Mechanotransduction of mesenchymal melanoma cell invasion into 3D collagen lattices: filopod-mediated extension-relaxation cycles and force anisotropy.

Mesenchymal cell migration in interstitial tissue is a cyclic process of coordinated leading edge protrusion, adhesive interaction with extracellular matrix (ECM) ligands, cell contraction followed by retraction and movement of the cell rear. During migration through 3D tissue, the force fields generated by moving cells are non-isotropic and polarized between leading and trailing edge, however the integration of protrusion formation, cell-substrate adhesion, traction force generation and cell translocation in time and space remain unclear. Using high-resolution 3D confocal reflectance and fluorescence microscopy in GFP/actin expressing melanoma cells, we here employ time-resolved subcellular coregistration of cell morphology, interaction and alignment of actin-rich protrusions engaged with individual collagen fibrils. Using single fibril displacement as sensitive measure for force generated by the leading edge, we show how a dominant protrusion generates extension-retraction cycles transmitted through multiple actin-rich filopods that move along the scaffold in a hand-over-hand manner. The resulting traction force is oscillatory, occurs in parallel to cell elongation and, with maximum elongation reached, is followed by rear retraction and movement of the cell body. Combined live-cell fluorescence and reflection microscopy of the leading edge thus reveals step-wise caterpillar-like extension-retraction cycles that underlie mesenchymal migration in 3D tissue.

Toxicogenomics applied to in vitro carcinogenicity testing with Balb/c 3T3 cells revealed a gene signature predictive of chemical carcinogens.

Information on the carcinogenic potential of chemicals is primarily available for High Production Volume (HPV) products. Because of the limited knowledge gain from routine cancer bioassays and the fact that HPV chemicals are tested only, there is the need for more cost-effective and informative testing strategies. Here we report the application of advanced genomics to a cellular transformation assay to identify toxicity pathways and gene signatures predictive for carcinogenicity. Specifically, genome-wide gene expression analysis and quantitative real time polymerase chain reaction (qRT-PCR) were applied to untransformed and transformed mouse fibroblast Balb/c 3T3 cells that were exposed to either 2, 4-diaminotoluene, benzo(a)pyrene, 2-acetylaminoflourene, or 3-methycholanthrene at IC20 conditions for 24 and 120 h, respectively. Then, bioinformatics was applied to define toxicity pathways and a gene signature predictive of the carcinogenic risk of these chemicals. Although bioinformatics revealed distinct differences for individual chemicals at the gene-level pathway, analysis identified common perturbation that resulted in an identification of 14 genes whose regulation in cancer tissue had already been established. Strikingly, this gene signature was identified in short-term (24 and 120 h) untransformed and transformed cells (3 weeks), therefore demonstrating robustness for its predictive power. The developed testing strategy thus identified commonly regulated carcinogenic pathways and a gene signature that predicted the risk for carcinogenicity for three well-known carcinogens. Overall, the testing strategy warrants in-depth validation for the prediction of carcinogenic risk of industrial chemicals in in vitro carcinogenicity assay.

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

BACKGROUND AND AIMS: Neuroendocrine 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 METHODS: Specimens 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. RESULTS: p53 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). CONCLUSION: Disruption 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.

Targeting the epidermal growth factor receptor by erlotinib (Tarceva) for the treatment of esophageal cancer.

Esophageal cancer is the sixth most common cause of cancer-related death worldwide. Because of very poor 5-year survival new therapeutic approaches are mandatory. Erlotinib (Tarceva), an inhibitor of epidermal growth factor receptor tyrosine kinase (EGFR-TK), potently suppresses the growth of various tumors but its effect on esophageal carcinoma, known to express EGFR, remains unexplored. We therefore studied the antineoplastic potency of erlotinib in human esophageal cancer cells. Erlotinib induced growth inhibition of the human esophageal squamous cell carcinoma (ESCC) cell lines Kyse-30, Kyse-70 and Kyse-140, and the esophageal adenocarcinoma cell line OE-33, as well as of primary cell cultures of human esophageal cancers. Combining erlotinib with the EGFR-receptor antibody cetuximab, the insulin-like growth factor receptor tyrosine kinase inhibitor tyrphostin AG1024, or the 3-hydroxy-3-methylglutaryl coenzyme. A reductase (HMG-CoAR) inhibitor fluvastatin resulted in additive or even synergistic antiproliferative effects. Erlotinib induced cell cycle arrest at the G1/S checkpoint. The erlotinib-mediated signaling involved the inactivation of EGFR-TK and ERK1/2, the upregulation of the cyclin-dependent kinase inhibitors p21(Waf1/CIP1) and p27(Kip1), and the downregulation of the cell cycle promoter cyclin D1. However, erlotinib did not induce immediate cytotoxicity or apoptosis in esophageal cancer cells. The inhibition of EGFR-TK by erlotinib appears to be a promising novel approach for innovative treatment strategies of esophageal cancer, as it powerfully induced growth inhibition and cell cycle arrest in human esophageal cancer cells and enhanced the antineoplastic effects of other targeted agents.

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.

Cell cycle arrest and apoptosis induction in hepatocellular carcinoma cells by HMG-CoA reductase inhibitors. Synergistic antiproliferative action with ligands of the peripheral benzodiazepine receptor.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is the fifth most common cause of cancer deaths worldwide. Inhibitors of cholesterol biosynthesis ('statins') have been proposed as promising adjunctive anticancer agents to treat HCC, but their mode of action is yet poorly characterized. We additionally investigated the potential benefit of a combination of peripheral benzodiazepine receptor (PBR) ligands and statins. METHODS: We analyzed the growth inhibitory effects of PBR ligands, statins, and their combination in two human HCC cell lines. Moreover, we investigated the regulation of cellular cholesterol levels and the expression of 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG-CoAR), the target of statins. RESULTS: Statins inhibited the proliferation of HCC cells by inducing apoptosis and G1/S cell cycle arrest. Statin-induced apoptosis was characterized by a breakdown of the mitochondrial membrane potential, caspase activation and nuclear degradation. Furthermore, activation of ERK1/2 was downregulated while p38MAPK was activated. Synergistic growth inhibition was obtained by the combination of the PBR ligand FGIN-1-27 with statins. PBR ligands induced a decrease of HMG-CoAR expression. This downregulation may be responsible for the enhanced sensitivity of HCC cells to statins. CONCLUSIONS: Our data shed light on the signaling cascades mediating statin-induced growth inhibition of HCC cells. Moreover, PBR ligands sensitized HCC cells to statins, suggesting a new strategy to treat HCC.

Mechanisms of mitochondrial apoptosis induced by peripheral benzodiazepine receptor ligands in human colorectal cancer cells.

Specific ligands of the peripheral benzodiazepine receptor (PBR) have been shown to induce apoptosis in gastrointestinal cancers. The aim of this study was to characterize the signaling pathways of PBR ligand-induced apoptosis. FGIN-1-27 but not PK 11195-induced apoptosis was associated with a decrease of mitochondrial membrane potential and an increase of mitochondrial volume in HT29 colorectal cancer cells. However, PK 11195-elicited apoptosis was associated with a downregulation of Bcl-2, translocation of Bax to the mitochondria including subsequent oligomerization, and activation of caspase-9, indicating the involvement of mitochondria in PK 11195-induced apoptosis. Moreover, PK 11195-induced apoptosis was associated with the generation of reactive oxygen species. This study demonstrates a novel mechanism of PK 11195-induced mitochondrial apoptosis without alteration of the mitochondrial membrane potential. The characterization of signaling pathways associated with PBR ligand-induced apoptosis will build the base for a future use of these ligands in anti-neoplastic therapeutic approaches.

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.

Peripheral benzodiazepine receptor ligands induce apoptosis and cell cycle arrest in human hepatocellular carcinoma cells and enhance chemosensitivity to paclitaxel, docetaxel, doxorubicin and the Bcl-2 inhibitor HA14-1.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is one of the most common causes of cancer deaths worldwide. Thus, novel therapies are urgently needed. A promising approach is the use of peripheral benzodiazepine receptor (PBR) ligands which inhibit the proliferation of various tumors. METHODS: PBR expression both in human HCC cell lines and in tumor specimens of HCC patients was analyzed by RT-PCR and immunostaining. To evaluate PBR ligands for the treatment of HCC, we tested their effects on human HCC cells. RESULTS: PBR was localized to the mitochondria both of HCC cell lines and tumor tissues of HCC patients. In contrast, normal liver did not express PBR. PBR ligands inhibited the proliferation of HCC cell lines by inducing apoptosis and cell cycle arrest. Apoptosis was characterized by a breakdown of the mitochondrial membrane potential, caspase-3 activation and nuclear degradation. Furthermore, pro-apoptotic Bax was overexpressed while anti-apoptotic Bcl-2 and Bcl-X(L) were suppressed. Cell cycle was arrested both at the G1/S- and G2/M-checkpoints. Synergistic anti-neoplastic effects were obtained by a combination of PBR ligands with cytostatic drugs (paclitaxel, docetaxel, doxorubicin), or with an experimental Bcl-2 inhibitor. CONCLUSIONS: This is the first report on the induction of apoptosis and cell cycle arrest by PBR ligands in HCC cells. Moreover, PBR ligands sensitized HCC cells to taxans and doxorubicin.

Cell cycle-related signaling pathways modulated by peripheral benzodiazepine receptor ligands in colorectal cancer cells.

Specific ligands of the peripheral benzodiazepine receptor (PBR) have been shown to induce both apoptosis and G1/G0 cell cycle arrest in colorectal cancers. The signaling pathways leading to cell cycle arrest are still unknown. Using cDNA array technology, we identified signaling molecules involved in cell cycle arrest induced by the PBR ligands FGIN-1-27 and PK 11195. Differential gene expression was confirmed by semi-quantitative RT-PCR or Western blot analysis of gene products. The PBR ligand-mediated signaling involved the upregulation of the cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p27Kip1, cdc16, and the cell cycle inhibitors gadd45 and gadd153, the downregulation of the cyclins D1 and B1, as well as the inactivation of ERK1/2. The p21-deficient colorectal cancer cell line HCT116 p21-/- was significantly less sensitive to PBR ligands than the parental HCT116 wild-type cells, demonstrating the functional involvement of p21WAF1/CIP1 in PBR ligand-mediated G1 arrest. This study thus revealed PBR ligand-triggered signaling pathways leading to cell cycle arrest. Moreover, we showed the functional implication and interaction of differentially expressed gene products and provided a model of signaling pathways involved in PBR ligand-induced G1 arrest. These results form the basis for future PBR ligand-mediated therapeutic approaches.

Enhancement of peripheral benzodiazepine receptor ligand-induced apoptosis and cell cycle arrest of esophageal cancer cells by simultaneous inhibition of MAPK/ERK kinase.

Specific ligands of the peripheral benzodiazepine receptor (PBR) activate pro-apoptotic and anti-proliferative signaling pathways. Previously, we found that PBR ligands activated the p38 mitogen-activated protein kinase (MAPK) pathway in esophageal cancer cells, and that the activation of p38MAPK contributed to tumor cell apoptosis and cell cycle arrest. Here, we report that PBR ligands also activate the pro-survival MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway in esophageal cancer cells, which might compromise the efficacy of PBR ligands. Hence, a combination treatment of PBR ligands and MEK inhibitors, which are emerging as promising anticancer agents, was pursued to determine whether this treatment could lead to enhanced apoptosis and cell cycle arrest. Using Western blotting we demonstrated a time- and dose-dependent phosphorylation of ERK1/2 in response to PBR ligands. Apoptosis was investigated by assessment of mitochondrial alterations and caspase-3 activity. Cell cycle arrest was measured by flow cytometric analysis of stained isolated nuclei. The inhibition of MEK/ERK with a pharmacologic inhibitor, 2'-amino-3'-methoxyflavone (PD 98059), resulted in a synergistic enhancement of PBR-ligand-induced growth inhibition, apoptosis and cell cycle arrest. Specifity of the pharmacologic inhibitor was confirmed by the use of 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U 0126), a second MEK/ERK inhibitor, and 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene (U 0124), a structural analogue of it which does not display any affinity to MEK. Enhanced pro-apoptotic and anti-proliferative effects were observed both in KYSE-140 esophageal squamous cancer and OE-33 adenocarcinoma cells, suggesting that this effect was not cell-type specific. In addition, the PBR-mediated overexpression of the stress response gene (growth arrest and DNA-damage-inducible gene gadd153) was synergistically enhanced by MEK inhibition. This is the first report of enhanced PBR-ligand-mediated apoptosis and cell cycle arrest by simultaneous MEK inhibition, suggesting a new anticancer strategy.

Release of cell fragments by invading melanoma cells.

Tumor cell invasion requires coordinated cell adhesion to an extracellular matrix (ECM) substrate at the leading edge and concomitant detachment at the cell rear. Known detachment mechanisms include the slow sliding of focal contacts, the detachment of adhesion receptors by affinity and avidity regulation, as well as the shedding of adhesion receptors, most notably integrins. In highly invasive melanoma cells migrating within 3D collagen matrices, beta1 integrins and CD44 are released upon retraction of the trailing edge, together with ripping-off complete cell fragments to become deposited along the migration trail of remodeled matrix. Cell fragments reach a size up to 12 microm in diameter, contain cytoplasm and occasionally polymerized actin enclosed by intact cell membrane including surface beta1 integrins, but do not include nuclear material. The release of cell fragments was migration dependent, as impairment of motility by a blocking anti-beta1 integrin antibody also blocked cell particle release. Invasion-associated deposition of cell fragments combines the secretory-type release of vesicles with a physical mechanism of rear retraction and migration efficiency. The deposition of cell fragments may further represent a disregulated detachment strategy with implications for neoplastic cell behavior, such as the paracrine effects on neighbor cells or a negative impact on immune effector cells.

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.

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

Esophageal cancer is the most markedly increasing tumor entity in Western countries. Due to very poor 5-year-survival, new therapeutic approaches are mandatory. Peripheral benzodiazepine receptors (PBR) have been implicated in growth control of various tumor models, but they have not been studied yet in esophageal cancer. We used esophageal cancer cell lines and primary cell cultures of human esophageal cancers and evaluated (i) expression and localization of PBR; (ii) PBR-ligand-induced inhibition of cell growth; (iii) induction of apoptosis; and (iv) alterations in cell cycle. Expression of PBR was detected both in cell lines and in primary cell cultures of human esophageal cancers. PBR was localized in the mitochondria. The PBR-specific ligands FGIN-1-27 and PK 11195, but not the centrally acting benzodiazepine clonazepam or the indolacetamide FGIN-1-52, neither of which displaying any affinity to the PBR, inhibited cell proliferation. FGIN-1-27 and PK 11195, but not clonazepam, potently induced apoptosis. FGIN-1-27 was shown to sequentially decrease the mitochondrial membrane potential, then to activate caspase-3 and finally to cause DNA fragmentation. In addition, PBR-specific ligands induced cell cycle arrest in the G1/G0 phase. Our data qualify PBR-specific ligands as innovative proapoptotic and antiproliferative substances. They might prove suitable for the treatment of esophageal cancer.

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.