Activation of K-ras and inactivation of p16 are the most frequently identified genetic alterations in human pancreatic epithelial adenocarcinoma (PDAC). Mouse models engineered with mutant K-ras and deleted p16 recapitulate key pathological features of PDAC. However, a human cell culture transformation model that recapitulates the human pancreatic molecular carcinogenesis is lacking. In this study, we investigated the role of p16 in hTERT-immortalized human pancreatic epithelial nestin-expressing (HPNE) cells expressing mutant K-ras (K-rasG12V). We found that expression of p16 was induced by oncogenic K-ras in these HPNE cells and that silencing of this induced p16 expression resulted in tumorigenic transformation and development of metastatic PDAC in an orthotopic xenograft mouse model. Our results revealed that PI3K/Akt, ERK1/2 pathways and TGF? signaling were activated by K-ras and involved in the malignant transformation of human pancreatic cells. Also, p38/MAPK pathway was involved in p16 up-regulation. Thus, our findings establish an experimental cell-based model for dissecting signaling pathways in the development of human PDAC. This model provides an important tool for studying the molecular basis of PDAC development and gaining insight into signaling mechanisms and potential new therapeutic targets for altered oncogenic signaling pathways in PDAC.
The most common genetic lesions in pancreatic ductal adenocarcinoma (PDAC) have been identified. However, significant gaps still exist in our understanding of how such genetic alterations act in concert to induce PDAC development. In this study, we investigated the mechanism of tumorigenic transformation in the immortalized human pancreatic ductal epithelial (HPDE) cell line by sequentially introducing PDAC signature alterations into this cell line.
Feedback regulation of transcription factor NF-kappaB by its inhibitor IkappaBalpha plays an essential role in control of NF-kappaB activity. To understand the biological significance of IkappaBalpha-mediated feedback regulation of NF-kappaB, we generated mice harboring mutated kappaB enhancers in the promoter of the IkappaBalpha gene (IkappaBalpha(M/M)) to inhibit NF-kappaB-regulated IkappaBalpha expression. Here, we report that these mutant mice are defective in NF-kappaB-induced expression of IkappaBalpha. This defective feedback regulation of NF-kappaB by IkappaBalpha not only altered activity of NF-kappaB, but also the expression of NF-kappaB-regulated genes. As a result, IkappaBalpha(M/M), the homozygous knock-in mice with mutated kappaB enhancers in the IkappaBalpha promoter, acquire shorten life span, hypersensitivity to septic shock, abnormal T-cell development and activation, and Sjögrens Syndrome. These findings therefore demonstrate that the IkappaBalpha-mediated feedback regulation of NF-kappaB has an essential role in controlling T-cell development and functions, provide mechanistic insight into the development of Sjögrens Syndrome, and suggest the potential of NF-kappaB signaling as a therapeutic target for Sjögrens Syndrome and other autoimmune diseases.
Many genetic and molecular alterations, such as K-ras mutation and NF-kappaB activation, have been identified in pancreatic cancer. However, the mechanisms by which pancreatic cancer metastasizes still remain to be determined. Although we previously showed that the tropomyosin-related kinase B (TrkB) was significantly correlated with the development of liver metastasis, its function in pancreatic cancer metastasis remained unresolved. In the present study, we showed that overexpressed TrkB is an alternatively spliced transcript variant of TrkB (TrkBT1) with a unique COOH-terminal 12-amino acid sequence and is mainly localized in the cytoplasm. Our results showed that overexpression of Flag-tagged TrkBT1 but not a Flag-tagged TrkBT1 COOH-terminal deletion mutant (Flag-TrkBT1DeltaC) in nonmetastatic pancreatic cancer cells enhanced cell proliferation, promoted formation of colonies in soft agar, stimulated tumor cell invasion, and induced liver metastasis in an orthotopic xenograft mouse model of pancreatic cancer. TrkBT1 interacted with Rho GDP dissociation inhibitor (GDI) in vivo, but Flag-TrkBT1DeltaC did not. Furthermore, overexpression of Flag-TrkBT1 and knockdown of RhoGDI expression by RhoGDI short hairpin RNAs promoted RhoA activation, but Flag-TrkBT1DeltaC overexpression did not. Therefore, our results showed that TrkBT1 overexpression induces liver metastasis of pancreatic cancer and uncovered a unique signaling mechanism by which TrkBT1 sequesters GDI and activates RhoA signaling.
The aim of this study was to investigate if there were any effects on the electroencephalogram (EEG) of human brain by the manual stimulation of Neiguan (PC 6) acupuncture site. In this paper, two groups of six healthy male volunteers of ages 27.6 +/- 14.2 (mean +/- SD) and 28.5 +/- 13.0 (mean +/- SD) and no neurological disease participated in this study. A digital storage of 12-channel EEG recorder was used and spectral analyses of the data set of 18 trials were obtained before, during, and after sham/ manual acupuncture. To minimize artefacts, all data were collected with the subjects alert but eyes closed. No significant changes (P > 0.05) were obtained for the sham acupuncture group. As for the manual acupuncture group, the needle was inserted perpendicularly into the PC 6 acupuncture site and manually stimulated about 15 to 30 seconds to achieve De Qi sensation. Needles were left in place for 30 min and then removed. Analysis of the EEG data due to acupuncture was compared to the baseline data and changes were obtained. First, all trials had an increase in the amplitude and power of the alpha band during manual acupuncture (P < 0.05) when compared with the baseline data. Secondly, in the mean time, the frequency peaks in alpha band of 12-channels were all synchronized with much smaller standard deviation (P < 0.01). Thirdly, the manual acupuncture effects of higher power and synchronized frequencies persisted for at least 10 minutes after the experiment (P < 0.05) and did not disappear immediately for all 18 experiments. Finally, we hypothesized that the higher power and synchronized rhythms in brain oscillations may have to do with autonomic nervous system.
Transcription factor nuclear factor-kappaB (NF-kappaB) is constitutively activated in most pancreatic cancer tissues and cell lines but not in normal pancreas nor in immortalized/nontumorigenic human pancreatic ductal epithelial cells. Inhibition of constitutive NF-kappaB activation in pancreatic cancer cell lines suppresses tumorigenesis and tumor metastasis. Recently, we identified autocrine secretion of proinflammatory cytokine interleukin (IL)-1alpha as the mechanism of constitutive NF-kappaB activation in metastatic pancreatic cancer cell lines. However, the role of IL-1alpha in determining the metastatic potential of pancreatic tumor remains to be further investigated. In the current study, we stably expressed IL-1alpha in the nonmetastatic, IL-1alpha-negative MiaPaCa-2 cell lines. Our results showed that the secretion of IL-1alpha in MiaPaCa-2 cells constitutively activated NF-kappaB and increased the expression of NF-kappaB downstream genes involved in the different steps of the metastatic cascade, such as urokinase-type plasminogen activator, vascular endothelial growth factor, and IL-8. MiaPaCa-2/IL-1alpha cells showed an enhanced cell invasion in vitro compared with parental MiaPaCa-2 cells and induced liver metastasis in an orthotopic mouse model. The metastatic phenotype induced by IL-1alpha was inhibited by the expression of phosphorylation-defective IkappaB (IkappaB S32, 36A), which blocked NF-kappaB activation. Consistently, silencing the expression of IL-1alpha by short hairpin RNA in the highly metastatic L3.6pl pancreatic cancer cells completely suppressed their metastatic spread. In summary, these findings showed that IL-1alpha plays key roles in pancreatic cancer metastatic behavior through the constitutive activation of NF-kappaB. Our findings further support the possible link between inflammation and cancer and suggest that IL-1alpha may be a potential therapeutic target for treating pancreatic adenocarcinoma.
A novel tubular waveguide particle plasmon resonance (TW-PPR) sensor is demonstrated for label-free biochemical detection. The sensor itself is a microchamber of a defined sample volume, a mechanical support for sensor coating, a waveguide to provide evanescent wave interrogation, and it can be easily extended to a multi-channel format. The sensor resolution is estimated to be 2.6×10(-6) RIU in measuring solutions of various refractive indices. The sensing system can perform multiple measurements simultaneously and its limit of detection for anti-DNP antibody and streptavidin is 1.2×10(-10) g/ml (0.55 pM) and 2.3×10(-10) g/ml (3.5 pM), respectively. Accurate determination of these analytes with known concentration spiked in artificial urine were examined and the bias is less than ±7%, supporting the utility of the device for analyte screening in more complex media. The TW-PPR sensor can be inexpensively fabricated and has a special niche for monitoring biomolecular interactions in real-time, hence it is ideally suitable for disposable uses, especially promising for convenient high-throughput biochemical sensing applications.
Dynamics of resonant energy transfer of a single excitation in a molecular dimer system are studied in the simultaneous presence of diagonal and off-diagonal exciton-phonon coupling. It is found that, at given temperatures, the off-diagonal coupling can enhance both the coherence of the resonant energy transfer and the net quantity of energy transferred from an initially excited monomer to the other. Also studied is the dynamics of entanglement between the dimer system and the phonon bath as measured by the von Neumann entanglement entropy, and the inter-monomer entanglement dynamics for the excitonic system.
Constitutive Kras and NF-?B activation is identified as signature alterations in pancreatic ductal adenocarcinoma (PDAC). However, how NF-?B is activated in PDAC is not yet understood. Here, we report that pancreas-targeted IKK2/? inactivation inhibited NF-?B activation and PDAC development in Kras(G12D) and Kras(G12D);Ink4a/Arf(F/F) mice, demonstrating a mechanistic link between IKK2/? and Kras(G12D) in PDAC inception. Our findings reveal that Kras(G12D)-activated AP-1 induces IL-1?, which, in turn, activates NF-?B and its target genes IL-1? and p62, to initiate IL-1?/p62 feedforward loops for inducing and sustaining NF-?B activity. Furthermore, IL-1? overexpression correlates with Kras mutation, NF-?B activity, and poor survival in PDAC patients. Therefore, our findings demonstrate the mechanism by which IKK2/?/NF-?B is activated by Kras(G12D) through dual feedforward loops of IL-1?/p62.
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