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Holger Kalthoff

Publications and source records attributed to Holger Kalthoff.

At least 19 recordsLinked to original sources
PubMed2007

Antitumor activity of ALK1 in pancreatic carcinoma cells.

In this study, the authors investigated the expression of activin receptor-like kinase 1 (ALK1) in pancreatic carcinoma and evaluated its potential role as a tumor suppressor in vitro and in vivo. Endogenous ALK1 expression was demonstrated by immunohistochemistry in both pancreatic tumor tissue and peritumoral normal tissue from 6 patients and by RT-PCR in 8/12 established pancreatic cancer cell lines. Ectopic expression of a constitutively active (ca) ALK1 mutant in TGF-beta sensitive PANC-1 and COLO-357 cells augmented transcriptional activation of a Smad2/3 responsive reporter, and slowed down basal growth in vitro. Both effects were further enhanced by TGF-beta/ALK5 stimulation, suggesting largely independent nuclear Smad signaling by both type I receptors. Upon orthotopic transplantation of PANC-1-caALK1 into immunodeficient mice, tumor size was strongly reduced and was associated with a lower microvessel density in the PANC-1-caALK1-derived tumors. In vitro, this mutant efficiently blocked TGF-beta-induced epithelial-to-mesenchymal transdifferentiation and suppressed TGF-beta/ALK5-mediated activation of the p38 MAPK pathway. Mechanistically, caALK1 silenced MyD118, an immediate TGF-beta target gene whose protein product, GADD45beta, couples Smad signaling to p38 activation. These results show that ALK1 activation in pancreatic tumor cells is antioncogenic by inducing ALK5-independent growth inhibition and by blocking TGF-beta/ALK5-mediated epithelial-to-mesenchymal transdifferentiation and, possibly, invasion and metastatic progression.

Activin Receptors, Type I↗
PubMed2006

Combination phenylbutyrate/gemcitabine therapy effectively inhibits in vitro and in vivo growth of NSCLC by intrinsic apoptotic pathways.

BACKGROUND: Standard chemotherapy protocols in NSCLC are of limited clinical benefit. Histone deacetylase (HDAC) inhibitors represent a new strategy in human cancer therapy. In this study the combination of the HDAC inhibitor phenylbutyrate (PB) and the nucleoside analogue gemcitabine (GEM) was evaluated and the mechanisms underlying increased cell death were analyzed. METHODS: Dose escalation studies evaluating the cytotoxicity of PB (0.01-100 mM), GEM (0.01-100 microg/ml) and a combination of the two were performed on two NSCLC cell lines (BEN and KNS62). Apoptotic cell death was quantified. The involvement of caspase-dependent cell death and MAP-kinase activation was analyzed. Additionally, mitochondrial damage was determined. In an orthotopic animal model the combined effect of PB and GEM on therapy was analyzed. RESULTS: Applied as a single drug both GEM and PB revealed limited potential to induce apoptosis in KNS62 and Ben cells. Combination therapy was 50-80% (p = 0.012) more effective than either agent alone. On the caspase level, combination therapy significantly increased cleavage of the pro-forms compared to single chemotherapy. The broad spectrum caspase-inhibitor zVAD was able to inhibit caspase cleavage completely, but reduced the frequency of apoptotic cells only by 30%. Combination therapy significantly increased changes in MTP and the release of cyto-c, AIF and Smac/Diabolo into the cytoplasm. Furthermore, the inhibitors of apoptosis c-IAP1 and c-IAP2 were downregulated and it was shown that in combination therapy JNK activation contributed significantly to induction of apoptosis. The size of the primary tumors growing orthotopically in SCID mice treated for 4 weeks with GEM and PB was significantly reduced (2.2-2.7 fold) compared to GEM therapy alone. The Ki-67 (KNS62: p = 0.015; Ben: p = 0.093) and topoisomerase IIalpha (KNS62: p = 0.008; Ben: p = 0.064) proliferation indices were clearly reduced in tumors treated by combination therapy, whereas the apoptotic index was comparably low in all groups. CONCLUSION: Therapy combining GEM and the HDAC inhibitor PB initiates a spectrum of apoptosis-inducing mitochondrial and further JNK-dependent events, thereby overcoming the therapeutic resistance of NSCLC tumor cells. In vivo, the combination therapy substantially reduced tumor cell proliferation, suggesting that the well tolerated PB is a useful supplemental therapeutic agent in NSCLC.

Journal Article↗
PubMed2006

Adjuvant treatment of pancreatic carcinoma in a clinically adapted mouse resection model.

BACKGROUND: The high rate of local recurrence after radical resection of pancreatic adenocarcinoma fosters intensive efforts to develop new approaches for adjuvant treatment. The established animal models show significant limitations in simulating an adjuvant therapeutic setting. For optimal approximation to the clinical situation we therefore improved a murine orthotopic human xenotransplantation model. METHODS: Subtotal pancreatectomy in mice was performed after orthotopic inoculation of human pancreatic cancer cells and manifestation of solid tumours. The natural course of disease, tumour growth and metastases were analysed. Gemcitabine as a cytotoxic drug was tested in vitro on the cell line used in this model and the effect of adjuvant treatment with gemcitabine in vivo was investigated. RESULTS: All tumour-resected animals showed local recurrence. Organ metastases occurred in 67% in resected compared to 25% of non-resected animals. Gemcitabine in vitro was ineffective, but as adjuvant monotherapy resulted in a highly significant reduction of tumour weight and metastatic events. CONCLUSION: Subtotal pancreatectomy for xenotransplanted pancreatic cancer in SCID beige mice is feasible. Due to high rates of local recurrence and increased organ metastases, this model offers a relevant option for preclinical adjuvant testing, especially as in vitro and in vivo effects of cytotoxic drugs differ enormously.

Adenocarcinoma↗
PubMed2006

A modified random oligonucleotide-based combination therapy for adjuvant treatment of pancreatic ductal adenocarcinoma.

Anti-cancer therapy in pancreatic ductal adenocarcinoma (PDAC) is mostly based on surgical removal or palliative therapy using antimetabolites, like 5'-fluorouracil or gemcitabine. Adjuvant treatment using these chemotherapeutics has recently proven a beneficial concept, though general survival rates are still poor. Most recently, combination therapy of gemcitabine with other targeted drugs was evaluated in clinical trials. We present here a study performed in a mouse orthotopic xenotransplant model of PDAC, using an oligo-nucleotide-based approach. We have shown previously that antisense oligonucleotides against p53 reduce the weight of orthotopic pancreatic tumours in immune-deficient mice. We further characterised terminal modifications of phosphorothioate oligonucleotides in vitro and found a random, unrelated control sequence carrying a D,L-alpha-tocopherol modification at the 5' and 3' ends to be most efficient in induction of cell death in PancTu-1 cells. Modified random oligonucleotide (MRON) were thus further tested in vivo. MRON showed a reduction of tumour weight in established primary orthotopic tumours in SCID/bg mice. In a surgically adapted pre-clinical model, where primary tumours were resected and animals received adjuvant treatment, MRON was very efficient in suppression of relapse and metastasis, when combined with gemcitabine. While the exact molecular mechanism of MRON activity still needs to be elucidated, the compound showed a remarkable preference for uptake into tumour cells in vivo.

Adenocarcinoma↗
PubMed2006

Stable expression of temperature-sensitive p53: a suitable model to study wild-type p53 function in pancreatic carcinoma cells.

Pancreatic adenocarcinoma is an extremely aggressive malignancy with a dismal prognosis. Inactivation of the p53 tumor-suppressor gene occurs in approximately 50% of primary tumors and is thought to account for a failure of the tumor cells to undergo growth arrest and apoptosis in response to chemotherapy. Hence, it is of interest to study the consequences of the restoration of wild-type (wt) p53 function in pancreatic carcinoma cells. Therefore, we retrovirally transduced temperature-sensitive (ts) human p53 into the p53-null pancreatic carcinoma cell line AsPC-1. ts p53 has a mutant phenotype at 37.5 degrees C, and a wt conformation at 32.5 degrees C. Stable expression of p53 in wt conformation caused upregulation of the p53 responsive gene p21Waf1/Cip1, and G1 growth arrest, but failed to induce Bax expression or apoptosis. In addition, we examined the effect of wt p53 expression on DNA damaging treatment. Interestingly, the doxorubicin- and radiation-induced S-/G2-phase arrests were suppressed by p53 in wt conformation. These results demonstrate that the ts p53/AsPC-1 model is suitable to investigate the effect of wt p53 restoration in pancreatic carcinoma cells.

Adenocarcinoma↗
PubMed2005

Gemcitabine-mediated apoptosis is associated with increased CD95 surface expression but is not inhibited by DN-FADD in Colo357 pancreatic cancer cells.

This study investigates the role of caspase-8 and DN-FADD, an inhibitor of CD95-dependent caspase-8 activation, in gemcitabine-induced apoptosis of Colo357 pancreatic cancer cells. Gemcitabine-mediated apoptosis was monitored by the kinetics of caspase-8 activation and cytochrome c release. Gemcitabine treatment of Colo357 cells increased CD95 surface expression, raising the possibility of the involvement of CD95 in gemcitabine-mediated caspase-8 activation. However, ectopic expression of DN-FADD and treatment of cells with the antagonistic anti-CD95 antibody ZB4 both failed to suppress gemcitabine-induced apoptosis but substantially inhibited CD95-mediated apoptosis. DN-FADD, which surprisingly accumulated in nuclei of Colo357 cells, was unable to block caspase-8 activation mediated by either gemcitabine or CD95. These observations argue against a role of CD95 in gemcitabine-induced caspase-8 activation and reveal that the anti-apoptotic function of DN-FADD differs from caspase-8 inhibition in Colo357 cells.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Detection of tumor cell dissemination in pancreatic ductal carcinoma patients by CK 20 RT-PCR indicates poor survival.

PURPOSE: This prospective study evaluates the diagnostic potential of Cytokeratin 20 (CK 20) RT-PCR for the detection of disseminated tumor cells in bone marrow and blood of a large cohort of patients with ductal adenocarcinoma of the pancreas and the prognostic value on overall survival prediction. METHODS: Between 1994 and 2003, 172 patients (83 male, 89 female; 13-82 years) with pancreatic ductal adenocarcinoma underwent surgery. Bone marrow samples and venous blood were taken preoperatively and analyzed for disseminated tumor cells by nested CK 20 RT-PCR. RESULTS: Disseminated tumor cells were detected in 81 (47.1%) of the 172 patients in the bone marrow and/or the venous blood. Overall, in 45 of the 135 (33.3%) bone marrow samples and in 52 of the 154 (33.8%) blood samples, CK 20 positive cells were detected. Detection rates increased with the UICC-tumor stage. According to Kaplan-Meier, univariate survival analysis of all 172 patients (n = 78 R0-; n = 18 R1- and n = 5 R2-resected; n = 71 palliative surgery) showed a statistically significant relationship of overall survival to radicality of the operation (P < 0.0001), the UICC-stage of the tumors (P = 0.0011) and the detection of disseminated tumor cells in bone marrow and/or venous blood (P = 0.05). Patients with well- and moderately- differentiated tumors (G1 and G2) had a significantly longer survival (P = 0.045) than patients suffering from poorly differentiated tumors (G3). A positive CK 20 status in the bone marrow and/or blood within the group of patients with G1 and G2 tumors had a significantly negative prognostic impact on their survival (P = 0.046). CONCLUSIONS: Disseminated tumor cells can be detected in patients with pancreatic ductal adenocarcinoma by CK 20 RT-PCR. Detection rates are stage dependent, and survival analysis demonstrated statistically relevant data. From a clinical point of view, this finding is especially noteworthy for the group of well- and moderately-differentiated tumors.

Adenocarcinoma↗
PubMed2005

Meta-analysis of microarray data on pancreatic cancer defines a set of commonly dysregulated genes.

Pancreatic ductal adenocarcinoma is the eighth most common cancer with the lowest overall 5-year relative survival rate of any tumor type today. Expression profiling using microarrays has been widely used to identify genes associated with pancreatic cancer development. To extract maximum value from the available gene expression data, we applied a meta-analysis to search for commonly differentially expressed genes in pancreatic ductal adenocarcinoma. We obtained data sets from four different gene expression studies on pancreatic cancer. We selected a consensus set of 2984 genes measured in all four studies and applied a meta-analysis approach to evaluate the combined data. Of the genes identified as differentially expressed, several were validated using RT-PCR and immunohistochemistry. Additionally, we used a class discovery algorithm to identify a gene expression signature. Our meta-analysis revealed that the pancreatic cancer gene expression data sets shared a significant number of up- and downregulated genes, independent of the technology used. This interstudy crossvalidation approach generated a set of 568 genes that were consistently and significantly dysregulated in pancreatic cancer. Of these, 364 (64.1%) were upregulated and 204 (35.9%) were downregulated in pancreatic cancer. Only 127 (22%) were described in the published individual analyses. Functional annotation of the genes revealed that genes presumably associated with the cell adhesion-mediated drug resistance pathway are frequently overexpressed in pancreatic cancer. Meta-analysis is an important tool for the identification and validation of differentially expressed genes. These could represent good candidates for novel diagnostic and therapeutic approaches to pancreatic cancer.

Adenocarcinoma↗
PubMed2005

Adhesion and Rac1-dependent regulation of biglycan gene expression by transforming growth factor-beta. Evidence for oxidative signaling through NADPH oxidase.

Both transforming growth factor-beta (TGF-beta)-induced expression of biglycan (BGN) and activation of p38 MAPK have been implicated in cellular adhesion and migration. Here, we analyzed the role of adhesive events and the small GTPase Rac1 in TGF-beta regulation of BGN. TGF-beta1 induction of BGN expression and activation of p38 was abolished or strongly reduced when cells were kept in suspension or exposed to either the actin cytoskeleton-disrupting agent cytochalasin D or a specific chemical Rac1 inhibitor. Ectopic expression of a dominant negative mutant (T17N) of Rac1 abrogated both TGF-beta-induced p38 MAPK activation and BGN up-regulation but did not affect TGF-beta-induced phosphorylation of Smad3 or transcriptional induction of Growth Arrest DNA Damage 45beta, previously shown to be crucial for TGF-beta regulation of BGN. Overexpression of wild type Rac1 greatly enhanced the TGF-beta effect on BGN in adherent cells, whereas ectopic expression of constitutively active Rac1 (Q61L) activated p38 and in the presence of exogenous TGF-beta was able to rescue BGN expression in nonadherent cells. Endogenous Rac1 was activated by TGF-beta treatment in PANC-1 cells in an adhesion-dependent fashion. Like Rac1-T17N, the NADPH oxidase inhibitor diphenylene iodonium and the tyrosine kinase inhibitor herbimycin A blocked TGF-beta-induced p38 activation and BGN expression, suggesting that Rac1 exerts its effect on BGN and p38 through increasing NADPH oxidase activity and subsequent production of reactive oxygen species. These results show that the TGF-beta effect on BGN is dependent on cell adhesion and that activated Rac1, presumably acting through NADPH oxidase(s), is necessary but not sufficient for TGF-beta-induced BGN expression.

Biglycan↗
PubMed2005

Detection and clinical implications of minimal residual disease in gastro-intestinal cancer.

INTRODUCTION: Metastatic dissemination is an important factor for the prognosis of patients with gastro-intestinal cancer. Exact staging is crucial to determine appropriate multimodal therapeutic strategies. At present, the sensitivity of routinely performed diagnostic techniques is suboptimal for the detection of minimal residual disease (MRD) and occult metastases since the number of disseminated tumour cells (DTCs) is mostly marginal. To amend the verification of DTCs, immunohistochemical and molecular methods were applied to retrieve epithelial cell-specific proteins in non-epithelial tissue of different body compartments or fluids. Many groups have eagerly focussed on the identification of new markers and novel tests, yet specificity and sensitivity of these methods as well as robustness in the clinical setting are frequently missing. MATERIALS AND METHODS: This review critically evaluates the prognostic impact of MRD in patients with pancreatic, colorectal and gastric cancer by outlining those studies showing diagnostic results of DTC detection in lymph nodes, bone marrow, venous blood and peritoneal lavage, some of which present novel strategies. CONCLUSION: The analysed data concerning MRD in gastro-intestinal cancers reveal that results are undesirably heterogeneous. From a critical point of view, many clinical studies missed their chance because of small cohort size; moreover, methodological standardisation is generally lacking. On the other hand, the very encouraging results achieved so far, together with the comprehensive analyses of a few research groups, foster the prediction that DTC/MRD issues will soon expand the standard TNM classification.

Biomarkers, Tumor↗
PubMed2005

Increased expression of the E3-ubiquitin ligase receptor subunit betaTRCP1 relates to constitutive nuclear factor-kappaB 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.

Antineoplastic Agents, Phytogenic↗
PubMed2005

CD95 and TRAF2 promote invasiveness of pancreatic cancer cells.

Pancreatic adenocarcinoma represents a tumor type with extremely poor prognosis. High apoptosis resistance and a strong invasive and early metastatic potential contribute to its highly malignant phenotype. Here we identified the death receptor adaptor molecule TRAF2 as a key player in pancreatic cancer pathophysiology. Using immunohistochemistry and Western blot analysis we found TRAF2 overexpressed in 34 of 36 pancreatic tumor samples as well as in pancreatic tumor cell lines resistant to CD95-mediated apoptosis. The high TRAF2 protein level was not related to chromosomal changes, as monitored by FISH analysis. Instead, the NF-kappaB- and MEK-signaling pathways were involved. Introduction of a TRAF2 expression vector in CD95-sensitive Colo357 cells resulted in (i) resistance to CD95-induced apoptosis; (ii) increased constitutive NF-kappaB and AP-1 activity; and (iii) higher basal secretion of matrix metalloproteinases (MMPs), urokinase-type plasminogen activator (uPA), and IL-8, leading to increased invasiveness. High apoptosis resistance and uPA secretion could be reverted by TRAF2-specific siRNA. Stimulation of TRAF2-overexpressing cells with CD95 ligand led to induction of NF-kappaB and AP-1, enhanced IL-8- and uPA-secretion, and a further increased invasiveness. Thus, TRAF2 overexpression does not only block apoptosis induction by CD95 but also converts this death receptor into a mediator of invasiveness.

Adenocarcinoma↗
PubMed2005

Paraneoplastic leukemoid reaction and rapid progression in a patient with malignant melanoma: establishment of KT293, a novel G-CSF-secreting melanoma cell line.

Paraneoplastic leukemoid reaction (PLR) is a rare condition of leucocytosis in cancer patients. Here we report the rapid progression of a patient suffering from a metastasized malignant melanoma and PLR. The patient's white blood cell count exceeded 200,000 cells per mul and the serum level of Granulocyte Colony-Stimulating Factor (G-CSF) was elevated up to 780 pg/mul. A Tc-m99-labeled anti-NCA90/95 based granulocyte scan demonstrated reactive bone marrow expansion, splenomegaly and granulocyte infiltration into the tumor. KT293, a S100, gp100 and CD68 positive melanoma cell line derived from an axillary metastasis, produced large amounts of G-CSF in vitro and induced rapidly growing tumors and PLR after subcutaneous inoculation in SCID mice. In contrast to G-CSF-secreting cancer cells of other tissue origin, G-CSF-neutralizing antibodies failed to inhibit the growth of KT293 cells. In addition, KT293 cells did not express G-CSF-receptor. These observations suggest that paracrine effects of G-CSF-secretion and PLR might promote an aggressive melanoma phenotype, as seen in this patient.

Animals↗
PubMed2005

Lymphatic spread of ductal pancreatic adenocarcinoma is independent of lymphangiogenesis.

Early lymph node metastasis is common in pancreatic ductal adenocarcinoma (PDAC). The present study has examined the relationship of lymphatic spread to lymph vessel development and the expression of lymphangiogenic cytokines in a series of well-characterized PDACs. The hot spot method revealed the intratumoural and peritumoural lymphatic vessel density (LVD) to be slightly higher in PDACs than in the normal pancreas. The average intratumoural LVD, however, was strikingly decreased. There was no overexpression of vascular endothelial growth factor (VEGF)-C and VEGF-D in PDACs compared with the normal pancreas. LVD and expression of lymphangiogenic cytokines were not related to any of the biological tumour features or to patient survival. Three orthotopic nude mouse PDAC models did not reveal any increase in tumour-associated LVD, despite a high rate of lymph node metastasis. Lymph vessel proliferation was comparable in PDAC and chronic pancreatitis, in both humans and mice. In conclusion, increased lymphangiogenic activity is not required for and does not significantly affect the lymphatic spread of PDAC. The reduced number of human and murine intratumoural lymph vessels indicates that lymphatic metastasis takes place predominantly via peritumoural lymphatic vessels. The weak expression of lymphangiogenic cytokines in neoplastic cells and lymphatic vessel proliferation in peritumoural regions and chronic pancreatitis indicate that inflammation may be the reason for the low rate of lymphangiogenesis.

Adult↗
PubMed2004

Transforming growth factor-beta (TGF-beta) type I receptor/ALK5-dependent activation of the GADD45beta gene mediates the induction of biglycan expression by TGF-beta.

We have recently shown that induction of biglycan (BGN) expression by transforming growth factor-beta1 (TGF-beta1) required sequential activation of both Smad and p38 mitogen-activated protein kinase signaling (Ungefroren, H., Lenschow, W., Chen, W.-B., and Kalthoff, H. (2003) J. Biol. Chem. 278, 11041-11049). Here, we have analyzed the receptors through which TGF-beta1 controls expression of BGN and GADD45beta, the latter of which is postulated to link early Smad signaling to delayed activation of p38. Ectopic expression of a dominant-negative mutant of the TGF-beta type II receptor in PANC-1 cells abrogated TGF-beta-induced BGN up-regulation. Similarly, inhibition of the TGF-beta type I receptor/ALK5 with either SB431542 or by enforced stable expression of a kinase-dead mutant greatly attenuated the TGF-beta effect on both BGN and GADD45beta expression in PANC-1 and MG-63 cells. The enhancing effect of ALK5 on TGF-beta-mediated GADD45beta and BGN expression and on GADD45beta promoter activity was also dependent on its ability to activate Smad signaling, because an ALK5 mutant defective in Smad activation (TbetaRImL45) but with an otherwise functional kinase domain failed to mediate these responses. The TGF-beta/ALK5 effect on p38 activation and BGN expression was mimicked by overexpression of GADD45beta alone (in the absence of TGF-beta stimulation) and suppressed upon antisense inhibition of GADD45beta expression. These results show that TGF-beta induces BGN expression through (the Smad-activating function of) ALK5 and GADD45beta and suggest that the sensitivity of MyD118 to activation by TGF-beta, which varies between tissues, ultimately determines the strength of the TGF-beta effect on BGN.

Activin Receptors, Type I↗
PubMed2004

Resistance of pancreatic cancer to gemcitabine treatment is dependent on mitochondria-mediated apoptosis.

Palliative chemotherapy with gemcitabine, a common mode of treatment of pancreatic cancer, has little influence on patients' survival. We investigated the impact of anti-apoptotic Bcl-xL protein and its antagonist Bax on gemcitabine-induced apoptosis in human pancreatic carcinoma cells in vitro and in vivo. The level of Bcl-xL and Bax expression was determined in 3 established pancreatic cancer cell lines that differ in their sensitivity to gemcitabine-mediated apoptosis. Bcl-xL and Bax genes were transduced into Colo357 cells by retroviral infection. In addition, cells were transfected with c-FLIP to assess involvement of CD95 and caspase-8. The impact of Bax/Bcl-xL expression on gemcitabine-sensitivity in vivo was evaluated in orthotopic Colo357 tumors in SCID mice. The apoptotic index revealed a strong inverse correlation between Bcl-xL expression and gemcitabine-induced apoptosis in the pancreatic carcinoma cell lines tested. Caspase-8 and Bid were cleaved in Colo357 cells exposed to gemcitabine, and there was no correlation with either Bcl-xL or with Bax expression. In contrast, the lack of mitochondrial transmembrane potential transition, release of cytochrome-c and absence of caspase-9- and PARP-cleavage showed a strong correlation with Bcl-xL expression. Expression of c-FLIP significantly increased the resistance towards gemcitabine. Orthotopically growing Colo357-bcl-xl tumors in SCID mice were refractory to gemcitabine treatment, and in contrast to the in vitro data, Colo357-bax tumors exhibited a 12-fold greater tumor regression than Colo357-wild-type tumors in the control group. Gemcitabine-induced apoptosis involves the mitochondria-mediated signaling pathway. A functional restoration of this pathway appears to be essential to overcome the resistance mechanisms of pancreatic tumor cells and to improve the response to therapy as demonstrated by Bax overexpression in a clinically relevant tumor model.

Animals↗
PubMed2004

Tumor stroma interactions induce chemoresistance in pancreatic ductal carcinoma cells involving increased secretion and paracrine effects of nitric oxide and interleukin-1beta.

Pancreatic ductal carcinoma is characterized by a profound chemoresistance. As we have shown previously, these tumor cells can develop chemoresistance by interleukin (IL)-1beta in an autocrine and nuclear factor-kappaB-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-1beta in T3M4 and PT45-P1 cells was increased by the fibroblasts, and IL-1beta-receptor blockade abolished the resistance-inducing effect during cocultivation. This stimulated IL-1beta secretion could be attributed to nitric oxide (NO) released by the fibroblasts as an IL-1beta-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-1beta 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-1beta 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-1beta by the tumor cells. These findings substantiate the implication of tumor-stromal interactions in the chemoresistance of pancreatic carcinoma.

Adenocarcinoma↗