Identification of chloroquine diphosphate as an autophagy inhibitor to treat PANC-1 cells [mRNA, circRNA, lncRNA]
ABSTRACT: Autophagy has been reported to be involved in the occurrence and development of pancreatic cancer. However, the mechanism of autophagy-related non-coding RNAs in pancreatic cancer remains largely unknown. In this study, we used microarrays to detect differential expression of mRNAs, miRNAs, lncRNAs and circRNAs post autophagy suppression by chloroquine diphosphate in PANC-1 cells. Overall design: The autophagy-inhibited model was constructed with PANC-1 cells treated by chloroquine diphosphate. It was indicated that 100µM chloroquine diphosphate was the best choice to inhibit the autophagy of PANC-1 in humans.
INSTRUMENT(S): Agilent-078298 human ceRNA array V1.0 4X180K [Probe Name Version]
Project description:The function and mechanism of autophagy in pancreatic cancer remained elusive owing to its complex biological process and multi-molecular involvement including non-coding RNAs. Here, we, from the view of mRNA, circRNA, lncRNA and miRNA, investigate the molecular mechanism of autophagy induction on human pancreatic cancer cells Overall design: The autophagy-induced model was constructed with PANC-1 cells treated by rapamycin. It was indicated that 100µM rapamycin was the best choice to induce the autophagy of PANC-1 in humans.
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:Analysis of differentially expressing genes in whole genome wide analysis of aptamer SQ2 positive cells (Capan-1, Panc-1, Panc-1+ve) and SQ2 negative cells (Panc-1-ve and HPDE) Panc-1 +ve and Panc-1-ve cell lines were generated from Panc-1 cell line based upon its hetergenous binding to aptamer SQ2. Detailed procedure of generation of these cell lines are described in Pooja Dua, Hye Suk Kang, Seung-Mo Hong, Ming-Sound Tsao, Soyoun Kim, and Dong-ki Lee. 2012 Alkaline Phosphatase ALPPL2 is a novel pancreatic carcinoma-associated protein. Cancer Research A five chip study using total RNA recovered from Capan-1, Panc-1, Panc-1+ve, Panc-1-ve and HPDE cells. Each chip measures 45,033 genes with three 60 mer probe pairs per target.
Project description:Analysis of differentially expressing genes in whole genome wide analysis of ALPPL2 expressing Panc-1 cells (Panc-1+ve) via siRNA mediated knockdown of ALPPL2 Panc-1 +ve and Panc-1-ve cell lines were generated from Panc-1 cell line based upon its hetergenous binding to aptamer SQ2. Detailed procedure of generation of these cell lines are described in Pooja Dua, Hye Suk Kang, Seung-Mo Hong, Ming-Sound Tsao, Soyoun Kim, and Dong-ki Lee. 2012 Alkaline Phosphatase ALPPL2 is a novel pancreatic carcinoma-associated protein. Cancer Research A four chip study using total RNA recovered from Panc-1+ve cells transfected with siALPPL2-2, siALPPL2-3, siGFP control and Lipofectamine 2000 treatament . Each chip measures 45,033 genes with three 60 mer probe pairs per target.
Project description:The Panc-1 human pancreatic cancer line (ATCC) was constructed to stably express control or GSK3B shRNA. Control or GSK3B shRNA expressing Panc-1 cells were implanted subcutaneously into the flanks of 3-5 week old, female, athymic nude mice. Tumors were grown to at least 250mm3. Tumor tissue was microdissected for further analysis. We compared control shRNA (n=4) to GSK3B shRNA (n=3) Panc-1 xenografts. The array data with simple statistical calculations are also provided in a supplementary Excel workbook, with probe-set annotation that we used at the time (users may want newer annotation). We compared xenografts of Panc-1 (Human pancreatic carcinoma cell line, grown in mice) stably expressing GSK3B shRNA (n=3) to similar xenografts with control shRNA (n=4). Xenografts were grown in the flanks of female, athymic nude mice until they reached at least 250mm3. Tumor tissue was microdissected for further analysis. mRNA abundance assays were performed using Affymetrix HG_U133_plus_2 arrays, with 54675 probe-sets.
Project description:cGMP is well-known secound messanger involved in vascular homeostasis. However little is known the effect of cGMP inducer on mRNA expression in cancer cells. We performed microarrays to revealed the transcriptional change of a human pancreatic ductal adenocarcinoma (PDCA) cell line (Panc -1) to cGMP inducer Bay41-2272 in order to provide insight into impact of cGMP induction on Panc-1 cells. Panc-1 Human PDCA cells were treated with vehicle (DMSO) or 5 μM Bay41-2272 for 48 h and total RNA was extracted by using TRIzol Reagent. The microarray analysis was conducted on the Panc-1 cells expressing by using Agilent Microarray.
Project description:The main goal of this study is to characterize the transcriptional modulations induced by antiviral compounds derived from chloroquine on Huh7 cells infected with HCV. And thus to identify among the genes modulated by infection, those who are regulated by chloroquine and potentially involved in the antiviral activity of the compound.Two HCV cell models were used: a non-infectious HCV replicon and the infectious HCV cell culture (HCVcc). In addition, this study aimed to highlight the characteristics of derivatives of antiviral chloroquine, compared with chloroquine itself. Abstract from the associated publication: Autophagy is a process of self-degradation of cellular components in which double-membrane autophagosomes sequester organelles or portion of cytosol and fuse with lysosomes or vacuoles for breakdown by resident hydrolases. Autophagy is upregulated in response to extra- or intracellular stress and signals such as starvation, growth factor deprivation, ER stress, and pathogen infection. Indeed, infection with hepatitis C virus (HCV) was shown to induce autophagy through ER stress signaling and subsequent Unfolded Protein Response (UPR) activation. Moreover, a role of autophagy in promoting HCV infection has been suggested and chloroquine (CQ), a lysosomal protease inhibitor, has been seen blocking autophagy as well as inhibiting HCV replication. In the present report, mechanisms accounting for these inhibitory effects were investigated. Gene expression profiling was performed on CQ treated JFH-1-infected Huh7 cells to identify the host cellular genes that are transcriptionally regulated by infection, and silenced by CQ-based treatment. Herein, we demonstrate that CQ reduces the expression of genes induced by the viral infection such as those encoding autophagic key factors triggering the turn-off of mTOR activity as well as factors involved in p53 and NF-KappaB activities. However, we present several lines of evidence demonstrating that the CQ repressive effect observed on the HCV-induced pathways results from a decrease of ER stress due to the upstream pH-dependent inhibitory effect of CQ on viral replication. Gene expression modulations were measured in Huh7 harboring replicon cells stimulated with a 10µM of a Chloroquine derivative compound called CQd, during 6h, 12h and 24h. Two independent experiments were performed at each time. An untreated control condition was performed in each experiment, expression was measured at the 24h time point. For the HCVcc model, gene expression analysis was analyzed in two conditions: infection and treatment of infected cells. To characterize modulations in time course of infection, Huh7 naïve cells were infected with JFH1/CS-N6-A4 viral stock and gene expression was measured at 6h, 24h and 48h postinfection. For each kinetic time point, an uninfected condition was performed and gene expression was measured. An additional control testing the interferon (IFN) induced modulations on infected cells was included and gene expression was measured after a 48h stimulation with 100 IU of IFN (alpha 2 b). To characterize the antiviral modulations resulting from treatment with Chloroquine (CQ) or a derivative compound (CQd), gene expression was measured in JFH1/CS-N6-A4 infected Huh7 cells treated with 40µM of CQ or CQd during 12h or 48h post-treatment and infection. For each kinetic time point 12h and 48h, an untreated infected control was also performed and gene expression measured.