Concerted BAG3 and SIRPα blockade impairs pancreatic tumor growth.

The BAG3- and SIRPα- mediated pathways trigger distinct cellular targets and signaling mechanisms in pancreatic cancer microenvironment. To explore their functional connection, we investigated the effects of their combined blockade on cancer growth in orthotopic allografts of pancreatic cancer mt4-2D cells in immunocompetent mice. The anti-BAG3 + anti-SIRPα mAbs treatment inhibited (p = 0.007) tumor growth by about the 70%; also the number of metastatic lesions was decreased, mostly by the effect of the anti-BAG3 mAb. Fibrosis and the expression of the CAF activation marker α-SMA were reduced by about the 30% in animals treated with anti-BAG3 mAb compared to untreated animals, and appeared unaffected by treatment with the anti-SIRPα mAb alone; however, the addition of anti-SIRPα to anti-BAG3 mAb in the combined treatment resulted in a > 60% (p < 0.0001) reduction of the fibrotic area and a 70% (p < 0.0001) inhibition of CAF α-SMA positivity. Dendritic cells (DCs) and CD8+ lymphocytes, hardly detectable in the tumors of untreated animals, were modestly increased by single treatments, while were much more clearly observable (p < 0.0001) in the tumors of the animals subjected to the combined treatment. The effects of BAG3 and SIRPα blockade do not simply reflect the sum of the effects of the single blockades, indicating that the two pathways are connected by regulatory interactions and suggesting, as a proof of principle, the potential therapeutic efficacy of a combined BAG3 and SIRPα blockade in pancreatic cancer.

Different mechanisms underlie IL-6 release in chemosensitive and chemoresistant ovarian carcinoma cells.

Interleukin (IL)-6 has been detected in serum and ascites from patients affected by epithelial ovarian cancers, and also in some human ovarian cancer cell lines. To investigate the role of IL-6 in ovarian lesions, we first measured its levels in serum samples of 24 healthy donors and in 17 and 9 patients affected by ovarian carcinomas and ovarian benign cysts respectively. IL-6 levels were significantly higher than healthy donors in serum samples from ovarian cancer patients, but not in benign ovarian cysts. We then investigated the mechanism of IL-6 production in two cell lines obtained from the same patient with high grade serous ovarian carcinoma before (PEA1) and after (PEA2) development of cisplatinum resistance. The levels of intracellular IL-6, analysed by western blotting, did not relevantly differ in the two cell lines, and they did not change after the cell treatment with an AKT inhibitor. Although the interleukin was present in supernatants from both cell lines, its concentration in the supernatant of chemoresistant cells was significantly higher than chemosensitive cells. Interestingly, exposure to the AKT inhibitor resulted in a reduced IL-6 release in PEA1, but not in PEA2 cells. These results let infer different mechanisms of IL-6 release in chemoresistant and chemosensitive cell lines, and contribute new insights in ovarian cancer biology that suggest more in depth studies about the role of AKT in IL-6 release and in development of chemoresistance.

BAG3 Protein Is Involved in Endothelial Cell Response to Phenethyl Isothiocyanate.

Phenethyl isothiocyanate (PEITC), a cruciferous vegetable-derived compound, is a versatile cancer chemopreventive agent that displays the ability to inhibit tumor growth during initiation, promotion, and progression phases in several animal models of carcinogenesis. In this report, we dissect the cellular events induced by noncytotoxic concentrations of PEITC in human umbilical vein endothelial cells (HUVECs). In the early phase, PEITC treatment elicited cells' morphological changes that comprise reduction in cell volume and modification of actin organization concomitantly with a rapid activation of the PI3K/Akt pathway. Downstream to PI3K, PEITC also induces the activity of Rac1 and activation of c-Jun N-terminal kinase (JNK), well-known regulators of actin cytoskeleton dynamics. Interestingly, PEITC modifications of the actin cytoskeleton were abrogated by pretreatment with JNK inhibitor, SP600125. JNK signaling led also to the activation of the c-Jun transcription factor, which is involved in the upregulation of several genes; among them is the BAG3 protein. This protein, a member of the BAG family of heat shock protein (Hsp) 70 cochaperones, is able to sustain survival in different tumor cell lines and neoangiogenesis by directly regulating the endothelial cell cycle. Furthermore, BAG3 is involved in maintaining actin folding. Our findings indicate that BAG3 protein expression is induced in endothelial cells upon exposure to a noncytotoxic concentration of PEITC and its expression is requested for the recovery of normal cell size and morphology after the stressful stimuli. This assigns an additional role for BAG3 protein in the endothelial cells after a stress event.

Steroidal glycosides with antiproliferative activities from Digitalis trojana.

The phytochemical investigation of Digitalis trojana led to the isolation of two cardiac glycosides (1, 2), one pregnane glycoside (3), three furostanol type saponins (4-6), along with three cleroindicins (7-9), four phenylethanoid glycosides (10-13), two flavonoids (14, 15) and two phenolic acid derivatives (16, 17). The structure elucidation of the isolates was carried out by NMR experiments as well as ESI-MS. The cytotoxic activity of compounds 1-13 against a small panel of cancer cell lines, namely MCF-7, T98G, HT-29, PC-3, A375 and SH-SY5Y, was investigated. Compounds 1-6 showed antiproliferative activity against human breast MCF-7 and colon HT-29 cancer cell lines with IC50 values ranging from 8.3 to 50 µM. In order to understand the mechanism involved in the cell death, the active compounds were tested as pro-apoptotic agents using propidium iodide staining by flow cytometry method. No significant increase was observed in the apoptosis of the MCF-7 and HT-29 cancer cells. Moreover, the effects of the active compounds on cell proliferation were assessed on the same cancer cell lines by cell cycle analysis of DNA content using flow cytometry. No significative changes were observed in the cell cycle of MCF-7, while significant changes in G2 /M cell cycle phase of HT-29 cells were observed after treatment with digitalin (1), cariensoside (3) and 22-O-methylparvispinoside B (6) at 10 µM.

Cyclohexa-2,5-diene-1,4-dione-based antiproliferative agents: design, synthesis, and cytotoxic evaluation.

BACKGROUND: Tumors are diseases characterized by uncontrolled cell growth and, in spite of the progress of medicine over the years, continue to represent a major threat to the health, requiring new therapies. Several synthetic compounds, such as those derived from natural sources, have been identified as anticancer drugs; among these compounds quinone represent the second largest class of anticancer agents in use. Several studies have shown that these act on tumor cells through several mechanisms. An important objective of this work is to develop quinoidscompounds showing antitumor activity, but with fewer side effects. The parachinone cannabinol HU-331, is a small molecule that with its core 4-hydroxy-1,4-benzoquinone, exhibits a potent and selective cytotoxic activity on different tumor cell lines. A series of derivatives 3-hydroxy-1,4-benzochinoni were thus developed through HU-331 chemical modifications. The purpose of the work is to test the ability of the compounds to induce proliferative inhibition and study the mechanisms of cell death. METHODS: The antitumor activities were evaluated in vitro by examining their cytotoxic effects against different human cancer cell lines. All cell lines tested were plated in 96-multiwell and treated with HU-100-V at different concentrations and cell viability was evaluated byMTT assay. Subsequently via flow cytometry (FACS) it was possible to assess apoptosis by the system of double labeling with PI and Annexin-V, and the effect of the compounds on ROS formation by measuring the dichlorofluorescein fluorescence. RESULTS: The substitution by n-hexyl chain considerably enhanced the bioactivity of the compounds. In details, 2-hexyl-5-hydroxycyclohexa-2,5-diene-1,4-dione (V), 2,5-Dimethoxy-3-hexyl-2,5-cyclohexadiene-1,4-dione (XII) and 2-hydroxy-5-methoxy-3-hexyl-cyclohexa-2,5-diene-1,4-dione (XIII) showed most prominent cytotoxicity against almost human tumour cell lines. Compound V was further subjected to downstream apoptotic analysis, demostrating a time-dependent pro-apoptotic activity on human melanoma M14 cell line mediated by caspases activation and poly-(ADP-ribose)-polymerase (PARP) protein cleavage. CONCLUSIONS: These findings indicate that 2-hexyl-5-idrossicicloesa-2,5-diene-1,4-dione can be a promising compound for the design of a new class of antineoplastic derivatives.Carmen Petronzi, Michela Festa, Antonella Peduto and Maria Castellano: equally contributed equally to this work.

Chemical composition and in vitro cytotoxic activity of the essential oils of Stachys rupestris and Salvia heldreichiana, two endemic plants of Turkey.

The chemical composition of the essential oils of two endemic plants of Turkey, Stachys rupestris Montbret et Aucher ex Benth. and Salvia heldreichiana Boiss. ex Benth., were obtained by hydrodistillation and studied by GC and GC-MS. In all, 46 compounds were identified, 22 for S. rupestris accounting for 94.6% of the total oil and 30 for S. heldreichiana, accounting for 91.9% of the total oil. The presence of diterpenoids (50.7%) characterized the oil from S. rupestris, while S. heldreichiana oil was rich in oxygenated sesquiterpenes (78.9%).The essential oils were evaluated for their in vitro potential cytotoxic activity on three human cancer cell lines. The oil of S. rupestris showed the higher antiproliferative activity against PC-3 and MCF-7 cancer cell lines.

HPLC-ESIMS(n) profiling, isolation, structural elucidation, and evaluation of the antioxidant potential of phenolics from Paepalanthus geniculatus.

The methanol extract of the flowers of Paepalanthus geniculatus Kunth. showed radical-scavenging activity in the TEAC assay. An analytical approach based on HPLC-ESIMS(n) was applied to obtain the metabolite profile of this extract and led to the rapid identification of 19 polyphenolic compounds comprising flavonoids and naphthopyranones. The new naphthopyranone (10, 16), quercetagetin (1, 5, 7, 13), and galetine derivatives (9, 11, 17, 19), and a flavonol glucoside cyclodimer in the truxillate form (12), were identified. Compounds 2, 6, and 7 showed the highest antioxidant capacity and ability to affect the levels of intracellular ROS in human prostate cancer cells (PC3).

Antiproliferative steroidal glycosides from Digitalis ciliata.

Two new compounds, a furostanol glycoside (1) and a pregnane glycoside (4), along with eight known compounds, belonging to the classes of spirostane (2,3), pregnane (5-7) and cardenolide (8-10) glycosides, were isolated from the seeds of Digitalis ciliata. Their structures were elucidated by 1D and 2D-NMR experiments as well as ESI-MS analysis. For the first time pregnane glycosides of the diginigenin series have been isolated from D. ciliata. The cytotoxic effects of compounds 1-10 on cell viability of several cancer cell lines, namely human breast cancer (MCF-7), human glioblastoma (T98G), human lung adenocarcinoma (A549), human colon carcinoma (HT-29), and human prostate cancer (PC-3) cell lines were evaluated. Compounds 1, 4, 7 and 8 showed antiproliferative effects against MCF-7, HT-29 and A549 cancer cells with IC50 values ranging from 8.3 to 20 µM. The effects of compounds 1-10 on cell proliferation were evaluated on these three cancer cell lines by cell cycle analysis of DNA content using flow cytometry. Compounds 7, 8 and 10 induced significant changes in G2/M cell cycle phase of all analyzed cells. The obtained results indicate that compounds 7, 8 and 10 are cytostatic compounds effective in reducing cell proliferation by inducing accumulation of the cells in the G2/M phase of the cell cycle.

Iridoid, phenylethanoid and flavonoid glycosides from Sideritis trojana.

From the MeOH extract of Sideritis trojana, a new iridoid glycoside, 10-O-(E)-feruloylmelittoside (1) was obtained in addition to four known iridoid glycosides [melittoside (2), 10-O-(E)-p-coumaroylmelittoside (3), stachysosides E (4) and G (5)]. Moreover, five phenylethanoid glycosides [verbascoside (6), isoacteoside (7), lamalboside (8), leonoside A (9), isolavandulifolioside (10), three flavone glycosides (isoscutellarein 7-O-[6'''-O-acetyl-ß-allopyranosyl-(1→2)]-ß-glucopyranoside (11), 4'-O-methyisoscutellarein 7-O-[6'''-O-acetyl-ß-allopyranosyl-(1→2)]-ß-glucopyranoside (12), 3'-hydroxy-4'-O-methyisoscutellarein 7-O-[6'''-O-acetyl-ß-allopyranosyl-(1→2)]-ß-glucopyranoside (13) and a benzylalcohol derivative (di-O-methylcrenatin) were obtained and identified. The structures were elucidated on the basis of NMR and HRMS data. All compounds were tested for their antioxidant activity by in vitro TEAC assay and some of them exhibited moderate activity (0.97-1.44 mM) when compared with the reference compound (quercetin 1.86 mM). Glycosides 6-13, the most active compounds in the TEAC assay, were also tested by flow cytometry to evaluate their ability to affect the levels of reactive oxygen species (ROS) in human prostate cancer cells (PC3).

Xanthohumol induces apoptosis in human malignant glioblastoma cells by increasing reactive oxygen species and activating MAPK pathways.

The effect of the biologically active prenylated chalcone and potential anticancer agent xanthohumol (1) has been investigated on apoptosis of the T98G human malignant glioblastoma cell line. Compound 1 decreased the viability of T98G cells by induction of apoptosis in a time- and concentration-dependent manner. Apoptosis induced by 1 was associated with activation of caspase-3, caspase-9, and PARP cleavage and was mediated by the mitochondrial pathway, as exemplified by mitochondrial depolarization, cytochrome c release, and downregulation of the antiapoptotic Bcl-2 protein. Xanthohumol induced intracellular reactive oxygen species (ROS), an effect that was reduced by pretreatment with the antioxidant N-acetyl-L-cysteine (NAC). Intracellular ROS production appeared essential for the activation of the mitochondrial pathway and induction of apoptosis after exposure to 1. Oxidative stress due to treatment with 1 was associated with MAPK activation, as determined by ERK1/2 and p38 phosphorylation. Phosphorylation of ERK1/2 and p38 was attenuated using NAC to inhibit ROS production. After treatment with 1, ROS provided a specific environment that resulted in MAPK-induced cell death, with this effect reduced by the ERK1/2 specific inhibitor PD98059 and partially inhibited by the p38 inhibitor SB203580. These findings suggest that xanthohumol (1) is a potential chemotherapeutic agent for the treatment of glioblastoma multiforme.

Triterpenoid constituents from the roots of Paeonia rockii ssp. rockii.

An investigation of a chloroform-soluble extract from the roots of Paeonia rockii ssp. rockii yielded three new noroleanane triterpenoids (1-3) together with 19 known compounds. Their structures were established by analysis of the spectroscopic data. The effects of this chloroform-soluble extract and its major constituents on cell proliferation and apoptosis of a panel of human cancer cell lines (melanoma M-14, colon cancer HT-29, breast cancer MCF-7) were evaluated by the MTT bioassay and propidium iodide staining, respectively, in comparison with normal human embryonic kidney cells (HEK-293). Two of the triterpenoids, betulinic acid (4) and oleanolic acid (5), and the crude extract were cytotoxic and induced apoptosis selectively in the M-14 melanoma cell line. This effect was reversed by the caspase-inhibitor z-VAD-fmk, suggesting that such action is mediated by caspase-3 activation.

Interconverting flavanone glucosides and other phenolic compounds in Lippia salviaefolia Cham. ethanol extracts.

Four interconverting flavanone glycosides [(2R)- and (2S)-3',4',5,6-tetrahydroxyflavanone 7-O-ß-D-glucopyranoside, and (2R)- and (2S)-3',4',5,8-tetrahydroxyflavanone 7-O-ß-D-glucopyranoside], in addition to eight known flavonoids [naringenin, asebogenin, sakuranetin, 6-hydroxyluteolin 7-O-ß-D-glucoside, (2R)- and (2S)-eriodictyol 7-O-ß-D-glucopyranoside, aromadendrin and phloretin], three phenylpropanoid glycosides [forsythoside B, alyssonoside and verbascoside] and the epoxylignan lariciresinol 4'-O-ß-D-glucopyranoside were isolated and identified in the EtOH extract of the aerial parts of Lippia salviaefolia Cham. The phytochemical study herein was guided by preliminary antioxidant tests, namely, ß-carotene protection and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity. The crude extracts, their active fractions and the isolated compounds were assayed against intracellular reactive oxygen species (ROS) and human embryonic kidney HEK-293 and human melanoma M14 cancer cell growth. Aromadendrin and phloretin were able to counteract elevation of ROS induced by the oxidant t-butylhydroperoxide (t-BOOH) in HEK-293 cells, whereas phloretin strongly protected HEK-293 cells from ROS damage at 1 µM. Additionally, phloretin exhibited a significant growth inhibitory effect at 20-40 µM in both HEK-293 and M14 cells and induced a concentration dependent apoptosis at 20 µM in M14 cells, suggesting a selective action towards malignant cells. Due to their equilibria, the four interconverting flavanone glycosides were studied using 1D and 2D NMR, HPLC-CD-PDA and HRMS analyses.

Neuroinflammation and ageing: current theories and an overview of the data.

The increase in the average lifespan and the consequent proportional growth of the elderly segment of society has furthered the interest in studying ageing processes. Ageing may be considered a multifactorial process derived from the interaction between genetic and environmental factors including lifestyle. There is ample evidence in many species that the maximum age attainable (maximum lifespan potential, MLSP) is genetically determined and several mitochondrial DNA polymorphisms are associated with longevity. This review will address the current understanding of the relationship between ageing and several factors both genetics and life style related. Firstly we focused on the most reliable and commonly shared theories which attempt to explain the phenomenon of ageing as the genetic, cellular, neuroendocrine, immunological and free-radicals related theories. Many studies have shown that most of the phenotypic characteristics observed in the aging process are the result of the occurrence, with age, of a low grade chronic pro-inflammatory status called "inflammaging", partially under genetic control. The term indicate that aging is accompanied by a low degree of chronic inflammatory, an up-regulation of inflammatory response and that inflammatory changes are common to many age-related diseases. In this review special attention was dedicated to diseases related to age as atherosclerosis, cancer and Alzheimer disease. Despite the fact that in recent years many theories about ageing have been developed, we are still far from a full understanding of the mechanisms underlying the ageing process.

Sesquiterpene coumarins from Ferula gumosa.

A new sesquiterpene coumarin, gumosin (1), two new sesquiterpene coumarin glycosides, gumosides A (2) and B (3), and 10 known compounds, namely, cauferoside (4), feselol (5), conferoside, ferilin, ferocaulidin, ligupersin A, conferol, and daucosterol, and the phenolic compounds acantrifoside E and 4-hydroxybenzoic acid 4-(6-O-sulfo)glucopyranoside, were isolated from a methanolic extract of Ferula gumosa roots. The structures of 1-3 were elucidated by spectroscopic data interpretation. The cytotoxic activity of the sesquiterpene coumarin derivatives was evaluated against a small panel of cancer cell lines.

Potential therapeutic effects of vitamin e and C on placental oxidative stress induced by nicotine: an in vitro evidence.

There have been a few studies that examined the oxidative stress effects of nicotine during pregnancy and lactation. The adverse effect of prenatal smoking exposure on human fetal development and growth has been a major public health issue. Active or passive smoking during pregnancy can result in a wide variety of adverse outcomes, including intrauterine growth retardation (IUGR), prematurity, stillbirth, and the sudden infant death syndrome. Smoking in pregnancy has also been associated with an increased risk of attention deficit and learning problems in childhood. Some studies argued that as a principal component of tobacco smoke, nicotine alone is responsible for the majority of negative reproductive outcomes. Nicotine and its major metabolite cotinine can cross the placental barrier. The level of nicotine in fetal tissues was found to be equal to or greater than the plasma nicotine level in the mothers. The oxidative stress induce by nicotine has been increasingly postulated as a major contributor to endothelial dysfunction. A large body of research has investigated the potential role of antioxidant nutrients in the prevention of endothelial dysfunction in women. Therefore, the present study was undertaken to assess the potential benefit of antioxidant supplementation on markers of placental oxidative stress in an in vitro model of endothelial dysfunction induced by nicotine, since it was previously found that nicotine is able to trigger the placental secretion of stress molecules. In this regard, we evaluated the effects of vitamin C, vitamin E and N-acetylcysteine (NAC), alone or in combination, in placental villi culture after exposure to nicotine. The effect of antioxidant nutrients on trophoblast cells proliferation and vitality was also evaluated. The results obtained suggest that in a patho-physiological condition, such as endothelial dysfunction induced by nicotine, the deleterious effect of reactive oxygen species may be counteracted by an antioxidant therapy, and there is the need to investigate the optimum dosing and timing of antioxidants administration, since an inappropriate antioxidant treatment in pregnant women may have deleterious consequences, reducing placental cells proliferation until to cell death.

IKK{gamma} protein is a target of BAG3 regulatory activity in human tumor growth.

BAG3, a member of the BAG family of heat shock protein (HSP) 70 cochaperones, is expressed in response to stressful stimuli in a number of normal cell types and constitutively in a variety of tumors, including pancreas carcinomas, lymphocytic and myeloblastic leukemias, and thyroid carcinomas. Down-regulation of BAG3 results in cell death, but the underlying molecular mechanisms are still elusive. Here, we investigated the molecular mechanism of BAG3-dependent survival in human osteosarcoma (SAOS-2) and melanoma (M14) cells. We show that bag3 overexpression in tumors promotes survival through the NF-kappaB pathway. Indeed, we demonstrate that BAG3 alters the interaction between HSP70 and IKKgamma, increasing availability of IKKgamma and protecting it from proteasome-dependent degradation; this, in turn, results in increased NF-kappaB activity and survival. These results identify bag3 as a potential target for anticancer therapies in those tumors in which this gene is constitutively expressed. As a proof of principle, we show that treatment of a mouse xenograft tumor model with bag3siRNA-adenovirus that down-regulates bag3 results in reduced tumor growth and increased animal survival.

The cyclin-dependent kinase inhibitor R-roscovitine down-regulates Mcl-1 to override pro-inflammatory signalling and drive neutrophil apoptosis.

Successful resolution of inflammation requires inflammatory cells such as neutrophils to undergo apoptosis prior to non-inflammatory phagocytosis by professional phagocytes. Recently, cyclin-dependent kinase (CDK) inhibitors (e.g. R-roscovitine) have been shown to induce neutrophil apoptosis and enhance the resolution of inflammation. Interestingly, NF-kappaB and MAPK pathways and key endogenous survival proteins (typified by Mcl-1) are involved in the regulation of neutrophil apoptosis and, in cancer-cell lines, have been implicated as possible targets of CDK inhibitors. Here, we demonstrate that R-roscovitine over-rides TNF-alpha and LPS-induced survival (determined by morphological examination and binding of fluorescently labelled annexin-V) of isolated peripheral blood neutrophils. This effect did not appear to be mediated via effects on early markers of neutrophil activation (e.g. surface marker expression, shape change, aggregation and superoxide anion generation), by direct inhibition of NF-kappaB activation (assessed by cytoplasmic IkappaBalpha proteolysis and NF-kappaB p65 subunit translocation) and ERK activation (determined by specific ERK phosphorylation) but due to down-regulation (at protein and mRNA level) of the survival protein Mcl-1 but not the pro-apoptotic bcl-2 homologue Bim. These findings suggest that key endogenous survival proteins may be the targets of CDK inhibitors and consequently may be of critical importance in the resolution of inflammation.

Saponins and polyphenols from Fadogia ancylantha (makoni tea).

Three new saponins (1-3) and a known saponin, together with four known polyphenolic compounds, have been isolated from the fermented and dried leaves of Fadogia ancylantha (Makoni tea). The structures of compounds 1-3 were established by analysis of their spectroscopic data. Both an ethanol-water extract of F. ancylantha and its phenolic constituents showed significant free-radical-scavenging and antimicrobial activities. No cytotoxicity, as evaluated by analysis of hypodiploid nuclei in HUVEC cells using propidium iodide staining, was observed for either the plant crude extract or its constituents.

R-roscovitine sensitizes anaplastic thyroid carcinoma cells to TRAIL-induced apoptosis via regulation of IKK/NF-kappaB pathway.

Among thyroid carcinomas, highly aggressive undifferentiated or anaplastic carcinomas still await effective therapeutic strategies. R-roscovitine is a novel cyclin-dependent kinase inhibitor in clinical trials as anti-cancer agent. We have investigated the effects of R-roscovitine on proliferation and apoptosis of 4 thyroid cancer cell lines with different degrees of malignancy. R-roscovitine induced cell cycle arrest in G2/M phase in all cells analyzed possibly by inhibiting the CDK1-cyclin B1 complex. However, the compound was unable to induce a significant cell apoptosis. R-roscovitine has been shown to sensitize cancer cells to TRAIL-induced apoptosis. We report that R-roscovitine sensitized thyroid cell lines to TRAIL-induced apoptosis with the highest degree of synergism observed in the most undifferentiated cancer cells. Apoptosis was associated with the activation of caspases. In thyroid cancers, NF-kappaB is constitutively activated contributing to the proliferation of malignant cells. Accordingly, we observed that R-roscovitine inhibited p65 expression and nuclear translocation. Moreover, IKKbeta over-expression inhibited R-roscovitine- and TRAIL-induced apoptosis. The combined treatment also caused down-regulation of anti-apoptotic proteins transcriptionally regulated by NF-kappaB. Finally, R-roscovitine up-regulated expression of DR5 TRAIL receptors. These results demonstrate that undifferentiated thyroid carcinoma cells can be effectively killed by a combination treatment of subtoxic doses of R-roscovitine and TRAIL. R-roscovitine sensitization of TRAIL-induced apoptosis appears to be mediated by the inhibition of the IKK/NF-KB pathway leading to down-regulation of anti-apoptotic genes and up-regulation of TRAIL death receptors. The combination of R-roscovitine and TRAIL may represent a novel approach to the treatment of anaplastic thyroid carcinomas resistant to conventional chemotherapy.

Molecular modelling studies, synthesis and biological activity of a series of novel bisnaphthalimides and their development as new DNA topoisomerase II inhibitors.

A series of bisnaphthalimide derivatives were synthesized and evaluated for growth-inhibitory property against HT-29 human colon carcinoma. The N,N'-bis[2-(5-nitro-1,3-dioxo-2,3-dihydro-1H-benz[de]-isoquinolin-2-yl)]propane-2-ethanediamine (9) and the N,N'-Bis[2-(5-nitro-1,3-dioxo-2,3-dihydro-1H-benz[de]-isoquinolin-2-yl)]butylaminoethyl]-2-propanediamine (12) derivatives emerged as the most potent compounds of this series. Molecular modelling studies indicated that the high potency of 12, the most cytotoxic compound of the whole series, could be due to larger number of intermolecular interactions and to the best position of the naphthalimido rings, which favours pi-pi stacking interactions with purine and pyrimidine bases in the DNA active site. Moreover, 12 was designed as a DNA topoisomerase II poison and biochemical studies showed its effect on human DNA topoisomerase II. We then selected the compounds with a significant cytotoxicity for apoptosis assay. Derivative 9 was able to induce significantly apoptosis (40%) at 0.1 microM concentration, and we demonstrated that the effect on apoptosis in HT-29 cells is mediated by caspases activation.