Shiow-Lin Pan

YC-1 inhibits HIF-1 expression in prostate cancer cells: Contribution of Akt/NF-κB signaling to HIF-1α accumulation during hypoxia

Hypoxia-inducible factor 1 (HIF-1), a transcription factor that is critical for tumor adaptation to microenvironmental stimuli, represents an attractive chemotherapeutic target. YC-1 is a novel antitumor agent that inhibits HIF-1 through previously unexplained mechanisms. In the present study, YC-1 was found to prevent HIF-1α and HIF-1β accumulation in response to hypoxia or mitogen treatment in PC-3 prostate cancer cells. Neither HIF-1α protein half-life nor mRNA level was affected by YC-1. However, YC-1 was found to suppress the PI3K/Akt/mTOR/4E-BP pathway, which serves to regulate HIF-1α expression at the translational step. We demonstrated that YC-1 also inhibited hypoxia-induced activation of nuclear factor (NF)-κB, a downstream target of Akt. Two modulators of the Akt/NF-κB pathway, caffeic acid phenethyl ester and evodiamine, were observed to decrease HIF-1α expression. Additionally, overexpression of NF-κB partly reversed the ability of wortmannin to inhibit HIF-1α-dependent transcriptional activity, suggesting that NF-κB contributes to Akt-mediated HIF-1α accumulation during hypoxia. Overall, we identify a potential molecular mechanism whereby YC-1 serves to reduce HIF-1 expression.

Antroquinonol displays anticancer potential against human hepatocellular carcinoma cells: A crucial role of AMPK and mTOR pathways

5AMP-activated protein kinase (AMPK) and the mammalian target of rapamycin (mTOR) are two serine/threonine protein kinases responsible for cellular energy homeostasis and translational control, respectively. Evidence suggests that these two kniases are potential targets for cancer chemotherapy against hepatocellular carcinoma (HCC). Antroquinonol that is isolated from Antrodia camphorate, a well-known Traditional Chinese Medicine for treatment of liver diseases, displayed effective anticancer activity against both HBV DNA-positive and -negative HCC cell lines. The rank order of potency against HCCs is HepG2>HepG2.2.15>Mahlavu>PLC/PRF/5>SK-Hep1>Hep3B. Antroquinonol completely abolished cell-cycle progression released from double-thymidine-block synchronization and caused a subsequent apoptosis. The data were supported by down-regulation and reduced nuclear translocation of G1-regulator proteins, including cyclin D1, cyclin E, Cdk4 and Cdk2. Further analysis showed that the mRNA expressions of the G1-regulator proteins were not modified by antroquinonol, indicating an inhibition of translational but not transcriptional levels. Antroquinonol induced the assembly of tuberous sclerosis complex (TSC)-1/TSC2, leading to the blockade of cellular protein synthesis through inhibition of protein phosphorylation including mTOR (Ser(2448)), p70(S6K) (Thr(421)/Ser(424) and Thr(389)) and 4E-BP1 (Thr(37)/Thr(46) and Thr(70)). Furthermore, the AMPK activity was elevated by antroquinonol. Compound C, a selective AMPK inhibitor, significantly reversed antroquinonol-mediated effects suggesting the crucial role of AMPK. Besides, the loss of mitochondrial membrane potential and depletion of mitochondrial content indicated the mitochondrial stress caused by antroquinonol. In summary, the data suggest that antroquinonol displays anticancer activity against HCCs through AMPK activation and inhibition of mTOR translational pathway, leading to G1 arrest of the cell-cycle and subsequent cell apoptosis.

Pharmacological evaluation of several major ingredients of Chinese herbal medicines in human hepatoma Hep3B cells

Long-dan-tan (Chinese name) is one of the most common herbal medicines used by Chinese people with chronic liver disease. Accumulated anecdotal evidence suggests that Long-dan-tan may show a beneficial effect in patients with hepatocellular carcinoma. Long-dan-tan is made from five plants: Gentiana root, Scutellaria root, Gardenia fruit, Alisma rhizome, and Bupleurum root. In this study, we have examined the cytotoxic effects of the five major ingredients isolated from the above plants, i.e. gentiopicroside, baicalein, geniposide, alisol B acetate and saikosaponin-d, respectively, on human hepatoma Hep3B cells. Annexin V immunofluorescence detection, DNA fragmentation assays and FACScan analysis of propidium iodide-staining cells showed that gentiopicroside, baicalein, and geniposide had little effect, whereas alisol B acetate and saikosaponin-d profoundly induced apoptosis in Hep3B cells. Alisol B acetate, but not saikosaponin-d, induced G2/M arrest of the cell cycle as well as a significant increase in caspase-3 activity. Interestingly, baicalein by itself induced an increase in H(2)O(2) generation and the subsequent NF-kappaB activation; furthermore, it effectively inhibited the transforming growth factor-beta(1) (TGF-beta(1))-induced caspase-3 activation and cell apoptosis. We suggest that alisol B acetate and saikosaponin-d induced cell apoptosis through the caspase-3-dependent and -independent pathways, respectively. Instead of inducing apoptosis, baicalein inhibits TGF-beta(1)-induced apoptosis via increase in cellular H(2)O(2) formation and NF-kappaB activation in human hepatoma Hep3B cells.

The HDAC inhibitor, MPT0E028, enhances erlotinib-induced cell death in EGFR-TKI-resistant NSCLC cells.

Epidermal growth factor receptor (EGFR), which promotes cell survival and division, is found at abnormally high levels on the surface of many cancer cell types, including many cases of non-small cell lung cancer. Erlotinib (Tarceva), an oral small-molecule tyrosine kinase inhibitor, is a so-called targeted drug that inhibits the tyrosine kinase domain of EGFR, and thus targets cancer cells with some specificity while doing less damage to normal cells. However, erlotinib resistance can occur, reducing the efficacy of this treatment. To develop more effective therapeutic interventions by overcoming this resistance problem, we combined the histone deacetylase inhibitor, MPT0E028, with erlotinib in an effort to increase their antitumor effects in erlotinib-resistant lung adenocarcinoma cells. This combined treatment yielded significant growth inhibition, induced the expression of apoptotic proteins (PARP, γH2AX, and caspase-3), increased the levels of acetylated histone H3, and showed synergistic effects in vitro and in vivo. These effects were independent of the mutation status of the genes encoding EGFR or K-Ras. MPT0E028 synergistically blocked key regulators of the EGFR/HER2 signaling pathways, attenuating multiple compensatory pathways (e.g., AKT, extracellular signal-regulated kinase, and c-MET). Our results indicate that this combination therapy might be a promising strategy for facilitating the effects of erlotinib monotherapy by activating various networks. Taken together, our data provide compelling evidence that MPT0E028 has the potential to improve the treatment of heterogeneous and drug-resistant tumors that cannot be controlled with single-target agents.

YC-1 induces apoptosis of human renal carcinoma A498 cells in vitro and in vivo through activation of the JNK pathway.

The aim of this study was to elucidate the mechanism of YC‐1{3‐(5′‐hydroxy methyl‐2′‐furyl)‐1‐benzylindazole}‐induced human renal carcinoma cells apoptosis and to evaluate the potency of YC‐1 in models of tumour growth in mice.

Orally active microtubule-targeting agent, MPT0B271, for the treatment of human non-small cell lung cancer, alone and in combination with erlotinib

Microtubule-binding agents, such as taxanes and vinca alkaloids, are used in the treatment of cancer. The limitations of these treatments, such as resistance to therapy and the need for intravenous administration, have encouraged the development of new agents. MPT0B271 (N-[1-(4-Methoxy-benzenesulfonyl)-2,3-dihydro-1H-indol-7-yl]-1-oxy-isonicotinamide), an orally active microtubule-targeting agent, is a completely synthetic compound that possesses potent anticancer effects in vitro and in vivo. Tubulin polymerization assay and immunofluorescence experiment showed that MPT0B271 caused depolymerization of tubulin at both molecular and cellular levels. MPT0B271 reduced cell growth and viability at nanomolar concentrations in numerous cancer cell lines, including a multidrug-resistant cancer cell line NCI/ADR-RES. Further studies indicated that MPT0B271 is not a substrate of P-glycoprotein (P-gp), as determined by flow cytometric analysis of rhodamine-123 (Rh-123) dye efflux and the calcein acetoxymethyl ester (calcein AM) assay. MPT0B271 also caused G2/M cell-cycle arrest, accompanied by the up-regulation of cyclin B1, p-Thr161 Cdc2/p34, serine/threonine kinases polo-like kinase 1, aurora kinase A and B and the downregulation of Cdc25C and p-Tyr15 Cdc2/p34 protein levels. The appearance of MPM2 and the nuclear translocation of cyclin B1 denoted M phase arrest in MPT0B271-treated cells. Moreover, MPT0B271 induced cell apoptosis in a concentration-dependent manner; it also reduced the expression of Bcl-2, Bcl-xL, and Mcl-1 and increased the cleavage of caspase-3 and -7 and poly (ADP-ribose) polymerase (PARP). Finally, this study demonstrated that MPT0B271 in combination with erlotinib significantly inhibits the growth of the human non-small cell lung cancer A549 cells as compared with erlotinib treatment alone, both in vitro and in vivo. These findings identify MPT0B271 as a promising new tubulin-binding compound for the treatment of various cancers.

Radiation-induced VEGF-C expression and endothelial cell proliferation in lung cancer@@@Strahleninduzierte VEGF-C-Expression und endotheliale Zellteilung bei Lungenkrebs

BackgroundThe present study was undertaken to investigate whether radiation induces the expression of vascular endothelial growth factor C (VEGF-C) through activation of the PI3K/Akt/mTOR pathway,subsequently affecting endothelial cells.Materials and methodsRadiotherapy-induced tumor micro-lymphatic vessel density (MLVD) was determined in a lung cancer xenograft model established in SCID mice. The protein expression and phosphorylation of members of the PI3K/Akt/mTOR pathway and VEGF-C secretion and mRNA expression in irradiated lung cancer cells were assessed by Western blot analysis, enzyme-linked immunosorbent assays (ELISAs), and reverse transcriptase–polymerase chain reaction (RT-PCR). Moreover, specific chemical inhibitors were used to evaluate the role of the PI3K/Akt/mTOR signaling pathway. Conditioned medium (CM) from irradiated control-siRNA or VEGF-C-siRNA-expressing A549 cells was used to evaluate the proliferation of endothelial cells by the MTT assay.ResultsRadiation increased VEGF-C expression in a dose-dependent manner over time at the protein but not at the mRNA level. Radiation also up-regulated the phosphorylation of Akt, mTOR, 4EBP, and eIF4E, but not of p70S6K. Radiation-induced VEGF-C expression was down-regulated by LY294002 and rapamycin (both pu2009<u20090.05). Furthermore, CM from irradiated A549 cells enhanced human umbilical vein endothelial cell (HUVEC) and lymphatic endothelial cell (LEC) proliferation, which was not observed with CM from irradiated VEGF-C-siRNA-expressing A549 cells.ConclusionsRadiation-induced activation of the PI3K/Akt/mTOR signaling pathway increases VEGF-C expression in lung cancer cells, thereby promoting endothelial cell proliferation.ZusammenfassungHintergrund und ZielDie vorliegende Studie untersucht, ob die Strahlung die Expression von VEGF-C (vascular endothelial growth factor C) mittels Aktivierung des PI3K/Akt/mTOR-Signalwegs induziert und anschließend die endothelialen Zellen beeinflusst.Methoden und MaterialDie durch Strahlentherapie induzierte Mikrolymphgefäßdichte (MLVD) im Tumor wurde in einem Lungenkrebs-Xenograftmodell mit SCID-Mäusen ermittelt. Die Proteinexpression, die Phosphorylierung der Mitglieder des PI3K/Akt/mTOR-Signalwegs und die Expressionder VEGF-C-Sekretion und mRNA-Expression in bestrahlten Lungenkrebszellen wurden jeweils mittels Westernblot-Analyse, ELISA (enzyme-linked immunosorbent assays) und RT-PCR (Reverse-Transkriptase-Polymerasekettenreaktion)bewertet. Außerdem wurden spezifische chemische Inhibitoren verwendet, um die Rolle des PI3K/Akt/mTOR-Signalwegs auszuwerten. Konditioniertes Medium (CM) aus bestrahlten Kontroll-siRNA-exprimierenden und ausunbestrahlten VEGF-C-siRNA-exprimierenden A549-Zellen wurde benutzt, um die Proliferation von Endothelzellen mittels MTT-Assay zu bewerten.ErgebnisseStrahlung erhöht die dosisabhängige und zeitabhängige VEGF-C-Expression auf Protein- anstatt auf mRNA-Ebene. Durch die Bestrahlung wurde die Phosphorylierung von Akt, mTOR, 4EBP und eIF4E hochreguliert, nicht jedoch von p70S6K. Die trahleninduzierte VEGF-C-Expression wurde durch LY294002 und Rapamycin herunterreguliert (beide pu2009<u20090,05). Des Weiteren fördert CM aus den unbestrahlten A549-Zellen die Proliferation der menschlichen Nabelvenenendothelzellen (HUVECs) und der lymphatischen Endothelzellen (LECs). CM aus den unbestrahlten und VEGF-C-iRNA-exprimierenden A549-Zellen hat jedoch keine proliferationsfördernde Wirkung.SchlussfolgerungenDie strahleninduzierte Aktivierung des PI3K/Akt/mTOR-Signalwegs kann die Expression des VEGF-C in Lungenkrebszellen erhöhen und die Proliferation der Endothelzellen fördern.

Abstract 4236: Moscatilin inhibits invasion by suppressing urokinase plasminogen activator expression through Akt inactivation in human hepatocellular carcinoma cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FLnnPurpose: Hepatocellular carcinoma (HCC) frequently shows early invasion into blood vessels as well as intrahepatic metastasis. The innovations of novel small-molecule agents to block HCC invasion and subsequent metastasis are urgently needed. Moscatilin is a bibenzyl derivative extracted from the stems of a traditional Chinese medicine, Orchid Dendrobrium loddigesii. Previous studies demonstrated that moscatilin exerted potent cytotoxic effects against numerous lines of cancer cells. Recently, moscatilin has been reported to suppress tumor angiogenesis. However, the anti-metastatic property of moscatilin has not been elucidated.nnMethods: Multiple molecular and pharmacological approaches such as invasion assay, MTT assay, ELISA assay, zymography, real-time PCR, Western blot, luciferase reporter gene assay, and chorioallantoic membrane (CAM) intravasation assay were used to determine the anti-metastatic effects of moscatilin in SK-Hep-1 cells, a highly metastatic human HCC cell line.nnResults: We found moscatilin inhibited cell invasion in a concentration-dependent manner in SK-Hep-1 cells in the absence of cytotoxicity. Moscatilin did not affect proteolytic activities of matrix metalloproteinase (MMP) -2 and MMP-9, but profoundly suppressed urokinase plasminogen activator (uPA) activity. We next found that moscatilin substantially inhibited protein expression of uPA but did not obviously impair uPAR and PAI-1 expression. To examine the upstream signaling of uPA suppression, the ERK1/2, Akt, mTOR, and NF-κB signaling pathways were evaluated in moscatilin-treated cells. We demonstrated that moscatilin dramatically inhibited Akt phosphorylation, but not ERK1/2 phosphorylation. Moscatilin did not reduce the phosphorylation of mTOR as well as of components of the translational machinery including p70S6K, 4E-BP1 and eIF4E. Additionally, IKKα/β phosphorylation, IκBα phosphorylation, p65 phosphorylation at Ser536, and κB-luciferase activity were not inhibited by moscatilin. However, Moscatilin repressed the expression of uPA mRNA, suggesting that moscatilin regulated the uPA production in transcriptional level. Transfection of constitutively active Akt (Myr-Akt) partly restored the moscatilin induced Akt inactivation. The inhibition of cell invasion, uPA activity and gene expression in response to moscatilin were also attenuated. Moreover, we found that moscatilin significantly inhibited cell metastasis using CAM intravasation assay, a well established in vivo model for metastasis.nnConclusions: This study provides evidence that moscatilin induces Akt inactivation and uPA suppression, which leads to inhibition of invasion and metastasis in human HCC cells. Our results suggest that moscatilin is a novel anti-metastatic agent, which has great potential for further development for treating cancer.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4236. doi:10.1158/1538-7445.AM2011-4236