Kerstin Maaser

Specific ligands of the peripheral benzodiazepine receptor induce apoptosis and cell cycle arrest in human colorectal cancer cells

The peripheral benzodiazepine receptor (PBR) has been implicated in growth control of various tumour models. Although colorectal cancers were found to overexpress PBR, the functional role of PBR in colorectal cancer growth has not been addressed to date. Using primary cell cultures of human colorectal cancers and the human colorectal carcinoma cell lines HT29, LS174T, and Colo320 DM we studied the involvement of PBR in the growth control and apoptosis of colorectal cancers. Both mRNA and protein expression of PBR were detected by RT-PCR and flow cytometry. Using confocal laser scanning microscopy and immunohistochemistry the PBR was localized in the mitochondria. The specific PBR ligands FGIN-1-27, PK 11195, or Ro5-4864 inhibited cell proliferation dose-dependently. FGIN-1-27 decreased the mitochondrial membrane potential, which indicates an early event in apoptosis. Furthermore, FGIN-1-27, PK 11195 or Ro5-4864 increased caspase-3 activity. In addition to their apoptosis-inducing effects, PBR ligands induced cell cycle arrest in the G1/G0-phase. Thus, our data demonstrate a functional involvement of PBR in colorectal cancer growth and qualify the PBR as a possible target for innovative therapeutic approaches in colorectal cancer.

Growth inhibition and apoptosis induced by P2Y2 receptors in human colorectal carcinoma cells: involvement of intracellular calcium and cyclic adenosine monophosphate.

Abstract. Extracellular nucleotides induce apoptosis and inhibit growth of colorectal cancer cells. To understand the underlying signaling pathways, we investigated the role of nucleotide-sensitive P2 receptors and focused on the receptor-mediated signaling of intracellular Ca2+ and cyclic adenosine monophosphate (cAMP) in two colorectal carcinoma cell lines (HT29, Colo320 DM). Expression and functionality of P2 receptor subtypes evaluated by RT-PCR and [Ca2+]i imaging revealed that solely metabotropic P2 receptors of the subtype P2Y2 were expressed on a functional level in both cell lines. Short-term stimulation of P2Y2 receptors caused Ca2+ mobilization from intracellular stores and a subsequent transmembrane Ca2+ influx. The receptor-induced [Ca2+]i elevation was shown to increase basal-stimulated [cAMP]i moderately and to potentiate forskolin-stimulated [cAMP]i vigorously, since the effects were dose-dependently inhibited by preloading the cells with the [Ca2+]i chelator BAPTA. In contrast, activation of protein kinase C (PKC) did not contribute to a receptor-mediated rise in [cAMP]i, since the PKC inhibitor staurosporine completely failed to reduce P2Y2 receptor-induced increases in [cAMP]i. Prolonged application of P2Y2 receptor agonists induced a time-dependent increase in apoptosis (up to 50% above control values) in both cell lines and caused dose-dependent inhibition of cell proliferation of up to 85% (Colo320 DM) or 64% (HT29). Chelating [Ca2+]i with BAPTA almost completely abolished P2Y2 receptor-induced cell death. Rises in [cAMP]i elicited by either forskolin or cAMP derivatives inhibited growth in both cell lines, too. In line with the potentiating effect of P2Y2 receptors on forskolin-stimulated [cAMP]i increases, costimulation with forskolin and P2Y2 receptor agonists led to synergistic antiproliferative effects. Moreover, a synergistic growth inhibition was observed when coincubating the cells with the P2Y2 receptor agonist ATP and the cytostatic drug 5-fluorouracil, which forms the basis for most currently applied chemotherapeutic regimes in colorectal cancer treatment. Our results demonstrate the growth inhibitory potency of P2Y2 receptors in colorectal carcinoma cells. Receptor-induced [Ca2+]i signaling appears to play a major role in the observed antiproliferative and apoptosis-inducing effects.

Extracellular nucleotides inhibit growth of human oesophageal cancer cells via P2Y 2 -receptors

Extracellular ATP is known to inhibit growth of various tumours by activating specific purinergic receptors (P2-receptors). Since the therapy of advanced oesophageal cancer is unsatisfying, new therapeutic approaches are mandatory. Here, we investigated the functional expression and potential antiproliferative effects of P2-purinergic receptors in human oesophageal cancer cells. Prolonged incubation of primary cell cultures of human oesophageal cancers as well as of the squamous oesophageal cancer cell line Kyse-140 with ATP or its stable analogue ATPγS dose-dependently inhibited cell proliferation. This was due to both an induction of apoptosis and cell cycle arrest. The expression of P2-receptors was examined by RT-PCR, immunocytochemistry, and [Ca2+]i-imaging. Application of various extracellular nucleotides dose-dependently increased [Ca2+]i. The rank order of potency was ATP=UTP>ATPγS>ADP=UDP. 2-methylthio-ATP and α,β-methylene-ATP had no effects on [Ca2+]i. Complete cross-desensitization between ATP and UTP was observed. Moreover, the phospholipase C inhibitor U73122 dose-dependently reduced the ATP triggered [Ca2+]i signal. The pharmacological features strongly suggest the functional expression of G-protein coupled P2Y2-receptors in oesophageal squamous cancer cells. P2Y2-receptors are involved in the antiproliferative actions of extracellular nucleotides. Thus, P2Y2-receptors are promising target proteins for innovative approaches in oesophageal cancer therapy.

Oesophageal squamous cell neoplasia in head and neck cancer patients: upregulation of COX-2 during carcinogenesis

Patients with (previous) head and neck cancer (HNC) are at high risk for developing second squamous cell cancer of the oesophagus. The role of cyclooxygenase-2 (COX-2) in oesophageal squamous carcinogenesis has not yet been investigated in this high-risk group. Therefore, this study examined COX-2 mRNA and protein expression in oesophageal biopsies and resected tissues of 44 HNC patients. The evaluation covered 55 oesophageal tissue samples (18 invasive oesophageal squamous cell cancers, four high- and eight low-grade dysplasias, 25 normal squamous epithelia) from the 44 patients. mRNA levels of COX-2 were measured by real-time PCR using a LightCycler. COX-2 protein expression was studied immunohistochemically and graded by a staining score. COX-2 mRNA was detected in all samples, and its levels correlated positively with the immunohistochemical staining score (P<0.05). COX-2 expression was upregulated during oesophageal squamous carcinogenesis in HNC patients, that is COX-2 expression increased significantly from normal oesophageal squamous epithelium to low- and high-grade dysplasia and finally to invasive squamous cell cancer (P<0.001). Our findings suggest that COX-2 upregulation contributes to oesophageal squamous carcinogenesis in HNC patients. Prospective studies are needed to evaluate the chemopreventive potential of COX-2 inhibitors in this high-risk group.

Ligands of the peripheral benzodiazepine receptor induce apoptosis and cell cycle arrest in oesophageal cancer cells: involvement of the p38MAPK signalling pathway

Specific ligands of the peripheral benzodiazepine receptor (PBR) are known to induce apoptosis and cell cycle arrest in oesophageal cancer cells. However, the underlying mechanisms are still unknown. Here, we investigated the transcriptional alterations and activation of protein kinases in response to PBR-specific ligands. Using cDNA arrays, we examined the transcriptional effects of the PBR-specific ligand FGIN-1-27 in two oesophageal cancer cell lines, KYSE-140 (squamous cell carcinoma) and OE-33 (adenocarcinoma). In oesophageal cancer cells, FGIN-1-27 induced extensive changes in the expression of genes involved in the regulation of apoptosis and cell cycle. Both in oesophageal cancer cell lines (KYSE-140, OE-33) we observed a strong upregulation of the growth arrest and DNA-damage-inducible genes, gadd45 and gadd153, in response to PBR ligands. gadd genes are known to be induced by p38MAPK activation. Using Western blotting we detected a time- and dose-dependent phosphorylation of p38MAPK, which was found to be functionally involved in gadd induction, apoptosis, and cell cycle arrest. In conclusion, our data indicate that PBR-specific ligands cause apoptosis and cell cycle arrest by activation of the p38MAPK pathway and induction of gadd45 and gadd153.

Up-Regulation of the Peripheral Benzodiazepine Receptor during Human Colorectal Carcinogenesis and Tumor Spread

The peripheral benzodiazepine receptor (PBR) is overexpressed in a variety of cancers. In Unio Internationale Contra Cancrum (UICC) III colorectal cancers, a high level of PBR overexpression correlates with poor prognosis. However, little is known about the role of PBR in the development and progression of colorectal cancer. This study addresses the up-regulation of PBR during colorectal carcinogenesis and tumor spread. One hundred sixteen consecutive patients undergoing surgery for colorectal cancer with either regional (59 patients) or distant metastases (57 patients) were followed-up for 5 years or until death. Twenty-four of the 59 patients with initial UICC stage III cancers later developed distant metastases. PBR overexpression in tumor specimens was determined by immunohistochemistry. UICC stage III patients with colorectal primaries highly overexpressing PBR developed metastases significantly more often than patients with low PBR overexpression in their primary carcinoma. In 54 of the 116 patients adenomas and/or metastases and/or recurrences were available to be studied for PBR up-regulation during colorectal carcinogenesis and tumor spread. PBR was found to be overexpressed in 86% of early and late adenomas. Furthermore, 85% of primaries and of 86% of metastases displayed PBR overexpression. PBR overexpression was also detected at the mRNA level as revealed by real-time PCR. The extent of PBR protein overexpression was equivalent in colorectal adenomas and carcinomas but slightly increased in metastases. These data suggest a functional role of PBR during colorectal carcinogenesis and tumor spread. Thus, PBR qualifies as a target for innovative diagnostic and therapeutic approaches.

Meta-iodobenzylguanidine induces growth inhibition and apoptosis of neuroendocrine gastrointestinal tumor cells

Neuroendocrine gastrointestinal tumors take up, decarboxylate and store large amounts of monoamines. Radioactive‐labeled monoamines like the norepinephrine analogue meta‐iodobenzylguanidine (MIBG) have been used for the imaging of neuroendocrine tumors for many years. MIBG is selectively taken up via norepinephrine transporters (NETs) localized in the plasma membrane of neuroendocrine gastrointestinal tumor cells and thereby offers the possibility for specific and innovative therapeutic approaches. We investigated the antiproliferative, cytotoxic, cell cycle‐arresting and apoptosis‐inducing effects of MIBG in the neuroendocrine gastrointestinal tumor cell line STC‐1 and for control in the nonneuroendocrine colorectal cancer cell line HT‐29. RT‐PCR revealed the expression of NET in STC‐1 but not in HT‐29 cells. MIBG dose‐dependently induced cytotoxicity and growth inhibition of STC‐1 cells. It potently induced apoptosis in STC‐1 cells as assessed by changes in the mitochondrial membrane potential, activation of caspase‐3 and DNA fragmentation. Moreover, MIBG altered the expression of several genes involved in proliferation, apoptosis and stress responses as shown by cDNA arrays. In contrast, neither cytotoxicity, nor growth inhibition nor induction of apoptosis were detected in response to MIBG in the NET‐deficient colorectal cancer cell line HT‐29. Our data show that MIBG induces growth inhibition and apoptosis in neuroendocrine gastrointestinal tumor cells. MIBG did not arrest the cell cycle in either cell line. Thus, monoamine transporters in the plasma membrane of neuroendocrine gastrointestinal tumor cells are promising targets for innovative and specific treatment strategies of these tumors.

Analysis of neuroendocrine differentiation and the p53/BAX pathway in UICC stage III colorectal carcinoma identifies patients with good prognosis

Background and aimsNeuroendocrine differentiation is an independent prognostic factor in colorectal cancer. Moreover, an altered p53/BAX pathway is associated with a poor clinical outcome in Union Internationale Contre le Cancer (UICC) stage III disease. Because these markers are involved in different genetic events disrupted in colorectal cancer, we investigated the prognostic power of a multimarker analysis.Patients and methodsSpecimens were analyzed from 59 patients with UICC stage III disease who underwent surgery for colorectal adenocarcinoma at our institution and were followed up for 5 years or until death. Tumors were studied for both p53 mutation and BAX protein expression as well as for the expression of neuroendocrine markers. Statistical analysis of each marker alone or in combination was performed.Resultsp53 status/BAX expression and neuroendocrine differentiation are not correlated in stage III colorectal cancers. However, the combination of both independent events identified a subgroup of patients with an excellent prognosis: Patients whose tumors were neuroendocrine marker-negative and who exhibited an intact p53/BAX pathway lived longer (mean survival, 93 months; range, 82–104 months) than patients whose tumors were either neuroendocrine marker-positive or whose tumors had a completely disrupted apoptotic pathway (41 months; range, 26–57 months; p<0.00001). In multivariate regression analysis, neuroendocrine marker-positive, p53 mutated, low-BAX-expressing tumors revealed an almost fivefold higher risk for earlier death (p<0.0001).ConclusionDisruption of the p53/BAX pathway is not pathognomonic for colorectal cancers with neuroendocrine differentiation. Both represent independent prognostic markers in UICC stage III disease. Therefore, the combined analysis of p53 status, BAX expression and neuroendocrine differentiation allows one to identify subgroups of patients with either very good or very poor prognosis.

Prognostic value of multimarker analysis in stage III colorectal cancer: one step forward towards an individualized therapy decision.

Background: Recently, we have analyzed new prognostic markers in colorectal cancer including neuroendocrine differentiation, overexpression of the sialyl-Lex antigen, overexpression of the peripheral benzodiazepine receptor (PBR), BAX protein expression and p53 mutational status. The predictive power of all markers in combination has not yet been evaluated. Patients and Methods: Between 1989 and 1991, 48 consecutive patients underwent surgery for stage III colorectal cancer at our hospital. All patients received a complete 5-year follow-up. Paraffin-embedded tumor samples were analyzed for all 5 markers. Multivariate discriminant analysis was performed to determine the prognostic value of all markers in combination. Results: Based on these prognostic markers a mathematical discriminant function was obtained. This function allowed to correctly predict the further course of disease in 77% of the patients (specificity: 83.3%, sensitivity: 70.8%). The discriminant function was confirmed in another group of 19 patients. Single marker analysis allowed the prediction of the further course of disease only in 58-70%. Conclusion: Our study shows that in colorectal cancer, multimarker analysis is superior to unimarker analysis in predicting prognosis. The derived discriminant function allows patient stratification according to risk. Therefore, a multimarker analysis provides a rationale for future individualized risk-adapted therapies in stage III colorectal cancer.

Overexpression of the peripheral benzodiazepine receptor is a relevant prognostic factor in stage III colorectal cancer

PURPOSE The peripheral benzodiazepine receptor (PBR) has been implicated in the growth control of colorectal cancer, where PBR-specific ligand-binding is increased 3-4-fold. However, the prognostic relevance of PBR (over) expression has not yet been evaluated in colorectal cancer. EXPERIMENTAL DESIGN A 5-year follow-up was performed in 116 consecutive patients undergoing surgery for colorectal cancer with regional or distant metastases [Union International Contre le Cancer (UICC) stage III, 59 patients; UICC stage IV, 57 patients]. The monoclonal anti-PBR antibody 8 D7 was used for immunohistochemical examination of paraffin-embedded sections. PBR-specific staining was compared in cancer tissues and normal mucosa. Kaplan-Meier survival curves were calculated. RESULTS Twenty-eight % of the colorectal cancers strongly overexpressed PBR. The mean survival of patients with stage III cancer was 56.2 +/- 9.2 months with and 86.8 +/- 6.6 months without high overexpression of PBR (P = 0.006). Univariate and multivariate analyses revealed that high PBR overexpression is an independent unfavorable prognostic factor in stage III colorectal cancer. In stage IV, however, the PBR status did not correlate with different survival times. CONCLUSIONS Strong PBR overexpression is a new independent prognostic marker in stage III colorectal cancer. Evaluating PBR overexpression may be useful for stratifying risk and developing risk-adapted strategies of adjuvant therapy.