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.

Anticancer activity of novel plant extracts from Trailliaedoxa gracilis (W. W. Smith & Forrest) in human carcinoid KRJ-I Cells.

BACKGROUND: Small intestinal (SI) neuroendocrine tumors (NETs) are rare neoplasms derived from neuroendocrine cells presenting distinct clinical symptoms according to the ability to secrete neuroamines. Nevertheless, many are asymptomatic and misdiagnosed. As response rates to chemotherapy are low, surgery remains the only effective treatment. Because many tumors have metastasized at the time of diagnosis, curative surgery is rarely achieved. Consequently, a substantial need for new therapeutic options has emerged. MATERIALS AND METHODS: The effects of novel plant extracts from Trailliaedoxa gracilis (W.W. Smith & Forrest) were investigated in the SI-NET cell line KRJ-I and in KRJ-I transplanted mice. Proliferation and viability were analyzed using cell counting and WST-1 cell proliferation assay. Apoptosis was determined by DAPI staining and electron microscopy, and quantified by luminescence assays for caspases 3/7, 6, 8, 9 and 2. RESULTS: Extracts of Trailliaedoxa gracilis showed a dose-dependent reduction of proliferation and induction of apoptosis in the KRJ-I cells. Normal fibroblasts were not impaired. Tumor growth inhibition was also observed in heterotransplanted SCID (severe combined immunodeficiency) mice. CONCLUSION: The in vitro and in vivo outcomes suggest a potential clinical effect of Trailliaedoxa gracilis in SI-NETs.

Establishment of stably transfected rat neuronal cell lines expressing alpha-synuclein GFP fusion proteins.

Mutations in the alpha-synuclein gene have been linked to rare cases of familial Parkinson's disease (PD). alpha-Synuclein, a 140 amino acid polypeptide, is a major component of Lewy bodies (LB), a pathological hallmark of PD. Transgenic mice, Drosophila and marmosets (Challitrix jacchus) expressing either wild type (WT) or mutant human alpha-synuclein develop motor deficits, LB-like inclusions in some neurons and neuronal degeneration. The effects of human alpha-synuclein were investigated in a neuronal rat cell line (B103). Plasmids expressing WT and mutant human alpha-synuclein regulated by the cytomegalovirus (CMV) promoter were prepared and used for creating stably transfected neuronal rat cell lines. For localizing alpha-synuclein expression, stably transfected neuronal rat cell lines, expressing alpha-synuclein enhanced green fluorescent protein fusion proteins, regulated by either the CMV or the human platelet-derived growth factor ss promoter were generated. Over-expression of WT and A53T alpha-synuclein regulated by CMV promoter in stable transfectants resulted in formation of alpha-synuclein-immunopositive inclusion-like structures and mitochondrial alterations. Taken together, these results suggest that abnormal accumulation of alpha-synuclein could lead to mitochondrial alterations that might result in oxidative stress and eventually, cell death.

Establishment and characterization of three novel cell lines - P-STS, L-STS, H-STS - derived from a human metastatic midgut carcinoid.

Carcinoids are rare tumors derived from enterochromaffin (EC) cells of the embryonic neural crest. They have malignant potential and their incidence is steadily increasing. The only curative treatment option is surgery. We have focused on cultivation of human neuroendocrine tumors (NET) as relevant models for the study of potential therapy. Only a few cell lines from human carcinoids have been established so far, among them our earlier KRJ-I cell line from a human ileal carcinoid. The reason for the poor success in establishing carcinoid cell lines is due to the small amount of tissue available and the low mitotic activity in primary cultures. We have successfully established three continuously growing cell lines from tissue obtained from a metastatic human carcinoid of the terminal ileum (midgut carcinoid): P-STS was derived from the primary tumor, L-STS from a lymph node metastasis and H-STS from a hepatic metastasis. Immunocytochemistry proved the maintenance of characteristic neuroendocrine properties. Electron microscopy confirmed the presence of neuroendocrine granules. The three cell lines were tumorigenous in SCID-mice. Cytogenetic analyses revealed clonal tetraploidy, inversion and deletion in chromosome 18q, and non-clonal numerical and structural aberrations. Array CGH did not show notable imbalances. Mutation screening of P-STS excluded a MEN1-gene-associated genetic predisposition with high probability. The novel cell lines P-STS, L-STS and H-STS may be useful in vitro and in vivo models for further studies of biological characteristics and the development of new therapeutic agents.

Anticancer activity of novel extracts from Cautleya gracilis (Smith) Dandy: apoptosis in human medullary thyroid carcinoma cells.

BACKGROUND: Medullary thyroid carcinoma (MTC) is a calcitonin-producing tumor of the thyroid arising from the parafollicular C-cells. MTC is poorly responsive to chemotherapy and radiotherapy, hence the only effective therapy is surgery. Based on this fact, alternative strategies have been sought. MATERIALS AND METHODS: The effects of Cautleya gracilis (Smith) Dandy were investigated for the first time in three human MTC cell lines and in MTC-transplanted mice. Proliferation and viability were quantified by cell counting, WST-1 tests, and ATP luminescent cell viability assays. Apoptosis was studied by DAPI staining, flow cytometry and luminescent assays for caspases 3/7, 8 and 9. RESULTS: A dose-dependent reduction of proliferation and an induction of apoptosis were found in all MTC cell lines, while normal fibroblasts were not impaired. Similar tumor inhibition was seen in heterotransplanted mice. CONCLUSION: Our in vitro and in vivo findings suggest a new potential clinical effect of Cautleya.

Structural and biochemical properties of lipid particles from the yeast Saccharomyces cerevisiae.

The two most prominent neutral lipids of the yeast Saccharomyces cerevisiae, triacylglycerols (TAG) and steryl esters (SE), are synthesized by the two TAG synthases Dga1p and Lro1p and the two SE synthases Are1p and Are2p. In this study, we made use of a set of triple mutants with only one of these acyltransferases active to elucidate the contribution of each single enzyme to lipid particle (LP)/droplet formation. Depending on the remaining acyltransferases, LP from triple mutants contained only TAG or SE, respectively, with specific patterns of fatty acids and sterols. Biophysical investigations, however, revealed that individual neutral lipids strongly affected the internal structure of LP. SE form several ordered shells below the surface phospholipid monolayer of LP, whereas TAG are more or less randomly packed in the center of the LP. We propose that this structural arrangement of neutral lipids in LP may be important for their physiological role especially with respect to mobilization of TAG and SE reserves.

The non-competitive metabotropic glutamate receptor-1 antagonist CPCCOEt inhibits the in vitro growth of human melanoma.

Five decades ago, the dicarboxylic amino acid glutamate became recognized as the major excitatory neurotransmitter in the central nervous system. In recent years, the expression of glutamate receptors was detected also in peripheral, non-neuronal tissues. Furthermore, it was found that glutamate stimulated the proliferation and migration of several peripheral tumor cells, and that glutamate receptor antagonists limited tumor growth. Most of these studies, however, used broad spectrum compounds and/or group-specific antagonists. Here we report that a selective, non-competitive metabotropic glutamate receptor-1 antagonist, CPCCOEt (7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethyl ester), significantly inhibited the proliferation and modified the morphology of two human melanoma cell lines. These effects were independent of the external glutamate level in the culture medium. In addition, CPCCOEt significantly enhanced the tumoricidal effects of cytostatic drugs. Thus, selective non-competitive metabotropic glutamate receptor antagonists may be used alone and/or with the synergistic effects of chemotherapy, thus enhancing existing therapies of melanoma and possibly other malignancies.

Biocompatibility of ultra-high molecular weight polyethylene (UHMW-PE) stabilized with alpha-tocopherol used for joint endoprostheses assessed in vitro.

Adding the natural antioxidant alpha-tocopherol to ultra-high molecular weight polyethylene (UHMW-PE) can remarkably delay the oxidation of hip cups made thereof. However, alpha-tocopherol is likely to undergo different chemical transformations during manufacturing and sterilization of hip cups than in human metabolism. Therefore, the biocompatibility of the putative transformation products has to be investigated. In-vitro tests with L929 mice fibroblast-cells gave no evidence for cytotoxicity. To further ensure the biocompatibility, in-vitro tests with human cells were carried out in this study. Two different human cell lines, one adherent cell line, HF-SAR, and one suspension culture, GSJO, were tested on UHMW-PE-tablets (diameter: 15 mm; thickness: 2 mm; processed according to standard procedures for artificial hip-cups) with and without alpha-tocopherol with respect to cell viability, proliferation and morphology by means of cell counting, WSt-1 proliferation assay and scanning electron microscopy. Similar proliferation rates were found with both polyethylene samples. Further, we found intact morphology in light and electron microscopy on each substrate. The morphologic characteristics of skin fibroblasts were not changed by any material. Normal adherence and spreading of the fibroblasts was found on controls of glass, as well as on polystyrene and on stabilized and unstabilized polyethylene. The characteristic behaviour as suspension of the GSJO cells remained unchanged. The mitochondrial activity, as studied by WST-1 cell proliferation reagent, was identical on each substrate during the whole observation period of 7 days.

Lipid composition of peroxisomes from the yeast Pichia pastoris grown on different carbon sources.

Highly purified peroxisomes from the yeast Pichia pastoris grown on methanol or oleic acid, respectively, were used to characterize the lipid composition of this organelle. For this purpose, an isolation procedure had to be adapted which yielded highly purified P. pastoris peroxisomes. When peroxisome proliferation was induced by growth on methanol, alcohol oxidase was the predominant peroxisomal protein. Cultivation of P. pastoris on oleic acid led to induction of a family of peroxisomal enzymes catalyzing fatty acid beta-oxidation, whose most prominent members were identified by mass spectrometry. On either carbon source, phosphatidylcholine and phosphatidylethanolamine were the major peroxisomal phospholipids, and cardiolipin was present in peroxisomal membranes at a substantial amount, indicating that this phospholipid is a true peroxisomal component. Ergosterol was the most abundant sterol of P. pastoris peroxisomal membranes irrespective of the culture conditions. The fatty acid composition of whole cells and peroxisomes was highly affected by cultivation of P. pastoris on oleic acid. Under these conditions, oleic acid became the predominant fatty acid in phospholipids from total cell and peroxisomal extracts. Thus, oleic acid was not only utilized as an appropriate carbon source but also as a building block for complex membrane lipids. In summary, our data provide first insight into biochemical properties of P. pastoris peroxisomal membranes, which may become important for the biotechnological use of this yeast.

The oxidized phospholipids POVPC and PGPC inhibit growth and induce apoptosis in vascular smooth muscle cells.

Oxidized phospholipids, including 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) are typically present in oxidatively modified low density lipoprotein (oxLDL) and have been found in atherosclerotic lesions. These compounds are gaining increasing importance as inducers of different cellular responses like inflammation, proliferation, or cell death. The aim of this study was to elicit the type and outcome of the cellular response of vascular smooth muscle cells (VSMC) upon treatment with POVPC and PGPC. Both oxidized phospholipids led to inhibition of cell proliferation and showed cytotoxic effects in VSMC. Several morphological criteria, the presence of typical DNA fragments, and a phosphatidylserine shift towards the outer leaflet of the cell membrane revealed that apoptosis was the predominant mode of cell death. In all experiments, POVPC was found to be a more potent inducer of apoptosis than PGPC. Interestingly, in the presence of high levels of serum in the growth media the proapoptotic but not the antiproliferative effects of both oxidized phospholipids were abolished. Thus, we conclude that under low serum conditions both intact POVPC and PGPC are proapoptotic mediators. Under high serum conditions, these lipids are hydrolyzed and the resultant lipid mixture containing the degradation products is no longer apoptotic but antiproliferative. Thus, the direct and indirect effects of POVPC and PGPC on cell viability may account for the detrimental actions of oxLDL on VSMC.

Glutamate receptor-mediated effects on growth and morphology of human histiocytic lymphoma cells.

Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS) and binds to a variety of receptors, which recently have also been detected in peripheral, non-excitable cells. New research suggests that this abundant amino acid might also be involved in the growth of tumor cells acting via novel receptor-mediated autocrine/paracrine signal transduction pathways. We report here that glutamate, as well as glutamate receptor reactive drugs, differentially modulate growth and morphology of human histiocytic lymphoma-derived U937 cells. These effects were different depending on the culture milieu: in glutamine-free medium the glutamate receptor agonists, kainate (KA), and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), but also the antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), significantly decreased the proliferation of U937 cells. In contrast, in cultures devoid of glutamate, glutamine and serum, the agonists significantly increased cell proliferation whereas the antagonist CNQX showed no effect. These data point to a significant role of peripheral glutamate receptors in tumor cell proliferation.

Lysosomal pathology associated with alpha-synuclein accumulation in transgenic models using an eGFP fusion protein.

Disorders with Lewy body (LB) formation, such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB), are characterized by alpha-synuclein accumulation in the neuronal cell body. Recent studies have suggested that in addition to LBs, alpha-synuclein might accumulate more widely throughout the neurons and their processes, leading to neurodegeneration and functional impairment. The precise patterns of alpha-synuclein accumulation in vivo, however, and its relationship with subcellular neuronal alterations such as lysosomal pathology are not completely clear. To this end, we developed transgenic (tg) in vivo and in vitro models expressing a stable enhanced green fluorescent protein (eGFP) tagged in the C-terminal site of a human (h)alpha-synuclein construct under the regulatory control of the platelet-derived growth factor-beta (PDGFbeta) promoter and carried out confocal, ultrastructural, and biochemical studies. In tg mice, confocal studies demonstrated a wide distribution of halpha-synuclein-eGFP in the neuronal cell bodies, axons, and presynaptic terminals. In several neuronal cell bodies and their neurites, halpha-synuclein-eGFP was found not only as inclusions but also as discrete granular structures that in double-labeling studies colocalized with antibodies against halpha-synuclein and the lysosomal marker cathepsin D. Consistent with these findings, ultrastructural analysis showed that halpha-synuclein-eGFP overexpression resulted in the accumulation of electrodense inclusions and laminated bodies suggestive of lysosomal pathology, and that the halpha-synuclein-eGFP protein was more abundant in the lysosomal fractions of the tg animals. Taken together, these findings support the notion that enhanced visualization of alpha-synuclein utilizing a hybrid eGFP molecule reveals a more widespread accumulation of this molecule in several neuronal compartments, promoting lysosomal dysfunction. Furthermore, the PDGFbeta-halpha-synuclein-eGFP tg model might be a valuable tool in testing new treatments for LBD in a fast and reliable manner.

YEH2/YLR020c encodes a novel steryl ester hydrolase of the yeast Saccharomyces cerevisiae.

Previous work from our laboratory (Zinser, E., Paltauf, F., and Daum, G. (1993) J. Bacteriol. 175, 2853-2858) demonstrated steryl ester hydrolase activity in the plasma membrane of the yeast Saccharomyces cerevisiae. Here, we show that the gene product of YEH2/ YLR020c, which is homologous to several known mammalian steryl ester hydrolases, is the enzyme catalyzing this reaction. Deletion of yeast YEH2 led to complete loss of plasma membrane steryl ester hydrolase activity whereas overexpression of the gene resulted in a significant elevation of the activity. Purification of enzymatically active Yeh2p close to homogeneity unambiguously identified this protein as a steryl ester hydrolase and thus as the first enzyme of this kind characterized in S. cerevisiae. In addition to evidence obtained in vitro experiments in vivo contributed to the characterization of this novel enzyme. Sterol analysis of yeh2Delta unveiled a slightly elevated level of zymosterol suggesting that the esterified form of this sterol precursor is a preferred substrate of Yeh2p. However, in strains bearing hybrid proteins with strongly enhanced Yeh2p activity decreased levels of all steryl esters were observed. Thus, it appears that Yeh2p activity is not restricted to distinct steryl esters but rather has broad substrate specificity. The fact that in a yeh2Delta deletion strain bulk steryl ester mobilization occurred at a similar rate as in wild type suggested that Yeh2p is not the only steryl ester hydrolase but that other enzymes with overlapping function exist in the yeast.

Role of ceramide in activation of stress-associated MAP kinases by minimally modified LDL in vascular smooth muscle cells.

Interaction of oxidized low-density lipoprotein (LDL) with arterial smooth muscle cells (SMC) is believed to play a key role in the development of atherosclerosis. Depending on the extent of oxidation, apolipoproteins and/or lipids in the particle may be modified and thus lead to different cellular responses (e.g. proliferation or cell death). Here we report on the signaling effects of LDL, in which only the lipids were oxidized. This so-called minimally modified LDL (mmLDL) mainly activated components involved in stress response and apoptotic cell death including p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase/stress-activated protein kinase (JNK) as well as neutral and acid sphingomyelinase. In contrast, proliferative signaling elements such as extracellular regulated kinase, AKT-kinase and phospho-BAD seem to play a minor role as they were only slightly stimulated by mmLDL. Ceramide, the hydrolysis product of sphingomyelin, seems to be a key mediator as it mimics mmLDL by inducing activation of the same signaling components. Moreover, mmLDL- and ceramide-associated effects on apoptotic protein kinases were abolished by NB6, a specific inhibitor of acid sphingomyelinase. Thus, acid sphingomyelinase is very likely to be primarily responsible for triggering intracellular signal transduction in SMC after exposure to mmLDL via formation of ceramide by an autocatalytic mechanism.

Medullary thyroid carcinomas in cell culture--models for future therapies.

We report the successful establishment of seven human medullary thyroid carcinomas (MTC) as continuous cell lines. Characteristic features--such as the presence of neuroendocrine granules--and the positive immunoreactivity to antibodies to CT, CGRP, GRP, SRIF, 5-HT, NSE, PHE, LK2H10, ER and Pgr were followed throughout the cultivation. An overexpression of the antiapoptotic gene bcl-2 was detected in the cell lines. Deregulation of apoptosis plays an important role in multistep tumorigenesis. MTCs are known for the phenomenon of bcl-2-based chemo- and radioresistance. Our studies focus on influencing the growth rates and modulating the apoptotic rates by treatment with proliferation-modifying substances and anticancer drugs. Our MTC cell lines are useful models for these in vitro studies.