Molecular genetics and mechanisms of apoptosis in carcinomas of the lung and pleura: therapeutic targets.

Cancers of the lung and pleura remain a major cause of cancer deaths, both in men and women, with strong causal relationships between cigarette smoking and asbestos fibres, and deaths from lung cancer and mesothelioma, respectively. The poor survival rates for small cell lung cancer and mesotheliomas argue powerfully for greater understanding of mechanisms of carcinogenesis, genetic abnormalities and the role of tumour suppressor genes and proteins in carcinomas of the lung and pleura. Despite progress in the development of newer cytotoxic drugs, lung cancer remains a lethal disease. Chemotherapy and radiotherapy produce only a modest improvement in survival of patients with advanced disease. Increased knowledge of molecular mechanisms of lung cancer and apoptosis are providing opportunities for treating lung cancer with new classes of molecularly targeted drugs. These novel therapies should target the abnormalities in lung cancer by maximizing the effects of anti-tumour molecules, with minimal side effects on normal tissues. Of the several molecular targets, those receiving attention are p53 gene replacement, Bcl-2 downregulation, apoptosis by induced by TNF, the FAS/CD95 receptor system and TRAIL, and inhibition of NF-kappaB. Although several studies have shown benefits, there is a need for well planned clinical trials of drugs that target the apoptotic cascade. Stem cell therapy and gene replacement offer the prospect of novel approaches that are likely in the near future to play a definitive role in the treatment of advanced lung cancer. Furthermore, with their apparent minimal toxicity to normal tissues, the newer molecular targets represent attractive investigational directions for innovative cancer therapies.

Activation of kinin B1 receptors induces chemotaxis of human neutrophils.

Kinins are biologically active peptides that are powerful mediators of cellular inflammation. They mimic the cardinal signs of inflammation by inducing vasodilatation and by increasing vascular permeability and pain. Neutrophils are chemoattracted to sites of inflammation by several stimuli. However, the evidence concerning the chemotactic effect of kinin peptides has been contradictory. We analyzed the chemotactic effect of kinin B(1) receptor agonists on neutrophils isolated from peripheral blood of human healthy subjects. Chemotaxis was performed using the migration under agarose technique. To test the effect of B(1) receptor agonists, each assay was carried out overnight at 37 degrees C in 5% CO(2)-95% air on neutrophils primed with 1 ng/ml interleukin-1beta. Simultaneous experiments were performed using unprimed cells or cells challenged with formyl-Met-Leu-Phe (fMLP). A clear chemotactic activity was observed when primed neutrophils were challenged with Lys-des[Arg(9)]-bradykinin (LDBK) or des[Arg(9)]-bradykinin at 10(-10) M but not when unprimed cells were used. A reduction in the chemotactic response was observed after priming of cells in the presence of 0.5 mM cycloheximide and 10 mug/ml brefeldin A, suggesting that some protein biosynthesis is required. Techniques such as reverse transcriptase-polymerase chain reaction and in situ hybridization confirmed the expression of the B(1) receptor mRNA, and immunocytochemistry and autoradiography demonstrated the expression of the B(1) receptor protein. In contrast to other chemoattractants such as fMLP, cytosolic intracellular calcium did not increase in response to the B(1) receptor agonist LDBK. A generation of kinin B(1) receptor agonists during the early phase of acute inflammation may favor the recruitment of neutrophils to the inflammatory site.

Upregulation of tissue kallikrein, kinin B1 receptor, and kinin B2 receptor in mast and giant cells infiltrating oesophageal squamous cell carcinoma.

BACKGROUND: The mitogenic kinin peptides formed by the serine protease, tissue kallikrein (TK1), stimulate the proliferation of tumour cells and, by increasing vascular permeability, enhance metastasis. Oesophageal mucosal epithelial cells are derived from the epithelial cell germ layer, which expresses the kallikrein-kinin cascade. AIM: To determine the cellular distribution of active TK1, prokallikrein, and the kinin B(1) and B(2) receptors in oesophageal carcinoma by immunocytochemistry and in situ hybridisation (ISH). METHODS: Fifty oesophageal specimens (33 biopsies and 17 resections) and 10 control specimens adjacent to tumour or normal oesophageal biopsies were studied. Specific antibodies were used to determine the cellular localisation of TK1, prokallikrein, and the kinin B(1) and B(2) receptors in normal and oesophageal specimens by standard immunohistochemical techniques. The intensity of immunolabelling was quantified by image analysis. Antisense probes for TK1 and the kinin B(1) and B(2) receptors were also used to localise mRNA. RESULTS: TK1 (active and prokallikrein) was expressed in the mucosa of normal and tumour oesophageal epithelium. In general, expression was highest in activated mast cells, followed by giant tumour cells. Immunolabelling results were confirmed by ISH experiments. CONCLUSIONS: This is the first demonstration that TK1 and kinin B(1) and B(2) receptors are expressed in oesophageal carcinoma. Because TK1 released from tumour cells enzymatically generates mitogenic kinins from its endogenous substrate, kininogen, it is possible that third generation kinin receptor antagonists, which have been shown to be cytotoxic to cancer cells, may be useful therapeutic agents in this disease.

Natural products for cancer prevention: a global perspective.

The control of cancer, the second leading cause of death worldwide, may benefit from the potential that resides in alternative therapies. The primary carcinogens stem from a variety of agricultural, industrial, and dietary factors. Conventional therapies cause serious side effects and, at best, merely extend the patient's lifespan by a few years. There is thus the need to utilise alternative concepts or approaches to the prevention of cancer. This review focuses on the many natural products that have been implicated in cancer prevention and that promote human health without recognisable side effects. These molecules originate from vegetables, fruits, plant extracts, and herbs.

Genealogy, expression, and cellular function of transforming growth factor-beta.

The transforming growth factor-beta (TGF-beta) gene superfamily expresses a large set of structurally and functionally related polypeptides. Three TGF-beta isoforms are regulated by specific genes and have been identified in mammals (TGF-beta1, -beta2, and -beta3). All three-protein isoforms are observed abundantly during development and display overlapping and distinct spatial and temporal patterns of expressions. Each isoform plays a distinct role, the nature of which depends on the cell type, its state of differentiation, and growth conditions, and on the other growth factors present. TGF-beta regulates many of the processes common to both tissue repair and disease, including angiogenesis, chemotoxins, fibroblast proliferation and the controlled synthesis, and degradation of matrix proteins, such as collagen and fibronectin. This review will examine the genealogy and mode of actions of TGF-beta on the cell types involved in inflammation and repair, as well as in carcinoma.

Visualization of the sequential changes in immunolabelled tissue kininogenase which accompany follicular development and luteinization of angiogenic granulosa cells of the ovary.

The serine protease, tissue kininogenase (kallikrein), belongs to a unique family of enzymes that cleaves the decapeptide, kallidin, from the endogenous substrate kininogen. By analysis of genealogy patterns, rat KLK gene family members have been detected in ovarian luteinizing granulosa cells of both gonadotrophin-treated and nontreated control rats. Preliminary experiments suggest that when granulosa and endothelial cells are co-cultured, granulosa cells participate in the formation of vascular capillary tubes. This inherent capacity of granulosa cells to behave and respond like endothelial cells may be of importance in the aetiology of ovarian angiogenesis, which drives new blood vessel formation in the ovary. Recently, we demonstrated that tissue kininogenase showed intense immunolabelling in angiogenic endothelial cells isolated from bovine mature and regressing corpora lutea. Therefore, the question to answer was whether granulosa cells possess the same capacity to express the kallikrein-kinin cascade as do microvascular endothelial cells. As a first step, experiments were designed to determine the expression and visualization of tissue kininogenase (both active and pro-forms) as well as kininogen and kinin receptors in granulosa cells of different developmental stage and segments of the ovarian follicle by immunoperoxidase, fluorescent microscopy (confocal) and in situ hybridization.

Endothelins: vasoactive modulators of renal function in health and disease.

Vasoactive autocoids with directly opposing actions on the renal vasculature, glomerular function, and in salt and water homeostasis have been demonstrated in the kidney. In the renal cortex, endothelin (ET)-1 and angiotensin-II cause vasoconstriction, decreasing renal blood flow, and glomerular filtration rate, whereas bradykinin and atrial natriuretic peptide cause vasodilation and increase glomerular capillary permeability. ET-1 causes vasoconstriction of the afferent and efferent arteries and outer medullary descending vasa recta, thereby decreasing vasa recta and papillary blood flow, while bradykinin has the opposite effect. ET-1 stimulates cell proliferation, increasing the expression of several genes, including collagenase, prostaglandin endoperoxidase synthase, and platelet-derived growth factor. ET-1 promotes natriuresis via the ET-B receptor, causing down-regulation of the epithelial Na(+) channel in the renal tubule. Thus, ETs affect three major aspects of renal physiology: vascular and mesangial tone, Na(+) and water excretion, and cell proliferation and matrix formation.

Kallikrein and kinin receptor genes.

Recent evidence increasingly supports the view that kinins exercise an important regulatory control in inflammation and in the growth and proliferation of cancer cells. The induction of tissue kallikrein (TK) gene results in either increased or new expression of this protease, resulting in an increased capacity to form kinins. The cellular actions of kinins are initiated and controlled by kinin B1 and B2 receptors. This review collates in detail current knowledge on the molecular profile and status of TK (hKLK1, hKLK2, and hKLK3) and the kinin B1 and B2 receptor genes. The development of TK inhibitors, as well as kinin receptor antagonists, for use in immune-modulated disorders and in tumours may provide a new generation of drugs of therapeutic value.

Expression of tissue kallikrein and kinin receptors in angiogenic microvascular endothelial cells.

Angiogenesis is the sprouting of new capillary blood vessels from pre-existing ones. The kinin family of vasoactive peptides, formed by the serine protease tissue kallikrein from its endogenous multifunctional protein substrate kininogen, is believed to regulate the angiogenic process. The aim of this study was to determine the expression of tissue kallikrein and kinin receptors in an in vitro model of angiogenesis. Microvascular endothelial cells from the bovine mature and regressing corpus luteum were used only if they reacted with known endothelial cell markers. At first the cultured endothelial cells began sprouting, and within four weeks formed three-dimensional, capillary-like structures. Immunolabelling for tissue prokallikrein and the mature enzyme was intense in the angiogenic endothelial cells derived from mature corpora lutea. Immunoreactivity was lower in non-angiogenic endothelial cells and least in angiogenic endothelial cultures of the regressing corpus luteum. Additionally, using specific antisense DIG-labelled probes, tissue kallikrein mRNA was demonstrated in cells of the angiogenic phenotype. Immunolabelled kinin B2 receptors, but not kinin B1 receptors, were visualised on angiogenic endothelial cells. Our results suggest an important regulatory role for kinins in the multiple steps of the angiogenic cascade that may occur in wound healing and cancer cell growth.

Endothelin-1 and endothelin receptor status in kidney transplants undergoing acute rejection.

Endothelin-1 (ET-1) is a potent vasoconstrictor with vasopressor and mitogenic effects. Blood samples were collected from 21 renal transplant patients undergoing acute rejection at the time of diagnostic kidney biopsy: there were 20 men and one woman, mean age 35.6 years. All patients were on triple immunosuppressive therapy with cyclosporine A, azathioprine and methylprednisolone. Twenty living kidney donors pre-uninephrectomy (11 men and nine women, mean age 34 years) served as controls. Control kidney was obtained from fresh autopsy material and normal kidney tissue from nephrectomies for malignancy. Mean plasma ET-1 was significantly increased at 1.56 +/- 0.2 pg ml(-1) during acute rejection compared to 0.74 +/- 0.06 pg ml(-1) in donors (p = 0.0009 unpaired t-test). ET(A) receptor immunolabelling was visualised in distal tubules and collecting ducts with minimal labelling in the glomeruli and blood vessels of control kidney tissue ET(A) receptor labelling was similar in kidney biopsies with acute rejection. ET(B) receptor immunolabelling was significantly increased in glomeruli (p = 0.002) and decreased in distal tubules (p = 0.004) in kidneys with acute rejection compared to control kidney tissue. While these findings may account for the oedema and hypertension observed during acute rejection, the exact significance needs to be studied further.

Identification of immunoreactive tissue prokallikrein on the surface membrane of human neutrophils.

Putative binding sites for prokallikrein, the endogenous zymogen of the vasoactive and pro-inflammatory tissue kallikrein-kinin system, were recently demonstrated on human neutrophils. However, the occurrence and distribution of neutrophil-bound prokallikrein itself have so far not been examined. In this study, a specific anti-peptide antibody directed against the propart of the zymogen was used to localize the kallikrein precursor by confocal laser-scanning microscopy on unstimulated human blood neutrophils. Our results describe, for the first time, the presence of tissue prokallikrein on the membrane of circulating neutrophils. Immunoreactive prokallikrein was associated into punctate clusters occupying the external surface of the neutrophil membrane and, after addition of exogenous zymogen, immunolabeling was enhanced four-fold. In contrast, only moderate immunoreactivity to prokallikrein was observed intracellularly. These results suggest that resting neutrophils provide a circulating platform for tissue prokallikrein whose surface density may be upregulated as part of the inflammatory process.

Urinary levels of tissue kallikrein in the South African Black and Indian hypertensives.

It is accepted that Black subjects differ from White and Indian hypertensives in their response to hypotensive agents. Black hypertensives in the USA have a lower urinary tissue kallikrein (TK) excretion levels compared to White hypertensives. It has been suggested that Black patients respond better to thiazide diuretics compared to beta-blockers because thiazides increase whereas beta-blockers decrease tissue kallikrein excretion. This study compares the excretion of urinary TK in Black and Indian hypertensive and normotensive subjects. Urinary TK levels were measured with the selective, synthetic peptic substrate with the sequence of H-D-Val-Leu-Arg-pNA. Ten hypertensive patients on placebo therapy and 10 normotensive Black and Indian subjects provided three samples at weeks 0, 2 and 4 for the determination of urinary TK. The results were analysed using analysis of variance to compare the two racial groups. There were no significant differences in urinary TK values of the three bi-weekly individual samples. Urinary tissue kallikrein values (ng TK/microg protein) in Indian hypertensives were in general lower than Black hypertensives.

Hydrolysis of kininogens by degranulated human neutrophils and analysis of bradykinin as chemotactic factor for cells isolated from peripheral blood.

Human neutrophils play a pivotal role in acute inflammation including the regulation of vascular permeability. We have examined the capacity of neutrophil enzymes to hydrolyse human kininogens in vitro and have also explored the potentiality of bradykinin to induce chemotactic migration on neutrophils isolated from peripheral blood. Isolated neutrophils were stimulated with either f-Met-Leu-Phe, thrombin or silica particles coated with human IgG. Neutrophil enzymes obtained by degranulation produced, after 45 min of incubation with high and low molecular weight kininogens, the complete transformation of both proteins in polypeptides ranging from 20 to less than 10 kDa in molecular mass. Supernatants obtained from nonstimulated neutrophils did not modify the molecular size of kininogens. The assay used to test the chemoattractant capacity of synthetic bradykinin on human neutrophils showed that this peptide has no chemotactic activity on cells isolated from healthy subjects. Our results show that stimulation of human neutrophils with opsonized silica, thrombin and the chemotactic peptide f-Met-Leu-Phe induces release of kininogen-hydrolyzing enzymes from these cells.

Kinin receptors are expressed in human astrocytic tumour cells.

Tissue kallikrein (TK) is known to be present in several tumours in which increased KLK1 (TK) gene expression has been demonstrated. By degrading components of the extracellular matrix, TK may facilitate tumour proliferation and invasion. The vasodilatory effect of the bioactive kinin peptides causes an increase in vascular permeability, thereby enhancing metastasis. Since kinins act by receptor-linked signal transduction mechanisms, the aim of this study was to elucidate the localization and expression of kinin B1 and B2 receptors in surgical samples of human astrocytic tumours. Tumour tissue collected was processed for light, confocal and electron microscopy (EM) and RNA extraction. The mean high intensity of immunolabeling in tumour cells was quantified in pixels per square micrometer using the Analysis 2.1 Prosystem (Soft-Imaging Software, Germany, 1996). The ultrastructural localization of B1 and B2 kinin receptors was performed on ultrathin sections of the resin-embedded tissue, using immunogold-labeled probes. In the human brain, immunoreactive B2 occurs in cortical neurones but not in glial cells, and immunolabeling for B1 receptors is absent in cortical areas. In the present study, in all of the tumours studied so far, immunolabeling for B2 (28.42 pixels/microm2, n = 12) and B1 (14.07 pixels/ microm2, n = 10) was observed on the astrocytic cells. Immunoreactive kinin receptors were also present in endothelial cells of the stromal blood vessels. At EM, the average number of immunogold particles was 14 for B2 receptors and eight for B1 receptors. The immunoreactive B2 receptors were located closer to the periphery of the tumour cells while B1 immunolabeling was observed throughout the cell.

Cellular visualization of tissue prokallikrein in human neutrophils and myelocytes.

The vasoactive peptides bradykinin and kallidin (lysyl-bradykinin) have been implicated in diapedesis, a cellular process by which neutrophils migrate through endothelial cell gap junctions. The kinin peptides are released from their precursor moiety, kininogen, by the specific action of endoproteinases, the kallikreins. Kininogens have been demonstrated on the surface of neutrophils, and the presence of a competent processing enzyme such as tissue prokallikrein in neutrophils has been postulated, but firm evidence for this is still lacking. We have raised antibodies to a synthetic peptide that is a sequence copy of the activation segment of human TK and demonstrated that the anti-peptide antibodies specifically recognized the zymogen but not the active form of kallikrein. Using these anti-peptide antibodies, we showed by Western blotting, immunocytochemistry and electron microscopy that the tissue prokallikrein antigen was localized in neutrophils and their precursor cells, the myelocytes. We further demonstrated by in situ hybridization the presence of tissue kallikrein mRNA in the mature neutrophils and myelocytes. Our findings lend credence to the hypothesis that upon release and activation, neutrophil-borne TK acts on cell-associated kininogens to trigger the release of kinins, which may open endothelial gates for neutrophil diapedesis.

Not more than three tissue kallikreins identified from organs of the guinea pig.

The large and varied multigene families of tissue kallikreins of rat and mouse are considered to selectively release as many bioactive peptides. In order to determine whether a similar family of enzymes is expressed in the organs of the guinea pig purification studies were performed. Tissue kallikreins from the submandibular gland, coagulating gland/prostate complex and the pancreas were separated by affinity chromatography on benzamidine-Sepharose. Amino-terminal sequences, the patterns of hydrolysis rates of a number of peptide p-nitroanilides, inactivation rates by active site-directed irreversible inhibitors, specific kininogenase activities and types of kinin released were used to probe the identity of the isolated enzymes. Guinea pig tissue kallikreins 1 and 2 have been reported previously. In the present study we have identified a third type, designated tissue kallikrein 1a because of its sequence similarity to kallikrein 1, which differs from the latter in the catalytic properties. The inferred occurrence of not more than two or three independent tissue kallikrein genes in the guinea pig contrasts with the varied family of enzymes expressed by the large number of such genes present in rats and mice. Expression in the guinea pig (and also in humans) of only a small number of tissue kallikreins makes specific processing of a multitude of biologically active peptides by such enzymes unlikely.

Traumatic brain contusions: a clinical role for the kinin antagonist CP-0127.

Focal cerebral contusions can be dynamic and expansive, leading to delayed neurological deterioration. Due to the high mortality associated with such cerebral contusions, our standard practice had evolved into evacuating contusions in patients who had a deterioration in level of consciousness, lesions > 30 cc and CT suggestion of raised ICP. Experimental brain edema studies have implicated kinins in causing 2 degrees brain swelling. CP-0127 (Bradycor), a specific bradykinin antagonist, has been found to reduce cerebral edema in a cold lesion model in rats. In a randomized, single blind pilot study, a 7 day infusion of CP-0127 (3.0 micrograms/kg/min) was compared to placebo in patients with focal cerebral contusions presenting within 24-96 hours of closed head injury with an initial GCS 9-14. The ICP, GCS, and vital signs were monitored hourly. The total lesion burden (TLB) was measured on serial CT scans. There were no differences in age, baseline GCS, TLB, initial ICP, or laboratory findings between the two groups (n = 20). The mean (+/- s.d.) rise in peak ICP from baseline was greater in the placebo group than with CP-0127 (21.9 +/- 4.7 vs 9.5 +/- 2.0, P = 0.018). In addition, the mean reduction in GCS in the placebo group was significantly greater than in the CP-0127 group (4 +/- 1.0 vs 0.6 +/- 0.4, P = 0.002). Significantly raised ICP and clinically significant neurological deterioration occurred in 7/9 patients on placebo (77%) and only in 1 patient (9%; n = 11) on CP-0127, mandating surgery (P = 0.005). There were no adverse drug reactions, significant changes in vital signs or variations in the laboratory values. The cerebral perfusion pressure was adequately maintained in all patients irrespective of therapy. These preliminary results with CP-0127 provide supporting evidence that the kinin-kallikrein system could be involved in cerebral edema. In this study, treatment with CP-0127 appeared to alter the natural history of traumatic brain contusions by preventing the 2 degrees brain swelling. In addition, CP-0127 obviated the need for surgery in the majority of treated patients. CP-0127 could act on the cerebral vasculature to limit dys-autoregulation and brain swelling or on the blood brain barrier to reduce cerebral edema.

Pathophysiology of the kallikrein-kinin system in mammalian nervous tissue.

The nervous system and peripheral tissues in mammals contain a large number of biologically active peptides and proteases that function as neurotransmitters or neuromodulators in the nervous system, as hormones or cellular mediators in peripheral tissue, and play a role in human neurological diseases. The existence and possible functional relevance of bradykinin and kallidin (the peptides), kallikreins (the proteolytic enzymes), and kininases (the peptidases) in neurophysiology and neuropathological states are discussed in this review. Tissue kallikrein, the major cellular kinin-generating enzyme, has been localised in various areas of the mammalian brain. Functionally, it may assist also in the normal turnover of brain proteins and the processing of peptide-hormones, neurotransmitters, and some of the nerve growth factors that are essential for normal neuronal function and synaptic transmission. A specific class of kininases, peptidases responsible for the rapid degradation of kinins, is considered to be identical to enkephalinase A. Additionally, kinins are known to mediate inflammation, a cardinal feature of which is pain, and the clearest evidence for a primary neuronal role exists so far in the activation by kinins of peripherally located nociceptive receptors on C-fibre terminals that transmit and modulate pain perception. Kinins are also important in vascular homeostasis, the release of excitatory amino acid neurotransmitters, and the modulation of cerebral cellular immunity. The two kinin receptors, B2 and B1, that modulate the cellular actions of kinins have been demonstrated in animal neural tissue, neural cells in culture, and various areas of the human brain. Their localisation in glial tissue and neural centres, important in the regulation of cardiovascular homeostasis and nociception, suggests that the kinin system may play a functional role in the nervous system.