Todd L. Kiefer

Involvement of the mt1 melatonin receptor in human breast cancer.

Two putative melatonin receptors have been described including the cell surface G-protein-linked receptors, mt1 and MT2, and the nuclear retinoic orphan receptor alpha (RORalpha). The mt1 receptor, but not the MT2 receptor, is expressed in human breast tumor cell lines, and melatonin-induced growth suppression can be mimicked by the mt1 and MT2 agonist, AMMTC, and blocked by the antagonist, CBPT. RORalpha receptors are also expressed in MCF-7 breast cancer cells and the putative RORalpha agonist CPG-52608 inhibits MCF-7 cell growth but with a very different dose-response than melatonin. Finally, melatonin and AMMTC, but not CPG-52608, can repress RORalpha transcriptional activity in MCF-7 cells.

Association Between Valvular Surgery and Mortality Among Patients With Infective Endocarditis Complicated by Heart Failure

CONTEXT Heart failure (HF) is the most common complication of infective endocarditis. However, clinical characteristics of HF in patients with infective endocarditis, use of surgical therapy, and their associations with patient outcome are not well described. OBJECTIVES To determine the clinical, echocardiographic, and microbiological variables associated with HF in patients with definite infective endocarditis and to examine variables independently associated with in-hospital and 1-year mortality for patients with infective endocarditis and HF, including the use and association of surgery with outcome. DESIGN, SETTING, AND PATIENTS The International Collaboration on Endocarditis-Prospective Cohort Study, a prospective, multicenter study enrolling 4166 patients with definite native- or prosthetic-valve infective endocarditis from 61 centers in 28 countries between June 2000 and December 2006. MAIN OUTCOME MEASURES In-hospital and 1-year mortality. RESULTS Of 4075 patients with infective endocarditis and known HF status enrolled, 1359 (33.4% [95% CI, 31.9%-34.8%]) had HF, and 906 (66.7% [95% CI, 64.2%-69.2%]) were classified as having New York Heart Association class III or IV symptom status. Within the subset with HF, 839 (61.7% [95% CI, 59.2%-64.3%]) underwent valvular surgery during the index hospitalization. In-hospital mortality was 29.7% (95% CI, 27.2%-32.1%) for the entire HF cohort, with lower mortality observed in patients undergoing valvular surgery compared with medical therapy alone (20.6% [95% CI, 17.9%-23.4%] vs 44.8% [95% CI, 40.4%-49.0%], respectively; P < .001). One-year mortality was 29.1% (95% CI, 26.0%-32.2%) in patients undergoing valvular surgery vs 58.4% (95% CI, 54.1%-62.6%) in those not undergoing surgery (P < .001). Cox proportional hazards modeling with propensity score adjustment for surgery showed that advanced age, diabetes mellitus, health care-associated infection, causative microorganism (Staphylococcus aureus or fungi), severe HF (New York Heart Association class III or IV), stroke, and paravalvular complications were independently associated with 1-year mortality, whereas valvular surgery during the initial hospitalization was associated with lower mortality. CONCLUSION In this cohort of patients with infective endocarditis complicated by HF, severity of HF was strongly associated with surgical therapy and subsequent mortality, whereas valvular surgery was associated with lower in-hospital and 1-year mortality.

Estrogen receptor transactivation in MCF-7 breast cancer cells by melatonin and growth factors

The pineal hormone, melatonin, inhibits proliferation of estrogen receptor (ER)-positive MCF-7 human breast cancer cells, modulates both ER mRNA and protein expression, and appears to be serum dependent, indicating interaction between melatonin and serum components. To examine the effects of melatonin on ER activity, ER transactivation assays were performed by transiently transfecting MCF-7 cells with an ERE-luciferase reporter construct. MCF-7 cells pre-treated with melatonin for as little as 5 min followed by either epidermal growth factor (EGF) or insulin resulted in the estrogen-independent transactivation of the ER. None of the compounds when used alone transactivated the ER. The ability of melatonin and EGF to transactivate the ER was abolished by the addition of the antiestrogen, ICI 164384, suggesting that melatonin and EGF co-operate to transactivate the ER. The modulation of ER transactivation was associated with changes in mitogen activated protein kinase activity and ER phosphorylation. This ER transactivation was blocked by pertussis toxin, a Galpha i-protein-coupled receptor inhibitor, suggesting cross talk between the G-protein-coupled melatonin receptor pathway and the EGF/insulin tyrosine kinase receptor pathways in modulating ER transactivation. Exactly how the ability of melatonin in combination with EGF to transactivate the ER relates to melatonins observed growth suppressive effects is not clear. It is possible that, although melatonin and EGF transactivate the ER, this transactivation does not result in the full transcription of estrogen-responsive genes, but rather, makes the ER refractory to activation by estradiol, thus, blocking the mitogenic actions of estradiol.

Melatonin inhibits estrogen receptor transactivation and cAMP levels in breast cancer cells.

We have previously demonstrated that the pineal hormone, melatonin, can inhibit the growth of estrogen receptor-alpha (ERα)-positive breast cancer cells and suppress ERα gene transcription. To investigate the relationship between the estrogen response pathway and melatonins growth inhibition, ERα-positive MCF-7 human breast cancer cells were transiently transfected with an estrogen response element (ERE) luciferase reporter construct and then treated with melatonin (10−9-10−6 M) for 30 min followed by 10−9 M 17-β-estradiol (E2) or treated with each compound alone. Melatonin pre-treatment significantly reduced E2-induced ERα transactivation and ERα-ERE binding activity. We also conducted experiments to determine if melatonin modulates cAMP levels in MCF-7 cells. Melatonin inhibited the forskolin-induced and E2-induced elevation of cAMP levels by 57 and 45%, respectively. These data indicate that melatonin can act as a biological modifier to affect ERα transcriptional activity by regulating signal transduction pathways which impinge on the ERα and by altering E2-mediated ERα transactivation and ERα DNA binding activity.

Overexpression of the MT1 melatonin receptor in MCF-7 human breast cancer cells inhibits mammary tumor formation in nude mice

Overexpression of the MT1 melatonin receptor in MCF-7 human breast cancer cells significantly enhances the response of these cells to the growth-inhibitory actions of melatonin. Athymic nude mice implanted with MT1-overexpressing MCF-7 cells developed significantly fewer palpable tumors (60% reduction) compared to mice receiving vector-transfected MCF-7 cells (vt-MCF-7). In response to exogenous melatonin, tumor incidence in the mice receiving the MT1-overexpressing MCF-7 cells was decreased by 80% compared to mice receiving vt-MCF-7 cells. Interestingly, daily melatonin administration did not decrease tumor incidence in mice receiving vt-MCF-7 cells, but rather stimulated overall tumor formation.

Differential regulation of estrogen receptor alpha, glucocorticoid receptor and retinoic acid receptor alpha transcriptional activity by melatonin is mediated via different G proteins.

Abstract:  Melatonin has been shown to bind to the MT1 G protein‐coupled receptor (GPCR) in MCF‐7 breast cancer cells to modulate the estrogen response pathway suppressing estrogen‐induced estrogen receptor alpha (ERα) transcriptional activity, blunting ER/DNA binding activity and suppressing cell proliferation. In these studies we have examined the effect of melatonin on the transcriptional activity of the ERα and other members of the steroid/thyroid hormone receptor superfamily, namely, the glucocorticoid receptor (GR) and the retinoic acid receptor alpha (RARα). As with the ERα, melatonin represses ligand (dexamethasone)‐induced activation of the GR. This effect of melatonin on ERα and GR is blocked by pertussis toxin (PTX) suggesting that melatonins actions may be mediated via a PTX‐sensitive Gαi protein. In contrast, melatonin potentiates the action of all‐trans‐retinoic acid on RARα transcriptional activation and enhances RARα/DNA binding activity, an action which is not PTX‐sensitive. Expression of a dominant‐positive Gαi2 protein, with which the MT1 receptor has been shown to couple, is able to mimic the effect of melatonin on ERα but not RARα transcriptional activation in breast cancer cells. This demonstrates that GPCRs can modulate the transcriptional activity of various steroid receptors in response to their ligand through activation of different G protein signaling pathways.

Pathways through which a regimen of melatonin and retinoic acid induces apoptosis in MCF-7 human breast cancer cells.

It has been established that melatonin (Mlt) and retinoic acid, individually, inhibit the proliferation of the estrogen receptor-alpha (ERα)-positive MCF-7 breast cancer cell line. Our laboratory has previously demonstrated that Mlt and all-trans-retinoic acid (atRA) not only inhibit the proliferation, but also induce apoptosis of MCF-7 cells when used in a sequential regimen of Mlt followed 24 h later by atRA. Using this same MCF-7 breast cancer cell line, we investigated the potential pathways through which apoptosis is being induced. We found that treatment of MCF-7 cells with Mlt for 24 h before the addition of atRA decreased the protein levels of the death suppressor, Bcl-2, and increased, although with different time courses, the levels of the death promoters, Bax and Bak; however, there was no change in the levels of the tumor suppressor gene, p53. MCF-7 cells treated sequentially with Mlt and atRA also demonstrated an enhanced sensitivity to the apoptotic effects of atRA, which did not appear to be due to increased expression of the retinoic acid receptors, RARα or RXRα, but rather to enhanced transcriptional activity of the RARα. These data suggest that the sequential treatment regimen of Mlt and atRA may induce apoptosis by modulation of members of the Bcl-2 family of proteins. Thus, this combinatorial regimen, which reduces the concentration of atRA needed for clinical efficacy while enhancing its anti-tumorigenic activity, could be of great therapeutic benefit, and may, in fact, specifically induce the regression of established breast tumors due to its apoptosis-promoting effects.

Differential responsiveness of MCF‐7 human breast cancer cell line stocks to the pineal hormone, melatonin

The estrogen receptor (ER)‐positive MCF‐7 human breast cancer cell line has been used extensively for the study of estrogen‐responsive human breast cancer. However, various levels of estrogen responsiveness have been described in different stocks of MCF‐7 cells. Because we have previously shown that the pineal hormone, melatonin, inhibits proliferation of MCF‐7 cells and can modulate ER expression and transactivation, we investigated if various stocks of MCF‐7 cells exhibit a differential responsiveness to the anti‐proliferative effects of melatonin and the possible mechanisms involved. The MCF‐7 stocks (M, O, H) were examined for: (1) mitogenic response to estradiol; (2) steady‐state ER mRNA levels; (3) expression of the mt1 melatonin membrane receptor; (4) growth inhibition by melatonin; and (5) melatonins modulation of expression of the ER and the estrogen‐regulated genes, PgR, TGFβ and pS2. For all of these parameters, there was a stock‐specific response which showed: MCF‐7M>MCF‐7O>MCF‐7 H. These results demonstrate that there are significant differences in the responsiveness of various stocks of MCF‐7 breast cancer cells to the growth‐inhibitory effects of melatonin which can be correlated with both the level of ER mRNA expression and the degree of estrogen‐responsiveness. These findings suggest that not only may these differences have some impact on the cells’ estrogen‐response pathway, but also that the primary growth‐inhibitory effects of melatonin are transduced through the membrane‐associated G‐protein coupled mt1 melatonin receptor.

Thrombolytic Therapy for Thrombosis of Continuous Flow Ventricular Assist Devices

BACKGROUND Despite chronic systemic anticoagulation, advanced heart failure patients treated with a continuous-flow left ventricular assist device (LVAD) remain at risk for pump thrombosis. Pump thrombosis may initially be suspected in the setting of clinical and biochemical evidence for intravascular hemolysis, putatively related to shear stress on red blood cells propelled through a partially occluded pump. Limited data exist to guide management in these patients. METHODS AND RESULTS We present a series of 8 LVAD patients who presented with intravascular hemolysis secondary to pump thrombosis who were treated with intraventricular thrombolytic therapy. In 3 patients, thrombolytic therapy led to complete and lasting resolution of hemolysis, suggesting successful dissolution of pump thrombus. In the remaining 5 patients, thrombolytic therapy ultimately failed to halt or reverse pump thrombosis and hemolysis: 1 patient required emergent pump exchange, 2 patients progressed to cardiogenic shock and died, 1 patient suffered a debilitating stroke after which care was withdrawn, and 1 patient underwent cardiac transplantation. CONCLUSIONS In the setting of LVAD thrombosis, thrombolytic therapy is an alternate treatment strategy in a subset of patients. Candidacy for this alternate procedure must carefully weigh the risks of complications, including hemorrhage and thromboembolism.

Pulmonary Hypertension Related to Left-Sided Cardiac Pathology

Pulmonary hypertension (PH) is the end result of a variety of diverse pathologic processes. The chronic elevation in pulmonary artery pressure often leads to right ventricular pressure overload and subsequent right ventricular failure. In patients with left-sided cardiac disease, PH is quite common and associated with increased morbidity and mortality. This article will review the literature as it pertains to the epidemiology, pathogenesis, and diagnosis of PH related to aortic valve disease, mitral valve disease, left ventricular systolic and diastolic dysfunction, and pulmonary veno-occlusive disease. Moreover, therapeutic strategies, which focus on treating the underlying cardiac pathology will be discussed.