Colon cancer cell-derived 12(S)-HETE induces the retraction of cancer-associated fibroblast via MLC2, RHO/ROCK and Ca2+ signalling.

Retraction of mesenchymal stromal cells supports the invasion of colorectal cancer cells (CRC) into the adjacent compartment. CRC-secreted 12(S)-HETE enhances the retraction of cancer-associated fibroblasts (CAFs) and therefore, 12(S)-HETE may enforce invasivity of CRC. Understanding the mechanisms of metastatic CRC is crucial for successful intervention. Therefore, we studied pro-invasive contributions of stromal cells in physiologically relevant three-dimensional in vitro assays consisting of CRC spheroids, CAFs, extracellular matrix and endothelial cells, as well as in reductionist models. In order to elucidate how CAFs support CRC invasion, tumour spheroid-induced CAF retraction and free intracellular Ca2+ levels were measured and pharmacological- or siRNA-based inhibition of selected signalling cascades was performed. CRC spheroids caused the retraction of CAFs, generating entry gates in the adjacent surrogate stroma. The responsible trigger factor 12(S)-HETE provoked a signal, which was transduced by PLC, IP3, free intracellular Ca2+, Ca2+-calmodulin-kinase-II, RHO/ROCK and MYLK which led to the activation of myosin light chain 2, and subsequent CAF mobility. RHO activity was observed downstream as well as upstream of Ca2+ release. Thus, Ca2+ signalling served as central signal amplifier. Treatment with the FDA-approved drugs carbamazepine, cinnarizine, nifedipine and bepridil HCl, which reportedly interfere with cellular calcium availability, inhibited CAF-retraction. The elucidation of signalling pathways and identification of approved inhibitory drugs warrant development of intervention strategies targeting tumour-stroma interaction.

Hsp90 stabilizes Cdc25A and counteracts heat shock-mediated Cdc25A degradation and cell-cycle attenuation in pancreatic carcinoma cells.

Pancreas cancer cells escape most treatment options. Heat shock protein (Hsp)90 is frequently over-expressed in pancreas carcinomas and protects a number of cell-cycle regulators such as the proto-oncogene Cdc25A. We show that inhibition of Hsp90 with geldanamycin (GD) destabilizes Cdc25A independent of Chk1/2, whereas the standard drug for pancreas carcinoma treatment, gemcitabine (GEM), causes Cdc25A degradation through the activation of Chk2. Both agents applied together additively inhibit the expression of Cdc25A and the proliferation of pancreas carcinoma cells thereby demonstrating that both Cdc25A-destabilizing/degrading pathways are separated. The role of Hsp90 as stabilizer of Cdc25A in pancreas carcinoma cells is further supported by two novel synthetic inhibitors 4-tosylcyclonovobiocic acid and 7-tosylcyclonovobiocic acid and specific Hsp90AB1 (Hsp90ß) shRNA. Our data show that targeting Hsp90 reduced the resistance of pancreas carcinoma cells to treatment with GEM.

Inhibition of tumour spheroid-induced prometastatic intravasation gates in the lymph endothelial cell barrier by carbamazepine: drug testing in a 3D model.

Metastatic breast cancer is linked to an undesired prognosis. One early and crucial metastatic step is the interaction of cancer emboli with adjacent stroma or endothelial cells, and understanding the mechanisms of this interaction provides the basis to define new targets as well as drugs for therapy and disease management. A three-dimensional (3D) co-culture model allowing the examination of lymphogenic dissemination of breast cancer cells was recently developed which facilitates not only the study of metastatic processes but also the testing of therapeutic concepts. This 3D setting consists of MCF-7 breast cancer cell spheroids (representing a ductal and hormone-dependent subtype) and of hTERT-immortalised lymph endothelial cell (LEC; derived from foreskin) monolayers. Tumour spheroids repel the continuous LEC layer, thereby generating "circular chemorepellent-induced defects" (CCIDs) that are reminiscent to the entry gates through which tumour emboli intravasate lymphatics. We found that the ion channel blocker carbamazepine (which is clinically used to treat epilepsy, schizophrenia and other neurological disorders) inhibited CCID formation significantly. This effect correlated with the inhibition of the activities of NF-κB, which contributes to cell motility, and with the inactivation of the mobility proteins MLC2, MYPT1 and FAK which are necessary for LEC migration. NF-κB activity and cell movement are prerequisites of CCID formation. On the other hand, the expression of the motility protein paxillin and of the NF-κB-dependent adhesion mediator ICAM-1 was unchanged. Also the activity of ALOX12 was unaffected. ALOX12 is the main enzyme synthesising 12(S)-HETE, which then triggers CCID formation. The relevance of the inhibition of CYP1A1, which is also involved in the generation of mid-chain HETEs such as 12(S)-HETE, by carbamazepine remains to be established, because the constitutive level of 12(S)-HETE did not change upon carbamazepine treatment. Nevertheless, the effect of carbamazepine on the inhibition of CCID formation as an early step of breast cancer metastasis was significant and substantial (~30 %) and achieved at concentrations that are found in the plasma of carbamazepine-treated adults (40-60 µM). The fact that carbamazepine is a drug approved by the US Food and Drug Administration facilitates a "from-bench-to-bedside" perspective. Therefore, the here presented data should undergo scrutiny in vivo.

Digalloylresveratrol, a novel resveratrol analog inhibits the growth of human pancreatic cancer cells.

Digalloylresveratrol (DIG) is a recently synthesized substance aimed to combine the effects of the natural polyphenolic compounds gallic acid and resveratrol, which both are excellent free radical scavengers with anticancer activity. In this study, we investigated the effects of DIG in the human AsPC-1 and BxPC-3 pancreatic adenocarcinoma cell lines. Treatment with DIG dose-dependently attenuated cells in the S phase of the cell cycle and led to a significant depletion of the dATP pool in AsPC-1 cells. The incorporation of (14)C-cytidine into nascent DNA of tumor cells was significantly inhibited at all DIG concentrations due to inhibition of ribonucleotide reductase, a key enzyme of DNA synthesis in tumor cells. Furthermore, Erk1/2 became inactivated and moderated p38 phosphorylation reflecting increased replication stress. DIG also activated ATM and Chk2, and induced the phosphorylation and proteasomal degradation of the proto-oncogene Cdc25A, which contributed to cell cycle attenuation. Taken together, DIG is an excellent free radical scavenger, strongly inhibits RR in situ activity, cell cycle progression, and colony formation in AsPC-1 and BxPC-3 cells thus warranting further investigations.

Xanthohumol attenuates tumour cell-mediated breaching of the lymphendothelial barrier and prevents intravasation and metastasis.

Health beneficial effects of xanthohumol have been reported, and basic research provided evidence for anti-cancer effects. Furthermore, xanthohumol was shown to inhibit the migration of endothelial cells. Therefore, this study investigated the anti-metastatic potential of xanthohumol. MCF-7 breast cancer spheroids which are placed on lymphendothelial cells (LECs) induce "circular chemorepellent-induced defects" (CCIDs) in the LEC monolayer resembling gates for intravasating tumour bulks at an early step of lymph node colonisation. NF-κB reporter-, EROD-, SELE-, 12(S)-HETE- and adhesion assays were performed to investigate the anti-metastatic properties of xanthohumol. Western blot analyses were used to elucidate the mechanisms inhibiting CCID formation. Xanthohumol inhibited the activity of CYP, SELE and NF-kB and consequently, the formation of CCIDs at low micromolar concentrations. More specifically, xanthohumol affected ICAM-1 expression and adherence of MCF-7 cells to LECs, which is a prerequisite for CCID formation. Furthermore, markers of epithelial-to-mesenchymal transition (EMT) and of cell mobility such as paxillin, MCL2 and S100A4 were suppressed by xanthohumol. Xanthohumol attenuated tumour cell-mediated defects at the lymphendothelial barrier and inhibited EMT-like effects thereby providing a mechanistic explanation for the anti-intravasative/anti-metastatic properties of xanthohumol.

In vitro characterisation of the anti-intravasative properties of the marine product heteronemin.

Metastases destroy the function of infested organs and are the main reason of cancer-related mortality. Heteronemin, a natural product derived from a marine sponge, was tested in vitro regarding its properties to prevent tumour cell intravasation through the lymph-endothelial barrier. In three-dimensional (3D) cell cultures consisting of MCF-7 breast cancer cell spheroids that were placed on lymph-endothelial cell (LEC) monolayers, tumour cell spheroids induce "circular chemorepellent-induced defects" (CCIDs) in the LEC monolayer; 12(S)-Hydroxyeicosatetraenoic acid (12(S)-HETE) and NF-κB activity are major factors inducing CCIDs, which are entry gates for tumour emboli intravasating the vasculature. This 3D co-culture is a validated model for the investigation of intravasation mechanisms and of drugs preventing CCID formation and hence lymph node metastasis. Furthermore, Western blot analyses, NF-κB reporter, EROD, SELE, 12(S)-HETE, and adhesion assays were performed to investigate the properties of heteronemin. Five micromolar heteronemin inhibited the directional movement of LECs and, therefore, the formation of CCIDs, which were induced by MCF-7 spheroids. Furthermore, heteronemin reduced the adhesion of MCF-7 cells to LECs and suppressed 12(S)-HETE-induced expression of the EMT marker paxillin, which is a regulator of directional cell migration. The activity of CYP1A1, which contributed to CCID formation, was also inhibited by heteronemin. Hence, heteronemin inhibits important mechanisms contributing to tumour intravasation in vitro and should be tested in vivo.

Fractionation of an Extract of Pluchea odorata Separates a Property Indicative for the Induction of Cell Plasticity from One That Inhibits a Neoplastic Phenotype.

Introduction. Several studies demonstrated that anti-inflammatory remedies exhibit excellent anti-neoplastic properties. An extract of Pluchea odorata (Asteraceae), which is used for wound healing and against inflammatory conditions, was fractionated and properties correlating to anti-neoplastic and wound healing effects were separated. Methods. Up to six fractionation steps using silica gel, Sephadex columns, and distinct solvent systems were used, and eluted fractions were analysed by thin layer chromatography, apoptosis, and proliferation assays. The expression of oncogenes and proteins regulating cell migration was investigated by immunoblotting after treating HL60 cells with the most active fractions. Results. Sequential fractionations enriched anti-neoplastic activities which suppressed oncogene expression of JunB, c-Jun, c-Myc, and Stat3. Furthermore, a fraction (F4.6.3) inducing or keeping up expression of the mobility markers MYPT, ROCK1, and paxillin could be separated from another fraction (F4.3.7), which inhibited these markers. Conclusions. Wound healing builds up scar or specific tissue, and hence, compounds enhancing cell migration support this process. In contrast, successful anti-neoplastic therapy combats tumour progression, and thus, suppression of cell migration is mandatory.

Short 42 degrees C heat shock induces phosphorylation and degradation of Cdc25A which depends on p38MAPK, Chk2 and 14.3.3.

The effects of heat shock (HS; 42 degrees C) on the cell cycle and underlying molecular mechanisms are astonishingly unexplored. Here, we show that HS caused rapid Cdc25A degradation and a reduction of cell cycle progression. Cdc25A degradation depended on Ser75-Cdc25A phosphorylation caused by p38MAPK and Chk2, which phosphorylated Ser177-Cdc25A that is specific for 14.3.3 binding. Upon HS, Cdc25A rapidly co-localized with 14.3.3 in the perinuclear space that was accompanied with a decrease of nuclear Cdc25A protein levels. Consistently, a 14.3.3 binding-deficient Cdc25A double mutant (Ser177/Ala-Tyr507/Ala) was not degraded in response to HS and there was no evidence for an increased co-localization of Cdc25A with 14.3.3 in the cytosol. Therefore, upon HS, p38, Chk2 and 14.3.3 were antagonists of Cdc25A stability. On the other hand, Cdc25A was protected by Hsp90 in HEK293 cells because the specific inhibition of Hsp90 with Geldanamycin caused Cdc25A degradation in HEK293 implicating that Cdc25A is an Hsp90 client. Specific inhibition of Hsp90 together with HS caused and accelerated degradation of Cdc25A and was highly cytotoxic. The results presented here show for the first time that Cdc25A is degraded by moderate heat shock and protected by Hsp90. We describe the mechanisms explaining HS-induced cell cycle retardation and provide a rationale for a targeted hyperthermia cancer therapy.

Pro- and anticarcinogenic mechanisms of piceatannol are activated dose dependently in MCF-7 breast cancer cells.

Estrogenic procarcinogenic effects of piceatannol (PIC) contrast reports about anticarcinogenic activities of PIC. To explain this contradiction, we investigated PIC in estrogen-dependent MCF-7 breast cancer cells and elucidated those cellular mechanisms that correlated with the observed cell effects induced by PIC. Low PIC concentrations (50 nM) induced c-Myc that depended on progesterone receptor (PR) and estrogen receptor (ER). PR-mediated c-Myc induction by PIC was independent of nuclear PR activity but depended on mitogen-activated protein kinase (MAPK) signaling and was associated with an acceleration of cancer cell proliferation. In contrast, 25 µM PIC inhibited deoxynucleotide triphosphate synthesis, activated Chk2 and p38-MAPK and this was accompanied by an attenuation of cancer cell growth. Apoptosis was most probably inhibited due to activation of Akt; however, high PIC concentrations (>100 µM) permitted apoptosis-like cell death in consequence to disruption of orchestrated mitotic signaling. The presented results show for the first time that nanomolar PIC concentrations signal through PR and Erk1/2 and provide a mechanistic explanation why moderate wine consumption-but not other alcoholic beverages-increases the breast cancer risk in women. In contrast, higher PIC concentrations in the micromolar range are considered for adjuvant anticancer therapeutic concepts.

Berberine and a Berberis lycium extract inactivate Cdc25A and induce alpha-tubulin acetylation that correlate with HL-60 cell cycle inhibition and apoptosis.

Berberis lycium Royle (Berberidacea) from Pakistan and its alkaloids berberine and palmatine have been reported to possess beneficial pharmacological properties. In the present study, the anti-neoplastic activities of different B. lycium root extracts and the major constituting alkaloids, berberine and palmatine were investigated in p53-deficient HL-60 cells. The strongest growth inhibitory and pro-apoptotic effects were found in the n-butanol (BuOH) extract followed by the ethyl acetate (EtOAc)-, and the water (H(2)O) extract. The chemical composition of the BuOH extract was analyzed by TLC and quantified by HPLC. 11.1 microg BuOH extract (that was gained from 1mg dried root) contained 2.0 microg berberine and 0.3 microg/ml palmatine. 1.2 microg/ml berberine inhibited cell proliferation significantly, while 0.5 microg/ml palmatine had no effect. Berberine and the BuOH extract caused accumulation of HL-60 cells in S-phase. This was preceded by a strong activation of Chk2, phosphorylation and degradation of Cdc25A, and the subsequent inactivation of Cdc2 (CDK1). Furthermore, berberine and the extract inhibited the expression of the proto-oncogene cyclin D1. Berberine and the BuOH extract induced the acetylation of alpha-tubulin and this correlated with the induction of apoptosis. The data demonstrate that berberine is a potent anti-neoplastic compound that acts via anti-proliferative and pro-apoptotic mechanisms independent of genotoxicity.

12(S)-HETE increases intracellular Ca(2+) in lymph-endothelial cells disrupting their barrier function in vitro; stabilization by clinical drugs impairing calcium supply.

Secretion of 12(S)-HETE by breast cancer emboli provokes "circular chemorepellent induced defects" (CCIDs) in the adjacent lymphatic vasculature facilitating their intravasation and lymph node metastasis which determines prognosis. Therefore, elucidating the mechanism of lymph endothelial cell (LEC) wall disintegration may provide cues for anti-metastatic intervention. The role of intracellular free Ca(2+) for CCID formation was investigated in LECs using MCF-7 or MDA-MB231 breast cancer cell spheroids in a three-dimensional cell co-culture model. 12(S)-HETE elevated the Ca(2+) level in LEC by activating PLC/IP3. Downstream, the Ca(2+)-calmodulin kinase MYLK contributed to the phosphorylation of Ser19-MLC2, LEC contraction and CCID formation. Approved clinical drugs, lidoflazine, ketotifen, epiandrosterone and cyclosporine, which reportedly disturb cellular calcium supply, inhibited 12(S)-HETE-induced Ca(2+) increase, Ser19-MLC2 phosphorylation and CCID formation. This treatment strategy may reduce spreading of breast cancer through lymphatics.

The effect of organic anion-transporting polypeptides 1B1, 1B3 and 2B1 on the antitumor activity of flavopiridol in breast cancer cells.

The contribution of organic anion transporting polypeptides (OATPs) to the cellular uptake of flavopiridol was investigated in OATP1B1-, OATP1B3- and OATP2B1-expressing Chinese hamster ovary (CHO) cells. Uptake of flavopiridol into these cells showed typical Michaelis-Menten kinetics with much higher transport capacity for OATP1B3 compared to OATP1B1 and OATP2B1 (Vmax/Km, 33.9 vs. 8.84 and 2.41 µl/mg/min, respectively). The predominant role of OATPs was further supported by a dramatic inhibition of flavopiridol uptake in the presence of the OATP substrate rifampicin. Uptake of flavopiridol by OATPs also seems to be an important determinant in breast cancer cells. The much higher mRNA level for OATP1B1 found in wild-type compared to ZR-75-1 OATP1B1 knockdown cells correlated with higher flavopiridol initial uptake leading to 4.6-fold decreased IC50 values in the cytotoxicity assay (IC50, 1.45 vs. 6.64 µM). Cell cycle profile also showed a clear incidence for a stronger cell cycle arrest in the G2/M phase for ZR-75-1 wild-type cells compared to OATP1B1 knockdown cells, further indicating an active uptake via OATP1B1. In conclusion, our results revealed OATP1B1, OATP1B3 and OATP2B1 as uptake transporters for flavopiridol in cancer cells, which may also apply in patients during cancer therapy.

Epigallocatechin gallate, ellagic acid, and rosmarinic acid perturb dNTP pools and inhibit de novo DNA synthesis and proliferation of human HL-60 promyelocytic leukemia cells: Synergism with arabinofuranosylcytosine.

Epigallocatechin gallate (EGCG), ellagic acid (EA) and rosmarinic acid (RA) are natural polyphenols exerting cancer chemopreventive effects. Ribonucleotide reductase (RR; EC 1.17.4.1) converts ribonucleoside diphosphates into deoxyribonucleoside diphosphates being essential for DNA replication, which is why the enzyme is considered an excellent target for anticancer therapy. EGCG, EA, and RA dose-dependently inhibited the growth of human HL-60 promyelocytic leukemia cells, exerted strong free radical scavenging potential, and significantly imbalanced nuclear deoxyribonucleoside triphosphate (dNTP) concentrations without distinctly affecting the protein levels of RR subunits (R1, R2, p53R2). Incorporation of (14)C-cytidine into nascent DNA of tumor cells was also significantly lowered, being equivalent to an inhibition of DNA synthesis. Consequently, treatment with EGCG and RA attenuated cells in the G0/G1 phase of the cell cycle, finally resulting in a pronounced induction of apoptosis. Sequential combination of EA and RA with the first-line antileukemic agent arabinofuranosylcytosine (AraC) synergistically potentiated the antiproliferative effect of AraC, whereas EGCG plus AraC yielded additive effects. Taken together, we show for the first time that EGCG, EA, and RA perturbed dNTP levels and inhibited cell proliferation in human HL-60 promyelocytic leukemia cells, with EGCG and RA causing a pronounced induction of apoptosis. Due to these effects and synergism with AraC, these food ingredients deserve further preclinical and in vivo testing as inhibitors of leukemic cell proliferation.

Resveratrol and its major sulfated conjugates are substrates of organic anion transporting polypeptides (OATPs): impact on growth of ZR-75-1 breast cancer cells.

SCOPE: Resveratrol is a naturally occurring polyphenolic compound with various pharmacological activities. These effects are observed despite its low bioavailability, which is particularly caused by extensive phase II metabolism. It is unknown whether resveratrol and its metabolites can accumulate to bioactive levels in organs and tissues through protein-mediated transport mechanisms. Because organic anion transporting polypeptides (OATPs) mediate the uptake of many clinically important drugs, we investigated their role in the cellular transport of resveratrol and its major glucuronides and sulfates. METHODS AND RESULTS: Uptake experiments were performed with resveratrol and its glucuronides and sulfates in OATP-expressing Chinese hamster ovary (CHO) and breast cancer (ZR-75-1) cells. The uptake rates for resveratrol in OATP1B1-, OATP1B3-, and OATP2B1-transfected Chinese hamster ovary cells were four- to sixfold higher compared to wild-type cells. Resveratrol-3-O-4'-O-disulfate was transported by OATP1B1 and OATP1B3, while resveratrol-3-O-sulfate was exclusively transported by OATP1B3. However, resveratrol-4'-O-sulfate, resveratrol-3-O-glucuronide, and resveratrol-4'-O-glucuronide did not show any affinity for these OATPs. OATP-dependent uptake of resveratrol was also confirmed in ZR-75-1 cells. CONCLUSION: Our data revealed that OATPs act as cellular uptake transporters for resveratrol and its major sulfates, which must be considered in humans following oral uptake of dietary resveratrol.

The dichloromethane extract of the ethnomedicinal plant Neurolaena lobata inhibits NPM/ALK expression which is causal for anaplastic large cell lymphomagenesis.

The present study investigates extracts of Neuolaena lobata, an anti-protozoan ethnomedicinal plant of the Maya, regarding its anti-neoplastic properties. Firstly, extracts of increasing polarity were tested in HL-60 cells analyzing inhibition of cell proliferation and apoptosis induction. Secondly, the most active extract was further tested in anaplastic large cell lymphoma (ALCL) cell lines of human and mouse origin. The dichloromethane extract inhibited proliferation of HL-60, human and mouse ALCL cells with an IC50 of ~2.5, 3.7 and 2.4 µg/ml, respectively and arrested cells in the G2/M phase. The extract induced the checkpoint kinases Chk1 and Chk2 and perturbed the orchestrated expression of the Cdc25 family of cell cycle phosphatases which was paralleled by the activation of p53, p21 and downregulation of c-Myc. Importantly, the expression of NPM/ALK and its effector JunB were drastically decreased, which correlated with the activation of caspase 3. Subsequently also platelet derived growth factor receptor ß was downregulated, which was recently shown to be transcriptionally controlled by JunB synergizing with ALK in ALCL development. We show that a traditional healing plant extract downregulates various oncogenes, induces tumor suppressors, inhibits cell proliferation and triggers apoptosis of malignant cells. The discovery of the 'Active Principle(s)' is warranted.

Effects of Scrophularia extracts on tumor cell proliferation, death and intravasation through lymphoendothelial cell barriers.

Different studies describe the anti-inflammatory effects of Scrophularia species, a medicinal plant widely used in folk medicine since ancient times. As knowledge regarding the anti-neoplastic properties of this species is rather limited, we investigated the influence of methanol extracts of different Scrophularia species on cell proliferation, cell death, and tumour cell intravasation through the lymph endothelial barrier. HL-60 leukaemia cells were treated with methanol extracts of different Scrophularia species leading to strong growth inhibition and high cell death rates. The expression of cell cycle regulators, oncogenes and cell death inducers was determined by Western blot analysis. Furthermore the effect of S. lucida was studied in an NF-κB reporter assay, and in a novel assay measuring 'circular chemo-repellent-induced defects' (CCID) in lymph endothelial monolayers that were induced by MCF-7 breast cancer spheroids. Methanol extracts of Scrophularia species exhibited strong anti-proliferative properties. S. floribunda extract inhibited G2/M- and later on S-phase and S. lucida inhibited S-phase and in both cases this was associated with the down-regulation of c-Myc expression. Extracts of S. floribunda and S. lucida led to necrosis and apoptosis, respectively. Furthermore, S. lucida, but not S. floribunda, effectively attenuated tumour cell intravasation through lymph endothelial cell monolayers, which correlated with the inhibition of NF-κB. S. lucida exhibited promising anti-neoplastic effects and this was most likely due to the down-regulation of various cell cycle regulators, proto-oncogenes and NF-κB and the activation of caspase-3.

Methanol extract of the ethnopharmaceutical remedy Smilax spinosa exhibits anti-neoplastic activity.

Plants have been the source of several effective drugs for the treatment of cancer and over 60% of anticancer drugs originate from natural sources. Therefore, extracts of the rhizome of Smilax spinosa, an ethnomedicinal plant from Guatemala which is used for the treatment of inflammatory conditions, were investigated regarding their anti-neoplastic activities. By using several solvents the methanol extract was by far the most potent against HL60 cell proliferation (50% inhibition at 60 µg/ml). Furthermore, fractionation of this extract yielded fraction F2, which exhibited enforced pro-apoptotic activity, and activated CYP1A1. Proteins that are relevant for cell cycle progression and apoptosis, as well as proto-oncogenes were investigated by western blotting. This revealed that the methanol extract increased the levels of p21 and this may have caused cell cycle attenuation. The derivative fraction F2 induced apoptosis through the intrinsic pathway, which correlated with the inhibition of Stat3 phosphorylation and concomitant induction of caspase 9, then caspase 8 and caspase 3. In summary, the methanol extract and the derivative fraction F2 of S. spinosa showed anti-neoplastic effects in HL-60 cells and CYP1A1 activation in estrogen receptor-positive MCF-7 breast cancer cells but not in estrogen-negative MDA-MB231 breast cancer cells. Based on our data Smilax spinosa may be a promising source for novel anticancer agents.

An apolar extract of Critonia morifolia inhibits c-Myc, cyclin D1, Cdc25A, Cdc25B, Cdc25C and Akt and induces apoptosis.

Investigating the bioactivity of traditional medical remedies under the controlled conditions of a laboratory is an option to find additional applications, novel formulations or lead structures for the development of new drugs. The present work analysed the anti­neoplastic activity of increasing polar extracts of the rainforest plant Critonia morifolia (Asteraceae) that has been successfully used as traditional remedy to treat various inflammatory conditions in the long-lasting medical tradition of the Central American Maya, which was here also confirmed in vitro. The apolar petroleum ether extract exhibited the most potent anti­proliferative and pro­apoptotic effects in HL­60 cells and triggered down-regulation of Cdc25C and cyclin D1 within 30 min followed by the inhibition of c-Myc expression and the onset of caspase-3 activation within 2 h. Subsequent to these very rapid molecular responses Chk2 and H2AX became phosphorylated (γ­H2AX) after 4 h. Analysis of the cell cycle distribution showed an accumulation of cells in the G2-M phase within 8 h and after 24 h in S-phase. This was temporally paralleled by the down-regulation of Cdc25A, Cdc25B, Wee1 and Akt. Therefore, the attenuation of cell cycle progression in the G2-M phase was consistent with the known role of Chk2 for G2-M arrest and with the role of Cdc25B in S-phase progression. These findings suggest the presence of two distinct active principles in the petroleum ether extract of C. moriflia. These facilitated the strong apoptotic response evidenced by the rapid activation of caspase-3 that was later enforced by the inhibition of the survival kinase Akt. Importantly, the efficient down-regulation of Akt, which is successfully tested in current clinical trials, is a unique property of C. morifolia.

Metabolomic analysis of resveratrol-induced effects in the human breast cancer cell lines MCF-7 and MDA-MB-231.

Resveratrol is a naturally occurring anticancer compound present in grapes and wine with antiproliferative properties against breast cancer cells and xenografts. Our objective was to investigate the metabolic alterations that characterize the effects of resveratrol in the human breast cancer cell lines MCF-7 and MDA-MB-231 using high-throughput liquid chromatography-based mass spectrometry. In both cell lines, growth inhibition was dose dependent and accompanied by substantial metabolic changes. For all 21 amino acids analyzed levels increased more than 100-fold at a resveratrol dose of 100 µM with far lower concentrations in MDA-MB-231 compared to MCF-7 cells. Among the biogenic amines and modified amino acids (n = 16) resveratrol increased the synthesis of serotonin, kynurenine, and spermindine in both cell lines up to 61-fold indicating that resveratrol strongly interacts with cellular biogenic amine metabolism. Among the eicosanoids and oxidized polyunsaturated fatty acids (n = 17) a pronounced increase in arachidonic acid and its metabolite 12S-HETE was observed in MDA-MB-231 and to a lesser extent in MCF-7 cells, indicating release from cell membrane phospholipids upon activation of phospholipase A2 and subsequent metabolism by 12-lipoxygenase. In conclusion, metabolomic analysis elucidated several small molecules as markers for the response of breast cancer cells to resveratrol.

Antiproliferative effects of some novel synthetic solanidine analogs on HL-60 human leukemia cells in vitro.

There is increasing evidence of the direct antiproliferative effects of various steroidal structures, including cardenolides, steroidal alkaloids and sexual hormones. The aim of the present study was to characterize the antiproliferative effects of three synthetic solanidine analogs (1-3) on HL-60 human leukemia cells. The three compounds exerted similar cytostatic effects (IC(50) values: 1.27-2.94 µM after a 72-h exposure) and the most effective (2) was selected for further investigations. Incubation with compound 2 resulted in a marked chromatin condensation followed by a gradual increase in cell membrane permeability detected by Hoechst dye 33258-propidium iodide double staining. A flow cytometric analysis revealed a marked decrease in the G1 phase and substantial increases in the S and G2/M phases after 24-h incubation, while after 48 h the proportion of cells in the subG1 phase was increased significantly with a concomitant decrease in cells in the G1 and G2/M phases. Compound 2 at 6.0 µM significantly decreased the activity of ribonucleotide reductase and proved to be a potent antioxidant in the lipid peroxidation and DPPH assays (IC(50) values: 2.0 and 13.1 µM, respectively). The antiproliferative effect of the test compound on the non-cancerous human lung fibroblast cell line (MRC-5) was significantly weaker than that on the leukemia cells. These results lead to the conclusion that compound 2 induces a marked disturbance in the cell cycle, which is, at least partially, a consequence of the inhibition of DNA synthesis.