Maike Witt

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.

Drug-induced expression of the cellular adhesion molecule L1CAM confers anti-apoptotic protection and chemoresistance in pancreatic ductal adenocarcinoma cells

Pancreatic ductal adenocarcinoma (PDAC) is characterized by rapid tumor progression, high metastatic potential and profound chemoresistance. We recently reported that induction of a chemoresistant phenotype in the PDAC cell line PT45-P1 by long-term chemotherapy involves an increased interleukin 1 beta (IL1β)-dependent secretion of nitric oxide (NO) accounting for efficient caspase inhibition. In the present study, we elucidated the involvement of L1CAM, an adhesion molecule previously found in other malignancies, in this NO-dependent chemoresistance. Chemoresistant PT45-P1res cells, but not chemosensitive parental PT45-P1 cells, express high levels of L1CAM in an ILβ-dependent fashion. PT45-P1res cells subjected to short interfering RNA (siRNA)-mediated L1CAM knock-down exhibited reduced inducible nitric oxide synthase expression and NO secretion, as well as a significant increase of anti-cancer drug-induced caspase activation, an effect reversed by the NO donor S-nitroso-N-acetyl-D,L-penicillamine. Conversely, overexpression of L1CAM in PT45-P1 cells conferred anti-apoptotic protection to anti-cancer drug treatment. Interestingly, L1CAM ectodomain shedding, in example, by ADAM10, as reported for other L1CAM-related activities, seemed to be dispensable for anti-apoptotic protection by L1CAM. Neither the shedded L1CAM ectodomain was detected in chemoresistant L1CAM-expressing PT45-P1 cells nor did the administration of various metalloproteinase inhibitors affect L1CAM-dependent chemoresistance. Immunohistochemical analysis revealed L1CAM expression in 80% of pancreatic cancer specimens, supporting a potential role of L1CAM in the malignancy of this tumor. These findings substantiate our understanding of the molecular mechanisms leading to chemoresistance in PDAC cells and indicate the importance of L1CAM in this scenario.

Increased Expression of the E3-Ubiquitin Ligase Receptor Subunit βTRCP1 Relates to Constitutive Nuclear Factor-κB Activation and Chemoresistance in Pancreatic Carcinoma Cells

The permanent activation of the transcription factor nuclear factor-kappaB (NF-kappaB) in pancreatic cancer cells is associated with a profound resistance towards chemotherapy. In the present study, we show that chemoresistant pancreatic cancer cell lines exhibiting constitutive NF-kappaB activity (i.e., PancTu-1, BxPc3, and Capan-1) express significantly elevated levels of the E3-ubiquitin ligase receptor subunit betaTRCP1, compared with pancreatic carcinoma cell lines lacking constitutive NF-kappaB activity and chemoresistance (i.e., PT45-P1 and T3M4). If transfected with betaTRCP1, PT45-P1 cells exhibit an elevated NF-kappaB activity and become less sensitive towards anticancer drug treatment (i.e., etoposide). Conversely, blockade of betaTRCP1 expression in PancTu-1 cells by transfection with a vector-expressed small interfering RNA reduces NF-kappaB activation and chemoresistance. In PancTu-1 cells, betaTRCP1 expression is inhibited, at least in part, by the interleukin-1 (IL-1) receptor(I) antagonist, whereas stimulation of PT45-P1 cells with IL-1beta resulted in an increased expression of betaTRCP1, and transfection of this cell line with betaTRCP1 induced IL-1beta secretion in a NF-kappaB-dependent fashion. Thus, via its close and mutual link to IL-1beta secretion, betaTRCP1 expression might substantially contribute to the persistent, IL-1beta-dependent activation of NF-kappaB in pancreatic carcinoma cells. In support of this, betaTRCP1 expression is detectable at considerable levels in a great number of pancreatic ductal adenocarcinoma specimens, along with an intense staining for activated NF-kappaB. Altogether, our findings of the elevated betaTRCP1 expression in pancreatic carcinoma cells pinpoint to another important mediator of constitutive NF-kappaB activation and thereby of chemoresistance.

Usage of the NF-κB inhibitor sulfasalazine as sensitizing agent in combined chemotherapy of pancreatic cancer

Sulfasalazine is commonly used as an anti inflammatory agent and is known as a potent inhibitor of NF‐κB. Some pancreatic carcinomas are characterized by a constitutively elevated NF‐κB activity accounting for chemoresistance. To elucidate whether blockade of NF‐κB activity with sulfasalazine is suitable for overcoming this chemoresistance in vivo, we employed a mouse model with subcutaneously xenotransplanted human Capan‐1 pancreatic carcinoma cells. Fourteen days upon tumor inoculation, animals were randomized in 6 groups, receiving no treatment, treatment with gemcitabine or with etoposide, either alone or in combination with sulfasalazine, or with sulfasalazine alone. Two therapy regimens were given with a 7‐day interval in between. Upon treatment with etoposide or gemcitabine alone, tumor sizes were moderately reduced to 65–68% and 50–65%, respectively, as compared to untreated tumors. Sulfasalazine alone only decreased temporarily the tumor sizes. Sulfasalazine in combination with gemcitabine showed only partially higher reduction in tumor sizes than gemcitabine alone, whereas the combination with etoposide reduced significantly the tumor sizes in all experiments (down to 20%). TUNEL‐staining showed higher numbers of apoptotic cells in tumors from the combination groups, in particular with etoposide, and proliferation as indicated by Ki67 staining was strongly reduced. Furthermore, combined treatment of sulfasalazine with the cytostatic drugs led to a decreased blood vessel density. Immunohistochemical staining of the activated p65 subunit showed that sulfasalazine treatment abolished the basal NF‐κB activity in tumor xenografts. These data imply that the well established anti‐inflammatory drug sulfasalazine sensitizes pancreatic carcinoma cells to anti cancer drugs, in particular to etoposide in vivo by inhibition of NF‐κB. This combined chemotherapy offers great potential for improved anti‐tumor responses in pancreatic carcinomas.

GLP-1/GIP chimeric peptides define the structural requirements for specific ligand-receptor interaction of GLP-1

The gastrointestinal hormones glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) strongly stimulate insulin release. Despite their high N-terminal sequence similarity, GLP-1 does not bind to the GIP receptor and vice versa. To characterize the domains required for interaction of the peptide ligands with their specific receptors, we performed displacement studies with various synthetic GLP-1/GIP hybrid peptides on RINm5F insulinoma cells. Displacement of 125I-GIP and 125I-GLP-1 was measured using GLP-1/GIP chimeras which comprised GIP and GLP-1 sequences at different positions. The binding affinity to the GLP-1 receptor was found to be sensitive to GIP-like exchanges in the N-terminal 22 amino acids as well as in positions 13 and 15 (loss of affinity 280-fold to more than 1000-fold). C-terminal substitution of the GLP-1 sequence by GIP diminished the affinity towards the GLP-1 receptor only 20-fold. All hybrid peptides investigated showed minimal binding affinity for the GIP receptor, indicating that the entire GIP-sequence (1-31) is important for receptor recognition. These findings provide insight into the structural requirements for the specific interaction of two important insulinotropic peptides with their specific receptors.

Acquired chemoresistance in pancreatic carcinoma cells: induced secretion of IL-1|[beta]| and NO lead to inactivation of caspases

Correction to: Oncogene (2006) 25, 3973–3981. doi:10.1038/sj.onc.1209423 Since publication of the above manuscript, the authors have identified errors in Figure 6b and c. The revised version of the figure is given below.

The apoptosis-inducing effect of gastrin on colorectal cancer cells relates to an increased IEX-1 expression mediating NF-|[kappa]|B inhibition

Addressing the puzzling role of amidated gastrin17 (G17) and the gastrin/CCKB/CCK2 receptor in colorectal carcinogenesis, we analysed potential candidate genes involved in G17-dependent NF-κB inhibition and apoptosis. The colorectal carcinoma cell line Colo320 overexpressing the wild-type CCK2 receptor (Colo320wt) underwent G17-induced apoptosis along with suppressed NF-κB activation and decreased expression of the antiapoptotic NF-κB target genes cIAP1 and cIAP2, whereas G17 was without effect on Colo320 cells expressing a CCK2 receptor bearing a loss of function mutation (Colo320mut). Gene microarray analysis revealed an elevated expression of the stress response gene IEX-1 in G17-treated Colo320wt but not Colo320mut cells. Quantitative real-time PCR and conventional RT–PCR confirmed this G17-dependent increase of IEX-1 expression in Colo320wt cells. If these cells were subjected to IEX-1 knockdown by small interfering RNA transfection, the apoptosis-inducing effect of G17 was abolished. Moreover, tumor necrosis factor alpha (TNFα)- or 5-FU-induced apoptosis that is greatly enhanced by G17 treatment in Colo320wt cells was prevented if IEX-1 expression was repressed. Under these conditions of blocked IEX-1 expression, the NF-κB activity remained unaffected by G17, in particular in Colo320wt cells co-treated with TNFα and also the suppressive effect of G17 on cIAP1 and cIAP2 expression was not observed anymore if IEX-1 expression was blocked. Conversely, IEX-1 overexpression in Colo320mut cells caused an increase of basal and TNFα- or 5-FU-induced apoptosis, an effect not further triggered by G17 treatment. Using a xenograft tumor model in severe combined immune deficiency mice, we could show that experimental systemic hypergastrinemia induced by the administration of omeprazole led to enhanced apoptosis as well as to a marked increase of IEX-1 expression in Colo320wt tumors, but not in Colo320mut tumors. These observations indicate that the proapoptotic effect of G17 on human colon cancer cells expressing the wild-type CCK2 receptor is mediated by IEX-1, which modulates NF-κB-dependent antiapoptotic protection and thereby exerts tumor-suppressive potential.

[Autocrine IL-1beta secretion leads to NF-kappabeta-mediated chemoresistance in pancreatic carcinoma cells in vivo].

Zusammenfassung.Hintergrund und Ziel:Das duktale Pankreaskarzinom (PDAC) ist durch einen hochmalignen Phänotyp und eine profunde Chemoresistenz charakterisiert. In der vorliegenden Studie wurde die autokrine Sekretion von Interleukin-(IL)1β durch Pankreaskarzinomzellen in vivo als eine Ursache für einen chemoresistenten Phänotyp untersucht.Material und Methodik:Zellen der humanen PDAC-Zelllinie PancTu1 mit ausgeprägter IL-1β-Sekretion wurden subkutan in weibliche SCID-Mäuse inokuliert. Nach 10 Tagen wurden die Tiere randomisiert und entweder unbehandelt gelassen oder für 14 Tage mit einem IL-1β-RI-Antikörper, mit Etoposid oder einer Kombination aus beidem behandelt. Anschließend wurden die Tumorgrößen bestimmt und die Tumoren immunhistologisch auf Apoptose, Vaskularisierung sowie die Aktivität des Transkriptionsfaktors NF-κB untersucht.Ergebnisse:Die Kombination aus IL-1β-RI-Antikörper und Etoposid führte im Vergleich zu den entsprechenden Monotherapien bzw. keiner Behandlung zu einem deutlich verringerten Wachstum der PancTu1-Tumoren. Entsprechend konnte in Tumoren der Kombinationsbehandlung eine signifikant erhöhte Anzahl an apoptotischen Zellen detektiert werden. Nach der Behandlung mit dem IL-1β-RI-Antikörper wiesen die Tumoren eine deutlich verminderte Präsenz von aktiviertem NF-κB im Vergleich zu den Kontrolltumoren auf. Ferner war in den Tumoren nach Kombinationstherapie die Vaskularisierung (CD31-positive Zellen) weniger stark ausgeprägt.Schlussfolgerung:Die autokrine Sekretion von IL-1β ist an der Entstehung konstitutiver NF-κB-Aktivität und einer dadurch vermittelten Chemoresistenz in Pankreaskarzinomzellen beteiligt.Abstract.Background and Purpose:The pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly malignant phenotype and a profound chemoresistance. Thus, options for an effective treatment of this disease are still quite poor. In this study, it was investigated whether the autocrine secretion of interleukin-(IL-)1β is related to a chemoresistant phenotype of PDAC cells in vivo.Material and Methods:Human PancTu1 PDAC cells were inoculated subcutaneously into female SCID mice. After 10 days of outgrowth, animals were randomized and left untreated or treated with an IL-1β-RI antibody, etoposide, or a combination of both. After treatment for 14 days, tumor sizes were determined and each tumor was analyzed immunohistochemically for apoptosis (TUNEL), activated NF-κB (p65), and vascularization (CD31 staining).Results:The combination of IL-1β-RI antibody and etoposide led to a significantly reduced outgrowth of PancTu1 tumors in comparison to the monotherapies or no treatment. Accordingly, the number of apoptotic cells was significantly elevated in tumors of the combination group. After treatment with the IL-1β-RI antibody, less activated NF-κB was present in tumors compared to the control group. Moreover, tumors of the combination group showed a clearly reduced vascularization.Conclusion:The autocrine secretion of IL-1β contributes to a constitutively increased NF-κB activity in PDAC cells along with a chemoresistant phenotype.

Autokrine IL-1β-Sekretion führt zu erhöhter NF-κB-Aktivität und zu Chemoresistenz in Pankreaskarzinomzellen in vivo

Zusammenfassung.Hintergrund und Ziel:Das duktale Pankreaskarzinom (PDAC) ist durch einen hochmalignen Phänotyp und eine profunde Chemoresistenz charakterisiert. In der vorliegenden Studie wurde die autokrine Sekretion von Interleukin-(IL)1β durch Pankreaskarzinomzellen in vivo als eine Ursache für einen chemoresistenten Phänotyp untersucht.Material und Methodik:Zellen der humanen PDAC-Zelllinie PancTu1 mit ausgeprägter IL-1β-Sekretion wurden subkutan in weibliche SCID-Mäuse inokuliert. Nach 10 Tagen wurden die Tiere randomisiert und entweder unbehandelt gelassen oder für 14 Tage mit einem IL-1β-RI-Antikörper, mit Etoposid oder einer Kombination aus beidem behandelt. Anschließend wurden die Tumorgrößen bestimmt und die Tumoren immunhistologisch auf Apoptose, Vaskularisierung sowie die Aktivität des Transkriptionsfaktors NF-κB untersucht.Ergebnisse:Die Kombination aus IL-1β-RI-Antikörper und Etoposid führte im Vergleich zu den entsprechenden Monotherapien bzw. keiner Behandlung zu einem deutlich verringerten Wachstum der PancTu1-Tumoren. Entsprechend konnte in Tumoren der Kombinationsbehandlung eine signifikant erhöhte Anzahl an apoptotischen Zellen detektiert werden. Nach der Behandlung mit dem IL-1β-RI-Antikörper wiesen die Tumoren eine deutlich verminderte Präsenz von aktiviertem NF-κB im Vergleich zu den Kontrolltumoren auf. Ferner war in den Tumoren nach Kombinationstherapie die Vaskularisierung (CD31-positive Zellen) weniger stark ausgeprägt.Schlussfolgerung:Die autokrine Sekretion von IL-1β ist an der Entstehung konstitutiver NF-κB-Aktivität und einer dadurch vermittelten Chemoresistenz in Pankreaskarzinomzellen beteiligt.Abstract.Background and Purpose:The pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly malignant phenotype and a profound chemoresistance. Thus, options for an effective treatment of this disease are still quite poor. In this study, it was investigated whether the autocrine secretion of interleukin-(IL-)1β is related to a chemoresistant phenotype of PDAC cells in vivo.Material and Methods:Human PancTu1 PDAC cells were inoculated subcutaneously into female SCID mice. After 10 days of outgrowth, animals were randomized and left untreated or treated with an IL-1β-RI antibody, etoposide, or a combination of both. After treatment for 14 days, tumor sizes were determined and each tumor was analyzed immunohistochemically for apoptosis (TUNEL), activated NF-κB (p65), and vascularization (CD31 staining).Results:The combination of IL-1β-RI antibody and etoposide led to a significantly reduced outgrowth of PancTu1 tumors in comparison to the monotherapies or no treatment. Accordingly, the number of apoptotic cells was significantly elevated in tumors of the combination group. After treatment with the IL-1β-RI antibody, less activated NF-κB was present in tumors compared to the control group. Moreover, tumors of the combination group showed a clearly reduced vascularization.Conclusion:The autocrine secretion of IL-1β contributes to a constitutively increased NF-κB activity in PDAC cells along with a chemoresistant phenotype.

Autokrine IL-1-Sekretion fhrt zu erhhter NF-?B-Aktivitt und zu Chemoresistenz in Pankreaskarzinomzellen in vivo@@@Autocrine IL-1 Secretion Leads to NF-?-Mediated Chemoresistance in Pancreatic Carcinoma Cells in Vivo

Zusammenfassung.Hintergrund und Ziel:Das duktale Pankreaskarzinom (PDAC) ist durch einen hochmalignen Phänotyp und eine profunde Chemoresistenz charakterisiert. In der vorliegenden Studie wurde die autokrine Sekretion von Interleukin-(IL)1β durch Pankreaskarzinomzellen in vivo als eine Ursache für einen chemoresistenten Phänotyp untersucht.Material und Methodik:Zellen der humanen PDAC-Zelllinie PancTu1 mit ausgeprägter IL-1β-Sekretion wurden subkutan in weibliche SCID-Mäuse inokuliert. Nach 10 Tagen wurden die Tiere randomisiert und entweder unbehandelt gelassen oder für 14 Tage mit einem IL-1β-RI-Antikörper, mit Etoposid oder einer Kombination aus beidem behandelt. Anschließend wurden die Tumorgrößen bestimmt und die Tumoren immunhistologisch auf Apoptose, Vaskularisierung sowie die Aktivität des Transkriptionsfaktors NF-κB untersucht.Ergebnisse:Die Kombination aus IL-1β-RI-Antikörper und Etoposid führte im Vergleich zu den entsprechenden Monotherapien bzw. keiner Behandlung zu einem deutlich verringerten Wachstum der PancTu1-Tumoren. Entsprechend konnte in Tumoren der Kombinationsbehandlung eine signifikant erhöhte Anzahl an apoptotischen Zellen detektiert werden. Nach der Behandlung mit dem IL-1β-RI-Antikörper wiesen die Tumoren eine deutlich verminderte Präsenz von aktiviertem NF-κB im Vergleich zu den Kontrolltumoren auf. Ferner war in den Tumoren nach Kombinationstherapie die Vaskularisierung (CD31-positive Zellen) weniger stark ausgeprägt.Schlussfolgerung:Die autokrine Sekretion von IL-1β ist an der Entstehung konstitutiver NF-κB-Aktivität und einer dadurch vermittelten Chemoresistenz in Pankreaskarzinomzellen beteiligt.Abstract.Background and Purpose:The pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly malignant phenotype and a profound chemoresistance. Thus, options for an effective treatment of this disease are still quite poor. In this study, it was investigated whether the autocrine secretion of interleukin-(IL-)1β is related to a chemoresistant phenotype of PDAC cells in vivo.Material and Methods:Human PancTu1 PDAC cells were inoculated subcutaneously into female SCID mice. After 10 days of outgrowth, animals were randomized and left untreated or treated with an IL-1β-RI antibody, etoposide, or a combination of both. After treatment for 14 days, tumor sizes were determined and each tumor was analyzed immunohistochemically for apoptosis (TUNEL), activated NF-κB (p65), and vascularization (CD31 staining).Results:The combination of IL-1β-RI antibody and etoposide led to a significantly reduced outgrowth of PancTu1 tumors in comparison to the monotherapies or no treatment. Accordingly, the number of apoptotic cells was significantly elevated in tumors of the combination group. After treatment with the IL-1β-RI antibody, less activated NF-κB was present in tumors compared to the control group. Moreover, tumors of the combination group showed a clearly reduced vascularization.Conclusion:The autocrine secretion of IL-1β contributes to a constitutively increased NF-κB activity in PDAC cells along with a chemoresistant phenotype.