sub-G1 Fraction Research Articles

84 COMBINATION THERAPY WITH 17-ALLYLAMINO-17-DEMETHOXYGELDANAMYCIN AND RITONAVIR INHIBITS EXPRESSION OF HEAT SHOCK FACTOR 1 IN RENAL CANCER CELLS

You have accessJournal of UrologyKidney Cancer: Basic Research I1 Apr 201084 COMBINATION THERAPY WITH 17-ALLYLAMINO-17-DEMETHOXYGELDANAMYCIN AND RITONAVIR INHIBITS EXPRESSION OF HEAT SHOCK FACTOR 1 IN RENAL CANCER CELLS Akinori Sato, Takako Asano, Junichi Asakuma, Keiichi Ito, Akio Horiguchi, Makoto Sumitomo, and Tomohiko Asano Akinori SatoAkinori Sato More articles by this author , Takako AsanoTakako Asano More articles by this author , Junichi AsakumaJunichi Asakuma More articles by this author , Keiichi ItoKeiichi Ito More articles by this author , Akio HoriguchiAkio Horiguchi More articles by this author , Makoto SumitomoMakoto Sumitomo More articles by this author , and Tomohiko AsanoTomohiko Asano More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.132AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Phase II clinical trials have shown 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of heat shock protein (HSP) 90, to be only a moderately effective anticancer agent because it generally increases the expression of HSPs. In the present study we combined 17-AAG with the protease inhibitor ritonavir and found that this combination therapy increases the effectiveness of 17-AAG against renal cancer cells by decreasing the expression of heat shock factor (HSF)-1, a transcription factor of HSPs. METHODS Renal cancer cells (769P, A498, ACHN, Caki-1) and renal proximal tubule epithelial cells (RPTEC) were treated with 17-AAG (0-1 μM) alone and in combination with ritonavir (0-50 μM). Cell viability and clonogenicity were assessed by MTS assay and colony formation assay, changes in cell cycle were evaluated by flow cytometry, and the expression of HSPs 27, 70, and 90; HSF-1; phosphorylated retinoblastoma protein (Rb); cyclin D1; cyclin-dependent kinase 4 (CDK4); histone deacetylases (HDACs) 1, 2, 3, and 6; acetylated histone; X-linked inhibitor of apoptosis (XIAP); and survivin was assessed by Western blot analysis. Apoptosis was assayed using flow cytometry and detecting active caspase 3 and cleaved poly(ADP-ribose) polymerase (PARP). RESULTS Combinations of 17-AAG and ritonavir inhibited the proliferation of renal cancer cells synergistically in a time- and dose-dependent manner and suppressed colony formation. They increased the sub-G1 fraction to as much as 64.2%, decreased the expression of survivin and XIAP, and increased active caspase 3 and cleaved PARP, thus leading to apoptosis. They inhibited the proliferation of RPTEC less than that of renal cancer cells and caused only small changes in the cell cycle of RPTEC. The combination caused Rb dephosphorylation and suppressed the expression of CDK4; cyclin D1; and HDACs 1, 2, and 6 in all the cancer cell lines and also induced histone acetylation in Caki-1 cells. Expression of HSPs 27, 70, and 90 was increased by 17-AAG alone but reduced by 17-AAG combined with ritonavir. Interestingly, the combination therapy decreased the expression of HSF-1, which may be one of the mechanisms of the combined effect. CONCLUSIONS Combination therapy with 17-AAG and ritonavir might be effective for treating renal cancer because ritonavir decreases the expression of HSF-1 and thereby suppresses the 17-AAG-induced increase of HSPs. Tokorozawa, Japan© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e35 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Akinori Sato More articles by this author Takako Asano More articles by this author Junichi Asakuma More articles by this author Keiichi Ito More articles by this author Akio Horiguchi More articles by this author Makoto Sumitomo More articles by this author Tomohiko Asano More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

1151 DIRECT ANTITUMOR EFFECT OF BACILLUS CALMETTE-GUERIN IN HIGH GRADE UROTHELIAL CANCER CELLS WHICH EXPRESS VLA5 (ALPHA 5 BETA 1 INTEGRIN)

You have accessJournal of UrologyBladder Cancer: Basic Research III1 Apr 20101151 DIRECT ANTITUMOR EFFECT OF BACILLUS CALMETTE-GUERIN IN HIGH GRADE UROTHELIAL CANCER CELLS WHICH EXPRESS VLA5 (ALPHA 5 BETA 1 INTEGRIN) Tomoyuki Kato, Mayu Yagi, Atsushi Yamagishi, Noriyuki Hosoya, Toshihiko Sakurai, Hayato Nishida, Sei Naito, Masaaki Tsukigi, Hisashi Kawazoe, Akinori Muto, Akira Nagaoka, and Yoshihiko Tomita Tomoyuki KatoTomoyuki Kato More articles by this author , Mayu YagiMayu Yagi More articles by this author , Atsushi YamagishiAtsushi Yamagishi More articles by this author , Noriyuki HosoyaNoriyuki Hosoya More articles by this author , Toshihiko SakuraiToshihiko Sakurai More articles by this author , Hayato NishidaHayato Nishida More articles by this author , Sei NaitoSei Naito More articles by this author , Masaaki TsukigiMasaaki Tsukigi More articles by this author , Hisashi KawazoeHisashi Kawazoe More articles by this author , Akinori MutoAkinori Muto More articles by this author , Akira NagaokaAkira Nagaoka More articles by this author , and Yoshihiko TomitaYoshihiko Tomita More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.650AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Intravesical instillation of Bacillus Calmette-Guerin (BCG) is an effective treatment for superficial high grade bladder cancer. Although both immunological and direct effects on tumor cells have been proposed, the complete mechanisms for its effect are not fully understood. Presence of fibronectin has been shown to be necessary for binding and internalization of BCG into tumor cells to exert direct effect on them. We have previously demonstrated the correlation between high expression of fibronectin receptor VLA5 (alpha 5 beta 1 integrin) and high malignant potential of urothelial cancer (UC). Here, we assessed the direct antitumor effects of BCG for UC cell lines which beard various amount of VLA5 on the cell surface. METHODS The human UC cell lines (T24, HT1376 and RT4) and Tokyo 172 BCG strain (Immunobladder, Japan BCG Laboratory), BCG cell wall skeleton (BCG-CWS SMP105, Dainippon Sumitomo Pharma) were used in this study. Viability of cell lines was evaluated with crystal violet stainig assay. Fluorescence of apoptotic nuclei stained with Propidium Iodide (PI) was measured with a FACScan. DNA synthesis was evaluated with BrDU incorporation assay. RESULTS First, expression of VLA5 on the surface of cell lines was checked. T24 and HT1376, high grade UC, beard large amount of VLA5 on the cell surface and RT4, a low grade UC, had traces of VLA5. Treatment with BCG resulted in dose dependent decrease of relative viability in T24 (p<0.0001) and HT1376 (p<0.0001). This effect was partially reversed by coculturing cells with VLA5 blocking antibody. In RT4, viability was not altered by treatment with BCG. Sub-G1 fraction of PI stained T24 increased from less than 1% (untreated) to more than 4% (1 mg/ml of BCG) after exposure to BCG and it suppressed BrDU incorporation (p=0.004) in T24 cells. Autoclaved killed BCG also demonstrated cell growth retardation effect in T24 (p<0.0001) and HT1376 (p<0.0001). BCG-CWS SMP105 also delayed T24 and HT1376 cell growth (p<0.0001). CONCLUSIONS These findings suggest that BCG exerts direct antitumor effect on UC cells via VLA5 with induction of apoptosis and inhibition of DNA synthesis. Moreover, killed BCG and BCG cell wall skeleton were able to exert similar effect making this approach attractive for clinical application where potentially serious adverse effect of viable BCG can be avoided while maintaining antitumor effect using killed BCG or cell extracts. Yamagata, Japan© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e445-e446 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Tomoyuki Kato More articles by this author Mayu Yagi More articles by this author Atsushi Yamagishi More articles by this author Noriyuki Hosoya More articles by this author Toshihiko Sakurai More articles by this author Hayato Nishida More articles by this author Sei Naito More articles by this author Masaaki Tsukigi More articles by this author Hisashi Kawazoe More articles by this author Akinori Muto More articles by this author Akira Nagaoka More articles by this author Yoshihiko Tomita More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Potent anti-tumor activity of a macrocycle-quinoxalinone class pan-Cdk inhibitor in vitro and in vivo

Deregulation of cell-cycle control is a hallmark of cancer. Thus, cyclin-dependent kinases (Cdks) are an attractive target for the development of anti-cancer drugs. Here, we report the biological characterization of a highly potent pan-Cdk inhibitor with a macrocycle-quinoxalinone structure. Compound M inhibited Cdk1, 2, 4, 5, 6, and 9 with equal potency in the nM range and was selective against kinases other than Cdks. This compound inhibited multiple events in the cell cycle in vitro, including retinoblastoma protein (pRb) phosphorylation, E2F-dependent transcription, DNA replication (determined by bromodeoxyuridine incorporation), and mitosis completion (assayed by flow cytometry) in the 10 nM range. Moreover, this compound induced cell death, as determined by induction of the subG1 fraction, activated caspase-3, and anexin V. In vivo, Compound M showed anti-tumor efficacy at a tolerated dose. In a nude rat xenograft tumor model, an 8-h constant infusion of Compound M inhibited pRb phosphorylation and induced apoptosis in tumor cells at ~ 30 nM, which led to the inhibition of tumor growth. Immunosuppression was the only liability observed at this dose, but immune function returned to normal after 10 days. Suppression of pRb phosphorylation in tumor cells was clearly correlated with tumor cell growth inhibition and cell death in vitro and in vivo. In vivo, Compound M inhibited pRb phosphorylation in both tumor and gut crypt cells. Rb phosphorylation may be a suitable pharmacodynamic biomarker in both tumors and normal tissues for monitoring target engagement and predicting the efficacy of Compound M.

Abstract B226: Induction of growth arrest DNA damage-inducible gene 45 beta (GADD45b) expression contributes to sorafenib-induced apoptosis in hepatocellular carcinoma (HCC) cells

Abstract GADD45b, which is associated with cellular stress response and apoptosis regulation, was frequently under-expressed in HCC. In a previous study we found GADD45b belongs to a group of sorafenib-regulated genes that are independent of mitogen-activated protein kinase-extracellular signal-regulated kinase (MEK)/ extracellular signal-regulated kinase (ERK) signaling pathway. The present study explored the role of sorafenib-induced GADD45b expression in HCC cells. HCC cell lines tested included HepG2, Hep3B, Huh-7, and Huh-7R, a sorafenib-resistant cell line established by continuously exposure of Huh-7 cells to sorafenib. GADD45b expression was examined by quantitative real-time PCR and western blotting. Control of GADD45b transcription was examined by luciferase reporter assay using a series of reporter plasmids with deletions or site-directed mutants of the 5′-flanking region of GADD45b promoter. Apoptosis was analyzed by measuring the subG1 fraction and annexin V staining using flow cytometry. Both the mRNA and the protein levels of GADD45b was increased after sorafenib but not after the MEK inhibitor CI-1040 or U0126 treatment, suggesting that GADD45b induction was independent of cellular MEK/ERK activity. GADD45b induction was more prominent in sorafenib-sensitive HCC cells (Huh-7 and HepG2, IC50 6–7 µM) but less so prominent in sorafenib-resistant HCC cells (Hep3B and Huh-7R, IC50 12–15 µM). Knockdown of GADD45b expression by siRNA partially abrogated the pro-apoptotic effects of sorafenib in Huh-7 and HepG2 cells but not Hep3B cells. The region −339/−267 in the 5′-flanking region of GADD45b promoter, which contained AP-1 (−298/−292) and Sp1 (−285/−277) binding sites, was found crucial for GADD45b induction by sorafenib. Binding of c-Jun and Sp1 to GADD45b promoter was confirmed by chromatin immunoprecipitation. Sorafenib also increased the phosphorylation of JNK in sorafenib-sensitive but not in sorafenib-resistant HCC cells. Sorafenib-induced GADD45b expression can be alleviated by treatment with the specific JNK inhibitor SP600125. Our data indicated that induction of GADD45b expression, which is mediated by cellular JNK/c-Jun signaling pathway but not MEK/ERK signaling, contributes to sorafenib-induced apoptosis in HCC cells. Failure of GADD45b induction may confer sorafenib resistance. GADD45b may be used as a predictive biomarker for sorafenib sensitivity in HCC. (Supported by grants NSC97-3112-B-002-012, NSC98-3112-B-002-007, NSC98-3112-B-002-037) Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B226.

Abstract C189: Active compound isolated from human urine induced differentiation and apoptosis in K562 chronic myelogenous leukemia cells and enhanced the effects of imatinib

Abstract Leukemia is a serious disease that affects both children and adults. Although all-trans retinoic acid (ATRA) and targeted drug such as imatinib could effectively treat acute promyelocytic (APL) and chronic myelogenous leukemia (CML), respectively, rare therapeutics is available when drug resistance or recurrence occurred. Searching for alternative agent for refractory patient is urgently needed. Our previous studies had found that a human urine extract CDA-2 could induce differentiation of HL60 APL cells as well as potentiate the effects of ATRA and imatinib on HL60 and K562 CML cells, respectively. Purification of active compound possessing these anti-leukemic effects from CDA-2 was thus performed. Recently, an active compound named as P18.9 was isolated. It induced differentiation of K562 CML cells as evidenced by the nitroblue-tetrazolium (NBT) reduction test. At higher dose, flow cytometry analysis showed that P18.9 induced sub-G1 apoptotic fraction of K562 cells and enhanced the sub-G1 fraction induced by imatinib at a scale of about two folds. Although P18.9 did not induce differentiation of HL60 APL cells, it substantially enhanced the ATRA-induced differentiation. This is the first study to isolate an active compound from human urine, which exerts anti-leukemic effects alone or cooperatively with clinical used drugs. The chemical structure of P18.9 had been identified and further subsequent structure modification is ongoing. Continuing the effort to develop P18.9 and its derivatives as novel therapeutics for refractory APL or CML patients is warranted. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C189.

Abstract C108: A novel mitotic kinesin Eg5 inhibitor exerts the growth-inhibitory effect in cancer cells in a manner independent of induction of monoastral formation but dependent of KRAS mutation status

Abstract Mitotic kinesin Eg5 plays an important role in mitosis, as it is critical for proper bipolar spindle assembly. After the discovery of the first Eg5 inhibitor monastrol, a number of Eg5 inhibitors have been developed as anticancer drugs. We have previously synthesized a series of S-trityl-L-cysteine (STLC) derivatives as Eg5 inhibitors and demonstrated potent inhibitory effect on Eg5 ATPase activity and induction of mitotic arrest with characteristic monoastral spindles in HeLa cells. In this study, we evaluated the growth-inhibitory acitivities of novel STLC derivatives, Compound1, Compound2, and Compound3 by MTT assay in a wide range of human cancer cell lines derived from gastric (MKN1, MKN45, MKN74, NUGC3, NUGC4, NCI-N87), colon (C170, DLD1, HCT15, COLO205), pancreatic (AsPC1, BxPC3, SUIT2) and lung (PC-9, PC-14, H1975, H441, A549) cancers and investigated possible predictive biomarkers. Among the derivatives, Compound3 showed the most potent growth-inhibitory effect and sensitivity between cell lines showed marked differences, with IC50 values ranging from 0.134 to 7.14 M. Pancreatic cancer cell line AsPC1 and non-small cell lung cancer (NSCLC) cell lines H441 and A549 which harbor KRAS mutation showed relatively high IC50 values compared to other cell lines without KRAS mutation. Compound3 induced G2/M arrest in all cell lines as early as 8 hours after the treatment by flowcytometry, while the increase of the apoptotic sub-G1 fraction was not observed in these AsPC1, H441 and A549 cells harboring KRAS mutation. These results suggested that differential sensitivity to Compound3 and attenuated induction of apoptosis by Compound3 were attributed to KRAS mutation. Induction of monoastral spindles formation by Compound3 was observed as early as 8 hours after the treatment not only in sensitive cell lines but in refractory cell lines such as AsPC1 and H441 cells by immunocytochemistry, suggesting the induction of monoastral spindles formation does not work as a predictive biomarker for clinical development. There was no correlation observed between the growth-inhibitory effect and the basal Eg5 expression level of each cell line by western blot analysis. No significant difference was observed in the induction of a spindle checkpoint kinase BubR1 phosphorylation among the cells, a possible predictive biomarker in response to the Eg5 inhibitor treatment by western blot analysis. Taken together, a novel mitotic kinesin Eg5 inhibitor Compound3 should be considered for further exploration and development and KRAS mutation status was suggested to be a candidate for a predictive biomarker of poor response to Eg5 inhibitors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C108.

Abstract B97: Dichloroacetate (DCA) enhances activities of sorafenib against hepatocellular carcinoma (HCC) via modulation of aberrant cellular metabolism of HCC cells

Abstract To meet high energy demand for proliferation, cancer cells rely more on aerobic glycolysis than on oxidative phosphorylation (OXPHOS). This aberrant cellular metabolism of cancer, well-known as the “Warburg effect”, may confer survival advantages and drug resistance to cancer cells. Pyruvate dehydrogenase (PDH) is the gatekeeper of OXPHOS, and is inhibited by pyruvate dehyrogenase kinase (PDK) through phosphorylation. Dichloroacetate (DCA), a synthetic PDK inhibitor, can reverse glycosis to OXPHOS through PDH activation, and has been used for the treatment of congenital lactic acidosis for more than 30 years. The present study explored whether modulating the Warburg effect by DCA can improve the efficacy of sorafenib (a multi-kinase inhibitor of Raf, VEGFR and PDGFR), the current standard systemic therapy for HCC. Modulation of the Warburg effect was evaluated by measurement of lactate in culture medium and PDH activity. In vitro growth-inhibitory effects were measured by median-effect analyses in a panel of human HCC cell-lines (Hep3B, Huh-7, Sk-hep1, HCC36 and HA22T) cultured in normoxic condition. In vivo growth-inhibitory effects were measured in both subcutaneous and orthotopic murine HCC xenograft models. Apoptosis was evaluated by flow cytometry (annexin V staining and subG1 fraction analysis) and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Expression of apoptosis-related proteins was analyzed by Western blotting. We found that DCA reduced lactate production and increased PDH activity in HCC cells in a dose-dependent manner, indicating switching glucose metabolism from glycolysis to OXPHOS. DCA (10–60 mM) induced synergistic growth-inhibitory and apoptotic effects with sorafenib in most HCC cell-lines tested. Nude mice carrying subcutaneously or intrahepatically implanted Hep3B cells were treated with vehicle, DCA 100 mg/kg /day (equivalent to the dosage used to treat lactic acidosis in human), sorafenib 10 mg/kg /day, and DCA plus sorafenib, via gavage for 21 days. DCA-sorafenib combination synergistically suppressed the tumor growth without advertently affecting the body weight of mice. Synergistic induction of apoptosis was shown by TUNEL assay. Our data suggest that modulation of aberrant cellular metabolism of HCC cells by DCA may improve the clinical efficacy of sorafenib. (Supported by grants NSC97-3112-B-002-012, NSC98-3112-B-002-007, and NSC98-3112-B-002-037) Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B97.

Cytotoxic Effects on HL-60 Cells of Myosin Light Chain Kinase Inhibitor ML-7 Alone and in Combination with Flavonoids.

Uncontrolled cell growth and increased cell proliferation are major features of cancer that are dependent on the stable structure and dynamics of the cytoskeleton. Since stable cytoskeleton structure and dynamics are partly regulated by myosin light chain kinase (MLCK), many current studies focused on MLCK inhibition as a chemotherapeutic target. As a potent and selective MLCK inhibitor, ML-7 [1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazapine hydrochloride] is a promising candidate for an anticancer agent, which would induce apoptosis as well as prevents invasion and metastasis in certain types of cancer cells. This study assessed cytotoxic effects of ML-7 against HL-60 cells and therapeutic efficacy of ML-7 as a potential antileukemia agent. Trypan-blue exclusion assays showed dose- and time- dependent decreases in ML-7 treated HL-60 cells (p < 0.05). Comet assays revealed a significant increase in DNA damage in HL-60 cells after treatment with 40 µM ML-7 for 2 h. Sub-G1 fractions, analyzed by flow cytometry increased in a dose-dependent manner, suggesting that ML-7 can induce apoptotic cell death in HL-60 cells. ML-7 was selectively cytotoxic towards HL-60 cells; not affecting normal human lymphocytes. That selective effect makes it a promising potential anti-leukemia agent. In addition, anticancer efficacy of ML-7 in combination with flavonoids (genistein or quercetin) or anticancer drugs (cisplatin or AraC) against HL-60 cells was assessed. Combination of ML-7 with flavonoids increased the anticancer effect of ML-7 to a greater extent than combination with the anticancer drugs. This implies that ML-7 in combination with flavonoids could increase the efficacy of anticancer treatment, while avoiding side effects cansed by conventional anticancer drug-containing combination chemotherapy.

BMS-754807, a small molecule inhibitor of insulin-like growth factor-1R/IR

BMS-754807 is a potent and reversible inhibitor of the insulin-like growth factor 1 receptor/insulin receptor family kinases (Ki, <2 nmol/L). It is currently in phase I development for the treatment of a variety of human cancers. BMS-754807 effectively inhibits the growth of a broad range of human tumor types in vitro, including mesenchymal (Ewing's, rhabdomyosarcoma, neuroblastoma, and liposarcoma), epithelial (breast, lung, pancreatic, colon, gastric), and hematopoietic (multiple myeloma and leukemia) tumor cell lines (IC50, 5-365 nmol/L); the compound caused apoptosis in a human rhabdomyosarcoma cell line, Rh41, as shown by an accumulation of the sub-G1 fraction, as well as by an increase in poly ADP ribose polymerase and Caspase 3 cleavage. BMS-754807 is active in vivo in multiple (epithelial, mesenchymal, and hematopoietic) xenograft tumor models with tumor growth inhibition ranging from 53% to 115% and at a minimum effective dose of as low as 6.25 mg/kg dosed orally daily. Combination studies with BMS-754807 have been done on multiple human tumor cell types and showed in vitro synergies (combination index, <1.0) when combined with cytotoxic, hormonal, and targeted agents. The combination of cetuximab and BMS-754807 in vivo, at multiple dose levels, resulted in improved clinical outcome over single agent treatment. These data show that BMS-754807 is an efficacious, orally active growth factor 1 receptor/insulin receptor family-targeted kinase inhibitor that may act in combination with a wide array of established anticancer agents.

Pro-Apoptotic and Proliferative Effects of Bone Marrow Adipocytes on Myeloid Leukemia Cells.

Abstract 4572Bone marrow stromal cells (MSCs) from elderly subjects have a reduced capacity to differentiate into osteoblasts and an increased capacity to differentiate into adipocytes, which leads to progressive accumulation of fat in the bone marrow space with increasing age. Adipocytes are the prevalent stromal cell type in adult BM that play an important role in the leukemic bone marrow microenvironment (Tabe et al., Blood 2004 103:1815-22). In this study, we examined the role of BM-derived adipocytes at different stages of differentiation on proliferation and apoptosis of AML cells. U937 cells were co-cultured with BM-derived MSC, MSC-differentiated pre-adipocytes (containing few small lipid vesicles), and mature adipocytes (with multiple hypertrophic lipid vesicules). Under serum-starved conditions, MSC and premature/mature adipocytes induced cell cycle progression of U937 cells with increase in the proportion of cells in S- and G2/M-phase fractions, and inhibited spontaneous cell death with decrease in subG1 fractions. However, only pre-adipocytes inhibited Ara-C-induced cell killing (Table 1). [Display omitted] We next focused on lepin and plasminogen activator inhibitor 1(PAI-1) as potential mediators of these effects by adipocytes. Leptin mRNA and protein levels were upregulated during adipocytic differentiation (mRNA relative expression to GAPDH (PCR): MSC 0, premature adipocyte 2.0±0.5, mature adipocyte 123.3± 35.0; leptin secretion: MSC 23.1±2.9, premature adipocyte 49.3±11.3, mature adipocyte 110.0±4.6 pg/mL (ELISA). PAI-1 mRNA levels were increased in mature adipocyte (relative expression to GAPDH: MSC 314.9±46.5, premature adipocyte 215.1, mature adipocyte 3766.1±656.2). Since PPARÿ activation is known to promote maturation and re-generation of fat-derived adipocytes, we next examined the potential of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) on the BM adipocytes, leptin and PAI-1 production and the survival of the leukemic cells. CDDO induced adipocyte re-generation with significant increase of the number of Oil-Red(+) small sized lipid vesicles without apoptosis induction. This resulted in a markedly enhanced leptin release from adipocytes (10-fold and 23-fold increase at 0.5 μM and 1.0 μM CDDO, respectively, at 72 hrs) without change in leptin mRNA transcription. On the contrary, PAI-1 mRNA levels were significantly decreased by CDDO (6 fold decrease in MSC, 4 fold decrease in premature adipocyte, 6 fold decrease in mature adipocyte). Co-culture of U937 cells with CDDO-primed premature adipocytes and mature adipocytes resulted in increased spontaneous apoptosis of U937 cells compared to adipocytes not exposed to CDDO (% specific apoptosis, U937 co-cultured with CDDO-primed premature adipocytes 26.9 %, mature adipocytes 20.9 %). At the same time, CDDO-primed premature adipocytes induced significant cell cycle progression with decreased proportion of G0/G1-phase and increase in S-phase fractions in U937 cells (Table 2). Co-culture with CDDO-primed premature adipocytes or with premature adipocytes co-treated with recombinant leptin increased subG1- and S-phase fractions in Ara-C-treated U937 cells compared to U937 cells co-cultured with premature adipocytes (Table 3). [Display omitted] In mature adipocytes, which already produce high levels of leptin, CDDO or leptin treatment failed to modulate anti-apoptotic or proliferative effects of AraC on U937 cells. In contrast, human recombinant PAI-1 effectively inhibited spontaneous and Ara-C induced apoptosis of U937 cells (decreased % of Annexin V: spontaneous apoptosis 11.2±1.1%, Ara-C induced apoptosis 15.1± 1.7%).In summary, these results suggest that BM pre-adipocytes support proliferation and survival of myeloid leukemia cells in part through complementary effects of leptin and PAI-1. Our findings indicate that secretion of leptin during MSC differentiation or through PPARg ligation promotes cell cycle progression, while PAI-1 primarily inhibits apoptosis of AML cells. It is conceivable that increased adipocyte content of BM in elderly AML patients may negatively affect the responsiveness of AML cells to chemotherapy. Disclosures:No relevant conflicts of interest to declare.

Centrosomal Clustering – a Novel Therapeutic Target for Multiple Myeloma.

Abstract 300The lack of tumor-specific targets that would allow for the selective eradication of malignant cells without affecting healthy tissues is a major challenge in the development of novel therapies for multiple myeloma (MM). In contrast to normal cells, malignant plasma cells frequently contain multiple centrosomes, associated with the transient formation of multipolar mitotic spindles that lead to segregation defects and chromosomal instability. As in most tumor types, mitotic stability in these cells is maintained by coalescence of multiple centrosomes into two functional spindle poles, termed “centrosomal clustering”. As we have recently shown, this mechanism is an attractive therapeutic target with specificity for tumor cells. To identify potent and selective inhibitors of centrosomal clustering, we performed a phenotype-based small molecule screen in order to force tumor cells with supernumerary centrosomes to undergo multipolar mitoses resulting in apoptotic cell death. We here describe the characterization of a novel small molecule GF-15, a derivative of griseofulvin, as a potent inhibitor of centrosomal clustering, thereby inducing multipolar spindles followed by apoptosis in MM cells. We tested a wide array of MM cell lines, including those resistant to conventional chemotherapeutic agents, and primary patient cells. We found mean inhibitory concentrations (IC50) of proliferation and survival in the range of 1-5 mM, associated with annexin V conversion and activation of caspases 8, 9, and 3. Importantly, GF-15 also overcomes the tumor cell growth advantage conferred by both bone marrow stromal cell-MM, and endothelial cell-MM, co-culture systems. Moreover, non-malignant cells without supernumerary centrosomes like activated PBMCs, immortalized hepatocytes, and bone marrow stromal cells did not reach their IC50 at doses of up to 50 mM. To further demonstrate the specificity of GF-15, we generated resistant MM cell lines by long-term culture with sub-IC50 doses of GF-15. In resistant cell lines, therapeutic doses of GF-15 no longer induce multipolar spindles, consistent with a significant loss of centrosomal aberrations in these cells, as observed by immunoflourescence microscopy. Mechanistically, cell cycle analysis of synchronized MM cells showed marked G2/M arrest within 12-16h followed by a dramatic increase of the sub-G1 fraction after treatment with GF-15. In addition, short term treatment with GF-15 was associated with inhibition of VEGF- and IGF1-triggered MM cell migration. Co-treatment assays to assess potential partners for therapeutic combinations revealed at least additive effects for GF-15 together with bortezomib and marked synergism with paclitaxel at very low doses (1-5 nM), while the combination with melphalan resulted in antagonistic effects due to the S-phase arrest of tumor cells induced by melphalan. Finally and most importantly, i.p. as well as oral treatment of murine xenograft models of human MM resulted in tumor growth inhibition and significantly prolonged survival in vivo. Growth inhibition of xenograft tumor samples was associated with a dramatic increase of mitotic aberrations and multipolarity, as assessed by immunohistochemistry. In vivo biodistribution studies are ongoing. Taken together, our results demonstrate the in vitro and in vivo anti-tumor efficacy of a prototype small molecule inhibitor of centrosomal clustering with specificity for tumor cells, and therefore strongly support its further evaluation and development as a lead compound of a new class of therapeutics for human malignancies. Disclosures:No relevant conflicts of interest to declare.

Effect of the anti-neoplastic drug doxorubicin on XPD-mutated DNA repair-deficient human cells

Doxorubicin (DOX), a member of the anthracycline group, is a widely used drug in cancer therapy. The mechanisms of DOX action include topoisomerase II-poisoning, free radical release, DNA adducts and interstrand cross-link (ICL) formation. Nucleotide excision repair (NER) is involved in the removal of helix-distorting lesions and chemical adducts, however, little is known about the response of NER-deficient cell lines to anti-tumoral drugs like DOX. Wild type and XPD-mutated cells, harbouring mutations in different regions of this gene and leading to XP-D, XP/CS or TTD diseases, were treated with this drug and analyzed for cell cycle arrest and DNA damage by comet assay. The formation of DSBs was also investigated by determination of γH2AX foci. Our results indicate that all three NER-deficient cell lines tested are more sensitive to DOX treatment, when compared to wild type cells or XP cells complemented by the wild type XPD cDNA, suggesting that NER is involved in the removal of DOX-induced lesions. The cell cycle analysis showed the characteristic G2 arrest in repair-proficient MRC5 cell line after DOX treatment, whereas the repair-deficient cell lines presented significant increase in sub-G1 fraction. The NER-deficient cell lines do not show different patterns of DNA damage formation as assayed by comet assay and phosphorylated H2AX foci formation. Knock-down of topoisomerase IIα with siRNA leads to increased survival in both MRC5 and XP cells, however, XP cell line still remained significantly more sensitive to the treatment by DOX. Our study suggests that the enhanced sensitivity is due to DOX-induced DNA damage that is subject to NER, as we observed decreased unscheduled DNA synthesis in XP-deficient cells upon DOX treatment. Furthermore, the complementation of the XPD-function abolished the observed sensitivity at lower DOX concentrations, suggesting that the XPD helicase activity is involved in the repair of DOX-induced lesions.

SiRNA-mediated knockdown against CDCA1 and KNTC2, both frequently overexpressed in colorectal and gastric cancers, suppresses cell proliferation and induces apoptosis

Ndc80 has been shown to play an important role in stable microtubule-kinetochore attachment, chromosome alignment, and spindle checkpoint activation in mitosis. It is composed of two heterodimers, CDCA1–KNTC2 and SPC24–SPC25. Overexpression of CDCA1 and KNTC2 is reported to be associated with poor prognosis in non-small cell lung cancers (NSCLC), and siRNA-mediated knockdown against CDCA1 or KNTC2 has been found to inhibit cell proliferation and induction of apoptosis in NSCLC, ovarian cancer, cervical cancer and glioma. Therefore, CDCA1 and KNTC2 can be considered good candidates for molecular target therapy as well as diagnosis in some cancers. However, the role of the Ndc80 complex in colorectal and gastric cancers (CRC and GC) still remains unclear. In the present study, we used qRT-PCR to evaluate the expression levels of CDCA1, KNTC2, SPC24 and SPC25 in CRC and GC and employed siRNA-mediated knockdown to examine cell proliferation and apoptosis. mRNA overexpression of these four genes was observed in CRCs and GCs when compared with the corresponding normal mucosae. Additionally, the expression levels of tumor/normal ratios of CDCA1, KNTC2, SPC24 and SPC25 correlated with each other in CRCs. MTT assays revealed that cell growths after the siRNA-mediated knockdown of either CDCA1 or KNTC2 were significantly suppressed, and flow cytometry analyses revealed significant increases of the subG1 fractions after knockdown against both genes. Our present results suggest that expressional control of component molecules of Ndc80 can be utilized for molecular target therapy of patients with CRC and GC.

Dehydroeburicoic Acid Induces Calcium- and Calpain-Dependent Necrosis in Human U87MG Glioblastomas

Dehydroeburicoic acid (DeEA) is a triterpene purified from medicinal fungi such as Antrodia camphorate, the crude extract of which is known to exert cytotoxic effects against several types of cancer cells. We aim to test the hypothesis that DeEA possesses significant cytotoxic effects against glioblastomas, one of the most frequent and malignant brain tumors in adults. 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase release assays indicated that DeEA inhibited the proliferation of the human glioblastoma cell U87MG. In addition, Annexin V and propidium iodide staining showed that DeEA treatment led to a rapid increase of glioblastomas in the necrotic/late apoptotic fraction, whereas cell cycle analysis revealed that DeEA failed to significantly enhance the population of U87MG cells in the hypodiploid (sub-G1) fraction. Using electron microscopy, we found that DeEA induced significant cell enlargements, massive cytoplasmic vacuolization, and loss of mitochondrial membrane integrity. DeEA treatment triggered an intracellular Ca(2+) increase, and DeEA-induced cell death was significantly attenuated by BAPTA-AM but not ethylenediaminetetraacetic acid or ethylene glycol tetraacetic acid. DeEA instigated a reduction of both mitochondrial transmembrane potential and intracellular ATP level. Moreover, DeEA induced proteolysis of alpha-spectrin by calpain, and DeEA cytotoxicity in U87MG cells was caspase-independent but was effectively blocked by calpain inhibitor. Interestingly, DeEA also caused autophagic response that was prevented by calpain inhibitor. Taken together, these results suggest that in human glioblastomas, DeEA induces necrotic cell death that involves Ca(2+) overload, mitochondrial dysfunction, and calpain activation.

Apoptosis signal-regulating kinase (ASK)-1 mediates apoptosis through activation of JNK1 following engagement of membrane immunoglobulin

Engagement of membrane immunoglobulin (mIg) on WEHI-231 mouse B lymphoma cells results in growth arrest at the G1 phase of the cell cycle, followed by a reduction of mitochondrial membrane potential (ΔΨm) and apoptosis. WEHI-231 cells resemble immature B cells in terms of the cell surface phenotype and sensitivity to mIg engagement. However, the molecular mechanisms underlying mIg-induced loss of ΔΨm and apoptosis have not yet been established. In this study, we show that apoptosis signal-regulating kinase 1 (ASK1)–c-Jun N-terminal kinase 1 (JNK1) signaling pathway participates in mIg-induced apoptosis through the generation of reactive oxygen species (ROS). Stimulation of WEHI-231 cells with anti-IgM induces phosphorylation and subsequent activation of ASK1, leading to JNK activation. Anti-IgM stimulation immediately (5 min) induces hydrogen peroxide (H 2O 2) production with a substantial increase during later time points (36–48 h), accompanied by loss of ΔΨm and an increase in cells with sub-G1 DNA content. The anti-IgM-induced late-phase H 2O 2 production, loss of ΔΨm, and increase in the sub-G1 fraction were all reduced substantially in WEHI-231 cells overexpressing a dominant-negative form of ASK1, compared with control vector alone, but enhanced substantially in cells overexpressing a constitutively active form of ASK1. These mIg-mediated events were also partially abrogated by ROS scavenger N-acetyl- l-cysteine (NAC). Taken together, these results suggest that mIg engagement induces H 2O 2 production leading to activation of ASK1–JNK1 pathway, creating a feedback amplification loop of ROS-ASK/JNK that leads to loss of ΔΨm and finally apoptosis.

N-Propionyl-Cysteaminylphenol-Magnetite Conjugate (NPrCAP/M) Is a Nanoparticle for the Targeted Growth Suppression of Melanoma Cells

A magnetite nanoparticle, NPrCAP/M, was produced for intracellular hyperthermia treatment of melanoma by conjugating N-propionyl-cysteaminylphenol (NPrCAP) with magnetite and used for the study of selective targeting and degradation of melanoma cells. NPrCAP/M, like NPrCAP, was integrated as a substrate in the oxidative reaction by mushroom tyrosinase. Melanoma, but not non-melanoma, cells incorporated larger amounts of iron than magnetite from NPrCAP/M. When mice bearing a B16F1 melanoma and a lymphoma on opposite flanks were given NPrCAP/M, iron was observed only in B16F1 melanoma cells and iron particles (NPrCAP/M) were identified within late-stage melanosomes by electron microscopy. When cells were treated with NPrCAP/M or magnetite and heated to 43 degrees C by an external alternating magnetic field (AMF), melanoma cells were degraded 1.7- to 5.4-fold more significantly by NPrCAP/M than by magnetite. Growth of transplanted B16 melanoma was suppressed effectively by NPrCAP/M-mediated hyperthermia, suggesting a clinical application of NPrCAP/M to lesional therapy for melanoma. Finally, melanoma cells treated with NPrCAP/M plus AMF showed little sub-G1 fraction and no caspase 3 activation, suggesting that the NPrCAP/M-mediated hyperthermia induced non-apoptotic cell death. These results suggest that NPrCAP/M may be useful in targeted therapy for melanoma by inducing non-apoptotic cell death after appropriate heating by the AMF.

Induction of Apoptosis and Inhibition of Invasion in Human Hepatoma Cells by Anthocyanins from Meoru

Anthocyanins belong to a class of flavonoids exhibiting antioxidant and anti-inflammatory actions as well as a variety of chemotherapeutic effects. However, little is known about the cellular and molecular mechanism of anticancer activity. In this study, we investigated if the anthocyanins (delphinidin-3,5-diglucoside: cyanidin-3,5-diglucoside: petunidin-3,5-diglucoside: delphinidin-3-glucoside: malvdin-3,5-diglucoside: peonidin-3,5-diglucoside: cyanidin-3-glucoside: petunidin-3-glucoside: peonidin-3- glucoside: malvidin-3- glucoside = 27:63:8.27:1:2.21:2.21:6.7:1.25:5.72:1.25) [corrected] isolated from meoru (Vitis coignetiae Pulliat) exerted antiproliferative and anti-invasive and apoptotic effects on human hepatoma Hep3B cells. It was found that the anthocyanins could inhibit cell growth by 75% at the concentration of 400 microg/mL for 48 h. Flow cytometric analysis showed that the anthocyanins increased the amount of DNA fragments (sub-G1 fraction) in a dose-dependent manner, which is closely related to mitochondrial dysfunction and reduction in antiapoptotic proteins (Bcl-2, xIAP, cIAP-1, and cIAP-2). The anthocyanins also significantly inhibited the migration and invasion of Hep3B cells through a matrigel-coated chamber. Taken together this study indicates that the anthocyanins from meoru have antiproliferative and anti-invasive effects and may induce apoptosis through the activation of the mitochondrial pathway and inhibition of antiapoptotic proteins. This study provides evidence that the anthocyanins isolated from meoru might be useful in the treatment of human hepatitis B-associated hepatoma.

Overexpression of Sp1 leads to p53‐dependent apoptosis in cancer cells

Numerous studies have documented that Sp1 expression level were elevated in various human cancers. However, the promoters of many pro-apoptotic genes have been found to contain the Sp1 binding elements and are activated by Sp1 overexpression. To better understand the role and the mechanism of increased Sp1 levels on apoptosis, we used adenovirus to ectopically express GFP-Sp1 protein in various cancer cell lines. First, in HeLa and A549 cells, we found that Sp1 overexpression suppressed the cell growth and increased the detection of sub-G1 fraction, caspase-3 cleavage, and annexin-V signal revealed that apoptosis occurred. Furthermore, when cells entered the mitotic stage, the cell apoptosis was induced by Sp1 overexpression through affecting mitotic chromatin packaging. We also verified that p53 protein was accumulated and activated the p53-dependent apoptotic pathways in the wild-type p53 cells but not in the p53-mutated or p53-deleted cell lines when these cells were infected with adeno-GFP-Sp1 virus. In addition, A549 (p53(+/+)) cells could be protected from apoptosis under Sp1 overexpression when p53 was knockdown by p53 shRNA. Finally, H1299 (p53(-/-)) cell viability was significantly inhibited by adeno-GFP-Sp1 virus infection in the expression of p53. In conclusion, p53 was an essential factor for Sp1 overexpression-induced apoptotic cell death in transforming cells.

Using YC-1 to overcome the radioresistance of hypoxic cancer cells

Targeting hypoxia-inducible factor-1 (HIF-1) active cells in tumors may be an excellent strategy to improve the outcome of radiation therapy. On the basis of the reported role of YC-1 as a HIF-1 inhibitor with anti-cancer activity, we tested the therapeutic efficacy of YC-1 against radioresistance in vitro. The AMC-HN3 cancer cell line, developed from squamous cell carcinoma of the larynx, was cultured under hypoxic conditions or in the presence of cobalt chloride. Both treatments induced nuclear accumulation of HIF-1alpha protein. Cells cultured under normoxic or hypoxic conditions with and without YC-1 treatment were irradiated and analyzed using flow cytometry and clonogenic assays. In the absence of YC-1 treatment, irradiation induced a greater cytotoxic effect in normoxic cells than in cobalt-treated cells. Treatment of cobalt-treated cells with YC-1 effectively inhibited HIF-1alpha expression, and enhanced the sensitivity of cells to radiation, decreasing the surviving fraction to that of normoxic cells. Flow cytometry confirmed these results, showing that the sub-G1 fraction was increased in YC-1-treated hypoxic cells after irradiation. Our results suggest that YC-1 treatment may be an effective therapeutic strategy for overcoming the radioresistance of HIF-1alpha-expressing, hypoxic cancer cells.

Apoptosis signal-regulating kinase (ASK)-1 mediates apoptosis in B cells (34.6)

Abstract Engagement of membrane immunoglobulin (mIg) on B cells results in reduction of the mitochondrial membrane potential (ƒ¢ƒμm) and apoptosis, which plays a crucial role in the regulation of immune responses. In this study, we show that the apoptosis signal-regulating kinase 1 (ASK1)-JNK1 signaling pathway participates in mIg-induced apoptosis in normal B cells as well as WEHI-231 B lymphoma cells. Stimulation of WEHI-231 cells with anti-IgM induces phosphorylation and subsequent activation of ASK1, leading to JNK activation. Anti-IgM stimulation produces superoxide anions (O2-), accompanied by loss of ƒ¢ƒμm and an increase in cells with sub-G1 DNA content. Anti-IgM-induced O2- production, loss of ƒ¢ƒμm, and increase in the sub-G1 fraction were all reduced substantially in WEHI-231 cells overexpressing a dominant-negative form of ASK1, compared with control vector alone. The mIg-mediated O2- production, loss of ƒ¢ƒμm, and increase in the sub-G1 fraction were partially abrogated by the reactive oxygen species scavenger N-acetyl-L-cysteine (NAC). Similarly to WEHI-231 B lymphoma cells, the mIg-mediated apoptotic features were moderately compromised in mouse ASK1-deficient B cells. Taken together, these results suggest that mIg engagement induces JNK activation through ASK1 activation, resulting in production of O2- that leads to loss of ƒ¢ƒμm and finally to apoptosis.