ABSTRACT
AIM: To examine whether beta-adrenoceptor (beta-AR) agonists can induce hypoxia-inducible factor (HIF)-1alpha accumulation which then up-regulate the expression of its target genes in pancreatic cancer cells at normoxia, and to further elucidate the mechanism involved. METHODS: Pulse-chase assay, RT-PCR, and Western blot were employed to detect the effects of beta-AR agonists and antagonists, siRNA as well as several inhibitors of signal transduction pathways on MIA PaCa2 and BxPC-3 pancreatic cancer cells. RESULTS: Treatment of pancreatic cancer cell lines with beta-AR agonists led to accumulation of HIF-1alpha and then up-regulated expression of its target genes independently of oxygen levels. The induction was partly or completely inhibited not only by beta-AR antagonists but also by inhibitors of PKA transduction pathways and by siHIF-1alpha. Both beta1-AR and beta2-AR agonists produced the above-mentioned effects, but beta2-AR agonist was more potent. CONCLUSION: Activation of beta-AR receptor transactivates epidermal growth factor receptor (EGFR) and then elicits Akt and ERK1/2 in a PKA-dependent manner, which together up-regulate levels of HIF-1alpha and downstream target genes independently of oxygen level. Our data suggest a novel mechanism in pancreatic cancer cells that links beta-AR and HIF-1alpha signaling under normoxic conditions, with implications for the control of glucose transport, angiogenesis and metastasis.
Subject(s)
Adrenergic beta-Agonists/pharmacology , Hypoxia-Inducible Factor 1, alpha Subunit/drug effects , Pancreatic Neoplasms/metabolism , Adrenergic beta-1 Receptor Agonists , Adrenergic beta-2 Receptor Agonists , Blotting, Western , Cell Line, Tumor , Cyclic AMP-Dependent Protein Kinases/metabolism , ErbB Receptors/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA, Small Interfering/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Up-Regulation/drug effectsABSTRACT
Stimulation of mice dorsal root ganglion neurons (DRGNs) activity by human pancreatic cancer (PanCa) cell line Mia PaCa-2 and its potential molecule mechanism has been investaged. DRGNs were cultured alone or along with the MIA PaCa-2. The effects of MIA PaCa-2 to DRGNs were determined by neurofilament (NF) immunocytochemical and Nissl staining. ELISA was used to detect the concentration of insulin-like growth factor-1 (IGF-1) in the culture supernatant. Cyton size, neurite outgrowth and neuronal activity in the experimental group were greater than in the control groups. However, the concentration of IGF-1 in the supernatants was not significantly different from those in the blank and non-cultured medium groups. In the presence of MIA PaCa-2 cell line, cyton size, neurite outgrowth and neuronal activity were enhanced, which may provide more routes for the invasion of cancer cells along nerves.
Subject(s)
Cell Communication/physiology , Ganglia, Spinal/metabolism , Growth Cones/metabolism , Insulin-Like Growth Factor I/metabolism , Neoplasm Invasiveness/physiopathology , Sensory Receptor Cells/metabolism , Animals , Animals, Newborn , Carcinoma/metabolism , Carcinoma/physiopathology , Cell Enlargement/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cell Survival/physiology , Culture Media, Conditioned/pharmacology , Ganglia, Spinal/cytology , Ganglia, Spinal/drug effects , Growth Cones/drug effects , Growth Cones/ultrastructure , Humans , Mice , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/physiopathology , Peripheral Nerves/metabolism , Peripheral Nerves/pathology , Peripheral Nerves/physiopathology , Sensory Receptor Cells/cytology , Sensory Receptor Cells/drug effectsABSTRACT
AIM: To discuss the expression of alpha-adrenoreceptors in pancreatic cancer cell lines PC-2 and PC-3 and the effects of alpha1- and alpha2-adrenoreceptor antagonists, yohimbine and urapidil hydrochloride, on the cell lines in vitro. METHODS: We cultured the human ductal pancreatic adenocarcinoma cell lines PC-2 and PC-3 and analyzed the mRNA expression of alpha1- and alpha2-adrenergic receptors by reverse transcription polymerase chain reaction (RT-PCR). The effects of yohimbine and urapidil hydrochloride on cell proliferation were assessed by 3-(4,5-dimethylthiasol-2-yl)-2,4,-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected using the terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labeling (TUNEL) assay and flow cytometry (FCM). RESULTS: PC-2 expressed mRNA in alpha1- and alpha2-adrenoreceptors. MTT assays showed that urapidil hydrochloride had no effect on PC-3 cell lines. However, exposure to urapidil hydrochloride increased DNA synthesis in PC-2 cell lines as compared to the control group. PC-2 cell lines were sensitive to both drugs. The proliferation of the 2 cell lines was inhibited by yohimbine. Cell proliferation was inhibited by yohimbine via apoptosis induction. CONCLUSION: The expression of alpha1- and alpha2-adrenoreceptors is different in PC-2 and PC-3 cell lines, which might be indicative of their different functions. The alpha2-adrenoceptor antagonist, yohimbine, can inhibit the proliferation of both cell lines and induce their apoptosis, suggesting that yohimbine can be used as an anticancer drug for apoptosis of PC-2 and PC-3 cells.
Subject(s)
Adrenergic alpha-2 Receptor Antagonists , Adrenergic alpha-Antagonists/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Proliferation/drug effects , Pancreatic Neoplasms/pathology , Yohimbine/pharmacology , Adrenergic alpha-1 Receptor Antagonists , Cell Line, Tumor , DNA Replication/drug effects , Dose-Response Relationship, Drug , Flow Cytometry , Gene Expression Regulation, Neoplastic , Humans , In Situ Nick-End Labeling , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Piperazines/pharmacology , RNA, Messenger/metabolism , Receptors, Adrenergic, alpha-1/metabolism , Receptors, Adrenergic, alpha-2/genetics , Receptors, Adrenergic, alpha-2/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
AIM: To construct the replication-incompetent recombinant adenovirus mediated shRNA to inhibit the neurite growth-promoting factor (Pleiotrophin, PTN) in pancreatic carcinoma and to study the inhibitory effect of shRNA-PTN recombinant adenoviruses on the neurite's growth of dorsal root ganglion neurons (DRGn). METHODS: Four pairs of complementary single-stranded oligonucleotides (ss oligo) were designed and synthesized and then they were annealed to create a double-stranded oligonucleotide (ds oligo). The ds oligos were cloned into pENTR/U6 vector to produce the shuttle plasmid pENTR/U6-shRNA, which was transduced into pancreatic carcinoma cells by liposome after sequencing. The plasmid with good silence effect was chosen by RT-PCR to perform the LR recombination reaction to the adenovirus backbone plasmid. The expression clone was transfected into HEK293A cell to produce adenovirus. The silence of the recombinant adenovirus against PTN was detected by Western blot. After DRGn was co-cultured with pc-2 cell infected by shRNA-PTN recombinant adenovirus, the morphological changes of DRGn were observed. RESULTS: The pENTR/U6-shRNA shuttle plasmid was constructed and confirmed by sequencing. The recombinant adenovirus mediated shRNA against PTN was constructed. The best silence effect of the adenovirus against PTN was detected by Western blot on the 7th day after the pancreatic carcinoma cell was transfected. The growth of DRGn neurites was inhibited after the co-culture of DRGn with pc-2 cell which was infected by shRNA-PTN recombinant adenovirus. CONCLUSION: The PTN SiRNA recombinant adenovirus has been constructed and the silence effect against PTN in pancreatic carcinoma cell has been confirmed. The growth of DRGn neurites can be inhibited by shRNA-PTN recombinant adenovirus.
