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Acute myeloid leukemia (AML) is a heterogeneous disease with poor overall survival characterized by various genetic changes. The continuous activation of oncogenic pathways leads to the development of drug resistance and limits current therapeutic efficacy. Therefore, a multi-targeting inhibitor may overcome drug resistance observed in AML treatment. Recently, groups of flavonoids, such as flavones and flavonols, have been shown to inhibit a variety of kinase activities, which provides potential opportunities for further anticancer applications.In this study, we evaluated the anticancer effects of flavonoid compounds collected from our in-house library and investigated their potential anticancer mechanisms by targeting multiple kinases for inhibition in AML cells.The cytotoxic effect of the compounds was detected by cell viability assays. The kinase inhibitory activity of the selected compound was detected by kinase-based and cell-based assays. The binding conformation and interactions were investigated by molecular docking analysis. Flow cytometry was used to evaluate the cell cycle distribution and cell apoptosis. The protein and gene expression were estimated by western blotting and qPCR, respectively.In this study, an O-methylated flavonol (compound 11) was found to possess remarkable cytotoxic activity against AML cells compared to treatment in other cancer cell lines. The compound was demonstrated to act against multiple kinases, which play critical roles in survival signaling in AML, including FLT3, MNK2, RSK, DYRK2 and JAK2 with ICO-methylated flavonol, compound 11, can target multiple kinases, which may provide potential opportunities for the development of novel therapeutics for drug-resistant AMLs. This work provides a good starting point for further compound optimization.

A series of 1-aroylindoline-hydroxamic acids have been synthesized in the present study. The results of the biological evaluation led to the identification of compound 12 as dual HDAC6/HSP90 inhibitor. Compound 12 displayed striking inhibitory effects towards the HDAC6 isoform and HSP 90 protein with IC50 values of 1.15 nM (HDAC6) and 46.3 nM (HSP90). Compound 12 also exhibited 113, 139 and 246 fold higher selectivity for HDAC6 over HDAC 1, HDAC 3 and HDAC 8 isoforms and was endowed with significant cytotoxic effects with GI50 values ranging 1.04–1.61 μM against lung A549, colorectal HCT116, leukemia HL60, and EGFR T790M mutant lung H1975 cell lines. Another interesting finding of the study was substantial cytotoxic effects of compounds particularly against lung H1975 (NSCLC) cell lines with IC50 = 0.26 μM which may be mediated through HSP90 inhibition. Compound 8 as such was devoid of HDAC inhibitory activity.

A series of 1,4-naphthoquinones (10a-10q) were synthesized and evaluated for anticancer activity. Compound 10e was identified as an inhibitor of Itch, a HECT domain-E3 ligase. In an evaluation of in vivo efficacy, 10e exhibited remarkable anticancer activity with TGI values of 98.3% and 100% at 25 mg/kg and 50 mg/kg orally daily, respectively, against human RPMI-8226 multiple myeloma xenograft. Treatment with 10e also showed a decrease of Itch level in human RPMI-8226 multiple myeloma cells. Thus 10e is a lead compound for further development of inhibitors targeting E3 ligase for treatment of multiple myeloma.

A series of ring-opened dihydroxybenzamides have been designed and synthesized as heat shock protein 90 inhibitors. One of derivatives, compound 6b ((N-ethyl-2,4-dihydroxy-5-isopropyl-N-(pyridin-3-yl)benzamide)) demonstrated remarkable antiproliferative activity against in human KRAS mutant A549 and EGFR T790 M mutant H1975 lung cancer cell lines with GI50 values of 0.07 and 0.05 μM, respectively. It is also active against in other cancer cell lines, such as colorectal HCT116 (GI50 = 0.09 μM), liver Hep3B (GI50 = 0.20 μM) and breast MDA-MB-231 (GI50 = 0.09 μM), and shows no evidence of toxicity in normal cell line. Compound 6b has an IC50 of 110.18 nM in HSP90α inhibitory activity, slightly better than reference compound 1 (17-AAG, IC50 = 141.62 nM) and achieves the degradation of multiple HSP90 client proteins in a dose- and time-dependent manner and downstream signaling of Akt in a concentration- and time-dependent manner in the human A549 lung cancer cell line. In the Boyden chamber assay, compound 6b can efficiently inhibit the migration of A549 cells when compared to the reference compound 1. It also induce significant activity through the apoptotic pathway. Treatment with 6b showed no vision toxicity (IC50 > 10 μM) on 661w photoreceptor cells as compared to AUY922 (3a) with a 0.04 μM values of IC50 and has no effect in hERG test. In a bidirectional Caco-2 permeability assay, compound 6b was classified as a highly permeable compound which is not a substrate of efflux transporters. In a pharmacokinetic study in rats, 6b showed an F = 17.8% of oral bioavailability. The effect of metabolic stability of compound 6b in human hepatocytes showed a T1/2 of 67.59 min. Compound 6b (50 mg/kg, po, daily) exhibits antitumor activity with a 72% TGD (tumor growth delay) in human A549 lung xenograft. The combination of 6b and afatinib, orally administered, showed tumor growth suppression with 67.5% of TGI in lung H1975 xenograft model. Thus compound 6b is a lead compound for further development of potential agents to treat lung cancer.

Aberrant alteration of epigenetic information disturbs chromatin structure and gene function, thereby facilitating cancer development. Several drugs targeting histone deacetylases (HDACs), a group of epigenetic enzymes, have been approved for treating hematologic malignancies in the clinic. However, patients who suffer from solid tumors often respond poorly to these drugs. In this study, we report a selective entinostat derivative, MPT0L184, with potent cancer-killing activity in both cell-based and mouse xenograft models. A time-course analysis of cell-cycle progression revealed that MPT0L184 treatment elicited an early onset of mitosis but prevented the division of cells with duplicated chromosomes. We show that MPT0L184 possessed potent inhibitory activity toward HDAC1 and 2, and its HDAC-inhibitory activity was required for initiating premature mitotic signaling. HDAC inhibition by MPT0L184 reduced WEE1 expression at the transcription level. In addition, MPT0L184 treatment also downregulated ATR-mediated CHK1 phosphorylation independent of HDAC inhibition. Furthermore, gastric cancer cells resistant to HDAC inhibitors were vulnerable to MPT0L184. Taken together, our study discovers MPT0L184 as a novel HDAC inhibitor that can trigger premature mitosis and potentially counteract drug resistance of cancers.

Multitarget agents simultaneously trigger molecules in functionally complementary pathways, and are therefore considered to have potential in effectively treating Alzheimer's disease (AD), which has a complex pathogenetic mechanism. In this study, the HDAC inhibitor core is incorporated into the acetylcholine esterase (ACE) inhibitor acridine-derived moiety and resulted in compounds that exhibited higher class IIa HDAC (4, 5, 7, and 9)- and class IIb HDAC6-inhibiting activity when compared to the pan-HDAC inhibitor SAHA in clinical practice. One of these compounds, 11b, displayed greater selectivity toward HDAC6 than other isoform enzymes. In contrast, the activity of compound 6a was selective toward class IIa HDAC and HDAC6. These two compounds exhibited strong activity against Aβ-aggregation as well as significantly disrupted Aβ-oligomer. Additionally, 11b and 6a strongly inhibited AChE. These experimental findings demonstrate that compounds 11b and 6a are HDAC-Aβ-aggregation-AChE inhibitors. Notably, they can enhance neurite outgrowth, but with no significant neurotoxicity. Further biological evaluation revealed the various cellular effects of multitarget compounds 11b and 6a, which have the potential to treat AD.

This study describes the development of a series of 1-arylsulfonyl-6-(N-hydroxyacrylamide)tetrahydroquinolines, potent histone deacetylase (HDAC) inhibitors which are cytotoxic to PC-3 cells. (E)-N-hydroxy-3-(1-(4-methoxyphenylsulfonyl)-1,2,3,4-tetrahydroquinolin-6-yl)acrylamide (11) exhibits marked anti-HDAC and antiproliferative activity, and is slightly more effective than N1-hydroxy-N8-phenyloctanediamide (SAHA, Vorinostat, 1). In a xenograft tumor model, 11, at doses of 100 or 200 mg/kg orally, suppresses the growth of PC-3 cells and leads to tumor growth inhibition of 38.8% and 57.9%, respectively. Compound 11 is a lead compound for further development of potential prostate cancer inhibitors.

Vorinostat, which is an extensively studied inhibitor against histone deacetylase (HDAC), shows limited clinical activity to solid tumors. WJ35435, a new hybrid of vorinostat and DACA (a topoisomerase inhibitor) potently inhibited HDAC activity (in particular HDAC1 and HDAC6) in kinase assay and cell-based examination. The anti-HDAC effect was confirmed by the induction of histone H3 acetylation and phosphorylation, α-tubulin acetylation and γ-H2AX formation. WJ35435 showed better potency than vorinostat and DACA against PC-3 and DU-145, two human hormone-refractory metastatic prostate cancer (HRMPC) cell lines, but not benign prostate cells. WJ35435 at differential concentrations induced G1- or G2-phase arrest of the cell cycle in HRMPCs but not in benign prostate cells. WJ35435 induced the formation of topoisomerase I-DNA cleavable complexes but not type-IIα or -IIβ. Topoisomerase activity assay confirmed the selective inhibition of topoisomerase I. WJ35435 induced profound DNA damage using comet tailing assay. WJ35435 was less effective than camptothecin and etoposide in inducing the phosphorylation and activation of Chk1, Chk2 and RPA32 which were crucial coordinators in DNA repair pathway, indicating a low DNA repair activity to WJ35435 action. Furthermore, WJ35435 showed an in vivo antitumor activity. A synergistic apoptosis (combination index = 0.55) was obtained in combination between WJ35435 and MG-132 (a proteasome inhibitor). In summary, WJ35435 is a dual-targeted anticancer hybrid induces anti-HDAC and anti-topoisomerase I activities that cause DNA damage associated with a low DNA repair capability, and induce cell cycle arrest at G1- and G2-phase. Ultimately, WJ35435 inhibits cell proliferation and induces apoptosis of HRMPCs.

Denbinobin, a phenanthraquinone derivative, was shown to exert antitumor activities in several types of cancer cell lines. However, the precise mechanism underlying denbinobin-induced cell death remains unclear. In this study, we investigated the apoptotic signaling cascade elicited by denbinobin in human glioblastoma multiforme (GBM) cells. Denbinobin concentration-dependently caused a decrease in the cell viability of GBM cells. A flow cytometric analysis of propidium iodide (PI)-stained cells demonstrated that denbinobin induced GBM cell apoptosis. Denbinobin evoked caspase-3 activation and degradation of poly (ADP-ribose) polymerase (PARP) and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor that prevented denbinobin-induced cell death. In addition, denbinobin-induced cell death was diminished by the transfection of wild-type (WT) Akt or IκB kinase (IKK) into GBM cells. Denbinobin reduced IKK phosphorylation in a time-dependent manner, and denbinobin-dephosphorylated IKK was accompanied by a decrease in Akt phosphorylation. The phosphorylation status of forkhead in rhabdomyosarcoma (FKHR), a downstream signal molecule of Akt, was also diminished by the presence of denbinobin. Furthermore, transfection of GBM cells with WT IKKα markedly suppressed the decreases in Akt and FKHR phosphorylation caused by denbinobin. In contrast, transfection with WT IKKβ only slightly affected denbinobin's action against IKK, Akt, and FKHR. These results suggest that IKKα inactivation, followed by Akt and FKHR dephosphorylation and caspase-3 activation, contributes to denbinobin-induced GBM cell apoptosis.

In this study, we investigated the anticancer effect of protopine on human hormone-refractory prostate cancer (HRPC) cells. Protopine exhibited an anti-proliferative effect by induction of tubulin polymerization and mitotic arrest, which ultimately led to apoptotic cell death. The data suggest that protopine increased the activity of cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex and that contributed to cell apoptosis by modulating mitochondria-mediated signaling pathways, such as Bcl-2 phosphorylation and Mcl-1 down-regulation. In conclusion, the data suggest that protopine is a novel microtubule stabilizer with anticancer activity in HRPC cells through apoptotic pathway by modulating Cdk1 activity and Bcl-2 family of proteins.

The aberrant regulation of epigenetic systems including histone acetylation contributes to inappropriate gene expression in cancer cells. In this study, we investigated the antitumor effects of the novel histone deacetylase inhibitor (S)-HDAC42 in oral squamous cell carcinoma (OSCC) cells. The antiproliferative effect of (S)-HDAC42 was multifold higher than that of suberoylanilide hydroxamic acid in a panel of oral squamous carcinoma cell lines examined. (S)-HDAC42 mediated caspase-dependent apoptosis by targeting multiple signaling pathways relevant to cell cycle progression and survival. We demonstrated that (S)-HDAC42 downregulated the levels of phospho-Akt, cyclin D1, and cyclin-dependent kinase 6, accompanied by increased p27 and p21 expression. In addition, (S)-HDAC42 suppressed NF-κB signaling by blocking tumor necrosis factor-α-induced nuclear translocation, and activated reactive oxygen species generation. Finally, (S)-HDAC42 exhibited high potency in suppressing OSCC tumor growth in a Ca922 xenograft nude mouse model. Together, these findings underscore the translational value of (S)-HDAC42 in fostering new therapeutic strategies for OSCC.

Attacking angiogenesis is considered an effective strategy for controls the expansion and metastasis of tumors and other related-diseases. The aim of this study was to assess the effects of moscatilin, a bibenzyl derivative, on VEGF and bFGF-induced angiogenesis in cultured human umbilical vein endothelial cells (HUVECs) in vitro and in vivo. Moscatilin significantly inhibited growth of lung cancer cell line A549 (NSCLC) and suppressed growth factor-induced neovascularization. In addition, VEGF- and bFGF-induced cell proliferation, migration, and tube formation of HUVECs was markedly inhibited by moscatilin. Western blotting analysis of cell signaling molecules indicated that moscatilin inhibited ERK1/2, Akt, and eNOS signaling pathways in HUVECs. These results suggest that inhibition of angiogenesis by moscatilin may be a major mechanism in cancer therapy.

CHM-1 (2′-fluoro-6,7-methylenedioxy-2-phenyl-4-quinolone) has been identified as a potent antitumor agent in human hepatocellular carcinoma; however, its role in tumor angiogenesis is unclear. This study investigated the effects of CHM-1 and the mechanisms by which it exerts its antiangiogenic and vascular disrupting properties. Using a xenograft model antitumor assay, we found that CHM-1 significantly inhibits tumor growth and microvessel formation. Flow cytometry, immunofluorescence microscopy, and cell death enzyme-linked immunosorbent assay kit revealed that CHM-1 inhibits growth of human umbilical vein endothelial cells (HUVEC) by induction of apoptotic cell death in a concentration-dependent manner. CHM-1 also suppresses HUVEC migration and capillary-like tube formation. We were able to correlate CHM-1-induced apoptosis in HUVEC with the cleavage of procaspase-3, -7, and -8, as well as with the cleavage of poly(ADP-ribose) polymerase by Western blotting assay. Such sensitization was achieved through up-regulation of death receptor 5 (DR5) but not DR4 or Fas. CHM-1 was also capable of increasing the expression level of p53, and most importantly, the induction of DR5 by CHM-1 was abolished by p53 small interfering RNA. Taken together, the results of this study indicate that CHM-1 exhibits vascular targeting activity associated with the induction of DR5-mediated endothelial cell apoptosis through p53 up-regulation, which suggests its potential as an antivascular and antitumor therapeutic agent.

Inhibition of angiogenesis controls the expansion and metastasis of many solid tumors and other related-diseases. KS-5 (1,7-dihydroxy-3-methoxyacridone), is an inactive analogue of the substituted 1-hydroxy acridone antiviral class. This study aimed at studying the effects of KS-5 on bFGF-induced angiogenesis in cultured human umbilical vein endothelial cells (HUVECs) in vitro and in vivo. KS-5 inhibited bFGF (10 ng/ml)-induced cell proliferation in a concentration-dependent manner, but did not exhibit significant cytotoxic effect examined by LDH release assay. KS-5 inhibited bFGF-induced angiogenesis was associated with decreasing DNA synthesis as evaluated by BrdU incorporation assay, and abrogating endothelial cell ERK1/2 and Akt protein phosphorylation, the major signaling pathways involved in cellular processes of angiogenesis. In addition, KS-5 also inhibited bFGF-induced phosphorylation of mTOR and the major downstream effectors, eIF4E and p70(S6K). Moreover, bFGF-induced protein synthesis was also inhibited by KS-5. Most importantly, KS-5 treatment in nude mice inhibited in vivo angiogenesis as revealed by Matrigel implant assay. In conclusion, the present study suggests that KS-5 has potential anti-angiogenetic effect for cancer therapy and other angiogenesis-dependent diseases.

Clinical observations suggest that hepatocyte growth factor (HGF) can promote invasion and metastasis in hepatocellular carcinoma. In this study, we found that HGF-stimulated invasion of SK-Hep-1 cells, together with increased expression of matrix metalloproteinase (MMP)-9. CHM-1 was identified from 2-phenyl-4-quinolone derivatives to potently inhibit HGF-induced cell invasion, proteolytic activity, and expression of MMP-9. CHM-1 significantly inhibited tyrosine autophosphorylation of c-Met induced by HGF. CHM-1 also suppressed HGF-induced Akt phosphorylation, and NF-κB activation, the downstream regulators of HGF/c-Met signaling, resulting in the inhibition of MMP-9. Thus, we suggest that CHM-1 is a potential therapeutic agent against tumor invasion.

Cancer chemotherapeutic agents that interfere with tubulin/microtubule function are in extensive use. Quinolone is a common structure in alkaloids and its related components exhibit several pharmacological activities. In this study, we have identified the anticancer mechanisms of 2-phenyl-4-quinolone. 2-Phenyl-4-quinolone displayed anti-proliferative effect in several cancer types, including hormone-resistant prostate cancer PC-3, hepatocellular carcinoma Hep3B and HepG2, non-small cell lung cancer A549 and P-glycoprotein-rich breast cancer NCI/ADR-RES cells. The IC(50) values were 0.85, 1.81, 3.32, 0.90 and 1.53 microM, respectively. 2-Phenyl-4-quinolone caused G2/M arrest of the cell-cycle and a subsequent apoptosis. The turbidity assay showed an inhibitory effect on tubulin polymerization. After immunochemical examination, the data demonstrated that the microtubules were arranged irregularly into dipolarity showing prometaphase-like states. Furthermore, 2-Phenyl-4-quinolone induced the Mcl-1 cleavage, the phosphorylation of Bcl-2 and Bcl-xL (12-h treatment), and the caspase activation including caspase-8, -2 and -3 (24-h treatment). The exposure of cells to 2-phenyl-4-quinolone caused Cdk1 activation by several observations, namely (i) elevation of cyclin B1 expression, (ii) dephosphorylation on inhibitory Tyr-15 of Cdk1, and (iii) dephosphorylation on Ser-216 of Cdc25c. Moreover, a long-term treatment (36h) caused the release reaction and subsequent nuclear translocation of AIF. In summary, it is suggested that 2-phenyl-4-quinolone displays anticancer effect through the dysregulation of mitotic spindles and induction of mitotic arrest. Furthermore, participation of cell-cycle regulators, Bcl-2 family of proteins, activation of caspases and release of AIF may mutually cross-regulate the apoptotic signaling cascades induced by 2-phenyl-4-quinolone.

The marine organisms produce many metabolic substances with numerous pharmacological activities. It has been suggested that ilimaquinone, a metabolite of sea sponge, can induce vesiculation of the Golgi apparatus and display several biological activities, such as anti-human immunodeficiency virus, anti-inflammation as well as anti-microbial activities. In this study, the sulforhodamine B assays showed that ilimaquinone induced a concentration-dependent anti-proliferative effect in several types of cancer cell lines, including prostate cancer PC-3 and LNCaP, non-small cell lung cancer A549 and hepatocellular carcinoma Hep3B cells. The anticancer mechanism of ilimaquinone in the representative PC-3 cells was identified. Ilimaquinone induced a time-dependent increase of G 1 phase arrest and a subsequent increase of hypodiploid sub-G 1 phase (apoptosis) of the cell cycle. The arrest of the cell cycle was associated with a sustained high level of nuclear cyclin E but the absence of DNA synthesis by flow cytometric analysis, indicating an incomplete S phase. Although ilimaquinone-induced Golgi vesiculation, the data showed that the inhibition of cancer cell growth was not through the Golgi fragmentation. Several biological kinases and transcription factors were examined in this study. The data demonstrated that ilimaquinone did not activate extracellular signal-regulated kinase and phosphatidylinositol 3-kinase but induce the up-regulation and nuclear translocation of growth arrest and DNA damage inducible gene 153 (CHOP/GADD153). Furthermore, ilimaquinone-mediated anti-proliferative effect is significantly reduced in the antisense CHOP/GADD153-overexpressing cells. Ilimaquinone also inhibited DNA binding of NF-κB; however, this inhibitory effect could not explain ilimaquinone-induced anticancer effect. In summary, it is suggested that ilimaquinone induces the anti-proliferative effect through the G 1 arrest of the cell cycle and the up-regulation and nuclear translocation of CHOP/GADD153.

Cyclopentenone prostaglandins (PGs) such as PGA1, PGA2 and delta12-PGJ2 have been shown to suppress tumor cell growth and to induce apoptosis in prostate cancer cells. Bromovulone III, which is isolated from the soft coral Clavularia viridis, is a cyclopentenone prostanoid. In this study, the anti-tumor activity as well as action mechanism of bromovulone III was identified in prostate cancer cells. Bromovulone III displayed anti-tumor activity of 30 to 100 times more effective than PGA1, PGA2 and delta12-PGJ2 in PC-3 cells. Several targets of caspases and Bcl-2 family of proteins were detected and the data demonstrated that bromovulone III induced the activation of caspase-8, -9 and -3, and Bid cleavage in which the caspase-8 activation occurred the first. Bromovulone III did not modify the protein levels of death receptors and ligands. Of note, the Fas clustering in PC-3 cells responsive to bromovulone III was observed by confocal immunofluorescence microscopy suggesting the involvement of Fas-mediated pathway. Bromovulone III also induced the cleavage of Mcl-1 in this study. The cleavage fragments (24, 19 and 17 kDa) may partly share the apoptotic insult. Although it has been suggested that Fas-mediated signaling may contribute to the caspase-8 activation induced by DNA-damaging agents; however, bromovulone III did not induce any DNA breakage, suggesting that bromovulone III-induced Fas/caspase-8-dependent signaling is not through the direct target on DNA damage. In summary, the data suggest that bromovulone III causes a rapid redistribution and clustering of Fas in PC-3 cells. Subsequently, the Fas event causes the activation and interaction of caspase-8/Bid/caspase-9 signaling cascades, and the activation of executor caspase-3.

Background/Aims Hepatocellular carcinoma is a very common malignancy and is highly chemoresistant to currently available chemotherapeutic agents. We isolated a marine prostanoid, bromovulone III, from soft coral Clavularia viridis and found that it displayed effective anti-tumor activity in human hepatocellular carcinoma. The anti-tumor mechanism has been delineated in this study. Methods Anti-tumor efficacy and apoptotic cell death were examined by sulforhodamine B and Hoechst 33342 assays. Rhodamine 123 was used to measure the change of mitochondrial membrane potential. Immunoprecipitation and Western blotting detect the involvement of several apoptosis-related proteins. Electron microscopic examination detects the morphological change of mitochondria and endoplasmic reticulum (ER). Results Bromovulone III primarily induced mitochondria-related activation of caspase-9 and -3 in several tumor types, such as prostate cancer PC-3 and acute promyelocytic leukemia HL-60 cells. However, it primarily induced the activation of m -calpain, caspase-12, and transcription factor CHOP/GADD153 in hepatocellular carcinoma Hep3B cells, suggesting the involvement of ER stress. Furthermore, a secondary mitochondrial swelling and depolarization of mitochondrial membrane potential were subsequently triggered after ER stress, suggesting the crosstalk between ER and mitochondria. Conclusions It is suggested that bromovulone III induces apoptosis in Hep3B cells through a mechanism that induces ER stress and leads to activation of CHOP/GADD153 and caspase-12.

Genistein, a natural isoflavone phytoestrogen present in soybeans, has been extensively studied as a chemopreventive or therapeutic agent in several types of cancer. The traditional Asian diet is rich in soy products may explain in part why the incidence of breast cancer in Asian women is relatively low. To improve therapeutic benefits, we investigated the combination of genistein with chemotherapeutic agents in phenotypically dissimilar human breast cancer cells, MCF-7 and MDA-MB-231, in which estrogen receptor expression is positive and negative, respectively. In the present study, genistein significantly decreased cell apoptosis induced by tubulin-binding agents, paclitaxel and vincristine. FACScan analysis revealed that genistein also diminished the accumulation of the G2/M phase in the cell cycle caused by tubulin-binding agents. In situ staining of microtubules revealed that genistein could decrease paclitaxel-induced tubulin polymerization. However, in vivo tubulin polymerization assay revealed that simultaneous treatment of genistein did not change the tubulin/microtubule dynamic. Genistein reduced Bcl-2 phosphorylation triggered by paclitaxel and vincristine without changing Bax protein expression. p53 and p21 expression, monitored by Western blotting, was not altered by genistein. However, the expression of cyclin B1 and CDC2 kinase was markedly decreased in combination with genistein. In conclusion, genistein inversely affected tubulin-binding agent-induced apoptosis via down-regulation of cyclin B1/CDC2 kinase expression resulting in reduced Bcl-2 phosphorylation.

The proliferation of vascular smooth muscle cells (VSMCs) induced by injury to the intima of arteries is an important etiologic factor in vascular proliferative disorders such as atherosclerosis and restenosis. Esculetin, derived from the Chinese herb Artemisia scoparia , is well known as a lipoxygenase inhibitor. We have investigated the inhibitory effects of esculetin on VSMC proliferation and intimal hyperplasia by balloon angioplasty in the rat. We determined, using [ 3 H ]thymidine incorporation and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, that esculetin inhibited the proliferation of VSMCs via a lipoxygenase-independent pathway. Three predominant signaling pathways were identified to be inhibited by esculetin: (a) the activation of p42/44 mitogen-activated protein kinase (MAPK) and the downstream effectors of c- fos and c- jun immediate early genes by means of western and reverse transcription–polymerase chain reaction (RT–PCR) analyses; (b) the activation of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), using the electrophoretic mobility shift assay; and (c) the activation of phosphoinositide 3-kinase (PI 3-kinase) and cell cycle progression, by western blot analysis and flow cytometric detection. Furthermore, esculetin also profoundly inhibited Ras activation, a shared upstream event of the above signaling cascades. In vascular injury studies, intraperitoneal administration of esculetin significantly suppressed intimal hyperplasia induced by balloon angioplasty. We conclude that esculetin blocks cell proliferation via the inhibition of an upstream effector of Ras and downstream events including p42/44 MAPK activation, PI 3-kinase activation, immediate early gene expression, as well as NF-κB and AP-1 activation. It also inhibits intimal hyperplasia after balloon vascular injury in the rat, indicating the therapeutic potential for treating restenosis after arterial injury.

In the present study, we investigated the effects and mechanisms of a novel potent antioxidant, octyl caffeate, on the induction of iNOS expression by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) in cultured primary rat aortic smooth muscle cells (RASMCs) in vitro and LPS-induced hypotension in vivo. Octyl caffeate (0.1-1.0 microM) exerted a concentration-dependent inhibition of iron-catalyzed lipid peroxidation in rat brain homogenates. Furthermore, octyl caffeate (20, 50, and 100 microM) concentration-dependently diminished the initial rate of superoxide-induced NBT reduction and the enzymatic activity of xanthine oxidase. It also concentration-dependently (1-50 microM) inhibited the NO production, iNOS protein and messenger RNA expressions upon stimulation by LPS (100 microg/mL)/IFN-gamma (100U/mL) in RASMCs. In addition, we found that octyl caffeate did not significantly affect IkappaBalpha degradation stimulated by LPS/IFN-gamma in RASMCs. On the other hand, octyl caffeate (10 and 50 microM) significantly suppressed activation of c-Jun-N-terminal kinase and extracellular signal-regulated kinase. Moreover, octyl caffeate (10mg/kg, i.v.) significantly inhibited the fall in mean arterial pressure stimulated by LPS (7.5mg/kg) in rats. In conclusion, we demonstrate that a novel potent antioxidant, octyl caffeate, significantly ameliorates circulatory failure of endotoxemia in vivo by a mechanism involving suppression of iNOS expression through inactivation of mitogen-activated protein kinases in RASMCs.

Oxidized low-density lipoprotein (OX-LDL) contributes significantly to the development of atherosclerosis. However, the mechanisms of OX-LDL-induced vascular smooth muscle cell (VSMC) proliferation are not completely understood. Therefore, we investigated the effect of OX-LDL on cell proliferation associated with a specific pattern of mitogen-activated protein kinase (MAPK) by [ 3 H]thymidine incorporation and p42/p44 MAPK phosphorylation in canine cultured VSMCs. OX-LDL-induced [ 3 H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in VSMCs. Pretreatment of these cells with pertussis toxin (PTX) for 24 hours attenuated the OX-LDL-induced [ 3 H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating that these responses were mediated through a receptor coupled to a PTX-sensitive G protein. In cells pretreated with PMA for 24 h and with either the PKC inhibitor staurosporine or the tyrosine kinase inhibitor genistein for 1h, substantially reduced the [ 3 H]thymidine incorporation and p42/p44 MAPK phosphorylation in response to OX-LDL. Removal of Ca 2+ by addition of BAPTA/AM plus EGTA significantly inhibited OX-LDL-induced [ 3 H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating the requirement of Ca 2+ for these responses. OX-LDL-induced [ 3 H]thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK). Furthermore, we also showed that overexpression of dominant negative mutants of Ras (RasN17) and Raf (Raf-301) completely suppressed MEK1/2 and p42/p44 MAPK activation induced by OX-LDL and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. Taken together, these results suggest that the mitogenic effect of OX-LDL is mediated through a PTX-sensitive G-protein-coupled receptor that involves the activation o Ras/Raf/MEK/MAPK pathway similar to those of PDGF-BB in canine cultured VSMCs.

Y. Chen, S. L. Pan, J. C. Wang, C. H. Chou, S. H. Kuo, J. C. H. Cheng, C. M. Teng Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan, Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, Graduate Institute of Clinical Medicine, National Taiwan University College of Medicine, Taipei, Taiwan, Department of Internal Medicine, National Taiwan University College of Medicine, Taipei, Taiwan