David B. Alexander

The Membrane-Anchored MMP Inhibitor RECK Is a Key Regulator of Extracellular Matrix Integrity and Angiogenesis

Matrix metalloproteinases (MMPs) are essential for proper extracellular matrix remodeling. We previously found that a membrane-anchored glycoprotein, RECK, negatively regulates MMP-9 and inhibits tumor invasion and metastasis. Here we show that RECK regulates two other MMPs, MMP-2 and MT1-MMP, known to be involved in cancer progression, that mice lacking a functional RECK gene die around E10.5 with defects in collagen fibrils, the basal lamina, and vascular development, and that this phenotype is partially suppressed by MMP-2 null mutation. Also, vascular sprouting is dramatically suppressed in tumors derived from RECK-expressing fibrosarcoma cells grown in nude mice. These results support a role for RECK in the regulation of MMP-2 in vivo and implicate RECK downregulation in tumor angiogenesis.

Transfer of Biologically Important Molecules Between Cells Through Gap Junction Channels

Gap junctions are unique intercellular channels that connect the cytoplasm of adjacent cells. They are the only channels that mediate the direct cytoplasmic exchange of small hydrophilic molecules between cells--a process called gap junctional communication. Formed by a family of integral membrane proteins called connexins, gap junctions are dynamic multifunctional complexes that are essential for healthy vertebrate development and physiology. Defects in connexin proteins, and, therefore, in gap junctional communication, are associated with a large variety of pathologies in humans and experimental animals. Thus, knowledge of the molecules that pass through gap junction channels is extremely important. However, aside from some notable cases, the repertoire of biologically important transjunctional molecules remains relatively unexplored. Indeed, the study of the intercellular transfer of endogenous molecules presents formidable challenges. Here we review developments in identifying biologically relevant molecules that pass between cells through gap junction channels.

Mutations in two matrix metalloproteinase genes, MMP-2 and MT1-MMP, are synthetic lethal in mice

The matrix metalloproteinase (MMP) family (∼25 members in mammals) has been implicated in extracellular matrix remodeling associated with embryonic development, cancer formation and progression, and various other physiological and pathological events. Inactivating mutations in individual matrix metalloproteinase genes in mice described so far, however, are nonlethal at least up to the first few weeks after birth, suggesting functional redundancy among MMP family members. Here, we report that mice lacking two MMPs, MMP-2 (nonmembrane type) and MT1-MMP (membrane type), die immediately after birth with respiratory failure, abnormal blood vessels, and immature muscle fibers reminiscent of central core disease. In the absence of MMP-2 and MT1-MMP, myoblast fusion in vitro is also significantly retarded. These findings suggest functional overlap in mice between the two MMPs with distinct molecular natures.

Multi-walled carbon nanotubes translocate into the pleural cavity and induce visceral mesothelial proliferation in rats

Multi‐walled carbon nanotubes have a fibrous structure similar to asbestos and induce mesothelioma when injected into the peritoneal cavity. In the present study, we investigated whether carbon nanotubes administered into the lung through the trachea induce mesothelial lesions. Male F344 rats were treated with 0.5 mL of 500 μg/mL suspensions of multi‐walled carbon nanotubes or crocidolite five times over a 9‐day period by intrapulmonary spraying. Pleural cavity lavage fluid, lung and chest wall were then collected. Multi‐walled carbon nanotubes and crocidolite were found mainly in alveolar macrophages and mediastinal lymph nodes. Importantly, the fibers were also found in the cell pellets of the pleural cavity lavage, mostly in macrophages. Both multi‐walled carbon nanotube and crocidolite treatment induced hyperplastic proliferative lesions of the visceral mesothelium, with their proliferating cell nuclear antigen indices approximately 10‐fold that of the vehicle control. The hyperplastic lesions were associated with inflammatory cell infiltration and inflammation‐induced fibrotic lesions of the pleural tissues. The fibers were not found in the mesothelial proliferative lesions themselves. In the pleural cavity, abundant inflammatory cell infiltration, mainly composed of macrophages, was observed. Conditioned cell culture media of macrophages treated with multi‐walled carbon nanotubes and crocidolite and the supernatants of pleural cavity lavage fluid from the dosed rats increased mesothelial cell proliferation in vitro, suggesting that mesothelial proliferative lesions were induced by inflammatory events in the lung and pleural cavity and likely mediated by macrophages. In conclusion, intrapulmonary administration of multi‐walled carbon nanotubes, like asbestos, induced mesothelial proliferation potentially associated with mesothelioma development.

Cancer prevention by bovine lactoferrin: from animal studies to human trial.

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers and, despite improved colonoscopic screening, CRC is a leading cause of death from cancer. Administration of bovine lactoferrin (bLF) suppresses carcinogenesis in the colon and other organs of test animals, and recently it was shown that ingestion of bLF inhibits the growth of adenomatous polyps in human patients. Here we review work which established bLF as an anti-carcinogenic agent in laboratory animals and the results of a clinical trial which demonstrated that bLF can reduce the risk of colon carcinogenesis in humans.

Effect of Orally Administered Bovine Lactoferrin on the Growth of Adenomatous Colorectal Polyps in a Randomized, Placebo-Controlled Clinical Trial

Lactoferrin (LF), a secreted, iron binding glycoprotein originally discovered as a component of milk, is found in a variety of exocrine secretions and in the secondary granules of polymorphonuclear leukocytes. Animal experiments have shown that oral administration of bovine lactoferrin (bLF) exerts anticarcinogenesis effects in the colon and other organs of the rat. The aim of this study was to determine whether oral bLF could inhibit the growth of adenomatous colorectal polyps in human patients. A randomized, double-blind, controlled trial was conducted in 104 participants, ages 40 to 75 years, with polyps ≤5 mm in diameter and likely to be adenomas. Participants were assigned to receive placebo, 1.5-g bLF, or 3.0-g bLF daily for 12 months. Target adenomatous polyps were monitored by colonoscopy. Ingestion of 3.0-g bLF significantly retarded adenomatous polyp growth in participants 63 years old or younger. Removal of adenomatous colorectal polyps is done as a preventative measure against colorectal cancer; however, polyps can be overlooked, and when detected, polypectomy is not always 100% effective in eradicating a polyp. Our study suggests that daily intake of 3.0 g of bLF could be a clinically beneficial adjunct to colorectal polyp extraction.

Normal cells control the growth of neighboring transformed cells independent of gap junctional communication and SRC activity.

The growth of many types of cancer cells can be controlled by surrounding normal cells. However, mechanisms underlying this phenomenon have not been defined. We used a layered culture system to investigate how nontransformed cells suppress the growth of neighboring transformed cells. Direct physical contact between transformed and nontransformed cells was required for growth suppression of transformed cells in this system; communication by diffusible factors was not sufficient. However, significant gap junctional communication was not required, indicating that other intercellular junctions mediated this growth regulatory response. We also report that the Src kinase activity in transformed cells was not directly inhibited by contact with nontransformed cells. Instead, nontransformed cells increased the expression of serum deprivation-response protein and the transcription factor four and a half LIM domain 1 in tumor cells. In addition, these results suggest mechanisms by which normal cells may block Wnt signaling, inhibit insulin-like growth factor activity, and promote host recognition of neighboring tumor cells.

Involvement of macrophage inflammatory protein 1α (MIP1α) in promotion of rat lung and mammary carcinogenic activity of nanoscale titanium dioxide particles administered by intra-pulmonary spraying

Titanium dioxide (TiO(2)) is evaluated by World Health Organization/International Agency for Research on Cancer as a Group 2B carcinogen. The present study was conducted to detect carcinogenic activity of nanoscale TiO(2) administered by a novel intrapulmonary spraying (IPS)-initiation-promotion protocol in the rat lung. Female human c-Ha-ras proto-oncogene transgenic rat (Hras128) transgenic rats were treated first with N-nitrosobis(2-hydroxypropyl)amine (DHPN) in the drinking water and then with TiO(2) (rutile type, mean diameter 20 nm, without coating) by IPS. TiO(2) treatment significantly increased the multiplicity of DHPN-induced alveolar cell hyperplasias and adenomas in the lung, and the multiplicity of mammary adenocarcinomas, confirming the effectiveness of the IPS-initiation-promotion protocol. TiO(2) aggregates were localized exclusively in alveolar macrophages and had a mean diameter of 107.4 nm. To investigate the underlying mechanism of its carcinogenic effects, TiO(2) was administered to wild-type rats by IPS five times over 9 days. TiO(2) treatment significantly increased 8-hydroxydeoxy guanosine level, superoxide dismutase activity and macrophage inflammatory protein 1alpha (MIP1alpha) expression in the lung. MIP1alpha, detected in the cytoplasm of TiO(2)-laden alveolar macrophages in vivo and in the media of rat primary alveolar macrophages treated with TiO(2) in vitro, enhanced proliferation of human lung cancer cells. Furthermore, MIP1alpha, also detected in the sera and mammary adenocarcinomas of TiO(2)-treated Hras128 rats, enhanced proliferation of rat mammary carcinoma cells. These data indicate that secreted MIP1alpha from TiO(2)-laden alveolar macrophages can cause cell proliferation in the alveoli and mammary gland and suggest that TiO(2) tumor promotion is mediated by MIP1alpha acting locally in the alveoli and distantly in the mammary gland after transport via the circulation.

The membrane-anchored metalloproteinase regulator RECK stabilizes focal adhesions and anterior-posterior polarity in fibroblasts

Accumulating evidence indicates that Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a membrane-anchored matrix metalloproteinase regulator, plays crucial roles in mammalian development and tumor suppression. Its mechanisms of action at the single cell level, however, remain largely unknown. In mouse fibroblasts, RECK is abundant around the perinuclear region, membrane ruffles and cell surface. Cells lacking Reck show decreased spreading, ambiguous anterior–posterior (AP) polarity, and increased speed and decreased directional persistence in migration; these characteristics are also found in transformed fibroblasts and fibrosarcoma cells with low RECK expression. RECK-deficient cells fail to form discrete focal adhesions, have increased levels of GTP-bound Rac1 and Cdc42, and a marked decrease in the level of detyrosinated tubulin, a hallmark of stabilized microtubules. RECK-deficient cells also show elevated gelatinolytic activity and decreased fibronectin fibrils. The phenotype of RECK-deficient cells is largely suppressed when the cells are plated on fibronectin-coated substrates. These findings suggest that RECK regulates pericellular extracellular matrix degradation, thereby allowing the cells to form proper cell–substrate adhesions and to maintain AP polarity during migration; this mechanism is compromised in malignant cells.

Lactoferrin: an alternative view of its role in human biological fluids.

Lactoferrin is a major component of biologically important mucosal fluids and of the specific granules of neutrophils. Understanding its biological function is essential for understanding neutrophil- and mucosal-mediated immunity. In this review, we reevaluate the in vivo functions of human lactoferrin (hLF) emphasizing in vivo studies and in vitro studies performed in biologically relevant fluids. We discuss the evidence in the literature that supports (or does not support) proposed roles for hLF in mucosal immunity and in neutrophil function. We argue that the current literature supports a microbiostatic role, but not a microbicidal role, for hLF in vivo. The literature also supports a role for hLF in inhibiting colonization and infection of epithelial surfaces by microorganisms and in protecting tissues from neutrophil-mediated damage. Using this information, we briefly discuss hLF in the context of the complex biological fluids in which it is found.