| Objectives: To establish gemcitabine (GEM)-resistance pancreatic cancer cell lineand provide the cell model for acquired drug-resistance study; to investigate theinhibition of gemcitabine-resistant human pancreatic cancer cell growth bycucurmosin (CUS) and its possible mechanism.Methods:1. Establishment of GEM-resistance human pancreatic cancer cell line: Agemcitabine-resistant human pancreatic cancer cell line (PANC-1RG7) wasestablished in vitro by gradually increased concentration of GEM and cloning culture.2. Identification of GEM-resistance human pancreatic cancer cell line: Weobserved the morphological changes of PANC-1RG7through an inverted microscopeand transmission electron microscope, draw the cell growth curve and calculate thepopulation doubling time of PANC-1RG7by cell counting. Cell cycle was examinedby flow cytometry analysis (FCM). The resistance to GEM and cross-resistance toadriamycin (ADM), mitomycin C (MMC), paclitaxel (PTX), methotrexate (MTX),vincristine (VCR), gefitinib (GEF), cisplatin (DDP) and5fluorouracil (5-FU) ofPANC-1RG7cells were detected by sulforhodamine B (SRB) assay. We establishednude mouse subcutaneous tumor models to observe GEM inhibition in vivo. Theknown resistance-associated gene and protein expressions, such as multidrugresistance (MDR1) gene and its protein product P-glycoprotein (P-gp), multidrugresistance-related protein (MRP) and breast cancer resistance protein (BCRP), andfactors have been indicated to involving in drug resistance in pancreatic cancer, suchas deoxycytidine kinase (dCK), cytidine deaminase (CDA),5’-nucleotidase (NT5),equilibrative nucleoside transporters (ENT), ribonucleotide reductase (RRM) andDNA polymerase α (POLA), were analyzed by realtime fluores-cence quantitativepolymerase chain reaction (qPCR) and western blot. Western blot was used to detect the expression of PI3K, Akt and mTOR from anti-apoptotic signal transductionpathways PI3K/Akt/mTOR.3. Proteomic analysis of differential expression proteins: Differential expressionproteins between gemcitabine-resistant cell line PANC-1RG7and the parental cellline PANC-1were analyzed by isobaric tags for relative and absolute quantitation(iTRAQ) and bioinformation analysis for probably new resistance related proteins.4. Probably mechanism of cucurmosin (CUS): Western blot was used to detectthe expression of EGFR and the proteins of its downstream, while qPCR used forEGFR in PANC-1after CUS intervention. We observed the inhibition of PANC-1cellgrowth when CUS was combined with gefitinib (GEF) by SRB and colony-formingexperiments.5. Effects of CUS in PANC-1RG7: We observed the morphological changes ofPANC-1RG7after CUS intervention through a transmission electron microscope. Cellcycle was examined by FCM. Cell proliferation inhibition, synergism and resistancereversal after intervention of CUS singly or combined with GEM were observed usingby SRB and colony-forming experiments in vitro and using by nude mousesubcutaneous tumor models in vivo. The reversal index and ratio were counted.Proteins changs were analyzed by western blot after CUS intervention.Results:1. We established GEM-resistance human pancreatic cancer cell linePANC-1RG7successfully, which could endure2μmol/L GEM for more than3d withnormal proliferation and passage.2. PANC-1RG7cells had special morphological changes, significant differentfrom the parental cells. The proliferation rate of PANC-1RG7cells slowed down(p<0.05), but cell cycle didn’t changed (p>0.05). PANC-1RG7, of which theresistance index to GEM was39.9, has cross-resistance characteristics to MTX, GEF,DDP and5-FU. The proliferation inhibition of GEM was significantly reduced in vivo(p<0.05). All detected genes and proteins were not change, except CDA, MRP andBCRP genes decreased and NT5, RRM1and RRM2proteins increased (p<0.05). 3.261differential proteins were detected by iTRAQ (p<0.05). These proteinsinvolve in many biological processes (like protein folding, reorganization ofcytoskeleton, translation and transcription, synthesis and reorganization of someprotein complexes, glucose synthesis, nucleotide-related extending functions), anumber of metabolic pathways of energy and genetic material and IL22SolubleReceptor Signaling Pathway.4. Protein expressions of EGFR, Akt, mTOR, PI3K, ras and c-raf in PANC-1were decreased after CUS intervention, while gene expression of EGFR was notchanged. A stronger inhibitory effect was observed when CUS was combined withGEF.5. After CUS intervention, significant apoptosis changes in PANC-1RG7cellswere observed under an electron microscope. Cell cycle was not changed butapoptotic peak apeared. The IC50of CUS in PANC-1and PANC-1RG7were0.0535±0.0100and0.0318±0.0090μmol/L respectively. An additive action wasobserved when CUS was combined with GEM. The reversal index and ratio were1.78±0.65and50.13±16.87%respectively with0.007813μmol/L CUS. Theproliferation inhibition of CUS was significantly enhanced in vivo (p<0.05). Proteinexpressions of RRM1, RRM2, PI3K, ras and c-raf in PANC-1were decreased afterCUS intervention in a dose-dependent manner.Conclusions:1. GEM-resistance human pancreatic cancer cell line we established by graduallyincreased concentration of GEM in vitro has a unique morphological and biologycharacteristics. The resistance was acquired through overexpressions of RRM1andRRM2probably.2.261differential proteins existed between GEM-resistance human pancreaticcancer cell line PANC-1RG7and its parental cell line PANC-1. These proteinsinvolve in many biological processes, a number of metabolic pathways of energy andgenetic material and IL22Soluble Receptor Signaling Pathway.3. CUS probably can inhibit Ras/Raf and PI3K/Akt/mTOR downstreamsignaling pathways of EGFR to induce cell apoptosis. 4. CUS can enhance the sensitivity of the EGFR-targeted drug GEF.5. CUS can induce PANC-1RG7cells apoptosis through inhibitingPI3K/Akt/mTOR signaling pathway. CUS can inhibit proliferation of PANC-1RG7cells stronger than its parental cells in vitro and in vivo.6. CUS combined with GEM shows an additive effect. CUS with lowconcentration can partially reversed the resistance to GEM in PANC-1RG7, probablythrough downregulating protein expressions of RRM1and RRM2. |
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