. Protein expression was detected by western blot analysis and mRNA expression was detected using q-PCR. A xenograft tumor model of pancreatic cancer was used to investigate the synergistic effects of gambogic acid and gemcitabine.Gambogic acid effectively inhibited the growth of pancreatic cancer cell lines by inducing S-phase cell cycle arrest and apoptosis. Synergistic activity of gambogic acid combined with gemcitabine was observed in PANC-1 and BxPC-3 cells based on the results of MTT, colony formation, and apoptosis assays. Western blot results demonstrated that gambogic acid sensitized gemcitabine-induced apoptosis by enhancing the expression of cleaved caspase-3, cleaved caspase-9, cleaved-PARP, and Bax, and reducing the expression of Bcl-2. In particular, gambogic acid reduced the expression of the ribonucleotide reductase subunit-M2 (RRM2) protein and mRNA, a trend that correlated with resistance to gemcitabine through inhibition of the extracellular signal-regulated kinase (ERK)/E2F1 signaling pathway. Treatment with gambogic acid and gemcitabine significantly repressed tumor growth in the xenograft pancreatic cancer model. Immunohistochemistry results demonstrated a downregulation of p-ERK, E2F1, and RRM2 in mice receiving gambogic acid treatment and combination treatment.These results demonstrate that gambogic acid sensitizes pancreatic cancer cells to gemcitabine in vitro and in vivo by inhibiting the activation of the ERK/E2F1/RRM2 signaling pathway. The results also indicate that gambogic acid treatment combined with gemcitabine might be a promising chemotherapy strategy for pancreatic cancer.
Project description:Pancreatic cancer is one of the most fatal cancers and is associated with limited diagnostic and therapeutic modalities. Currently, gemcitabine is the only effective drug and represents the preferred first-line treatment for chemotherapy. However, a high level of intrinsic or acquired resistance of pancreatic cancer to gemcitabine can contribute to the failure of gemcitabine treatment. To investigate the underlying molecular mechanisms for gemcitabine resistance in pancreatic cancer, we performed label-free quantification of protein expression in intrinsic gemcitabine-resistant and - sensitive human pancreatic adenocarcinoma cell lines using our improved proteomic strategy, combined with filter-aided sample preparation, single-shot liquid chromatography-mass spectrometry, enhanced spectral counting, and a statistical method based on a power law global error model. We identified 1931 proteins and quantified 787 differentially expressed proteins in the BxPC3, PANC-1, and HPDE cell lines. Bioinformatics analysis identified 15 epithelial to mesenchymal transition (EMT) markers and 13 EMT-related proteins that were closely associated with drug resistance were differentially expressed. Interestingly, 8 of these proteins were involved in glutathione and cysteine/methionine metabolism. These results suggest that proteins related to the EMT and glutathione metabolism play important roles in the development of intrinsic gemcitabine resistance by pancreatic cancer cell lines.