ABSTRACT
Smoking and chronic stress are well-documented risk factors that are associated with ß-adrenoceptors in the development of pancreatic cancer. Stimulation of ß-adrenoceptors can activate cyclic adenosine monophosphate (cAMP)/ protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) pathways in pancreatic cancer cells. Many recent studies have focused on the function of ß-adrenoceptors in cancer invasion. Thus, we hypothesized that ß-adrenoceptors may play a role in pancreatic cancer invasion, and ß-blockers may suppress the pancreatic cancer invasion and proliferation. MIA PaCa-2 and BxPC-3 cell lines express mRNA and protein of both ß1 and ß2-adrenoceptors. ß2-adrenergic antagonist ICI118,551 and ß1/2-adrenergic antagonist propranolol significantly suppressed cell invasion and proliferation in comparison to ß1-adrenergic antagonist metoprolol and control in a Matrigel invasion assay and subrenal capsular assay. Treatment with ß2-adrenoceptor antagonists inhibited activation of transcription factors nuclear factor κB (NF-κB), activator protein 1 (AP-1) and cAMP response element binding protein (CREB) as demonstrated by electrophoretic mobility shift assays and Western blotting. ß2-adrenoceptor antagonists also significantly altered vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2), matrix metalloproteinase 2 (MMP-2) and MMP-9 expression. The ß2-adrenergic antagonists suppressed invasion and proliferation by inhibiting both cAMP/PKA and Ras, which regulate activation of the MAPK pathway and transcription factors, such as NF-κB, AP-1 and CREB, as well as expression of its target genes, MMP-9, MMP-2 and VEGF. However, ß1-adrenergic antagonists suppressed invasion by inhibiting only the cAMP/PKA pathway, suggesting that they may be useful as novel preventive and therapeutic strategies for pancreatic cancer.
Subject(s)
Adrenergic beta-2 Receptor Antagonists/pharmacology , Cyclic AMP Response Element-Binding Protein/antagonists & inhibitors , NF-kappa B/antagonists & inhibitors , Pancreatic Neoplasms/drug therapy , Propanolamines/pharmacology , Transcription Factor AP-1/antagonists & inhibitors , Animals , Blotting, Western , Cell Line, Tumor , Cell Proliferation , Cyclic AMP Response Element-Binding Protein/metabolism , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Electrophoretic Mobility Shift Assay , Enzyme-Linked Immunosorbent Assay , Humans , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , NF-kappa B/metabolism , Neoplasm Invasiveness , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , RNA, Messenger/genetics , Receptors, Adrenergic, beta/genetics , Receptors, Adrenergic, beta/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factor AP-1/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
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 study the variabilities of serum proteomic spectra in patients with gastric cancer before and after operation in order to detect the specific protein markers that can be used for quick diagnosis of gastric cancer. METHODS: Proteomic spectra of 46 serum samples from patients with gastric cancer before and after operation and 40 from normal individuals were generated by IMAC-Cu protein chip and surface-enhanced laser desorption/ ionization time of flight mass spectrometry. RESULTS: Fourteen differentially expressed proteins in serum were screened by analysis of proteomic spectra of preoperative patients and normal individuals. We obtained 4 proteins (heat shock protein 27, glucose-regulated protein, prohibitin, protein disulfide isomerase A3) making up marker pattern which was able to class the patient-team and normal-team. These marker patterns yielded 95.7% sensitivity and 92.5% specificity, respectively. The proteins over-expressed in serum of preoperative patients were obviously down-regulated. CONCLUSION: Specific protein markers of gastric cancer can be used for the quick diagnosis of gastric cancer and judgment of prognosis. SELDI-TOF-MS is a useful tool for the detection and identification of new protein markers in serum.
