RNA interference inhibition of matrix metalloproteinase-1 prevents melanoma metastasis by reducing tumor collagenase activity and angiogenesis

Melanoma incidence is increasing worldwide, and metastatic melanoma is almost completely resistant to every known therapy. New approaches to treating melanoma are urgently needed, and a greater understanding of the biology of melanoma invasion and metastasis will aid in their creation. A high proportion of invasive melanomas have a constitutively active Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) signaling cascade; however, the downstream effectors of ERK signaling that contribute to melanoma invasion and metastasis are unknown. ERK signaling drives the production of the interstitial collagenase matrix metalloproteinase-1 (MMP-1), which is expressed specifically by invasive melanomas. Using short hairpin RNAs (shRNA) to knock down MMP-1 expression in a human melanoma cell line, we investigated the role of MMP-1 in melanoma metastasis in a xenograft model. Knockdown of MMP-1 had no effect on primary tumor growth, but reduction of MMP-1 expression significantly decreased the ability of the melanoma to metastasize from the orthotopic site in the dermis to the lung. Mechanistically, tumor cells expressing MMP-1 shRNAs had diminished collagenase activity, which is required for tumor cell invasion. Additionally, attenuation of MMP-1 expression reduced angiogenesis. These results show, for the first time, that targeted inhibition of MMP-1, a single effector of the Raf/MEK/ERK signaling cascade, prevents the progression of melanoma from a primary to metastatic tumor and, as such, may represent a useful therapeutic tool in controlling this disease.     

Early postnatal stimulation alters pregnane neurosteroids in the hippocampus

RATIONALE: The progesterone metabolite 5alpha-pregnane-3alpha-ol-20-one (3alpha,5alpha-THP) is an important modulator of the hypothalamic-pituitary-adrenal axis and stress-induced corticosterone response. Typically, 3alpha,5alpha-THP levels are increased in response to acute stress, which may then reduce corticosterone release from the adrenals. Early postnatal stimulation is a developmental stressor that can produce pervasive endocrine effects. OBJECTIVES: The present studies investigated the effects of early postnatal stimulation on plasma progestin and corticosterone levels and hippocampal progestin levels of rats. METHODS: On postnatal days 9 and 10, rats were either left in their home cage undisturbed or injected intraperitoneally as a means of early stimulation (ES). Tissues were collected on either postnatal day 10 (6 h after last handling experience) or adulthood. Plasma corticosterone, progesterone, and 3alpha,5alpha-THP and hippocampal progesterone and 3alpha,5alpha-THP were measured by radioimmunoassay. RESULTS: On postnatal day 10, plasma, but not hippocampal, levels of progesterone and 3alpha,5alpha-THP were significantly lower among rats exposed to ES than control rats. These effects occurred concomitant with a tendency for plasma corticosterone to be higher among ES compared to control rats. In adulthood, hippocampal 3alpha,5alpha-THP was significantly lower among ES vs control rats. CONCLUSIONS: Together, these data suggest that ES may influence immediate secretion of 3alpha,5alpha-THP and corticosterone and have pervasive effects in adulthood on the biosynthesis and/or metabolism of progestins in the hippocampus.     

Evaluation of [11C]GB67, a novel radioligand for imaging myocardial alpha 1-adrenoceptors with positron emission tomography

Dysfunction of the sympathetic nervous system underlies a number of myocardial disorders. Positron emission tomography (PET) offers a way of assessing receptor function non-invasively in humans, but there are no PET radioligands for assessing myocardial alpha-adrenoceptors. GB67, a structural and pharmacological analogue of the alpha 1-adrenoceptor antagonist prazosin, was labelled with positron-emitting carbon-11 (t1/2 = 20.4 min) by 11C-methylation of N-desmethylamido-GB67 (GB99). [11C]GB67 was injected intravenously into conscious rats. Serial arterial blood samples were taken. Rats were killed and tissues removed to determine radioactivity. The percentages of unchanged [11C]GB67 and its radioactive metabolites in plasma and tissues were assessed by HPLC. Plasma clearance of radioactivity was rapid. Myocardial uptake was maximal at 1-2 min and decreased slowly during 60 min. Predosing with adrenoceptor antagonists demonstrated selectivity for myocardial alpha 1-adrenoceptors. GB67 and prazosin blocked uptake of radioactivity; the non-selective antagonist, phentolamine, partially blocked uptake; the alpha 2-adrenoceptor antagonist, RX 821002, only blocked uptake at high dose and the beta-adrenoceptor antagonist, CGP 12177, had no effect. Additionally, injection of prazosin at 20 min after radioligand displaced radioactivity. In vivo competition curves obtained by injecting [11C]GB67 with varying amounts of either unlabelled GB67 or its precursor GB99 were fitted to a competitive binding model to provide estimates of the maximum number of binding sites (Bmax) and half saturation doses (K) for myocardium. Assuming a tissue protein content of 10%, the values of Bmax [approximately 13 pmol.(g tissue)-1[ were similar to those ]50-170 fmol.(mg protein)-1] reported for myocardial alpha 1-adrenoceptors assessed in vitro. Both GB67 and its precursor GB99 had high affinity for alpha 1-adrenoceptors [KGB67 = 1.5 nmol.(kg body weight)-1, KGB99 = 4.8 nmol.(kg body weight)-1]. HPLC demonstrated four radioactive metabolites in plasma. [11C]GB67 was 80% of the radioactivity at 5 min and 50% at 45 min. No radioactive metabolites were detected in myocardium up to 60 min after injection. [11C]GB67 was assessed in two male human volunteers. PET demonstrated high myocardial uptake. The profile of radioactive metabolites in plasma was comparable to that in the rat, although metabolism was slower in humans. Thus, [11C]GB67 is a promising radioligand for assessing alpha 1-adrenoceptors in human myocardium with PET.     

A review of the concept of the heart rate deflection point

The heart rate deflection point (HRDP) is a downward or upward change from the linear HR-work relationship evinced during progressive incremental exercise testing. The HRDP is reported to be coincident with the anaerobic threshold. In 1982, Conconi and colleagues suggested that this phenomenon could be used as a noninvasive method to assess the anaerobic threshold. These researchers developed a field test to assess the HRDP, which has become popularised as the 'Conconi test'. Concepts used to define and assess the anaerobic threshold as well as methodological procedures used to determine the HRDP are diverse in the literature and have contributed to controversy surrounding the HRDP concept. Although the HRDP may be assessed in either field or laboratory settings, the degree of HR deflection is highly dependent upon the type of protocol used. The validity of HRDP to assess the anaerobic threshold is uncertain, although a high degree of relationship exists between HRDP and the second lactate turnpoint. The HRDP appears to be reliable when a positive identification is made; however, not all studies report 100% reproducibility. Although the physiological mechanisms explaining the HRDP are unresolved, a relationship exists between the degree and direction of HRDP and left ventricular function. The HRDP has potential to be used for training regulation purposes. Clinically, it may be incorporated to set exercise intensity parameters for cardiac rehabilitation.     

Ethics in practice

H. K. is a ninety-two-year-old woman with Alzheimer's disease and mild hypertension. She resides at a nursing home, where she transfers from bed to chair with maximal assistance. She presents to our emergency department with a painful right hip. Physical examination demonstrates a confused, elderly patient with significant right hip pain and shortening and external rotation of the lower extremity. Radiographs demonstrate a displaced intertrochanteric hip fracture. The patient lacks the capacity for informed consent. Her family is contacted to obtain consent for insertion of a compression screw. The family refuses to give consent, stating that the patient is too old and the surgery is too dangerous.     

Do hospital E-codes consistently capture suicidal behaviour?

Hospital separation data are used to study suicidal behaviour; however, there is little information about the appropriateness of these data for research and planning activities. The study purpose is to examine how consistently hospital separation E-code data reflect suicidal behaviours. Expert clinicians reviewed medical records of individuals who had a separation for self-poisoning to determine whether the self-poisoning was deliberate. Agreement among clinicians was evaluated and latent class analysis performed to derive a summary estimate of the prevalence of deliberate self-poisoning. This estimate was then compared to the prevalence of deliberate self-poisoning based on the external cause of injury (E-codes). Clinicians estimated the prevalence to be 63% higher than the E-code based prevalence. Much larger discrepancies were apparent among older age groups, those whose care was primarily medical in nature and those with a longer length of hospital stay. In acute care settings, self-poisonings among the elderly may not receive adequate attention and/or documentation. Estimating the prevalence of admissions for suicidal behaviour using hospital separation data is of questionable validity, particularly among older age groups.