Is copper chelation an effective anti-angiogenic strategy for cancer treatment?

Angiogenesis and the acquisition of an angiogenic phenotype is important for cancer cell proliferation. Copper in an essential trace element that participates in many enzymatic complexes like the cytochrome c, superoxide dismutase and lysyl oxidase and it is involved in processes, like embryogenesis, growth, angiogenesis and carcinogenesis. In particular, its involvement in carcinogenesis was described for the first time in oral submucous fibrosis, where fibroblasts produce large amounts of collagen in the presence of copper. Copper's action in carcinogenesis is two-fold: (1) it participates in reactions with an increased redox potential that result in the production of oxidative products and oxidative stress. Through this mechanism, copper may cause DNA mutations in the nucleus and mitochondria or alterations to membrane phospholipids, (2) it participates in angiogenesis even in the absence of angiogenic molecules, as it was reported for the first time in rabbit cornea models with copolymer pellets charged with PGE1. Copper chelation regimens like penicillamine and tetrathiomolybdate are being described in the literature as having anti-angiogenic, anti-fibrotic and anti-inflammatory actions. Animal models of brain cancer that evaluated the anti-angiogenic properties of copper, have proven evidence of the reduction of tumor's microvascular supply, tumor volume and vascular permeability after plasma copper levels reduction. Interestingly, plasma copper levels reduction was shown to suppress micrometastases generation in mice models of breast cancer. We hypothesize that copper chelation therapy: increases oxidative stress in cancer cells to a level that does not allow survival because of the reduction of anti-oxidative enzymes production. It may also result in inhibition of angiogenesis and of the initiation of the angiogenic switch, because copper normally enhances endothelial cell migration and proliferation, improves binding of growth factors to endothelial cells and enhances the expression of angiogenic molecules. Copper chelation may also reduce extracellular matrix degradation and cancer spread, through reduction of MMP-9 production and probably of other collagenases and may inhibit propagation of micrometastases. However, copper chelation therapy may enhance angiogenesis through reduction of thrombospondin-1, that results into an increase in VEGF-VEGFR2 complexes and a high level of active MMP-9. These hypotheses help in understanding of the anti-angiogenic action of copper chelation therapies and of the complex network of interactions between copper and other molecules involved in angiogenesis. It may also stimulate further research regarding differences in copper metabolism, the effects of anti-copper regimens on organs, the development of resistance, and their possible angiogenic action through thrombospondin expression reduction.

Meniscofibular ligament: morphology and functional significance of a relatively unknown anatomical structure.

Purpose. A relatively unknown ligamentous structure of the posterolateral corner of the knee joint, the so-called meniscofibular ligament (MFL), was investigated as regards its macroscopic morphology, its histological features, and its reaction to knee movements. Material and Methods. MFL was exposed on 21 fresh-frozen unpaired knee joints. Its microscopic morphology was examined utilizing for comparison the fibular collateral and the popliteofibular ligament. Results. MFL was encountered in 100% of the specimens as a thin striplike fibrous band extending between the lower border of the lateral meniscus and the head of the fibula. MFL was tense during knee extension and external rotation of the tibia, whereas its histological features were similar to those of fibular collateral and popliteofibular ligament. Discussion. Its precise histological nature is studied as well as its tension alterations during knee movements. The potential functional significance of the MFL with respect to its role in avoidance of lateral meniscus and lateral coronary ligament tears is discussed. Conclusions. MFL presumably provides an additional protection to the lateral meniscus during the last stages of knee extension, as well as to the lateral coronary ligament reducing the possibility of a potential rupture.

Factors affecting chromatin stability of bovine spermatozoa.

The structural stability of transcriptionally inert paternal chromatin is of vital importance for the fertilization process and early embryonic development. Accordingly, a series of eight experiments were conducted during a 7-month period to investigate: (1) effects of bull breed, individuality, successive ejaculations, semen quality characteristics (SQC), semen dilution rates and hypothermic storage of semen in a Tris-egg yolk extender on incidence of sperm nuclear chromatin instability (NCI), and (2) effects of the interaction between variation of NCI within a frozen ejaculate and variation of oocytes quality due to maturation time and/or season on the efficiency of in vitro embryo production (IVEP). Semen samples were collected once a week from six bulls using an AV and only ejaculates (n=220) of >0.30x10(9) sperm/ml and >or=60% motility were used. NCI was measured by: (1) detection of lysine-rich histones in sperm chromatin using aniline blue staining, (2) sperm susceptibility to acid-induced nuclear DNA denaturation in situ using acridine orange test, and (3) sperm susceptibility to nuclear chromatin decondensation (NCD). Bovine oocytes (n=695) were matured in vitro for 18 or 24 h, fertilized after sperm selection through a swim-up procedure and cultured for 72 h. The results showed that the 2nd ejaculates were superior to the 1st ones with respect to chromatin stability. Dilution of semen to 49.67+/-8.56x10(6) sperm/ml (1:19) decreased resistance of sperm to NCD. Cooling of semen had no significant effect on chromatin stability. Cryopreservation of semen augmented sperm vulnerability to DNA denaturation. Improvement of SQC (semen volume, sperm motility, velocity, viability and morphological normalcy) was generally concomitant with increase of sperm resistance to NCI. While Blonde d'Aquitaine bulls had a resistance to NCD higher than Limousine bulls in fresh semen, the former showed a greater susceptibility to DNA denaturation than the latter in cooled semen. Individuality significantly influenced NCI. The variability of NCI within a frozen ejaculate affected efficiency of IVEP. Significant negative correlations were observed between incidence of NCI and both fertilization rate and developmental capacity of embryos after maturation of oocytes for 18 h. The significant variation in IVEP traits due to season was independent of the effect of sperm chromatin instability.

Histological evaluation of colonic anastomotic healing, during perioperative Capecitabine administration. Experimental study in rats.

BACKGROUND: Neoadjuvant chemotherapy is a highly promising treatment modality for colorectal cancer. One of the basic side effects of this method is the possible impact on anastomotic healing. Capecitabine is a tumor selective pro-drug of 5-fluorouracil, indicated for the therapy of colorectal cancer. The aim of this study is to estimate the effect of perioperative Capecitabine administration on the colonic anastomotic healing process, by evaluating histopathological findings. METHODS: We studied the effect of Capecitabine on hand sutured colonic anastomosis in rats. Sixty Wistar rats were randomized in two groups. In the study group (n=30) Capecitabine was given p.o. in therapeutic dose of 359 mg/kg, (2/3 of the mean toxic dose), 1 week prior the anastomosis and throughout the study. In the control group (n=30) placebo medication was administrated. Both groups were further subdivided into 3 groups, each consisting of 10 animals. Rats of each group were sacrificed on the 3rd, 7th and 14th postoperative day, in both study and control groups. RESULTS: No negative impact on the healing process of colonic anastomosis was found. Histological findings indicated a more effective healing during the early postoperative days, with lesser necrosis effects on the anastomotic line for the study group, in comparison with the control group. The median bursting pressure was found to be significantly higher in the subdivision of the study group sacrificed on the 3rd day, in comparison to respective control subdivision. CONCLUSION: Perioperative administration of Capecitabine, as neoadjuvant chemotherapy, does not impair the healing of colonic anastomosis in rats.

The role of ascorbic acid and exercise in chronic ischemia of skeletal muscle in rats.

This study was designed to investigate the effects of peripheral arterial insufficiency, exercise, and vitamin C administration on muscle performance, cross-sectional area, and ultrastructural morphology in extensor digitorum longus (EDL) and soleus (Sol) muscles in rats. Adult Wistar rats were assigned to ischemia alone (isch), ischemia-exercised (exe), ischemia-vitamin C (vit C), and ischemia-exercise-vitamin C (vit C + exe) groups. Ischemia was achieved via unilateral ligation of the right common iliac artery. Contralateral muscles within the same animal served as controls. Exercise protocol consisted of 50-min intermittent level running performed every other day for 5 days. Vitamin C (100 mg/kg body wt) was administered intraperitoneally on a daily basis throughout the 14 days of the experiment. With regard to the EDL muscle, ischemia alone reduced muscle strength, which was not recovered after vitamin C administration. Exercise alone following ischemia induced the most severe structural damage and cross-sectional area decrease in the muscle, yet the reduction in tetanic tension was not significant. Exercise in conjunction with vitamin C administration preserved ischemia-induced EDL muscle tetanic tension. In the Sol muscle, a significant reduction in single twitch tension after vitamin C administration was found, whereas the tetanic force of the ischemic Sol was not significantly decreased compared with the contralateral muscles in any group. Ischemic Sol muscle cross-sectional area was reduced in all but the exe groups. In Sol, muscle strength was reduced in the vit C group, and mean cross-sectional area of ischemic Sol muscles was reduced in all groups except the exe group. These results illustrate that mild exercise, combined with a low dose of vitamin C supplementation, may have beneficial effects on ischemic EDL muscle with a smaller effect on the Sol muscle.

Pathogenesis of bromodeoxyuridine-induced cleft palate in mice.

This study was designed to examine the pathogenesis of bromodeoxyuridine-induced (BrdU) clefts of the secondary palate in mice. Intraperitoneal injections of BrdU (500 mg/kg body weight) were given on days 11 and 12 to some pregnant mice and on days 12 and 13, and days 11, 12 and 13 to others. Evaluation of craniofacial relations and palate development in BrdU-treated mice revealed inhibition of vertical development of the palatal shelves, mandibular hypoplasia which led to failure of downward displacement of the tongue and the creation of an obstacle to reorientation of the palatal shelves. The results of this study demonstrate a strong correlation between induction of cleft palate and the presence of structural alterations in the mandible, and the mechanism of BrdU-induced cleft palate resembles the defect in the Pierre Robin anomaly.

Ultrastructural alterations of the rabbit sciatic nerve, spinal cord and cerebellum, following methionine sulphoximine administration.

Methionine sulphoximine (MSO) is a centrally acting neurotoxin which inhibits the glutamate metabolism enzymes and has convulsive properties. Small doses of MSO were administered to rabbits, either intravenously (i.v.) or intracerebroventricularly (ICV), and electron microscopic examination of the cerebellum, the spinal cord and the sciatic nerve was performed on the first day of rabbit hind leg rigid paralysis (myopathy with histological findings resembling myositis), which set in by the 2nd to 4th day after MSO administration. In the cerebellum focal minor alterations were found in the astrocytes (swelling and lucidity, diminution of glycogen granules) and sparsely in the presynaptic terminals (lucidity and clumping), whereas most of the neuron presented a normal appearance. In the spinal cord and in the sciatic nerve a dissociation of the axon from the myelin sheath was evident in a small number of myelinated nerve fibres, along with the appearance of vacuolated spaces. Mitochondrial disorganisation in the axons, as well as glial cell alterations, were also seen. The ultrastructural alterations were non specific, and since they were induced 2 to 4 days after the administration of either minimum doses (i.v.) or of extremely low doses (ICV) of MSO, they may be attributed to the inordinate increase of metabolism during the period of convulsions.

Ultrastructural alterations of the rat intestinal epithelium fed with polymeric, oligopeptidic or elementary full diet, following starvation.

In this study the ultrastructure of rat jejunal epithelial cells was examined, following a starvation period of 72 hours and an enteral refeeding period of 12 days, with either Nutrison, Pepti 2000, or Nutri 2000. Most changes occurred in the animals examined immediately after the 72-hour starvation period; these mainly included a significant decrease in microvilli population, occasional cell membrane disintegration, and a usual microvesicular appearance and degranulation of the rough endoplasmic reticulum. No alterations were found in the normally-fed animals (control group). This was also practically the same for the Pepti 2000 group. In the Nutrison group, a small amount of changes were found, while in the Nutri 2000 group many alterations were detected, which nevertheless were fewer than in the starved animals. The results demonstrate that the micromorphological alterations of the intestinal epithelium caused by starvation improve faster when an oligopeptidic formula is provided, which consequently results in faster and better absorption of the nutrients.

Ultrastructural study of foetal and adult mouse renal tissue implanted in testis.

Kidneys of mice foetuses 15, 17, 19 days old, as well as kidneys of mice 1 and 4 weeks old of the pure C3H/SY species, were implanted into the right testis of 40 adult mice 1.5 to 2 months old, of the same species. The animals were sacrificed after 30 and 60 days and the evolution and development of the renal implants within the testis ware studied. The findings in the light as well as in the electron microscope, showed that the renal implants presented the histologic characters of nephroblastoma, which became clearer the more prolonged the time of the implantation was.

Morphogenesis and ultrastructure of the peripolar cells in the mouse kidney.

Peripolar cells are located in the outer layer of the Bowman's capsule. They surround the vascular pole of the renal corpuscle and project into the urinary space. Morphologically they are characterized by the presence of secretory granules within their cytoplasm. In order to study their embryological development, we used 60 C57bl mice embryos (15th to 19th gestational day), 10 newborn mice (2 hours to 6 days old), 10 preadult mice (8-30 days old) and 4 adults (4 months old). Some granular cells, dispersed at the outer and inner layer of the Bowman's capsule, appear on the 17th gestational day. Later, these cells are found around the vascular pole of the renal corpuscle, located exclusively at the outer layer of the Bowman's capsule. Their granules are spherical and variously dense, they are surrounded by a membrane and their number increases progressively with time and reaches a maximum on the 4th postnatal day. Following that, there is a diminution and then their population stabilizes. By the end of the first month, there are only a few such cells (mean number 1 to 2). They become smaller and they always project into the urinary space.

[Ultrastructural and histochemical study of a pleomorphic adenoma of the palate]. / Hypermikroskopike kai histochemike melete polymorphou adenomatos tes hyperoas.

Histochemical and ultrastructural study of the epithelial regions forming lumina in one case of pleomorphic adenoma of the palate was undertaken. The ultrastructural findings suggest that these epithelial regions are derived from the intercalated ducts. The presence in the cytoplasm of periluminal cells of pinocytotic vesicles and vacuoles containing material similar in appearance of that seen in the luminal supports a reabsorptive activity of these cells. The microcalculi of calcium phosphate occasionally seen in the cytoplasm of these cells may be formed by the phagocytosis of the endocytotic material from the lumina.