Ursolic acid from Trailliaedoxa gracilis induces apoptosis in medullary thyroid carcinoma cells.

Medullary thyroid carcinoma (MTC) originates from the C­cells of the thyroid and is not sensitive to radiation or chemotherapy. Therefore, surgical removal of the tumor tissue in its entirety is the only curative treatment for MTC. The present study aimed to examine the potential mechanisms of action of extracts of Trailliaedoxa gracilis (TG; WW Smith & Forrest), a plant from the province of Sichuan, China, and of ursolic acid (UA), a pentacyclic triterpen present in TG, on the MTC­SK MTC cell line. A total of 13 TG fractions and UA were examined in vitro for their effects on cell morphology, cell number, proliferation and rates of apoptosis. Reverse transcription­quantitative polymerase chain reaction of nuclear factor­κB essential modifier (NEMO) was performed to delineate the role of the apoptotic pathway following treatment with UA. TG and UA were examined in vivo in xenotransplanted MTC­bearing severe combined immunodeficient mice. The TG fractions exhibited antiproliferative effects, with inhibition of mitochondrial activity in the tumor cells at concentrations, which caused no impairment of the normal control cells. The apoptotic rates of the MTC­SK cells treated with the TG fractions and UA were determined, in which no marked tumor inhibition was observed in the treated MTC­mice, and no change in the expression of NEMO was detected in the treated MTC­SK cells. The observation of early­onset activation of caspase 8 suggested that the responsible factor was linked to NEMO, an anti­apoptotic protein. However, no differences in the mRNA transcription levels of NEMO were detected in MTC­SK cells treated with UA, suggesting that this protein was not associated with the signal transducer and activator of transcription 3 pathway.

The anti-hypertensive drug prazosin induces apoptosis in the medullary thyroid carcinoma cell line TT.

BACKGROUND/AIM: Medullary thyroid carcinoma (MTC) is a tumor associated with poor prognosis since it exhibits high resistance against conventional cancer therapy. Recent studies have shown that quinazolines exhibit a pro-apoptotic effect on malignant cells. The aim of the present study was to elucidate whether MTC cells are affected by quinazolines, in particular prazosin. MATERIALS AND METHODS: Proliferation, apoptosis and cell morphology of the MTC cell line TT were analyzed by WST-1 assay, caspase 3/7 activation tests and microscopy. Fibroblasts were used as control for non-malignant cells. RESULTS: Prazosin potently inhibited the growth of TT cells, induced apoptosis and caused vacuolization, as well as needle-like filopodia. Fibroblasts were affected by prazosin in the same way as MTC cells. CONCLUSION: MTC cells are responsive to prazosin treatment similar to other malignancies. The fact that fibroblasts also respond to prazosin further highlights the importance to identify the unknown pro-apoptotic target of quinazolines.

The influence of glutamate receptors on proliferation and metabolic cell activity of neuroendocrine tumors.

Neuroendocrine tumors are relatively insensitive to radiation therapy, as well as chemotherapy. Thus, new approaches for alternative therapies are needed. We found that glutamate receptor antagonists are capable of suppressing tumor growth and cell activity of different peripheral malignancies. In the present article we review scientific literature in this field of science. Subtype-specific, non-competitive, metabotropic glutamate receptor-1 antagonists differently suppressed the growth and metabolic cell activity of one human medullary thyroid carcinoma cell line, as well as of four different human midgut neuroendocrine tumor cell lines. Furthermore, PCR analyses revealed that this subtype of glutamate receptor is expressed in these cell lines. These first results indicate that specific metabotropic glutamate receptor antagonists suppress the proliferation and cell activity of neuroendocrine tumor cells, which makes them possible targets in cancer therapy.

Christia vespertilionis plant extracts as novel antiproliferative agent against human neuroendocrine tumor cells.

Neuroendocrine tumors respond poorly to radiation and conventional chemotherapy, hence surgical removal of the neoplastic tissue is still the most effective way of treatment. In an attempt to find new therapeutic plant extracts of Christia vespertilionis (CV) their antitumor potential in human medullary thyroid carcinoma (MTC) and human small intestinal neuroendocrine tumor (SI-NET) cell lines were tested. Proliferation and viability were analyzed using cell counting and WST-1 assay. Apoptosis was determined by microscopy, luminescence assays for caspases 3/7, and expression studies of apoptosis-related genes. CV extracts showed antiproliferative and proapoptotic effects in all MTC and SI-NET cell lines, whereby high growth inhibition was observed by treatment with the ethylacetate-extracts (CV-45) in tumor cell lines but not in normal human fibroblasts. Furthermore CV-45 treatment resulted in alterations of gene expression of PDCD5, MTDH and TNFRSF10b in MTC as well as in SI-NET cells. The results indicate that Christia vespertilionis could serve as an anticancer therapeutic for treatment of neuroendocrine tumors.

Modification of the alkaline comet assay with human mesenchymal stem cells.

MSCs (mesenchymal stem cells) are planned foruse in regenerative medicine to offset age-dependent alterations. However, MSCs are affected by replicative senescence associated with decreasing proliferation potential, telomere shortening and DNA damage during in vitro propagation. To monitor in vitro senescence, we have assessed the integrity of DNA by the alkaline comet assay. For optimization of the comet assay we have enhanced the stability of comet slides in liquid and minimized the background noise of the method by improving adhesion of agarose gels on the comet slides and concentrating cells on a defined small area on the slides. The modifications of the slide preparation increase the overall efficiency and reproducibility of the comet assay and minimize the image capture and storage. DNA damage of human MSCs during in vitro cultivation increased with time, as assessed by the comet assay, which therefore offers a fast and easy screening tool in future efforts to minimize replicative senescence of MSCs in vitro.

α1-adrenergic drugs exhibit affinity to a thapsigargin-sensitive binding site and interfere with the intracellular Ca2+ homeostasis in human erythroleukemia cells.

Even though the erythroleukemia cell lines K562 and HEL do not express α1-adrenoceptors, some α1-adrenergic drugs influence both survival and differentiation of these cell lines. Since Ca2+ is closely related to cellular homeostasis, we examined the capacity of α1-adrenergic drugs to modulate the intracellular Ca2+ content in K562 cells. Because of morphological alterations of mitochondria following α1-adrenergic agonist treatment, we also scrutinized mitochondrial functions. In order to visualize the non-adrenoceptor binding site(s) of α1-adrenergic drugs in erythroleukemia cells, we evaluated the application of the fluorescent α1-adrenergic antagonist BODIPY® FL-Prazosin. We discovered that the α1-adrenergic agonists naphazoline, oxymetazoline and also the α1-adrenergic antagonist benoxathian are able to raise the intracellular Ca2+-content in K562 cells. Furthermore, we demonstrate that naphazoline treatment induces ROS-formation as well as an increase in Δψm in K562 cells. Using BODIPY® FL-Prazosin we were able to visualize the non-adrenoceptor binding site(s) of α1-adrenergic drugs in erythroleukemia cells. Interestingly, the SERCA-inhibitor thapsigargin appears to interfere with the binding of BODIPY® FL-Prazosin. Our data suggest that the effects of α1-adrenergic drugs on erythroleukemia cells are mediated by a thapsigargin sensitive binding site, which controls the fate of erythroleukemia cells towards differentiation, senescence and cell death through modulation of intracellular Ca2+.

α1-Adrenergic drugs modulate differentiation and cell death of human erythroleukemia cells through non adrenergic mechanism.

Preliminary data showed that α1-adrenergic antagonists induce apoptosis and a switch towards megakaryocytic differentiation in human erythroleukemia cells. To test the hypothesis whether survival and differentiation of erythroleukemia cells are under control of α1-adrenergic signalling, we examined α1-adrenoceptor expression of erythroleukemia cells and compared the in vitro effects of α-adrenergic antagonists with those of agonists. We discovered that α1-adrenergic agonists suppress both erythroid differentiation and growth of erythroleukemia cells concomitant with lipofuscin accumulation, autophagy and necrotic cell death. α1-adrenergic agonists also inhibit the in vitro growth of physiologic hematopoietic progenitors obtained from umbilical cord blood with high selectivity for the erythroid lineage. Interestingly, the observed effects could not be related to α1-adrenoceptors, even though agonists and antagonists displayed opposing effects regarding cellular growth and differentiation of erythroleukemia cells. Our data suggest that the effects of α1-adrenergic drugs are related to a non-adrenoceptor binding site, controlling the fate of erythroid progenitor cells towards differentiation and cell death. Since the observed effects are not mediated through adrenoceptors, the physiologic relevance of our data remains unclear, so far. Nevertheless, the identification of the still unknown binding site(s) might disclose new insights into regulation of erythroid differentiation and cell death.

Peripheral glutamate signaling in head and neck areas.

The major excitatory neurotransmitter glutamate is also found in the periphery in an increasing number of nonexcitable cells. In line with this it became apparent that glutamate can regulate a broad array of peripheral biological responses, as well. Of particular interest is the discovery that glutamate receptor reactive reagents can influence tumor biology. However, the knowledge of glutamate signaling in peripheral tissues is still incomplete and, in the case of head and neck areas, is almost lacking. The roles of glutamate signaling pathways in these regions are manifold and include orofacial pain, periodontal bone production, skin and airway inflammation, as well as salivation. Furthermore, the interrelations between glutamate and cancers in the oral cavity, thyroid gland, and other regions are discussed. In summary, this review shall strengthen the view that glutamate receptor reagents may also be promising targets for novel therapeutic concepts suitable for a number of diseases in peripheral tissues. The contents of this review cover the following sections: Introduction; The "Glutamate System"; The Taste of Glutamate; Glutamate Signaling in Dental Regions; Glutamate Signaling in Head and Neck Areas; Glutamate Signaling in Head and Neck Cancer; A Brief Overview of Glutamate Signaling in Other Cancers; and Conclusion.

The alpha1-adrenergic receptor antagonists, benoxathian and prazosin, induce apoptosis and a switch towards megakaryocytic differentiation in human erythroleukemia cells.

The erythroleukemia cell lines K562 and human erythroleukemia (HEL) are established models to study erythroid and megakaryocytic differentiation in vitro. In this study, we show that the alpha1-adrenergic antagonists, benoxathian and prazosin, inhibit the proliferation and induce apoptosis in K562 and HEL cells. Furthermore, both tested substances induced the expression of the megakaryocytic marker CD41a, whereas the expression of the erythroid marker glycophorin-a was decreased or unchanged. Even though the expression of differentiation markers was similar after benoxathian and prazosin treatment in both cell lines, endomitosis of erythroleukemia cells was observed only after prazosin treatment. So far, benoxathian and prazosin are the first described extracellular ligands, which cause megakaryocytic differentiation in K562 and HEL cells. In summary, these results indicate a possible role of alpha1-adrenergic receptor signaling in the regulation of erythroid and megakaryocytic differentiation, even though the receptor dependence of the observed effects needs further investigation.

Human endothelial cells of the placental barrier efficiently deliver cholesterol to the fetal circulation via ABCA1 and ABCG1.

Although maternal-fetal cholesterol transfer may serve to compensate for insufficient fetal cholesterol biosynthesis under pathological conditions, it may have detrimental consequences under conditions of maternal hypercholesterolemia leading to preatherosclerotic lesion development in fetal aortas. Maternal cholesterol may enter fetal circulation by traversing syncytiotrophoblast and endothelial layers of the placenta. We hypothesized that endothelial cells (ECs) of the fetoplacental vasculature display a high and tightly regulated capacity for cholesterol release. Using ECs isolated from human term placenta (HPECs), we investigated cholesterol release capacity and examined transporters involved in cholesterol efflux pathways controlled by liver-X-receptors (LXRs). HPECs demonstrated 2.5-fold higher cholesterol release to lipid-free apolipoprotein (apo)A-I than human umbilical vein ECs (HUVECs), whereas both cell types showed similar cholesterol efflux to high-density lipoproteins (HDLs). Interestingly, treatment of HPECs with LXR activators increased cholesterol efflux to both types of acceptors, whereas no such response could be observed for HUVECs. In line with enhanced cholesterol efflux, LXR activation in HPECs increased expression of ATP-binding cassette transporters ABCA1 and ABCG1, while not altering expression of ABCG4 and scavenger receptor class B type I (SR-BI). Inhibition of ABCA1 or silencing of ABCG1 decreased cholesterol efflux to apoA-I (-70%) and HDL(3) (-57%), respectively. Immunohistochemistry localized both transporters predominantly to the apical membranes of placental ECs in situ. Thus, ECs of human term placenta exhibit unique, efficient and LXR-regulated cholesterol efflux mechanisms. We propose a sequential pathway mediated by ABCA1 and ABCG1, respectively, by which HPECs participate in forming mature HDL in the fetal blood.

On the role of the kidneys in the pathogenesis of edema formation during permanent morphine application/an experimental study in rats.

In order to assess possible renal mechanisms causing the as yet unexplained side-effects of peripheral edema, weight gain and swollen limbs seen during the chronic administration of opioids in patients, a rat study was designed, mimicking a constant infusion (24 h) of morphine (CAS 57-27-2) using subcutaneous drug delivery device systems. Subcutaneous (s.c.) morphine infusions (0, 10, 30 mg/kg body weight/24 h) have been administered using implantable mini-osmotic pumps with a delivery rate of 10 microl/h. In addition, s.c. implantable inulin/PAH clearance depot systems were used in order to measure renal functions such as glomerular filtration rate (GFR = inulin clearance), renal plasma flow (RPF = PAH clearance), creatinine clearance and urea clearance in the conscious rat. Compared to controls, morphine caused an increase in PAH clearance, creatinine clearance (both dose dependent), urea clearance (higher dose), body weight, and urine potassium concentration (both groups). Urine minute volume was reduced in the 10 mg morphine group, a dose dependent highly significant decrease of urine sodium in the 30 mg morphine group compared to controls and a significant decrease compared to the 10 mg group could be found. Serum sodium was highly significant and food uptake was significantly increased in the 30 mg group compared to controls. The histological examination of kidneys revealed no differences among the various groups. These data indicate a role of the kidney in the induction of peripheral edema during permanent morphine administration.

[Transdermal buprenorphine for treatment of chronic tumor and non-tumor pain]. / Transdermales Buprenorphin für die Behandlung chronischer Tumor- und Nicht-Tumorschmerzen.

Patients with moderate to severe pain were treated with buprenorphine patches in one of 3 concentrations: 35 micrograms/h, 52.5 micrograms/h and 70 micrograms/h (= 0.8 mg/d, 1.2 mg/d and 1.6 mg/d respectively). The aim of this review was to assess the efficacy and tolerability of this transdermal system (TDS) in patients with chronic pain. A total of 445 patients were included in 3 double-blinded studies. The dosage titration with buprenorphine patches followed pretreatment with buprenorphine sublingual tablets, higher doses of weak opioids (level 2 substances), low dose morphine (level 3) or other analgesics. Patients with chronic tumour or non-tumour pain were recruited for these studies and treated with buprenorphine patches or placebo for 6 to 15 days. All patients were offered, in addition, buprenorphine sublingual tablets to be taken as required for supplementary pain relief. Pain intensity, analgesia, consumption of buprenorphine sublingual tablets and sleep duration were all assessed. All patients in the double-blinded studies were between the ages of 22 and 88. 249 patients suffered from tumour pain and 196 patients suffered from non-tumour pain. To examine long-term efficacy and tolerability of the transdermal system, treatment was expanded, if the patients were interested in participating in an open-label-study. In all 3 studies, the number of patients with moderate, severe and very severe pain increased in the placebo-patch treated group, while the patients in the buprenorphine transdermal system treated group had a greater incidence of mild or no pain. A further benefit in the buprenorphine transdermal system treated group was evidenced by a great number of patients with a daily sleep duration of more than 6 hours compared to the placebo group--an indicator of greater well-being. The systemic side-effects were typically opioid in nature and rare and usually only mild. Of particular note was the very low incidence of constipation in only 5.3% of cases. Dermatological reactions to the patches were only rarely encountered. The dermatological reactions consisted mainly of erythema and pruritus with a mild to moderate extent. Half the cases of erythema and more than on third of the cases of pruritus were spontaneously reversible. More than half the patients (53.7%) in the double blind studies wished to continued treatment with buprenorphine transdermal system. These results demonstrate that buprenorphine patches achieved a very good analgesic effect in all 3 studies and that in particular with respect to the quality of life of the patient these patches offer an exceptional alternative to other conventional therapies.

Determination and quantification of clonidine in human blood serum.

Clonidine ((2-[2,6-dichlorophenyl]amino)-2-imidazoline) preferentially stimulates central alpha(2)-adrenoceptors, which leads to inhibition of sympathetic tone, resulting in a lowering of arterial pressure and of heart rate. Additionally, many other desirable and undesirable effects are described, including analgesia, sedation and withdrawal reactions, which consist of a sudden rise in arterial pressure, nervousness, agitation and increased heart rate. The present study has the goal to develop a simple and effective method for the analysis of trace amounts of clonidine in human blood serum. Special emphasis is necessary to make application of electron impact ionization and separation of the analyte fragments in a quadruple mass analyzer suitable. The procedure comprises solid phase extraction followed by formation of the pentafluorobenzyl derivative. Further purification is achieved by phase transfer extraction into an acidic aqueous solution succeeded by re-extraction into dichloromethane. After solvent exchange, an aliquot is injected into the gas chromatograph equipped with a DB5 MS capillary column and a mass spectrometric detector. Chromatograms are recorded in single ion monitoring mode. Quantification is accomplished by internal standardization with moxonidine [4-chloro-5-(2-imidazolin-2-yl-amino)-6-methoxy-2-methylpyridine].