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 G(1)/G(0)-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.     

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.     

Modulation of intracellular calcium in human neutrophils by peripheral benzodiazepine receptor ligands

Abstract

From January 1991 to June 1997 217 patients undergoing monolateral or bilateral total knee replacement (TKR) were consecutively enrolled in a prospective study on the incidence of postoperative infections and related risk factors. Regional antimicrobial prophylaxis (teicoplanin 400 mg) was used in 263 (95%) prostheses implanted; in the remaining 14 implants (5%) periopera-tive antibiotic prophylaxis (teicoplanin 800 mg) was administered as usual by systemic route. None of the patients experienced local or systemic adverse effects. Over the 2-year follow-up period, 8 (2.9%) primary site infectious complications were recorded, i.e. 4 superficial infections, which were cured without involvement of the prostheses, and 4 deep infections, which required prosthesis removal. Six infections occurred in patients who had undergone previous surgery of the same knee joint, and 2 in patients undergoing primary TKR (p=0.0005); diabetic patients had infections (13%) more frequently than non-diabetic patients (1.9%, p=0.01). Staphylococci were the leading organisms isolated from infections; however 3 strains of Escherichia coli were isolated from patients who had undergone a previous prosthesis implantation at the same knee joint. Regional administration of teicoplanin appears to be a safe and valuable prophylactic technique; however, in patients at risk of infection a prophylactic regimen which is also active against Gram-negative bacteria should probably be considered.     

Pro-apoptotic interactions between XK469 and the peripheral benzodiazepine receptor

XK469 (2-[4-(7-chloro-2-quinoxalinyloxy) phenoxy]propionic acid) is a new anti-tumor agent with substantial activity against several drug-resistant cell lines. Using murine leukemia L1210 cells in culture, we found the chiral R(+) form of XK469 to be substantially more cytotoxic than the S(-) form, while the herbicide analog 'Assure' was essentially inactive. The cytotoxic response to these agents was accompanied by apoptosis, and was found to be correlated with drug binding to the peripheral benzodiazepine receptor in cell culture, suggesting that receptor binding may be a factor in drug-induced cytotoxicity.     

Disruption of oncogene/tumor suppressor networks during human carcinogenesis

Oncogenes were initially discovered as retrovirally transmitted tumor causing agents. The realization that such retroviral oncogenes constitute specifically altered versions of cellular genes—proto-oncogenes, was a landmark discovery that set the stage for the molecular and mechanistic era of cancer research. Moreover, the studies on oncogene functions have been instrumental in delineating many of the paradigms of cellular signal transduction. In contrast to the original studies in animals, oncogenic activation through retroviral transmission does not appear to be a major factor in human tumorigenesis. However, oncogenes are frequently activated by gain of function mutations or fusions with other genes, or they are aberrantly expressed due to amplification, increased promoter activity, or protein stabilization, and hence they play integral roles in the genesis of human tumors.     

Abnormal regulation of DNA methyltransferase expression during colorectal carcinogenesis.

Somatic changes in CpG dinucleotide methylation occur quite commonly in human cancer cell DNA. Relative to DNA from normal human colonic cells, DNA from human colorectal cancer cells typically displays regional CpG dinucleotide hypermethylation amid global CpG dinucleotide hypomethylation. The role of the maintenance DNA methyltransferase (DNMT1) in the acquisition of such abnormal CpG dinucleotide methylation changes in colorectal cancer cells remains controversial; in one study, 60-200-fold increases in DNMT1 mRNA expression were detected in colorectal polyps and cancers relative to normal colonic tissue [W. S. El-Deiry et al., Proc. Natl. Acad. Sci. USA, 88: 3470-3474, 1991], whereas in another study, only small increases in DNMT1 mRNA expression, commensurate with differences in cell proliferation accompanying colonic tumorigenesis, were observed [P. J. Lee et al., Proc. Natl. Acad. Sci. USA, 93: 10366-10370, 1996]. To definitively ascertain whether abnormal DNMT1 expression might accompany human colorectal carcinogenesis, we subjected a series of normal and neoplastic colonic tissues to immunohistochemical staining using a polyclonal antiserum raised against a DNMT1 polypeptide. A concordance of DNMT1 expression with the expression of PCNA and other cell proliferation markers, such as Ki-67 and DNA topoisomerase IIalpha, was observed in normal colonic epithelial cells and in cells comprising other normal epithelia and lymphoid tissues. The polypeptide p21, which has been reported to undermine DNMT1 binding to proliferating cell nuclear antigen at DNA replication sites, was not expressed by normal colonic cells containing DNMT1 and other cell proliferation markers. In adenomatous polyps, although DNMT1 expression coincided with the expression of other cell proliferation markers, many DNMT1-expressing cells also expressed p21. The fidelity of DNMT1 expression was further undermined in colorectal carcinomas, in which a striking heterogeneity in DNMT1 expression, with some carcinoma cells containing very high DNMT1 levels and others containing very low DNMT1 levels, was observed. These results indicate that human colorectal carcinogenesis is accompanied by a progressive dysregulation of DNMT1 expression and suggest that abnormalities in DNMT1 expression may contribute to the abnormal CpG dinucleotide methylation changes characteristic of human colorectal carcinoma cell DNA.     

Cell cycle-related expression and ligand binding of peripheral benzodiazepine receptor in human breast cancer cell lines

The aim of this study was to measure the expression of peripheral benzodiazepine receptors (PBR) as well as mitochondria content in different phases of the cell cycle of BT-20 and MCF-7 breast cancer cell lines, using two-parameter flow cytometric analyses. The PBR expression as well as mitochondria mass, were found to increase as cells pass through different stages of the cell cycle, whereas the amount of PBR in quiescent cells was very low. Binding capacity for the PBR ligand [3H]-Ro5-4864 was strongly related to the phase of the cell cycle with a positive correlation (r=0.98) with a high percentage of cells in S phase. Incubation of BT-20 cells in serum-deprived medium with nanomolar concentrations of Ro5-4864 caused an increase in S phase cells. This effect was not observed in MCF-7 cells. Using micromolar concentrations of Ro5-4864, both BT-20 and MCF-7 cells were reversibly arrested in the G(0/1) phase.     

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.     

Immunohistochemical assessment of the peripheral benzodiazepine receptor in breast cancer and its relationship with survival.

PURPOSE: The peripheral benzodiazepine receptor (PBR) expression has been shown dramatically increased in neoplastic tissues and tumor cell lines originated from ovary, liver, colon, breast, or brain relative to untransformed tissues. Its expression has been also associated with tumor progression and aggressiveness. To explore whether PBR expression level could be of prognostic value in invasive breast cancer, we studied a series of 117 patients who underwent surgery for primary breast carcinomas and were followed-up for 8 years. EXPERIMENTAL DESIGN: Using an immunohistochemical approach, we first compared PBR expression in normal and tumoral tissues, then we studied PBR expression together with clinicopathological variables (histological type, histological grade, lymph node, estrogen and progesterone receptor status), and biological markers such as BclII, Ki-67, and HER2/Neu. RESULTS: Our results revealed a significant increase of PBR expression in tumoral versus normal breast cells. We found a negative correlation between PBR expression and estrogen receptor status (P = 0.03) as well as a positive correlation between PBR and Ki-67 (P = 0.044). Although the disease-free survival was not affected by PBR in the whole population, high PBR expression level was significantly correlated with a shorter disease-free survival in the lymph node-negative patients, P = 0.038. CONCLUSIONS: As the axillary lymph node-negative status is generally considered as a good prognosis factor, the high expression of PBR in this patient subgroup may be used to identify a new high risk population, for which a more specific therapy would be beneficial.     

Up-regulation of fibroblast growth factor-binding protein, by beta-catenin during colon carcinogenesis

Fibroblast growth factor-binding protein (FGF-BP) releases immobilized FGFs from the extracellular matrix and can function as an angiogenic switch molecule in cancer. Here we show that FGF-BP is up-regulated in early dysplastic lesions of the human colon that are typically associated with a loss of adenomatous polyposis coli and up-regulation of beta-catenin. In addition, FGF-BP expression is induced in dysplastic lesions in ApcMin/+ mice in parallel with the up-regulation of beta-catenin. Also, in cell culture studies FGF-BP is induced by beta-catenin through direct activation of the FGF-BP gene promoter. We conclude that FGF-BP is a target gene of beta-catenin.     

Increased estrogen receptor betacx expression during mammary carcinogenesis.

Identification of proteins that markedly vary during early steps of mammary carcinogenesis may help to understand its pathophysiology and to develop a prevention strategy. The expression of total estrogen receptor beta (ERbeta) protein and of its COOH-terminally spliced variant ERbetacx (or ERbeta2) was compared in 43 invasive breast cancers and in 39 adjacent normal mammary glands and 26 ductal carcinoma in situ (DCIS). Thirty-six breast cancers were ER positive by radioligand binding assay. The analysis was done by immunohistochemistry on adjacent sections of formalin-fixed, paraffin-embedded tumors using polyclonal anti-ERbeta 503 IgY and sheep polyclonal ERbetacx antibodies that were previously validated. Nuclear staining was quantified using a computerized image analyzer in selected areas of normal and cancer epithelial cells. Total ERbeta expression was high in normal glands, decreased in DCIS (P = 0.0004), and increased from DCIS to invasive tumors (P = 0.029). In contrast, the ERbetacx expression was low in normal glands, increased significantly in DCIS (P = 0.0014), and continued to increase in invasive carcinomas (P = 0.0027) in both ERalpha-positive and ERalpha-negative tumors. This is the first study showing a significant increase of the ERbetacx variant protein in DCIS and invasive breast cancer compared with adjacent normal glands. This contrasts with the decrease of the total ERbeta level in the same patients and indicates different mechanisms to explain these variations during mammary carcinogenesis. It also suggests a role of the ERbetacx variant in carcinogenesis opposite to the protective effect of the wild-type ERbeta1.     

Expression of midkine in the early stage of carcinogenesis in human colorectal cancer.

It has been suggested that a heparin-binding growth factor, midkine (MK), plays an important role in carcinogenesis because of its frequent overexpression in various malignant tumours. To clarify whether or not MK contributes to the early stage of carcinogenesis, we examined the status of MK mRNA in 20 adenomas with moderate- and severe-grade dysplasia, 28 carcinomas and 28 corresponding normal tissues, by means of Northern blotting. The MK expression level was significantly more elevated in adenomas than in normal tissues (P < 0.001, unpaired Student's t-test). A difference was also observed between carcinomas and the corresponding normal tissues (P < 0.04, paired Student's t-test). Moreover, MK immunostaining was positive in the adenomas with moderate- and severe-grade dysplasia and in the carcinomas, but not in mild-grade dysplasia or in normal tissues. These findings were in line with those on Western blotting. In three patients with both adenomas with moderate- or severe-grade dysplasia and carcinomas, elevated MK expression was observed in the neoplastic lesions. This is the first report of the association of elevated MK expression with the early stage of carcinogenesis in humans.     

Expression of prostasin and its inhibitors during colorectal cancer carcinogenesis

Background  

Clinical trials where cancer patients were treated with protease inhibitors have suggested that the serine protease, prostasin, may act as a tumour suppressor. Prostasin is proteolytically activated by the serine protease, matriptase, which has a very high oncogenic potential. Prostasin is inhibited by protease nexin-1 (PN-1) and the two isoforms encoded by the mRNA splice variants of hepatocyte growth factor activator inhibitor-1 (HAI-1), HAI-1A, and HAI-1B.