Primary and hTERT-Transduced Mesothelioma-Associated Fibroblasts but Not Primary or hTERT-Transduced Mesothelial Cells Stimulate Growth of Human Mesothelioma Cells.

Pleural mesothelioma (PM) is an aggressive malignancy that develops in a unique tumor microenvironment (TME). However, cell models for studying the TME in PM are still limited. Here, we have generated and characterized novel human telomerase reverse transcriptase (hTERT)-transduced mesothelial cell and mesothelioma-associated fibroblast (Meso-CAF) models and investigated their impact on PM cell growth. Pleural mesothelial cells and Meso-CAFs were isolated from tissue of pneumothorax and PM patients, respectively. Stable expression of hTERT was induced by retroviral transduction. Primary and hTERT-transduced cells were compared with respect to doubling times, hTERT expression and activity levels, telomere lengths, proteomes, and the impact of conditioned media (CM) on PM cell growth. All transduced derivatives exhibited elevated hTERT expression and activity, and increased mean telomere lengths. Cell morphology remained unchanged, and the proteomes were similar to the corresponding primary cells. Of note, the CM of primary and hTERT-transduced Meso-CAFs stimulated PM cell growth to the same extent, while CM derived from mesothelial cells had no stimulating effect, irrespective of hTERT expression. In conclusion, all new hTERT-transduced cell models closely resemble their primary counterparts and, hence, represent valuable tools to investigate cellular interactions within the TME of PM.

Isolation and NMR Scaling Factors for the Structure Determination of Lobatolide H, a Flexible Sesquiterpene from Neurolaena lobata.

A new flexible germacranolide (1, lobatolide H) was isolated from the aerial parts of Neurolaena lobata. The structure elucidation was performed by classical NMR experiments and DFT NMR calculations. Altogether, 80 theoretical level combinations with existing 13C NMR scaling factors were tested, and the best performing ones were applied on 1. 1H and 13C NMR scaling factors were also developed for two combinations utilizing known exomethylene containing derivatives, and the results were complemented by homonuclear coupling constant (JHH) and TDDFT-ECD calculations to elucidate the stereochemistry of 1. Lobatolide H possessed remarkable antiproliferative activity against human cervical tumor cell lines with different HPV status (SiHa and C33A), induced cell cycle disturbance and exhibited a substantial antimigratory effect in SiHa cells.

Mesothelioma-associated fibroblasts enhance proliferation and migration of pleural mesothelioma cells via c-Met/PI3K and WNT signaling but do not protect against cisplatin.

BACKGROUND: Pleural mesothelioma (PM) is an aggressive malignancy with poor prognosis. Unlike many other cancers, PM is mostly characterized by inactivation of tumor suppressor genes. Its highly malignant nature in absence of tumor driving oncogene mutations indicates an extrinsic supply of stimulating signals by cells of the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are an abundant cell type of the TME and have been shown to drive the progression of several malignancies. The aim of the current study was to isolate and characterize patient-derived mesothelioma-associated fibroblasts (Meso-CAFs), and evaluate their impact on PM cells. METHODS: Meso-CAFs were isolated from surgical specimens of PM patients and analyzed by array comparative genomic hybridization, next generation sequencing, transcriptomics and proteomics. Human PM cell lines were retrovirally transduced with GFP. The impact of Meso-CAFs on tumor cell growth, migration, as well as the response to small molecule inhibitors, cisplatin and pemetrexed treatment was investigated in 2D and 3D co-culture models by videomicroscopy and automated image analysis. RESULTS: Meso-CAFs show a normal diploid genotype without gene copy number aberrations typical for PM cells. They express CAF markers and lack PM marker expression. Their proteome and secretome profiles clearly differ from normal lung fibroblasts with particularly strong differences in actively secreted proteins. The presence of Meso-CAFs in co-culture resulted in significantly increased proliferation and migration of PM cells. A similar effect on PM cell growth and migration was induced by Meso-CAF-conditioned medium. Inhibition of c-Met with crizotinib, PI3K with LY-2940002 or WNT signaling with WNT-C59 significantly impaired the Meso-CAF-mediated growth stimulation of PM cells in co-culture at concentrations not affecting the PM cells alone. Meso-CAFs did not provide protection of PM cells against cisplatin but showed significant protection against the EGFR inhibitor erlotinib. CONCLUSIONS: Our study provides the first characterization of human patient-derived Meso-CAFs and demonstrates a strong impact of Meso-CAFs on PM cell growth and migration, two key characteristics of PM aggressiveness, indicating a major role of Meso-CAFs in driving PM progression. Moreover, we identify signaling pathways required for Meso-CAF-mediated growth stimulation. These data could be relevant for novel therapeutic strategies against PM.

Isolation, Structure Determination of Sesquiterpenes from Neurolaena lobata and Their Antiproliferative, Cell Cycle Arrest-Inducing and Anti-Invasive Properties against Human Cervical Tumor Cells.

Seven new germacranolides (1-3, 5-8), among them a heterodimer (7), and known germacranolide (4), eudesmane (9) and isodaucane (10) sesquiterpenes were isolated from the aerial parts of Neurolaena lobata. Their structures were determined by using a combination of different spectroscopic methods, including HR-ESIMS and 1D and 2D NMR techniques supported by DFT-NMR calculations. The enantiomeric purity of the new compounds was investigated by chiral HPLC analysis, while their absolute configurations were determined by TDDFT-ECD and OR calculations. Due to the conformationally flexible macrocycles and difficulties in assigning the relative configuration, 13C and 1H NMR chemical shift and ECD and OR calculations were performed on several stereoisomers of two derivatives. The isolated compounds (1-10) were shown to have noteworthy antiproliferative activities against three human cervical tumor cell line with different HPV status (HeLa, SiHa and C33A). Additionally, lobatolide C (6) exhibited substantial antiproliferative properties, antimigratory effect, and it induced cell cycle disturbance in SiHa cells.

Micronucleus assays with the human derived liver cell line (Huh6): A promising approach to reduce the use of laboratory animals in genetic toxicology.

The inadequate representation of enzymes which catalyze the activation/detoxification of xenobiotics in cells that are currently used in genotoxicity testing of chemicals leads to a high number of false positive results and the number of follow up studies with rodents could be reduced by use of more reliable in vitro models. We found earlier that several xenobiotic drug metabolizing enzymes are represented in the human derived liver cell line Huh6 and developed a protocol for micronucleus (MN) experiments which is in agreement with the current OECD guideline. This protocol was used to test 23 genotoxic and non-genotoxic reference chemicals; based on these results and of earlier findings (with 9 chemicals) we calculated the predictive value of the assay for the detection of genotoxic carcinogens. We found a sensitivity of 80% and a specificity of 94% for a total number of 32 chemicals; comparisons with results obtained with other in vitro assays show that the validity of MN tests with Huh6 is higher as that of other experimental models. These results are promising and indicate that the use of Huh6 cells in genetic toxicology may contribute to the reduction of the use of laboratory rodents; further experimental work to confirm this assumption is warranted.

Antiproliferative and antimetastatic characterization of an exo-heterocyclic androstane derivative against human breast cancer cell lines.

Cancer in general, and specifically gynaecological neoplasms, represents a major public health issue worldwide. Based on the effect of sex hormones on breast tumorigenesis and prognosis, as well as on the development of breast cancer metastases, modification of the steroid skeleton is a hotspot of research for novel anticancer agents. Numerous recent studies support that minor modifications of the androstane skeleton yield potent antiproliferative and antimetastatic drug candidates. The aim of the present study was to assess the antitumor and antimetastatic properties, as well as the mechanism of action of a D-ring-modified exo-heterocyclic androstadiene derivative named 17APAD. The test compound was found to be highly selective towards human breast cancer-derived cell lines (MCF-7, T47D, MDA-MB-361, MDA-MB-231) compared to non-cancerous fibroblast cells (NIH/3T3), and exerted superior effect compared to the clinically applied reference drug cisplatin. Changes in MCF-7- and MDA-MB-231 cell morphology and membrane integrity induced by the test substance were assessed by fluorescent double staining. Cell cycle disturbances were analyzed by flow cytometry, and concentration-dependent alterations were detected on breast cancer cell lines. Mitochondrial apoptosis induced by the test compound was demonstrated by JC-1 staining. Inhibitory effects on metastasis formation, including the inhibition of migration, invasion and intravasation were investigated in 2D and 3D models. Significant anti-migratory and anti-invasive effects on MCF-7 and MDA-MB-231 cells were detected after 24 h exposure in 2D wound healing and Boyden-chamber assays. The anti-intravasative properties of 17APAD were evident after 4 h of incubation in a co-culture 3D circular chemorepellent-induced defects (CCID) assay, and the level of inhibition at concentrations ≥2 µM was comparable to that exerted by the focal adhesion kinase inhibitor defactinib. Single cell mass cytometry revealed that chemosensitive subpopulations of MDA-MB-231 cells engaged to apoptosis were less positive for EGFR, CD274, and CD326, while the percentage of cells positive for GLUT1, MCT4, Pan-Keratin, CD66(a,c,e), Galectin-3 and TMEM45A increased in response to 17APAD treatment. Finally, the novel androstane analogue 17APAD had an outstanding inhibitory effect on tumour growth in the 4T1 orthotopic murine breast cancer model in vivo after 2 weeks of intraperitoneal administration. These findings support that substitution of the androsta-5,16-diene framework with a N-containing heterocyclic moiety at C17 position yields a molecular entity rational to be considered for design and synthesis of novel, effective antitumor agents, and 17APAD is worth further investigation as a promising anticancer drug candidate.

Use of micronucleus assays for the prediction and detection of cervical cancer: a meta-analysis.

Cervical cancer (CC) is the fourth most common cancer in women; the survival rates depend strongly on its early detection. The Pap test is the most frequently used diagnostic tool, but due to its limited sensitivity/specificity, additional screening tests are needed. Therefore, we evaluated the use of micronucleus (MN) assays with cervical cells for the prediction and diagnosis of CC. MN reflects structural and numerical chromosomal aberrations. A search was performed in Pubmed, Scopus, Thomson ISI and Google Scholar. Subsequently, meta-analyses were performed for different grades of abnormal findings in smears and biopsies from patients which were diagnosed with CC. Results of 21 studies in which findings of MN experiments were compared with data from Pap tests show that higher MN frequencies were found in women with abnormal cells that are indicative for increased cancer risks. MN frequency ratios increased in the order inflammation (2.1) < ASC-US and ASC-H (3.3) < LGSIL (4.4) < HGSIL (8.4). Furthermore, results are available from 17 investigations in which MN were scored in smears from patients with neoplasia. MN rates increased with the degree of neoplasia [CIN 1 (4.6) < CIN 2 (6.5) and CIN 3 (10.8)] and were significantly higher (8.8) in CC patients. Our meta-analysis indicates that the MN assay, which is easy to perform in combination with Pap tests, may be useful for the detection/prediction of CC. However, standardization (including definition of the optimal cell numbers and stains) and further validation is necessary before the MN test can be implemented in routine screening.

Flavonoids Distinctly Stabilize Lymph Endothelial- or Blood Endothelial Disintegration Induced by Colon Cancer Spheroids SW620.

The health effects of plant phenolics in vegetables and other food and the increasing evidence of the preventive potential of flavonoids in "Western Diseases" such as cancer, neurodegenerative diseases and others, have gained enormous interest. This prompted us to investigate the effects of 20 different flavonoids of the groups of flavones, flavonols and flavanones in 3D in vitro systems to determine their ability to inhibit the formation of circular chemorepellent induced defects (CCIDs) in monolayers of lymph- or blood-endothelial cells (LECs, BECs; respectively) by 12(S)-HETE, which is secreted by SW620 colon cancer spheroids. Several compounds reduced the spheroid-induced defects of the endothelial barriers. In the SW620/LEC model, apigenin and luteolin were most active and acacetin, nepetin, wogonin, pinocembrin, chrysin and hispidulin showed weak effects. In the SW620/BEC model acacetin, apigenin, luteolin, wogonin, hispidulin and chrysin exhibited weak activity.

Feed­back loops integrating RELA, SOX18 and FAK mediate the break­down of the lymph­endothelial barrier that is triggered by 12(S)­HETE.

Metastatic cancer cells cross endothelial barriers and travel through the blood or lymphatic fluid to pre­metastatic niches, leading to their colonisation. 'S' stereoisomer 12S­hydroxy­5Z,8Z,10E,14Z­eicosatetraenoic acid [12(S)­HETE] is secreted by a variety of cancer cell types and has been indicated to open up these barriers. In the present study, another aspect of the endothelial unlocking mechanism was elucidated. This was achieved by investigating 12(S)­HETE­treated lymph endothelial cells (LECs) with regard to their expression and mutual interaction with v­rel avian reticuloendotheliosis viral oncogene homolog A (RELA), intercellular adhesion molecule 1, SRY­box transcription factor 18 (SOX18), prospero homeobox 1 (PROX1) and focal adhesion kinase (FAK). These key players of LEC retraction, which is a prerequisite for cancer cell transit into vasculature, were analysed using western blot analysis, reverse transcription­quantitative PCR and transfection with small interfering (si)RNA. The silencing of a combination of these signalling and executing molecules using siRNA, or pharmacological inhibition with defactinib and Bay11­7082, extended the mono­culture experiments to co­culture settings using HCT116 colon cancer cell spheroids that were placed on top of LEC monolayers to measure their retraction using the validated 'circular chemorepellent­induced defect' assay. 12(S)­HETE was indicated to induce the upregulation of the RELA/SOX18 feedback loop causing the subsequent phosphorylation of FAK, which fed back to RELA/SOX18. Therefore, 12(S)­HETE was demonstrated to be associated with circuits involving RELA, SOX18 and FAK, which transduced signals causing the retraction of LECs. The FAK­inhibitor defactinib and the NF­κB inhibitor Bay11­7082 attenuated LEC retraction additively, which was similar to the suppression of FAK and PROX1 (the target of SOX18) by the transfection of respective siRNAs. FAK is an effector molecule at the distal end of a pro­metastatic signalling cascade. Therefore, targeting the endothelial­specific activity of FAK through the pathway demonstrated herein may provide a potential therapeutic method to combat cancer dissemination via vascular routes.

High impact of miRNA-4521 on FOXM1 expression in medulloblastoma.

Medulloblastoma, an embryonal tumor of the cerebellum/fourth ventricle, is one of the most frequent malignant brain tumors in children. Although genetic variants are increasingly used in treatment stratification, survival of high-risk patients, characterized by leptomeningeal dissemination, TP53 mutation or MYC amplification, is still poor. FOXM1, a proliferation-specific oncogenic transcription factor, is deregulated in various solid tumors, including medulloblastoma, and triggers cellular proliferation, migration and genomic instability. In tissue samples obtained from medulloblastoma patients, the significant upregulation of FOXM1 was associated with a loss of its putative regulating microRNA, miR-4521. To understand the underlying mechanism, we investigated the effect of miR-4521 on the expression of the transcription factor FOXM1 in medulloblastoma cell lines. Transfection of this microRNA reduced proliferation and invasion of several medulloblastoma cell lines and induced programmed cell death through activation of caspase 3/7. Further, downstream targets of FOXM1 such as PLK1 and cyclin B1 were significantly reduced thus affecting the cell cycle progression in medulloblastoma cell lines. In conclusion, a restoration of miRNA-4521 may selectively suppress the pathophysiological effect of aberrant FOXM1 expression and serve as a targeted approach for medulloblastoma therapy.

Impact of Weight Loss Strategies on Obesity-Induced DNA Damage.

SCOPE: Obesity causes DNA damage, which is causally related to several disorders including cancer, infertility, and cognitive dysfunctions. The aim of this study is to investigate whether weight loss improves the integrity of the genetic material. METHODS AND RESULTS: Overweight mice are fed ad libitum either with a Western diet (WD), with a 40% caloric restricted WD, or with a high carbohydrate low protein (HCLP) diet. Caloric restriction and also the HCLP diet lead to ca. 30% weight loss, which is paralleled by decreased DNA damage ("comet" formation) and oxidative damage of purines in inner organs, additionally the activity of nucleotide excision repair increased. The effects are more pronounced in animals that have received the HCLP chow. Results of biochemical analyses indicate that the reduction of DNA damage is associated with a decrease of pro-inflammatory cytokines and lower insulin levels. CONCLUSION: The study indicates that weight loss may prevent obesity-associated adverse health effects due to reduction of overall DNA damage.

Organic anion­transporting polypeptides contribute to the uptake of curcumin and its main metabolites by human breast cancer cells: Impact on antitumor activity.

Curcumin is a natural polyphenolic compound with pronounced anticancer properties, despite its low bioavailability caused by extensive glucuronidation and sulfation. Information on the cellular uptake mechanisms and their contribution to the anticancer effects of curcumin and its biotransformation products is limited. The present study, therefore, investigated the role of organic anion­transporting polypeptides (OATPs) in the cellular uptake of curcumin and its major metabolites in OATP­expressing Chinese hamster ovary (CHO) and human ZR­75­1 breast cancer cells. The uptake rates for curcumin in OATP1B1­, OATP1B3­ and OATP2B1­transfected CHO cells were 2­ to 3­fold higher than wild­type cells. Curcumin sulfate was transported by all three OATPs, although to a much lesser extent, while uptake of tetrahydrocurcumin was the highest but only via OATP1B1 and OATP1B3. Notably, curcumin glucuronide did not exhibit any affinity for these OATPs. The increased mRNA levels of OATP1B1 in wild­type human breast cancer ZR­75­1 cells compared with OATP1B1 knockdown cells was associated with a higher initial uptake of curcumin and tetrahydrocurcumin leading to decreased IC50 values. In conclusion, our data revealed that OATPs act as cellular uptake transporters for curcumin and its major metabolites, and this may also be applicable to patients undergoing cancer therapy.

12(S)-HETE induces lymph endothelial cell retraction in vitro by upregulation of SOX18.

Metastasising breast cancer cells communicate with adjacent lymph endothelia, intravasate and disseminate through lymphatic routes, colonise lymph nodes and finally metastasize to distant organs. Thus, understanding and blocking intravasation may attenuate the metastatic cascade at an early step. As a trigger factor, which causes the retraction of lymph endothelial cells (LECs) and opens entry ports for tumour cell intravasation, MDA-MB231 breast cancer cells secrete the pro-metastatic arachidonic acid metabolite, 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid [12(S)-HETE]. In the current study, treatment of LECs with 12(S)-HETE upregulated the expression of the transcription factors SRY-related HMG-box 18 (SOX18) and prospero homeobox protein 1 (PROX1), which determine endothelial development. Thus, whether they have a role in LEC retraction was determined using a validated intravasation assay, small interfering RNA mediated knockdown of gene expression, and mRNA and protein expression analyses. Specific inhibition of SOX18 or PROX1 significantly attenuated in vitro intravasation of MDA-MB231 spheroids through the LEC barrier and 12(S)-HETE-triggered signals were transduced by the high and low affinity receptors, 12(S)-HETE receptor and leukotriene B4 receptor 2. In addition, the current findings indicate that there is crosstalk between SOX18 and nuclear factor κ-light-chain-enhancer of activated B cells, which was demonstrated to contribute to MDA-MB231/lymph endothelial intravasation. The present data demonstrate that the endothelial-specific and lymph endothelial-specific transcription factors SOX18 and PROX1 contribute to LEC retraction.

Chemical Composition of Scrophularia lucida and the Effects on Tumor Invasiveness in Vitro.

A detannified methanolic extract of Scrophularia lucida L. attenuated the formation of cancer cell-induced circular chemorepellent induced defects (CCIDs) in the lymph endothelial cell barrier, which resemble entry ports for the intravasating tumor into the vasculature as a prerequisite for lymph node metastasis. Therefore, the composition of this extract was studied in an activity-guided approach. Since no data on the secondary metabolites of this plant were available, first phytochemical data were collected in the course of the fractionation of the extract. The study resulted in the identification of 14 substances, among them very rare iridoids, such as scrovalentinoside or koelzioside, and several flavonoids (e.g., nepitrin and homoplantaginin). One of the latter group, 2″-O-acetyl-homoplantaginin, is a new natural compound. In the most active fraction, the flavonoid hispidulin was identified as major component and the assay of the pure compound confirmed a contribution of hispidulin to the CCID-inhibitory effects of S. lucida. The activity of the two major iridoids in this assay was less compared to hispidulin.

FGF2 and EGF induce epithelial-mesenchymal transition in malignant pleural mesothelioma cells via a MAPKinase/MMP1 signal.

Malignant pleural mesothelioma (MPM), an aggressive malignancy affecting pleural surfaces, occurs in three main histological subtypes. The epithelioid and sarcomatoid subtypes are characterized by cuboid and fibroblastoid cells, respectively. The biphasic subtype contains a mixture of both. The sarcomatoid subtype expresses markers of epithelial-mesenchymal transition (EMT) and confers the worst prognosis, but the signals and pathways controlling EMT in MPM are not well understood. We demonstrate that treatment with FGF2 or EGF induced a fibroblastoid morphology in several cell lines from biphasic MPM, accompanied by scattering, decreased cell adhesion and increased invasiveness. This depended on the MAP-kinase pathway but was independent of TGFß or PI3-kinase signaling. In addition to changes in known EMT markers, microarray analysis demonstrated differential expression of MMP1, ESM1, ETV4, PDL1 and BDKR2B in response to both growth factors and in epithelioid versus sarcomatoid MPM. Inhibition of MMP1 prevented FGF2-induced scattering and invasiveness. Moreover, in MPM cells with sarcomatoid morphology, inhibition of FGF/MAP-kinase signaling induced a more epithelioid morphology and gene expression pattern. Our findings suggest a critical role of the MAP-kinase axis in the morphological and behavioral plasticity of mesothelioma.

Apigenin and Luteolin Attenuate the Breaching of MDA-MB231 Breast Cancer Spheroids Through the Lymph Endothelial Barrier in Vitro.

Flavonoids, present in fruits, vegetables and traditional medicinal plants, show anticancer effects in experimental systems and are reportedly non-toxic. This is a favorable property for long term strategies for the attenuation of lymph node metastasis, which may effectively improve the prognostic states in breast cancer. Hence, we studied two flavonoids, apigenin and luteolin exhibiting strong bio-activity in various test systems in cancer research and are readily available on the market. This study has further advanced the mechanistic understanding of breast cancer intravasation through the lymphatic barrier. Apigenin and luteolin were tested in a three-dimensional (3-D) assay consisting of MDA-MB231 breast cancer spheroids and immortalized lymph endothelial cell (LEC) monolayers. The 3-D model faithfully resembles the intravasation of breast cancer emboli through the lymphatic vasculature. Western blot analysis, intracellular Ca2+ determination, EROD assay and siRNA transfection revealed insights into mechanisms of intravasation as well as the anti-intravasative outcome of flavonoid action. Both flavonoids suppressed pro-intravasative trigger factors in MDA-MB231 breast cancer cells, specifically MMP1 expression and CYP1A1 activity. A pro-intravasative contribution of FAK expression in LECs was established as FAK supported the retraction of the LEC monolayer upon contact with cancer cells thereby enabling them to cross the endothelial barrier. As mechanistic basis, MMP1 caused the phosphorylation (activation) of FAK at Tyr397 in LECs. Apigenin and luteolin prevented MMP1-induced FAK activation, but not constitutive FAK phosphorylation. Luteolin, unlike apigenin, inhibited MMP1-induced Ca2+ release. Free intracellular Ca2+ is a central signal amplifier triggering LEC retraction through activation of the mobility protein MLC2, thereby enhancing intravasation. FAK activity and Ca2+ levels did not correlate. This implicates that the pro-intravasative contribution of FAK and of Ca2+ release in LECs was independent of each other and explains the better anti-intravasative effects of luteolin in vitro. In specific formulations, flavonoid concentrations causing significant anti-intravasative effects, can certainly be achieved in vivo. As the therapeutic strategy has to be based on permanent flavonoid treatment both the beneficial and adverse effects have to be investigated in future studies.

Intravasation of SW620 colon cancer cell spheroids through the blood endothelial barrier is inhibited by clinical drugs and flavonoids in vitro.

Mechanisms how colorectal cancer (CRC) cells penetrate blood micro-vessel endothelia and metastasise is poorly understood. To study blood endothelial cell (BEC) barrier breaching by CRC emboli, an in vitro assay measuring BEC-free areas underneath SW620 cell spheroids, so called "circular chemorepellent induced defects" (CCIDs, appearing in consequence of endothelial retraction), was adapted and supported by Western blotting, EIA-, EROD- and luciferase reporter assays. Inhibition of ALOX12 or NF-κB in SW620 cells or BECs, respectively, caused attenuation of CCIDs. The FDA approved drugs vinpocetine [inhibiting ALOX12-dependent 12(S)-HETE synthesis], ketotifen [inhibiting NF-κB], carbamazepine and fenofibrate [inhibiting 12(S)-HETE and NF-κB] significantly attenuated CCID formation at low µM concentrations. In the 5-FU-resistant SW620-R/BEC model guanfacine, nifedipine and proadifen inhibited CCIDs stronger than in the naïve SW620/BEC model. This indicated that in SW620-R cells formerly silent (yet unidentified) genes became expressed and targetable by these drugs in course of resistance acquisition. Fenofibrate, and the flavonoids hispidulin and apigenin, which are present in medicinal plants, spices, herbs and fruits, attenuated CCID formation in both, naïve- and resistant models. As FDA-approved drugs and food-flavonoids inhibited established and acquired intravasative pathways and attenuated BEC barrier-breaching in vitro, this warrants testing of these compounds in CRC models in vivo.

Thiosemicarbazone derivatives, thiazolyl hydrazones, effectively inhibit leukemic tumor cell growth: Down-regulation of ribonucleotide reductase activity and synergism with arabinofuranosylcytosine.

Cellular growth inhibition exerted by thiosemicarbazones is mainly attributed to down-regulation of ribonucleotide reductase (RNR) activity, with RNR being responsible for the rate-limiting step of de novo DNA synthesis. In this study, we investigated the antineoplastic effects of three newly synthesized thiosemicarbazone derivatives, thiazolyl hydrazones, in human HL-60 promyelocytic leukemia cells. The cytotoxicity of compounds alone and in combination with arabinofuranosylcytosine (AraC) was determined by growth inhibition assays. Effects on deoxyribonucleoside triphosphate (dNTP) concentrations were quantified by HPLC, and the incorporation of radio-labeled 14C-cytidine into nascent DNA was measured using a beta counter. Cell cycle distribution was analyzed by FACS, and protein levels of RNR subunits and checkpoint kinases were evaluated by Western blotting. VG12, VG19, and VG22 dose-dependently decreased intracellular dNTP concentrations, impaired cell cycle progression and, consequently, inhibited the growth of HL-60 cells. VG19 also lowered the protein levels of RNR subunits R1 and R2 and significantly diminished the incorporation of radio-labeled 14C-cytidine, being equivalent to an inhibition of DNA synthesis. Combination of thiazolyl hydrazones with AraC synergistically potentiated the antiproliferative effects seen with each drug alone and might therefore improve conventional chemotherapeutic regimens for the treatment of human malignancies such as acute promyelocytic or chronic myelogenous leukemia.

Isolation of eudesmanes from Pluchea odorata and evaluation of their effects on cancer cell growth and tumor invasiveness in vitro.

The traditionally used Central American medicinal plant Pluchea odorata, known as an anti-inflammatory and cancer cell growth-inhibiting remedy, was subjected to bioassay-guided isolation. Structure elucidation by 1D- and 2D-NMR and MS techniques supported by ECD and UV spectroscopic data revealed seven structurally previously undescribed and eight known eudesmane-type sesquiterpenes. Furthermore, one previously undescribed and one known phytol-like alcohol were identified. All compounds were tested for their cytotoxicity in cancer cells and for their anti-invasive effects. Among the eudesmanes, 3α-(2',3'-epoxy-2'-methylbutyryloxy)-4α-hydroxy-11-hydroperoxy-eudesm-6-en-8-one exhibited the most potent cytotoxic activity with an IC50 value of 8.8 µM (after 48 h). Also in an in vitro model measuring the tumor-triggered breaching of the adjacent lymph endothelial cell barrier (3S*,4R*,5S*,10S*,2'R*,3'R*)-3-(2',3'-epoxy-2'-methylbutyryloxy)-4,7-dihydroxy-eudesm-11-en-8-one (IC75 = 47 µM) and (3S*,4R*,5R*,10S*,2'R*,3'R*)-3-(2',3'-epoxy-2'-methylbutyryloxy)-4-acetyloxy-6-methoxy-11-hydroxy-eudesm-6-en-8-one (IC75 = 73 µM) showed inhibitory activities. Furthermore, preliminary structure-activity relationships (SARs) of the eudesmanes were developed.

Fenofibrate inhibits tumour intravasation by several independent mechanisms in a 3-dimensional co-culture model.

Lymph node metastasis of breast cancer is a clinical marker of poor prognosis. Yet, there exist no therapies targeting mechanisms of intravasation into lymphatics. Herein we report on an effect of the antidyslipidemic drug fenofibrate with vasoprotective activity, which attenuates breast cancer intravasation in vitro, and describe the potential mechanisms. To measure intravasation in a 3-dimensional co-culture model MDA-MB231 and MCF-7 breast cancer spheroids were placed on immortalised lymphendothelial cell (LEC) monolayers. This provokes the formation of circular chemorepellent induced defects (CCIDs) in the LEC barrier resembling entry ports for the intravasating tumour. Furthermore, the expression of adhesion molecules ICAM-1, CD31 and FAK was investigated in LECs by western blotting as well as cell-cell adhesion and NF-κB activity by respective assays. In MDA-MB231 cells the activity of CYP1A1 was measured by EROD assay. Fenofibrate inhibited CCID formation in the MDA-MB231/LEC- and MCF-7/LEC models and the activity of NF-κB, which in turn downregulated ICAM-1 in LECs and the adhesion of cancer cells to LECs. Furthermore, CD31 and the activity of FAK were inhibited. In MDA-MB231 cells, fenofibrate attenuated CYP1A1 activity. Combinations with other FDA-approved drugs, which reportedly inhibit different ion channels, attenuated CCID formation additively or synergistically. In summary, fenofibrate inhibited NF-κB and ICAM-1, and inactivated FAK, thereby attenuating tumour intravasation in vitro. A combination with other FDA-approved drugs further improved this effect. Our new concept may lead to a novel therapy for cancer patients.