Design, Synthesis, and Characterization of I-BET567, a Pan-Bromodomain and Extra Terminal (BET) Bromodomain Oral Candidate.

Through regulation of the epigenome, the bromodomain and extra terminal (BET) family of proteins represent important therapeutic targets for the treatment of human disease. Through mimicking the endogenous N-acetyl-lysine group and disrupting the protein-protein interaction between histone tails and the bromodomain, several small molecule pan-BET inhibitors have progressed to oncology clinical trials. This work describes the medicinal chemistry strategy and execution to deliver an orally bioavailable tetrahydroquinoline (THQ) pan-BET candidate. Critical to the success of this endeavor was a potency agnostic analysis of a data set of 1999 THQ BET inhibitors within the GSK collection which enabled identification of appropriate lipophilicity space to deliver compounds with a higher probability of desired oral candidate quality properties. SAR knowledge was leveraged via Free-Wilson analysis within this design space to identify a small group of targets which ultimately delivered I-BET567 (27), a pan-BET candidate inhibitor that demonstrated efficacy in mouse models of oncology and inflammation.

Variant Transthyretin Amyloidosis (ATTRv) in Hungary: First Data on Epidemiology and Clinical Features.

BACKGROUND: Variant transthyretin amyloidosis (ATTRv) is an autosomal dominant inherited disease, where the mutation of the transthyretin gene (TTR) results in the deposition of pathogenic protein fibrils in various tissues. The mutation type influences the clinical course. Until now, no data were available on the genotype, phenotype, and prevalence of Hungarian ATTRv patients. The aim of our study was to assess the prevalence, regional distribution, genotypes, and phenotypes of Hungarian patients with ATTRv. METHODS: With the collaboration of Hungarian regional and university centers, we identified patients diagnosed with ATTRv. We also searched prior publications for case studies of Hungarian ATTRv patients. RESULTS: 40 individuals in 23 families with ATTRv were identified within the borders of Hungary. At the time of the diagnosis, 24 of them were symptomatic. The two most common mutations were ATTRHis88Arg (nine families) and ATTRIle107Val (8 families). ATTRVal30Met was demonstrated in 2 families, and ATTRVal122del, ATTRPhe33Leu, ATTRIle84Ser, and ATTRAsp18Gly in one family each. The median age of the symptomatic patients at the time of clinical diagnosis was 65 years. The most common clinically significant organ involvement was restrictive cardiomyopathy, found in 24 patients. Polyneuropathy was diagnosed in 20 patients. A total of 19 patients showed a mixed phenotype. The leading symptom was heart failure in 8 cases (3 of them had only cardiac symptoms), polyneuropathy in 11 cases (all of them also had cardiac symptoms), and equally severe cardiac and neuropathy symptoms were present in 3 cases. Out of 24 symptomatic patients, 10 received targeted pharmacological therapy. The follow-up period ranged from 1 to 195 months. At the time of the retrospective analysis, 12 patients had already died, and 1 patient underwent heart transplantation. CONCLUSIONS: As TTR genotype influences the phenotype and clinical course of ATTRv, it is important to know the regional data. In Hungary, ATTRHis88Arg and ATTRIle107Val are the most common mutations in ATTRv, both presenting with mixed phenotype, but the median age at the time of the diagnosis is 9 years lower in patients with ATTRHis88Arg than in patients with ATTRIle107Val.

Inhibition of ABCG2/BCRP-mediated transport-correlation analysis of various expression systems and probe substrates.

BCRP / ABCG2 is a key determinant of pharmacokinetics of substrate drugs. Several BCRP substrates and inhibitors are of low passive permeability, and the vesicular transport assay works well in this permeability space. Membranes were prepared from BCRP-HEK293, MCF-7/MX, and baculovirus-infected Sf9 cells with (BCRP-Sf9-HAM), and without (BCRP-Sf9) cholesterol loading. Km values for three substrates - estrone-3-sulfate, sulfasalazine, topotecan - correlated well between the four expression systems. In contrast, a 10-20-fold range in Vmax values was observed, with BCRP-HEK293 membranes possessing the largest dynamic range. IC50 values of the different test systems were similar to each other, with 94.4% of pairwise comparisons being within 3-fold. Substrate dependent inhibition showed somewhat greater variation, as 81.4% of IC50 values in the BCRP-HEK293 membranes were within 3-fold in pairwise comparisons. Overall, BCRP-HEK293 membranes demonstrated the highest activity. The IC50 values showed good concordance but substrate dependent inhibition was observed for some drugs.

Etiology-related Degree of Sprouting of Parvalbumin-immunoreactive Axons in the Human Dentate Gyrus in Temporal Lobe Epilepsy.

In the present study, we examined parvalbumin-immunoreactive cells and axons in the dentate gyrus of surgically resected tissues of therapy-resistant temporal lobe epilepsy (TLE) patients with different etiologies. Based on MRI results, five groups of patients were formed: (1) hippocampal sclerosis (HS), (2) malformation of cortical development, (3) malformation of cortical development + HS, (4) tumor-induced TLE, (5) patients with negative MRI result. Four control samples were also included in the study. Parvalbumin-immunoreactive cells were observed mostly in subgranular location in the dentate hilus in controls, in tumor-induced TLE, in malformation of cortical development and in MR-negative cases. In patients with HS, significant decrease in the number of hilar parvalbumin-immunoreactive cells and large numbers of ectopic parvalbumin-containing neurons were detected in the dentate gyrus' molecular layer. The ratio of ectopic/normally-located cells was significantly higher in HS than in other TLE groups. In patients with HS, robust sprouting of parvalbumin-immunoreactive axons were frequently visible in the molecular layer. The extent of sprouting was significantly higher in TLE patients with HS than in other groups. Strong sprouting of parvalbumin-immunoreactive axons were frequently observed in patients who had childhood febrile seizure. Significant correlation was found between the level of sprouting of axons and the ratio of ectopic/normally-located parvalbumin-containing cells. Electron microscopy demonstrated that sprouted parvalbumin-immunoreactive axons terminate on proximal and distal dendritic shafts as well as on dendritic spines of granule cells. Our results indicate alteration of target profile of parvalbumin-immunoreactive neurons in HS that contributes to the known synaptic remodeling in TLE.

Nephronectin promotes breast cancer brain metastatic colonization via its integrin-binding domains.

This study demonstrates a role for the extracellular matrix protein nephronectin (NPNT) in promoting experimental breast cancer brain metastasis, possibly through enhanced binding to- and migration through brain endothelial cells. With the introduction of more targeted breast cancer treatments, a prolonged survival has resulted during the last decade. Consequently, an increased number of patients develop metastasis in the brain, a challenging organ to treat. We recently reported that NPNT was highly expressed in primary breast cancer and associated with unfavourable prognosis. The current study addresses our hypothesis that NPNT promotes brain metastases through its integrin-binding motifs. SAGE-sequencing revealed that NPNT was significantly up-regulated in human breast cancer tissue compared to pair-matched normal breast tissue. Human brain metastatic breast cancers expressed both NPNT and its receptor, integrin α8ß1. Using an open access repository; BreastMark, we found a correlation between high NPNT mRNA levels and poor prognosis for patients with the luminal B subtype. The 66cl4 mouse cell line was used for expression of wild-type and mutant NPNT, which is unable to bind α8ß1. Using an in vivo model of brain metastatic colonization, 66cl4-NPNT cells showed an increased ability to form metastatic lesions compared to cells with mutant NPNT, possibly through reduced endothelial adhesion and transmigration.

[Investigation of sleep and fatigue in patients with cancer]. / Az alvás és a kimerültség vizsgálata daganatos betegek körében.

INTRODUCTION: The diagnosis of cancer elicits greater distress than any other diagnosis. The prevalence of psychological difficulties is high in cancer, and resources of the medical staff are limited. The development of efficient screening measures is therefore of utmost importance. Sleep is vital to all psychological functioning and poor sleep is a known problem in cancer. AIM: The main goal of the present study was testing of visual analogue scales assessing sleep quality and fatigue. METHOD: Sleep quality and fatigue were assessed with visual analogue scales. The Sleep Condition Indicator, the Athens Insomnia Scale and the Cancer Fatigue Scale were also included. Psychological distress was assessed with the Generalized Anxiety Disorder Scale, the Beck Depression Inventory and the Brief Difficulties in Emotion Regulation Scale. Pain and well-being was measured with the Faces of Pain Scale and the WHO Well-being Scale, respectively. A total of 71 patients with cancer were enrolled in this study. RESULTS: Insomnia and fatigue - measuring them with visual and several-item scales - showed high correlation with the measures of distress (anxiety, depression, emotion regulation difficulties) and pain. Distress and pain showed significant negative correlation with well-being. CONCLUSIONS: It has been affirmed that sleep quality is crucial in the changes of distress, pain and general well-being in cancer patients. It affirms that the visual analogue scales assessing sleep quality and fatigue - besides sleep quality and fatigue - are acceptable screening tools of distress and decreased well-being. Their use in clinical practice is recommended for screening in cancer patients and providing indications for onco-psychological treatment. Orv Hetil. 2018; 159(42): 1720-1726.

Histone Deacetylase Inhibitor Treatment Increases the Expression of the Plasma Membrane Ca2+ Pump PMCA4b and Inhibits the Migration of Melanoma Cells Independent of ERK.

Several new therapeutic options emerged recently to treat metastatic melanoma; however, the high frequency of intrinsic and acquired resistance among patients shows a need for new therapeutic options. Previously, we identified the plasma membrane Ca2+ ATPase 4b (PMCA4b) as a metastasis suppressor in BRAF-mutant melanomas and found that mutant BRAF inhibition increased the expression of the pump, which then inhibited the migratory and metastatic capability of the cells. Earlier it was also demonstrated that histone deacetylase inhibitors (HDACis) upregulated PMCA4b expression in gastric, colon, and breast cancer cells. In this study, we treated one BRAF wild-type and two BRAF-mutant melanoma cell lines with the HDACis, SAHA and valproic acid, either alone, or in combination with the BRAF inhibitor, vemurafenib. We found that HDACi treatment strongly increased the expression of PMCA4b in all cell lines irrespective of their BRAF mutational status, and this effect was independent of ERK activity. Furthermore, HDAC inhibition also enhanced the abundance of the housekeeping isoform PMCA1. Combination of HDACis with vemurafenib, however, did not have any additive effects on either PMCA isoform. We demonstrated that the HDACi-induced increase in PMCA abundance was coupled to an enhanced [Ca2+]i clearance rate and also strongly inhibited both the random and directional movements of A375 cells. The primary role of PMCA4b in these characteristic changes was demonstrated by treatment with the PMCA4-specific inhibitor, caloxin 1c2, which was able to restore the slower Ca2+ clearance rate and higher motility of the cells. While HDAC treatment inhibited cell motility, it decreased only modestly the ratio of proliferative cells and cell viability. Our results show that in melanoma cells the expression of both PMCA4b and PMCA1 is under epigenetic control and the elevation of PMCA4b expression either by HDACi treatment or by the decreased activation of the BRAF-MEK-ERK pathway can inhibit the migratory capacity of the highly motile A375 cells.

Direct mapping of melanoma cell - endothelial cell interactions.

The most life-threatening aspect of cancer is metastasis; cancer patient mortality is mainly due to metastasis. Among all metastases, presence of brain metastasis is one with the poorest prognosis; the median survival time can be counted in months. Therefore, prevention or decreasing their incidence would be highly desired both by patients and physicians. Metastatic cells invading the brain must breach the cerebral vasculature, primarily the blood-brain barrier. The key step in this process is the establishment of firm adhesion between the cancer cell and the cerebral endothelial layer. Using the atomic force microscope, a high-resolution force spectrograph, our aim was to explore the connections among the cell morphology, cellular mechanics, and biological function in the process of transendothelial migration of metastatic cancer cells. By immobilization of a melanoma cell to an atomic force microscope's cantilever, intercellular adhesion was directly measured at quasi-physiological conditions. Hereby, we present our latest results by using this melanoma-decorated probe. Binding characteristics to a confluent layer of brain endothelial cells was directly measured by means of single-cell force spectroscopy. Adhesion dynamics and strength were characterized, and we present data about spatial distribution of elasticity and detachment strength. These results highlight the importance of cellular mechanics in brain metastasis formation and emphasize the enormous potential toward exploration of intercellular dynamic-related processes.

Genetic background of the hereditary spastic paraplegia phenotypes in Hungary - An analysis of 58 probands.

BACKGROUND: Hereditary spastic paraplegias (HSPs) are a clinically and genetically heterogeneous group of neurodegenerative diseases with progressive lower limb spasticity and weakness. The aim of this study is to determine the frequency of different SPG mutations in Hungarian patients, and to provide further genotype-phenotype correlations for the known HSP causing genes. METHODS: We carried out genetic testing for 58 probands with clinical characteristics of HSP. For historical reasons, three different approaches were followed in different patients: 1) Sanger sequencing of ATL1 and SPAST genes, 2) whole exome, and 3) targeted panel sequencing by next generation sequencing. RESULTS: Genetic diagnosis was established for 20 probands (34.5%). We detected nine previously unreported mutations with high confidence for pathogenicity. The most frequently affected gene was SPAST with pathogenic or likely pathogenic mutations in 10 probands. The most frequently detected variant in our cohort was the SPG7 p.Leu78*, observed in four probands. Altogether five probands were diagnosed with SPG7. Additional mutations were detected in SPG11, ATL1, NIPA1, and ABCD1. CONCLUSION: This is the first comprehensive genetic epidemiological study of patients with HSP in Hungary. Next generation sequencing improved the yield of genetic diagnostics in this disease group even when the phenotype was atypical. However, considering the frequency of the HSP-causing gene defects, SPG4, the most common form of the disease, should be tested first to be cost effective in this economic region.

Investigation of the Antiproliferative Properties of Natural Sesquiterpenes from Artemisia asiatica and Onopordum acanthium on HL-60 Cells in Vitro.

Plants and plant extracts play a crucial role in the research into novel antineoplastic agents. Four sesquiterpene lactones, artecanin (1), 3ß-chloro-4α,10α-dihydroxy-1α,2α-epoxy-5α,7αH-guaia-11(13)-en-12,6α-olide (2), iso-seco-tanapartholide 3-O-methyl ether (3) and 4ß,15-dihydro-3-dehydrozaluzanin C (4), were isolated from two traditionally used Asteraceae species (Onopordum acanthium and Artemisia asiatica). When tested for antiproliferative action on HL-60 leukemia cells, these compounds exhibited reasonable IC50 values in the range 3.6-13.5 µM. Treatment with the tested compounds resulted in a cell cycle disturbance characterized by increases in the G1 and G2/M populations, while there was a decrease in the S phase. Additionally, 1-3 elicited increases in the hypodiploid (subG1) population. The compounds elicited concentration-dependent chromatin condensation and disruption of the membrane integrity, as revealed by Hoechst 33258-propidium staining. Treatment for 24 h resulted in significant increases in activity of caspases-3 and -9, indicating that the tested sesquiterpenes induced the mitochondrial pathway of apoptosis. The proapoptotic properties of the sesquiterpene lactones were additionally demonstrated withannexin V staining. Compounds 1 and 2 increased the Bax/Bcl-2 expression and decreased the expressions of CDK1 and cyclin B2, as determined at the mRNA level by means of RT-PCR. These experimental results indicate that sesquiterpene lactones may be regarded as potential starting structures for the development of novel anticancer agents.

Transmigration characteristics of breast cancer and melanoma cells through the brain endothelium: Role of Rac and PI3K.

Brain metastases are common and devastating complications of both breast cancer and melanoma. Although mammary carcinoma brain metastases are more frequent than those originating from melanoma, this latter has the highest tropism to the brain. Using static and dynamic in vitro approaches, here we show that melanoma cells have increased adhesion to the brain endothelium in comparison to breast cancer cells. Moreover, melanoma cells can transmigrate more rapidly and in a higher number through brain endothelial monolayers than breast cancer cells. In addition, melanoma cells have increased ability to impair tight junctions of cerebral endothelial cells. We also show that inhibition of Rac or PI3K impedes adhesion of breast cancer cells and melanoma cells to the brain endothelium. In addition, inhibition of Rac or PI3K inhibits the late phase of transmigration of breast cancer cells and the early phase of transmigration of melanoma cells. On the other hand, the Rac inhibitor EHT1864 impairs the junctional integrity of the brain endothelium, while the PI3K inhibitor LY294002 has no damaging effect on interendothelial junctions. We suggest that targeting the PI3K/Akt pathway may represent a novel opportunity in preventing the formation of brain metastases of melanoma and breast cancer.

Discovery of I-BRD9, a Selective Cell Active Chemical Probe for Bromodomain Containing Protein 9 Inhibition.

Acetylation of histone lysine residues is one of the most well-studied post-translational modifications of chromatin, selectively recognized by bromodomain "reader" modules. Inhibitors of the bromodomain and extra terminal domain (BET) family of bromodomains have shown profound anticancer and anti-inflammatory properties, generating much interest in targeting other bromodomain-containing proteins for disease treatment. Herein, we report the discovery of I-BRD9, the first selective cellular chemical probe for bromodomain-containing protein 9 (BRD9). I-BRD9 was identified through structure-based design, leading to greater than 700-fold selectivity over the BET family and 200-fold over the highly homologous bromodomain-containing protein 7 (BRD7). I-BRD9 was used to identify genes regulated by BRD9 in Kasumi-1 cells involved in oncology and immune response pathways and to the best of our knowledge, represents the first selective tool compound available to elucidate the cellular phenotype of BRD9 bromodomain inhibition.

A molecular understanding of D-homoestrone-induced G2/M cell cycle arrest in HeLa human cervical carcinoma cells.

2-Methoxyestradiol (ME), one of the most widely investigated A-ring-modified metabolites of estrone, exerts significant anticancer activity on numerous cancer cell lines. Its pharmacological actions, including cell cycle arrest, microtubule disruption and pro-apoptotic activity, have already been described in detail. The currently tested D-ring-modified analogue of estrone, D-homoestrone, selectively inhibits cervical cancer cell proliferation and induces a G2/M phase cell cycle blockade, resulting in the development of apoptosis. The question arose of whether the difference in the chemical structures of these analogues can influence the mechanism of anticancer action. The aim of the present study was therefore to elucidate the molecular contributors of intracellular processes induced by D-homoestrone in HeLa cells. Apoptosis triggered by D-homoestrone develops through activation of the intrinsic pathway, as demonstrated by determination of the activities of caspase-8 and -9. It was revealed that D-homoestrone-treated HeLa cells are not able to enter mitosis because the cyclin-dependent kinase 1-cyclin B complex loses its activity, resulting in the decreased inactivation of stathmin and a concomitant disturbance of microtubule formation. However, unlike 2-ME, D-homoestrone does not exert a direct effect on tubulin polymerization. These results led to the conclusion that the D-homoestrone-triggered intracellular processes resulting in a cell cycle arrest and apoptosis in HeLa cells differ from those in the case of 2-ME. This may be regarded as an alternative mechanism of action among steroidal anticancer compounds.

Regulation of NOD-like receptors and inflammasome activation in cerebral endothelial cells.

Cerebral endothelial cells (CECs) forming the blood-brain barrier are at the interface of the immune and the central nervous systems and thus may play an important role in the functional integration of the two systems. Here, we investigated how CECs recognize and respond to pathogen- and damage-associated molecular patterns to regulate the functions of the neurovascular unit. First we detected the expression of several NOD-like receptors (NLRs) - including NOD1, NOD2, NLRC4, NLRC5, NLRP1, NLRP3, NLRP5, NLRP9, NLRP10, NLRP12, NLRA, and NLRX - in human brain endothelial cells. Inflammatory cytokines, such as interferon-γ, tumor necrosis factor-α, and IL-1ß had stimulatory effects on the transcription of many of these receptors. Expression of key inflammasome components (NOD2, NLRP3, and caspase 1) along with caspase-cleaved interleukins IL-1ß and IL-33 could be induced by priming with lipopolysaccharide and activation with muramyl dipeptide. In addition, combined treatment with lipopolysaccharide and muramyl dipeptide resulted in IL-1ß secretion in a caspase- and ERK1/2 kinase-dependent manner. Our findings demonstrate that NLRs and inflammasomes can be activated in cerebral endothelial cells, which may confer a yet unexplored role to the blood-brain barrier in neuroimmune and neuroinflammatory processes.

Endothelial-mesenchymal transition of brain endothelial cells: possible role during metastatic extravasation.

Cancer progression towards metastasis follows a defined sequence of events described as the metastatic cascade. For extravasation and transendothelial migration metastatic cells interact first with endothelial cells. Yet the role of endothelial cells during the process of metastasis formation and extravasation is still unclear, and the interaction between metastatic and endothelial cells during transendothelial migration is poorly understood. Since tumor cells are well known to express TGF-ß, and the compact endothelial layer undergoes a series of changes during metastatic extravasation (cell contact disruption, cytoskeletal reorganization, enhanced contractility), we hypothesized that an EndMT may be necessary for metastatic extravasation. We demonstrate that primary cultured rat brain endothelial cells (BEC) undergo EndMT upon TGF-ß1 treatment, characterized by the loss of tight and adherens junction proteins, expression of fibronectin, ß1-integrin, calponin and α-smooth muscle actin (SMA). B16/F10 cell line conditioned and activated medium (ACM) had similar effects: claudin-5 down-regulation, fibronectin and SMA expression. Inhibition of TGF-ß signaling during B16/F10 ACM stimulation using SB-431542 maintained claudin-5 levels and mitigated fibronectin and SMA expression. B16/F10 ACM stimulation of BECs led to phosphorylation of Smad2 and Smad3. SB-431542 prevented SMA up-regulation upon stimulation of BECs with A2058, MCF-7 and MDA-MB231 ACM as well. Moreover, B16/F10 ACM caused a reduction in transendothelial electrical resistance, enhanced the number of melanoma cells adhering to and transmigrating through the endothelial layer, in a TGF-ß-dependent manner. These effects were not confined to BECs: HUVECs showed TGF-ß-dependent SMA expression when stimulated with breast cancer cell line ACM. Our results indicate that an EndMT may be necessary for metastatic transendothelial migration, and this transition may be one of the potential mechanisms occurring during the complex phenomenon known as metastatic extravasation.

Endothelial-mesenchymal transition of brain endothelial cells: possible role during metastatic extravasation.

Cancer progression towards metastasis follows a defined sequence of events described as the metastatic cascade. For extravasation and transendothelial migration metastatic cells interact first with endothelial cells. Yet the role of endothelial cells during the process of metastasis formation and extravasation is still unclear, and the interaction between metastatic and endothelial cells during transendothelial migration is poorly understood. Since tumor cells are well known to express TGF-ß, and the compact endothelial layer undergoes a series of changes during metastatic extravasation (cell contact disruption, cytoskeletal reorganization, enhanced contractility), we hypothesized that an EndMT may be necessary for metastatic extravasation. We demonstrate that primary cultured rat brain endothelial cells (BEC) undergo EndMT upon TGF-ß1 treatment, characterized by the loss of tight and adherens junction proteins, expression of fibronectin, ß1-integrin, calponin and α-smooth muscle actin (SMA). B16/F10 cell line conditioned and activated medium (ACM) had similar effects: claudin-5 down-regulation, fibronectin and SMA expression. Inhibition of TGF-ß signaling during B16/F10 ACM stimulation using SB-431542 maintained claudin-5 levels and mitigated fibronectin and SMA expression. B16/F10 ACM stimulation of BECs led to phosphorylation of Smad2 and Smad3. SB-431542 prevented SMA up-regulation upon stimulation of BECs with A2058, MCF-7 and MDA-MB231 ACM as well. Moreover, B16/F10 ACM caused a reduction in transendothelial electrical resistance, enhanced the number of melanoma cells adhering to and transmigrating through the endothelial layer, in a TGF-ß-dependent manner. These effects were not confined to BECs: HUVECs showed TGF-ß-dependent SMA expression when stimulated with breast cancer cell line ACM. Our results indicate that an EndMT may be necessary for metastatic transendothelial migration, and this transition may be one of the potential mechanisms occurring during the complex phenomenon known as metastatic extravasation.

A click approach to novel D-ring-substituted 16α-triazolylestrone derivatives and characterization of their antiproliferative properties.

A simple and efficient synthesis of novel, D-ring substituted estrone derivatives containing a 16α-triazolyl moiety is described. Two epimeric azido alcohols (16α-azido-17α-hydroxy and 16α-azido-17ß-hydroxy) of estra-1,3,5(10)-triene-3-methyl ether were prepared, followed by copper(I)-catalyzed azide-alkyne cycloaddition with various terminal alkynes. The steroidal triazoles were obtained in high yields and their activities against three human cancer cell lines (HeLa, MCF7 and A431) were screened. The most effective analogs were submitted to additional experiments in order to characterize their antiproliferative properties. As evidenced by flow cytometry, the selected steroids induced a disturbance in the HeLa cell cycle in a concentration- and exposure-dependent manner, through an increase of the hypodiploid population (subG1) and a cell cycle arrest in the G2/M phase. A noncancerous human fibroblast cell line (MRC5) was used to determine the selectivities of these compounds. Fluorescent microscopy after Hoechst 33258 - propidium iodide (HOPI) double staining revealed nuclear condensation and disturbed cell membrane integrity. The enhanced activities of caspase-3 and caspase-9 without activation of caspase-8 in the treated cells indicated the activation of the intrinsic pathway of apoptosis. The levels of cell cycle regulators (CDK1, cyclin B1/B2 and cdc25B) were decreased and the ratio Bax/Bcl-2 was increased 24 h after the treatment of HeLa cells (determined at an mRNA level by means of an RT-PCR technique). Under the same conditions, two agents elicited substantially increased degrees of phosphorylation of stathmin, as evidenced by Western blotting. The presented results demonstrate that these steroids can be regarded as appropriate structural scaffolds for the design and synthesis of further steroid analogs as innovative drug candidates with good efficacy.

Evaluation of lignans from Heliopsis helianthoides var. scabra for their potential antimetastatic effects in the brain.

Two new arylbenzofuran-type neolignans, 1"-dehydroegonol 3"-methyl ether (1) and egonol 3"-methyl ether (2), and four known lignan derivatives, namely, helioxanthin (3), (7E)-7,8-dehydroheliobuphthalmin (4), heliobuphthalmin (5), and 7-acetoxyhinokinin (6), were isolated from a chloroform-soluble partition of the methanol extract of the fresh roots of Heliopsis helianthoides var. scabra. These six compounds were evaluated in vitro in terms of their ability to inhibit the various steps involved in brain tumor metastasis formation. Compounds 3 and 4 inhibited the migration of both melanoma and brain endothelial cells, and 3 also reduced the adhesion of melanoma cells to the brain endothelium. Furthermore, 3 and 4 additionally enhanced the barrier function of the blood-brain barrier and the expression of the tight junction protein ZO-1 at the junctions of the endothelial cells. These findings suggest that 3 and 4 may have the potential to interfere with different steps of brain metastasis formation and to enhance the barrier function of cerebral endothelial cells.

Antiproliferative activity of Artemisia asiatica extract and its constituents on human tumor cell lines.

The extract of Artemisia asiatica herb with antiproliferative activity against four human tumor cell lines (A2780, A431, HeLa, and MCF7) was analyzed by the MTT assay, and bioassay-directed fractionation was carried out in order to identify the compounds responsible for the cytotoxic activity. Guaianolide (1-4), seco-guianolide (5), germacranolide (6) and eudesmanolide sesquiterpenes (7), monoterpenes (8, 9), including the new compound artemisia alcohol glucoside (8), and flavonoids (10-16) were isolated as a result of a multistep chromatographic procedure (CC, CPC, PLC, and gel filtration). The compounds were identified by means of UV, MS, and NMR spectroscopy, including (1)H-and (13)C-NMR, (1)H-(1)H COSY, NOESY, HSQC, and HMBC experiments. The isolated compounds 1-16 were evaluated for their tumor cell growth-inhibitory activities on a panel of four adherent cancer cell lines, and different types of secondary metabolites were found to be responsible for the cytotoxic effects of the extract. Especially cirsilineol (13), 3ß-chloro-4α,10α-dihydroxy-1α,2α-epoxy-5α,7αH-guai-11(13)-en-12,6α-olide (3), and iso-seco-tanapartholide 3-O-methyl ester (5) exerted marked cytotoxic effects against the investigated cell lines, while jaceosidin (12), 6-methoxytricin (15), artecanin (2), and 5,7,4',5'-tetrahydroxy-6,3'-dimethoxyflavone (14) were moderately active. All the sesquiterpenes and monoterpenes are reported here for the first time from this species, and in the case of artecanin (2), 3α-chloro-4ß,10α-dihydroxy-1ß,2ß-epoxy-5α,7αH-guai-11(13)-en-12,6α-olide (4), ridentin (6), and ridentin B (7), previously unreported NMR spectroscopic data were determined.

CB2 receptor activation inhibits melanoma cell transmigration through the blood-brain barrier.

During parenchymal brain metastasis formation tumor cells need to migrate through cerebral endothelial cells, which form the morphological basis of the blood-brain barrier (BBB). The mechanisms of extravasation of tumor cells are highly uncharacterized, but in some aspects recapitulate the diapedesis of leukocytes. Extravasation of leukocytes through the BBB is decreased by the activation of type 2 cannabinoid receptors (CB2); therefore, in the present study we sought to investigate the role of CB2 receptors in the interaction of melanoma cells with the brain endothelium. First, we identified the presence of CB1, CB2(A), GPR18 (transcriptional variant 1) and GPR55 receptors in brain endothelial cells, while melanoma cells expressed CB1, CB2(A), GPR18 (transcriptional variants 1 and 2), GPR55 and GPR119. We observed that activation of CB2 receptors with JWH-133 reduced the adhesion of melanoma cells to the layer of brain endothelial cells. JWH-133 decreased the transendothelial migration rate of melanoma cells as well. Our results suggest that changes induced in endothelial cells are critical in the mediation of the effect of CB2 agonists. Our data identify CB2 as a potential target in reducing the number of brain metastastes originating from melanoma.