Anna Trauzold

Bcl-XL protects pancreatic adenocarcinoma cells against CD95- and TRAIL-receptor-mediated apoptosis

In this study we sought to clarify the role of the pro-apoptotic potential of mitochondria in the death pathway emanating from the TRAIL (APO-2L) and CD95 receptors in pancreatic carcinoma cells. We focused on the role of the Bcl-2 family member Bcl-XL, using three pancreatic carcinoma cell lines as a model system, two of which have high (Panc-1, PancTuI) and one has low (Colo357) Bcl-XL expression. In these cell lines, the expression of Bcl-XL correlated with sensitivity to apoptosis induced by TRAIL or anti-CD95. Flow cytometric analysis revealed cell surface expression of TRAIL-R1 and TRAIL-R2 on PancTuI and Colo357, and TRAIL-R2 on Panc-1 cells. In Colo357 cells retrovirally transduced with Bcl-XL, caspase-8 activation in response to treatment with TRAIL or anti-CD95 antibody was not different from parental cells and EGFP-transfected controls, however, apoptosis was completely suppressed as measured by the mitochondrial transmembrane potential Δψm, caspase-3 activity (PARP cleavage) and DNA-fragmentation. Inhibition of Bcl-XL function by overexpression of Bax or administration of antisense oligonucleotides against Bcl-XL mRNA resulted in sensitization of Panc-1 cells to TRAIL and PancTuI cells to anti-CD95 antibody-induced cell death. The results show that Bcl-XL can protect pancreatic cancer cells from CD95- and TRAIL-mediated apoptosis. Thus, in these epithelial tumour cells the mitochondrially mediated ‘type II’ pathway of apoptosis induction is not only operative regarding the CD95 system but also regarding the TRAIL system.

TRAIL promotes metastasis of human pancreatic ductal adenocarcinoma.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has attracted considerable attention for its potential use in tumor therapy, as some recombinant variants of this ligand induce apoptosis in tumor cells without harming most normal cells. Here, we show that TRAIL strongly induces the expression of the proinflammatory cytokines interleukin-8 and monocyte chemoattractant protein 1 and enhances the invasion of apoptosis-resistant pancreatic ductal adenocarcinoma cells in vitro by upregulation of the urokinase-type plasminogen activator expression. Most importantly, we also demonstrate for the first time that TRAIL treatment results in strongly increased distant metastasis of pancreatic tumors in vivo. We orthotopically transplanted human pancreatic ductal adenocarcinoma cells to the pancreata of severe combined immunodeficiency mice and observed a dramatic increase in metastatic spread including a sixfold increase in the volume and fourfold increase in the number of liver metastases upon TRAIL treatment. Our results point to the necessity to carefully evaluate in vivo side effects of TRAIL and to select therapy conditions that not only enhance apoptosis induction but in addition prevent proinvasive and proinflammatory non-apoptotic TRAIL signaling.

CD95 and TRAIL receptor-mediated activation of protein kinase C and NF-kappaB contributes to apoptosis resistance in ductal pancreatic adenocarcinoma cells.

The molecular alterations in tumour cells leading to resistance towards apoptosis induced by CD95 and TRAIL-receptors are not fully understood. We report here that the stimulation of the CD95- and TRAIL-resistant human pancreatic adenocarcinoma cell line PancTuI with an agonistic anti-CD95 antibody or TRAIL resulted in activation of protein kinase C and NF-κB. Inhibition of protein kinase C by Gö6983 sensitized these cells to apoptotic challenges and strongly diminished activation of NF-κB by anti-CD95 and TRAIL. Similarly, inhibition of NF-κB by MG132 or by transient transfection with a dominant negative mutant of IκBα restored the responsiveness of PancTuI cells to both death ligands. In the CD95 and TRAIL-sensitive cell line Colo357 the induction of protein kinase C and NF-κB following activation of CD95 and TRAIL-R was very moderate compared with PancTuI cells. However, pre-incubation of these cells with PMA strongly reduced their apoptotic response to anti-CD95 and TRAIL. Taken together, we show that activation of protein kinase C operates directly in a death receptor-dependent manner in PancTuI cells and protect pancreatic tumour cells from anti-CD95 and TRAIL-mediated apoptosis by preventing the loss ΔΨm and Cytochrome c release as well as by induction of NF-κB.

Anti–Tumor Necrosis Factor Therapy Inhibits Pancreatic Tumor Growth and Metastasis

Chronic inflammation has been implicated in the pathogenesis of many severe autoimmune disorders, as well as in diabetes, pulmonary diseases, and cancer. Inflammation accompanies most solid cancers including pancreatic ductal adenocarcinoma (PDAC), one of the most fatal cancers with surgery being the only curative therapeutic approach currently available. In the present work, we investigated the role of the major proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) in the malignancy of PDAC cells in vitro and in vivo. In vitro, TNFalpha strongly increased invasiveness of Colo357, BxPc3, and PancTuI cells and showed only moderate antiproliferative effect. TNFalpha treatment of mice bearing orthotopically growing PDAC tumors led to dramatically enhanced tumor growth and metastasis. Notably, we found that PDAC cells themselves secrete TNFalpha. Although inhibition of TNFalpha with infliximab or etanercept only marginally affected proliferation and invasiveness of PDAC cells in vitro, both reagents exerted strong antitumoral effects in vivo. In severe combined immunodeficient mice with orthotopically growing Colo357, BxPc3, or PancTuI tumors, human-specific anti-TNF antibody infliximab reduced tumor growth and metastasis by about 30% and 50%, respectively. Importantly, in a PDAC resection model performed with PancTuI cells, we found an even stronger therapeutic effect for both anti-TNF compounds. Infliximab and etanercept reduced the number of liver metastases by 69% and 42%, respectively, as well as volumes of recurrent tumors by 73% and 51%. Thus, tumor cell-derived TNFalpha plays a profound role in malignancy of PDAC, and inhibition of TNFalpha represents a promising therapeutic option particularly in adjuvant therapy after subtotal pancreatectomy.

Anti-apoptotic and growth-stimulatory functions of CK1 delta and epsilon in ductal adenocarcinoma of the pancreas are inhibited by IC261 in vitro and in vivo

Background: Pancreatic ductal adenocarcinomas (PDACs) are highly resistant to treatment due to changes in various signalling pathways. CK1 isoforms play important regulatory roles in these pathways. Aims: We analysed the expression levels of CK1 delta and epsilon (CK1δ/∊) in pancreatic tumour cells in order to validate the effects of CK1 inhibition by 3-[2,4,6-(trimethoxyphenyl)methylidenyl]-indolin-2-one (IC261) on their proliferation and sensitivity to anti-CD95 and gemcitabine. Methods: CK1δ/∊ expression levels were investigated by using western blotting and immunohistochemistry. Cell death was analysed by FACS analysis. Gene expression was assessed by real-time PCR and western blotting. The putative anti-tumoral effects of IC261 were tested in vivo in a subcutaneous mouse xenotransplantation model for pancreatic cancer. Results: We found that CK1δ/∊ are highly expressed in pancreatic tumour cell lines and in higher graded PDACs. Inhibition of CK1δ/∊ by IC261 reduced pancreatic tumour cell growth in vitro and in vivo. Moreover, IC261 decreased the expression levels of several anti-apoptotic proteins and sensitised cells to CD95-mediated apoptosis. However, IC261 did not enhance gemcitabine-mediated cell death either in vitro or in vivo. Conclusions: Targeting CK1 isoforms by IC261 influences both pancreatic tumour cell growth and apoptosis sensitivity in vitro and the growth of induced tumours in vivo, thus providing a promising new strategy for the treatment of pancreatic tumours.

PKC|[mu]| prevents CD95-mediated apoptosis and enhances proliferation in pancreatic tumour cells

Loss of growth control and a marked resistance to apoptosis are considered major mechanisms driving tumour progression. Protein kinases C (PKC) have been shown to be important in the regulation of proliferation and apoptosis. In this report, we investigated the role of the PKC-like kinase PKCμ in the control of these processes in pancreatic adenocarcinoma cells. We demonstrate that in these cells, PKCμ expression strongly correlates with resistance to CD95-induced apoptosis. Inhibition of PKCμ with Goe6983 sensitized resistant cells to CD95-induced apoptosis. In CD95-sensitive Colo357 cells, forced overexpression of PKCμ strongly reduced CD95-mediated apoptosis, an effect that could be reversed by pretreatment with Goe6983. In addition, PKCμ overexpression led to a strongly enhanced cell growth and to a significant increase of telomerase activity. In an attempt to identify the signalling pathways affected by PKCμ, we identified the antiapoptotic proteins c-FLIPL and survivin to be strongly upregulated in PKCμ overexpressing cells. Immunohistochemical analysis of pancreatic tumour tissue of 48 patients and 10 normal pancreatic tissues revealed marked overexpression of PKCμ in tumours. In conclusion, we showed that PKCμ controls proliferative, as well as anti-apoptotic, signalling pathways and therefore plays an important role in acquiring the malignant phenotype of pancreatic tumours.

The promoter of human p22/PACAP response gene 1 (PRG1) contains functional binding sites for the p53 tumor suppressor and for NFκB

We describe functional binding sites for the tumor suppressor p53 and for NFκB residing in the promoter of the novel human early response gene p22/PRG1 (IEX‐1/DIF‐2). Gel shift and supershift assays demonstrate binding of p53 and NFκB to their corresponding sites in vitro. CAT‐reporter gene assays show transactivation of the human p22/PRG1 promoter by p53 in Hep3B cells stably transfected with a temperature‐sensitive mutant p53, but not in p53‐deficient Hep3B cells. TNFα induced NFκB dependent transactivation was shown in HepG2 cells or in 818‐4 pancreatic cancer cells. These data imply that human p22/PRG1 is a target gene for p53 and NFκB involved in growth regulation and stress response.

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‐κB‐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‐κB 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‐κB 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.

TRAIL signaling is mediated by DR4 in pancreatic tumor cells despite the expression of functional DR5

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) and agonistic anti-DR4/TRAIL-R1 and anti-DR5/TRAIL-R2 antibodies are currently under clinical investigation for treatment of different malignancies. TRAIL activates DR4 and DR5 and thereby triggers apoptotic and non-apoptotic signaling pathways, but possible different roles of DR4 or DR5 in these responses has poorly been addressed so far. In the present work, we analyzed cell viability, DISC formation as well as IL-8 and NF–κB activation side by side in responses to TRAIL and agonistic antibodies against DR4 (mapatumumab) and against DR5 (lexatumumab) in pancreatic ductal adenocarcinoma cells. We found that all three reagents are able to activate cell death and pro-inflammatory signaling. Death-inducing signaling complex (DISC) analysis revealed that mapatumumab and lexatumumab induce formation of homocomplexes of either DR4 or DR5, whereas TRAIL additionally stimulated the formation of heterocomplexes of both receptors. Notably, blocking of receptors using DR4- and DR5-specific Fab fragments indicated that TRAIL exerted its function predominantly via DR4. Interestingly, inhibition of PKC by Goe6983 enabled DR5 to trigger apoptotic signaling in response to TRAIL and also strongly enhanced lexatumumab-mediated cell death. Our results suggest the existence of mechanisms that silence DR5 for TRAIL- but not for agonistic-antibody treatment.

Epicatechin gallate and catechin gallate are superior to epigallocatechin gallate in growth suppression and anti-inflammatory activities in pancreatic tumor cells

Green tea catechins are considered as possible cancer preventive agents for several cancer types but little is known regarding their effects on pancreatic cancer cells. The best studied catechin and the major polyphenol present in green tea is epigallocatechin gallate (EGCG). In the present study, we investigated the in vitro anti‐tumoral properties of EGCG on human pancreatic ductal adenocarcinoma (PDAC) cells PancTu‐I, Panc1, Panc89 and BxPC3 in comparison with the effects of two minor components of green tea catechins, catechin gallate (CG) and epicatechin gallate (ECG). We found that all three catechins inhibited proliferation of PDAC cells in a dose‐ and time‐dependent manner. Interestingly, CG and ECG exerted much stronger anti‐proliferative effects than EGCG. Western blot analyses performed with PancTu‐I cells revealed catechin‐mediated modulation of cell cycle regulatory proteins (cyclins, cyclin‐dependent kinases [CDK], CDK inhibitors). Again, these effects were clearly more pronounced in CG or ECG than in EGCG‐treated cells. Importantly, catechins, in particular ECG, inhibited TNFα‐induced activation of NF‐κB and consequently secretion of pro‐inflammatory and invasion promoting proteins like IL‐8 and uPA. Overall, our data show that green tea catechins ECG and CG exhibit potent and much stronger anti‐proliferative and anti‐inflammatory activities on PDAC cells than the most studied catechin EGCG. (Cancer Sci 2011; 102: 728–734)