Marie-Luise Kruse

Role of NF-κB and Akt/PI3K in the resistance of pancreatic carcinoma cell lines against gemcitabine-induced cell death

Pancreatic cancer is resistant to almost all cytotoxic drugs. Currently, gemcitabine appears to be the only clinically active drug but, because of pre-existing or acquired chemoresistance of most of the tumor cells, it failed to significantly improve the outcome of pancreatic carcinoma patients. The current study examined the relevance of nuclear factor κB (NF-κB) and PI3K/Akt in the resistance of five pancreatic carcinoma cell lines towards gemcitabine. Treatment for 24 h with gemcitabine (0.04–20 μM) led to a strong induction of apoptosis in PT45-P1 and T3M4 cells but not in BxPc-3, Capan-1 and PancTu-1 cells. These resistant cell lines exhibited a high basal NF-κB activity in contrast to the sensitive cell lines. Furthermore, gemcitabine showed a dose-dependent induction of NF-κB. At a dose of 0.04 μM, gemcitabine still induced apoptosis in the sensitive cell lines, but did not induce NF-κB. In addition, NF-κB inhibition by MG132, sulfasalazine or the IκBα super-repressor strongly diminished the resistance against gemcitabine (0.04–20 μM). In contrast to this obvious correlation between basal NF-κB activity and gemcitabine resistance, PI3K/Akt seems not to be involved in gemcitabine resistance of these cell lines. Neither did the basal Akt activity correlate with the sensitivity towards gemcitabine treatment, nor did the inhibition of PI3K/Akt by LY294002 alter gemcitabine-induced apoptosis. These results indicate that constitutive NF-κB activity confers resistance against gemcitabine and that modulation of this activity by pharmacological or genetic approaches may have therapeutical potential when combined with gemcitabine in the treatment of pancreatic carcinoma.

Tumor Stroma Interactions Induce Chemoresistance in Pancreatic Ductal Carcinoma Cells Involving Increased Secretion and Paracrine Effects of Nitric Oxide and Interleukin-1β

Pancreatic ductal carcinoma is characterized by a profound chemoresistance. As we have shown previously, these tumor cells can develop chemoresistance by interleukin (IL)-1β in an autocrine and nuclear factor-κB-dependent fashion. Because pancreatic ductal carcinoma contains many mesenchymal stromal cells, we further investigated how tumor–stroma interactions contribute to chemoresistance by using a transwell coculture model, including murine pancreatic fibroblasts and the chemosensitive human pancreatic carcinoma cell lines T3M4 and PT45-P1. If cultured with fibroblast-conditioned medium or kept in coculture with fibroblasts, both cell lines became much less sensitive toward treatment with etoposide than cells cultured under standard conditions. Furthermore, the secretion of IL-1β in T3M4 and PT45-P1 cells was increased by the fibroblasts, and IL-1β-receptor blockade abolished the resistance-inducing effect during cocultivation. This stimulated IL-1β secretion could be attributed to nitric oxide (NO) released by the fibroblasts as an IL-1β-inducing factor. Although both tumor cells secreted only little NO, which was in line with undetectable inducible nitric oxide synthase (iNOS) expression, fibroblasts exhibited significant iNOS expression and NO secretion that could be further induced by the tumor cells. Incubation of T3M4 and PT45-P1 cells with the NO donor S-Nitroso-N-acetyl-D,l-penicillamine up-regulated IL-1β secretion and conferred resistance toward etoposide-induced apoptosis. Conversely, the resistance-inducing effect of the fibroblasts was significantly abolished, when the specific iNOS inhibitor aminoguanidine was added during coculture. Immunohistochemistry on tissue sections from human pancreatic ductal carcinoma also revealed iNOS expression in stromal cells and IL-1β expression in tumor cells, thus supporting the in vitro findings. These data clearly demonstrate that fibroblasts contribute to the development of chemoresistance in pancreatic carcinoma cells via increased secretion of NO, which in turn leads to an elevated release of IL-1β by the tumor cells. These findings substantiate the implication of tumor–stromal interactions in the chemoresistance of pancreatic carcinoma.

Inhibition of Receptor Internalization by Monodansylcadaverine Selectively Blocks p55 Tumor Necrosis Factor Receptor Death Domain Signaling

The 55-kDa receptor for tumor necrosis factor (TR55) triggers multiple signaling cascades initiated by adapter proteins like TRADD and FAN. By use of the primary amine monodansylcadaverine (MDC), we addressed the functional role of tumor necrosis factor (TNF) receptor internalization for intracellular signal distribution. We show that MDC does not prevent the interaction of the p55 TNF receptor (TR55) with FAN and TRADD. Furthermore, the activation of plasmamembrane-associated neutral sphingomyelinase activation as well as the stimulation of proline-directed protein kinases were not affected in MDC-treated cells. In contrast, activation of signaling enzymes that are linked to the “death domain” of TR55, like acid sphingomyelinase and c-Jun-N-terminal protein kinase as well as TNF signaling of apoptosis in U937 and L929 cells, are blocked in the presence of MDC. The results of our study suggest a role of TR55 internalization for the activation of select TR55 death domain signaling pathways including those leading to apoptosis.

Expression of stem cell markers in human astrocytomas of different WHO grades

According to new hypotheses astrocytomas/gliomas either arise from or attract neural stem cells. Biological markers, particularly antigenic markers, have played a significant role for the characterization of these tumour stem cells (TSCc). Because these studies have been performed with single experimental samples mostly from gliomas, we investigated the expression of the stem cell markers CD133/Prominin, Nestin, Sox-2, Musashi-1, CXCR4, Flt-4/VEGFR-3 and CD105/Endoglin in 72 astrocytomas of different WHO-grades and compared it to normal adult human brain. Expression of their mRNA was quantified by quantitative RT-PCR, of their protein by counting immunopositive cells. In contrast to normal brain, tumour samples showed a high variability for the expression of all markers. However, their mean expression was significantly increased in astrocytomas, but this depended on the WHO grade only for CD133, Nestin, Sox-2 and Musashi-1. Confocal microscopy revealed that these markers mostly could be co-stained with glial fibrillary acidic protein, a marker for astoglial cells, but less frequently with the proliferation marker Ki-67/MIB-1. These markers sometimes, but not necessarily could be co-stained with each other in complex patterns. Our results show that most astrocytomas contain considerable portions of cells expressing stem cell markers. It appears that some of these cells originate from tumour genesis (supporting the stem cell hypothesis) while others are attracted by the tumours. Further functional markers are required to differentiate these TSC-types.

Inhibition of the Nrf2 transcription factor by the alkaloid trigonelline renders pancreatic cancer cells more susceptible to apoptosis through decreased proteasomal gene expression and proteasome activity

Evidence accumulates that the transcription factor nuclear factor E2-related factor 2 (Nrf2) has an essential role in cancer development and chemoresistance, thus pointing to its potential as an anticancer target and undermining its suitability in chemoprevention. Through the induction of cytoprotective and proteasomal genes, Nrf2 confers apoptosis protection in tumor cells, and inhibiting Nrf2 would therefore be an efficient strategy in anticancer therapy. In the present study, pancreatic carcinoma cell lines (Panc1, Colo357 and MiaPaca2) and H6c7 pancreatic duct cells were analyzed for the Nrf2-inhibitory effect of the coffee alkaloid trigonelline (trig), as well as for its impact on Nrf2-dependent proteasome activity and resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and anticancer drug-induced apoptosis. Chemoresistant Panc1 and Colo357 cells exhibit high constitutive Nrf2 activity, whereas chemosensitive MiaPaca2 and H6c7 cells display little basal but strong tert-butylhydroquinone (tBHQ)-inducible Nrf2 activity and drug resistance. Trig efficiently decreased basal and tBHQ-induced Nrf2 activity in all cell lines, an effect relying on a reduced nuclear accumulation of the Nrf2 protein. Along with Nrf2 inhibition, trig blocked the Nrf2-dependent expression of proteasomal genes (for example, s5a/psmd4 and α5/psma5) and reduced proteasome activity in all cell lines tested. These blocking effects were absent after treatment with Nrf2 siRNA, a condition in which proteasomal gene expression and proteasome activity were already decreased, whereas siRNA against the related transcription factor Nrf1 did not affect proteasome activity and the inhibitory effect of trig. Depending on both Nrf2 and proteasomal gene expression, the sensitivity of all cell lines to anticancer drugs and TRAIL-induced apoptosis was enhanced by trig. Moreover, greater antitumor responses toward anticancer drug treatment were observed in tumor-bearing mice when receiving trig. In conclusion, representing an efficient Nrf2 inhibitor capable of blocking Nrf2-dependent proteasome activity and thereby apoptosis protection in pancreatic cancer cells, trig might be beneficial in improving anticancer therapy.

Expression of the NF-κB target gene IEX-1 (p22/PRG1) does not prevent cell death but instead triggers apoptosis in Hela cells

P22PRG1/IEX-1 is a putative NF-κB target gene implicated in the regulation of cellular viability. Here, we show that in HeLa cells TNFα induces expression of p22PRG1/IEX-1 in an NF-κB dependent fashion. Blockade of NF-κB activation by various NF-κB inhibitors abolished TNFα-induced p22PRG1/IEX-1 expression and increased the sensitivity to apoptosis induced by TNFα, an activating Fas-antibody or the anti-cancer drug etoposide. Surprisingly, ectopic expression of p22PRG1/IEX-1 in HeLa cells transfected with an inducible p22PRG1/IEX-1-expression vector augments the susceptibility to apoptosis initiated by death-receptor ligands or by etoposide. In addition, p22PRG1/IEX-1 expressing HeLa cells exhibit an accelerated progression through the cell cycle. Transfection of an antisense hammerhead ribozyme targeted to p22PRG1/IEX-1 reduced the speed in cell cycle progression and decreased the apoptotic response to death ligands. Our data demonstrate that p22PRG1/IEX-1 is specifically induced during NF-κB activation, but this seems not to be related to the anti-apoptotic actions of NF-κB. Instead, NF-κB dependent recruitment of p22PRG1/IEX-1 might be related to a modulation in the cell cycle, and hereby, p22PRG1/IEX-1 may accelerate cell growth on the one hand, but may trigger apoptosis on the other.

Inhibition of TNF receptor 1 internalization by adenovirus 14.7K as a novel immune escape mechanism

The adenoviral protein E3-14.7K (14.7K) is an inhibitor of TNF-induced apoptosis, but the molecular mechanism underlying this protective effect has not yet been explained exhaustively. TNF-mediated apoptosis is initiated by ligand-induced recruitment of TNF receptor-associated death domain (TRADD), Fas-associated death domain (FADD), and caspase-8 to the death domain of TNF receptor 1 (TNFR1), thereby establishing the death-inducing signaling complex (DISC). Here we report that adenovirus 14.7K protein inhibits ligand-induced TNFR1 internalization. Analysis of purified magnetically labeled TNFR1 complexes from murine and human cells stably transduced with 14.7K revealed that prevention of TNFR1 internalization resulted in inhibition of DISC formation. In contrast, 14.7K did not affect TNF-induced NF-kappaB activation via recruitment of receptor-interacting protein 1 (RIP-1) and TNF receptor-associated factor 2 (TRAF-2). Inhibition of endocytosis by 14.7K was effected by failure of coordinated temporal and spatial assembly of essential components of the endocytic machinery such as Rab5 and dynamin 2 at the site of the activated TNFR1. Furthermore, we found that the same TNF defense mechanisms were instrumental in protecting wild-type adenovirus-infected human cells expressing 14.7K. This study describes a new molecular mechanism implemented by a virus to escape immunosurveillance by selectively targeting TNFR1 endocytosis to prevent TNF-induced DISC formation.

Expression of Lymphangiogenic Factors and Evidence of Intratumoral Lymphangiogenesis in Pancreatic Endocrine Tumors

Lymphangiogenesis is thought to promote the progression of malignant tumors. Because the lymphangiogenic factors vascular endothelial factor (VEGF)-C and -D are expressed in endocrine cells, we investigated their expression in pancreatic endocrine tumors (PETs) and correlated these data and intratumoral lymph vessel density (iLVD) with clinicopathological features. Lymph vessels were identified with anti-podoplanin antiserum and with podoplanin/proliferating cell nuclear antigen double labeling. PETs (n = 104) were investigated by immunohistochemical staining for VEGF, basic fibroblast growth factor, and VEGF-C expression. VEGF-C and VEGF-D mRNA were quantified by real-time reverse transcriptase-polymerase chain reaction. PETs showed higher iLVD than normal pancreata, but iLVD did not discriminate between benign and malignant PETs. In PETs proliferating lymph vessels were identified. High iLVD was associated with lymph vessel invasion and it was more frequent in angioinvasive/metastatic tumors than in grossly invasive tumors. VEGF-C expression correlated with iLVD as well as with glucagon and pancreatic polypeptide expression. PETs show intratumoral lymphangiogenesis, which is associated with VEGF-C expression in tumor cells. The association between iLVD and lymph vessel invasion and angioinvasive/metastatic features in PETs suggests that lymphangiogenesis may promote malignant progression of PETs. PET is the first human tumor entity in which VEGF-C-related intratumoral lymphangiogenesis has been demonstrated.

Cellular expression of CCK-A and CCK-B/gastrin receptors in human gastric mucosa

Gastrin stimulates gastric acid secretion in various species, but the role of the structurally related CCK for the peripheral regulation of acid secretion in humans remains controversial. Moreover, species differences in CCK receptor function and expression have been reported. We therefore sought to identify the cellular targets of CCK and gastrin within the human gastric mucosa in situ. Gastric biopsies were collected from 15 patients without gastric disease. Expression of CCK receptor subtypes was detected in individual cells of the gastric mucosa by reverse transcription (RT)-PCR in situ, immunohistochemistry and confocal laser scanning microscopy, using antisera against the CCK-A or CCK-B/gastrin receptor subtype. Both CCK-A and CCK-B receptors were detected in antral and oxyntic mucosa at the mRNA and protein level. In fundic mucosa, CCK-A receptor mRNA and protein mapped to D cells (37.4+/-7.7). Besides, individual chief cells, mucous neck cells and parietal cells (12.3+/-4.7%) expressed CCK-A receptors. CCK-B/gastrin receptor mRNA and protein were detected in parietal cells (57.4+/-11.1%) and in neuroendocrine cells (33.2+/-4.4%) expressing chromogranin A. Furthermore, epithelial cells within the neck of the gastric gland were found to express the CCK-B/gastrin receptor. We conclude that (i) identification of CCK-A receptors on somatostatin producing D cells in humans provide the anatomical basis for a receptor-mediated mode of action of CCK on somatostatin release and (ii) detection of either CCK receptor subtype in the putative stem cell compartment implies a role of CCK in the maintenance of tissue homeostasis in human gastric mucosa.

TGFβ1 autocrine growth control in isolated pancreatic fibroblastoid cells/stellate cells in vitro

TGFbeta1 is a multifunctional factor, controlling cellular growth and extracellular matrix production. Deletion of the TGFbeta1 gene in mice results in multiple inflammatory reactions. Targeted overexpression of TGFbeta1 in pancreatic islet cells leads to fibrosis of the exocrine pancreas in transgenic mice. In pancreatic fibrosis interstitial fibroblasts are primary candidates for production and deposition of extracellular matrix. Still, little is known about regulation of these cells during development of pancreatic disease. We established primary cell lines of pancreatic fibroblastoid/stellate cells (PFC) from rat pancreas. Investigation of rPFCs in vitro shows TGFbeta1 expression by RT-PCR analysis. Mature TGFbeta1 was detected in culture supernatants by immunoassay. Rat PFCs in culture possess both receptors TGFbeta receptor type I, and type II, necessary for TGFbeta1 signal transduction. Inhibition of TGFbeta1 activity by means of neutralizing antibodies interferes with an autocrine loop and results in a 2-fold stimulation of cell growth. So far, pancreatic fibroblastoid/stellate cells in vitro were known as a target of TGFbeta1 action, but not as a source of TGFbeta1. Our data indicate TGFbeta1 activity in rat pancreas extends beyond regulation of matrix production, but appears to be important in growth control of pancreatic fibroblastoid cells.