bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4382: N-acetylcysteine interacts with Cu++ to induce cancer cell death through the generation of hydrogen peroxide
Authors: Zheng Jie , Lou Jessica R. , Benbrook Doris M. , Hanigan Marie H. , Lind Stuart E. , Ding Wei-Qun
Published: February 2011

Metal ionophores that transport copper or zinc into cells are toxic to cancer cells. Loading of metals into cells results in loss of protective intracellular thiols, which can contribute to cell death. We postulated that the metal-thiol interaction might also lead to the generation of cytotoxic compounds. We tested this hypothesis by adding N-acetylcysteine (NAC) to cancer cell lines in the presence of copper or zinc. NAC itself, at concentrations up to 10 mM, was not toxic to a variety of human cancer cell lines, including A2780 (ovarian), MCF-7 (breast), Panc-1 (pancreas), and A375 (melanoma). When added to cells in the presence of Cu++ (3-30 µM), NAC significantly enhanced the cytotoxicity induced by CuCl2 alone. The cytotoxicity of the NAC/copper combination was blocked by the copper-binding compounds tetrathiomolybdate and bathocuproinedisulfonic acid. NAC was not cytotoxic in the presence of zinc. This finding suggested that the redox activity of copper was an important determinant of cytotoxicity and raised the possibility that H2O2 was being generated. This was confirmed in vitro using a colorimetric assay (Biovision). The ability of other thiols to promote H2O2 generation when exposed to Cu++ was then examined. Reduced lipoic acid and L-cysteine generated higher level of H2O2 than NAC did; reduced glutathione was able to generate comparable levels of H2O2 as NAC, while oxidized glutathione generated significantly lower levels of H2O2; oxidized cysteine did not generate H2O2. To determine if H2O2 played a role in the observed cytotoxicity, catalase was added to cells treated with NAC/copper. Its addition completely blocked cell killing, indicating that significant amounts of extracellular H2O2 were being generated. These findings indicate that the interaction of copper with thiols leads to cytotoxic concentrations of H2O2. Thiols may differ in their reactivity with redox active compounds such as copper. Since the copper concentration of plasma is greater than that of tissue culture media, NAC's effects in vivo may differ from those seen in vitro.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4382.
Abstract 4389: Potential role of superoxide dismutase 3 in prevention of estrogen-induced breast cancer
Authors: Bhat Hari K. , Singh Bhupendra
Published: February 2011

Estrogens have been implicated in the development of human breast cancers. Literature and our studies strongly support the role of oxidative stress in estrogen-induced breast carcinogenesis. We have recently demonstrated for the first time that antioxidants vitamin C or butylated hydroxyanisole (BHA) attenuate 17β-estradiol (E2)-induced oxidative stress; increase the latency of tumor development; and, severely inhibit E2-induced breast tumor development in female ACI rats. The female ACI rat is an established animal model to study the mechanisms of E2-induced breast cancer. The objective of the present study was to identify genes that may provide an insight into the mechanism of inhibition of E2-induced breast carcinogenesis by vitamin C or BHA. Microarray analyses were performed on total RNA isolated from breast and breast tumor tissues of rats treated with E2 (3 mg, s.c.); vitamin C (1% in drinking water); BHA (0.7% in diet); vitamin C + E2 and BHA + E2 for 8 months. A number of genes were shown to be differentially expressed in breast tissue and tumor tissue of rats treated with E2 vs. other treatments. One gene of interest identified was extracellular superoxide dismutase (SOD3) that was 18-fold suppressed in mammary tumors vs. normal mammary tissue and more than 6-fold decreased in mammary tissue of E2 treated animals vs. normal age-matched controls. Protein expression data confirmed the suppression of SOD3 in mammary tissue of E2-treated rats and in mammary tumors. This suppression was overcome by co-treatment of rats with E2 and vitamin C or BHA. SOD3 was induced 2.9- and 2.1-fold respectively by treatments of rats with vitamin C or BHA compared to sham-treated controls. SOD3 expression was not regulated by vitamin C or BHA in the liver tissue suggesting that its expression may be target-organ specific. SOD3 is an extracellular antioxidant enzyme implicated in the prevention of oxidative stress-mediated tissue damage. Thus, SOD3 may play an important role in the prevention of breast cancer and this gene may have a great potential in the development of therapeutic strategies for the prevention of estrogen-induced neoplasia.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4389.
Abstract 4390: The p16INK4A tumor suppressor regulates cellular oxidative stress
Authors: Jenkins Noah , Liu Tong , Goodson Agnessa , Samadashwily George , Grossman Douglas
Published: February 2011

Melanoma, a malignant tumor of melanocytes, is a potentially lethal form of skin cancer linked to both environmental and genetic factors. Mutations or deletions in the cyclin-dependent kinase inhibitor p16INK4A are associated with multiple cancer types, but more commonly found in melanoma tumors and specifically associated with familial melanoma predisposition. Although p16 is thought to function as a tumor suppressor by negatively regulating the cell cycle, it remains unclear why genetic compromise of p16 predisposes to melanoma rather than other cancers. Here we describe a novel role for p16 in regulating oxidative stress in several cell types, including melanocytes. Compared to keratinocytes and fibroblasts, we also found increased ROS levels basally and following treatment with H2O2. Expression of p16 was rapidly upregulated following UV-irradiation in melanocytes, and in response to H2O2-induced oxidative stress in a p38 stress-activated protein kinase-dependent manner. Knockdown of p16 using siRNA increased intracellular ROS and oxidative (8-oxoguanine) DNA damage which was further enhanced by H2O2 treatment. Elevated ROS were also observed in p16-depleted human keratinocytes, and in whole skin and dermal fibroblasts from p16-deficient mice. ROS in p16-deficient fibroblasts were normalized by expression of exogenous p16. Finally, p16-mediated suppression of ROS could not be attributed to upregulation of antioxidant enzymes or the role of p16 in cell cycle regulation. These findings suggest a potential alternate tumor-suppressor function of p16 as an endogenous regulator of carcinogenic intracellular oxidative stress. The increased susceptibility of melanocytes to oxidative stress may explain why compromise of p16 predisposes to melanoma over other cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4390.
Abstract 4399: EphA2-targeting antibody-gelonin conjugate specifically binds and kills target-expressing tumor cells, including multi-drug resistant (MDR) lines
Authors: Marks John W. , Gooya John , Pore Nabendu , Mao Shenlan , Jackson Dowdy , Rosenblum Michael G.
Published: February 2011

The EphA2 receptor is over-expressed on the surface of many different human tumors, and has been shown to internalize upon ligand or antibody binding. The fully human monoclonal antibody anti-EphA2 antibody was developed by phage display methods and specifically binds both human and rodent EphA2 receptors. The type II ribosome-inactivating protein gelonin was genetically engineered with an extra cysteine residue to facilitate chemical cross-linking with targeting proteins. The fully human anti-EphA2 antibody, anti-EphA2 antibody, was conjugated to the recombinant gelonin (rGel) using the heterobifunctional cross-linker SPDP and partially reduced rGel. The conjugated antibody (anti-EphA2 antibody/rGel) was separated from free antibody and rGel by gel-permeation and affinity chromatography. The final material was composed of antibody+1 rGel (predominant) and antibody+2 rGel (minor) and was essentially free of contaminating rGel or unreacted anti-EphA2 antibody. The anti-EphA2 antibody/rGel conjugate was assessed for cell-free protein synthesis inhibitory activity and found to be similar to free rGel. The conjugate proved specifically cytotoxic to the EphA2 expressing PC-3, MDA-MB-231 and CT-26 cell lines, with IC50 values of 3.8, 6.1 and 11 ng/ml, respectively. The Targeting Index for the anti-EphA2 antibody/rGel conjugate (vs. rGel) was high, with values of 1909, 306 and 75 for PC-3, PC-3-MDR and MDA-MB-231, respectively. Confocal microscopy showed that the anti-EphA2 antibody/rGel conjugate was specifically and rapidly internalized. The maximal intracellular concentration was achieved 4 hours after exposure. Minimal contact time studies showed specific cytotoxicity with only 1 hour exposure while optimal cytotoxicity was achieved with an exposure time of 24 hours. Of special interest, the anti-EphA2 antibody/rGel conjugate demonstrated no significant loss in specific cytotoxicity against Dox-resistant (MDR expressing) cell lines derived from either MDA-MB-231 or PC-3 cells compared to their parental cell lines. These data suggest that the development of MDR-resistant phenotypes may not impair the successful utilization of rGel-based targeted therapeutics. These results support the continued evaluation of the rGel antibody conjugate technology. Research conducted, in part, by the Clayton Foundation for Research.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4399.
Abstract 4422: 2-AAPA: A novel anticancer agent that induces microtubule depolymerization and apoptosis
Authors: Chen Wei , Seefeldt Teresa , Young Alan , Zhang Xiaoying , Ruffolo John , Kaushik Radhey , Guan Xiangming
Published: February 2011

This study was aimed to investigate the anticancer activities and the mechanism of the anticancer activities of a novel anticancer agent - 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2-AAPA). 2-AAPA was found to inhibit various human cancer cell lines with a narrow range of IC50 values (28-75 μM). These cell lines include lung (NCI-H226), prostate (PC-3), breast (MCF-7), skin (A431), melanoma (UACC-62 and SK-MEL-2), and renal (UO-31) cancers, as well as human ovarian cancers (OVCAR-3 and NCI/ADR-RES) which are resistant toward doxorubicin. Flow cytometric studies revealed that 2-AAPA induced cell cycle arrest at G2/M phase and produced significant cell apoptosis in UACC-62 cells. Through immunofluorescence studies, 2-AAPA was found to depolymerize microtubules and cause cell morphological change. An HPLC (high performance liquid chromatography) assay demonstrated that proteins were glutathionylated in both OVCAR-3 and UACC-62 cells when treated with 2-AAPA. A Western blot analysis also showed that a band corresponding to the molecular weight of tubulins was significantly glutathionylated in cell lysate obtained from UACC-62 cells treated with 2-AAPA. Considering the rich thiol content of tubulin proteins and the nature of protein glutathionylation, it is likely that microtubule function might be affected through microtubule glutathionylation resulting in cell cycle arrest at G2/M phase. Immunoprecipitation is being employed to confirm that tubulins are glutathionylated in cells treated with 2-AAPA. Together, our findings could lead to an antimitotic agent with a novel mechanism which is different than those of current antimitotic drugs.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4422.
Abstract 4424: Molecular mechanism study of a novel microtubule-binding agent-induced mitotic cell death of human hepatocellular carcinoma
Authors: Liu Jun Jen , Ho Chun Te , Yang Li Xi , Chang Yu Jia , Liu Tsan Zon , Wei Po- Li
Published: February 2011

Polymerization and de-polymerization of microtubule are responsible for separation of chromosomes during cell division which has been considered to be one of major targets in cancer chemotherapy. In order to develop a newer generation of chemotherapeutic agents with higher efficacy and lower toxicity, a novel microtubule-binding agent (AD1), an analog of thiocolchicine, has been successfully synthesized. We here demonstrated AD1 could effectively kill a panel of seven human hepatocellular carcinoma (HCC) cell lines with varying degrees of differentiation. The IC50 values for all these HCC cell lines, using NCI standard protocol (sulforhodamine B, SRB), ranged from 2 to 50 nM. Cell cycle analysis study by flowcytometer unveiled that AD1 could evoke a dose-dependent cell cycle arrest in the G2/M phase. Further immunoflurorescence studies revealed that AD1 could also disrupt tubulin polymerization which would be responsible for the G2/M arrest. These prominent events provoked the disruption of cellular microtubule networks and prevented the proper formation of spindle apparatus and consequently resulted in the mitotic cell death. Interestingly, we also observed that AD1 could induce transcription factor ATF6 cleavage and subsequent reduction of the level of ER stress chaperone protein Grp78, and phosphorylated Akt expression. In vivo toxicity studies demonstrated that AD1, with maximum tolerance dose (MTD) of 150 mg/kg, was remarkably less toxic to normal C3H/HeN mice than colchicine (MTD of 1.6 mg/kg). The in vivo efficacy study of AD1 on HCC xenografts are now being investigated in nude mouse model in our laboratories.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4424.
Abstract 444: Hypoxia modulates EWS-FLI1 transcriptional signature and enhances invasiveness and anchorage-independent growth of Ewing's sarcoma cells in-vitro
Authors: Aryee Dave N. T , Niedan Stephan , Kauer Maximilian , Muehlbacher Karin , Walker Robert L. , Meltzer Paul , Poremba Christopher , Kofler Reinhard , Kovar Heinrich
Published: February 2011

Hypoxia is an important factor in the tumor cell microenvironment and approximately 1-1.5% of the genome is transcriptionally responsive to hypoxia. Hypoxia-inducible factor-1 (HIF-1) is the major mediator of transcriptional activation under hypoxia. Tumor hypoxia is associated with a more aggressive phenotype of many cancers in adults, but data on pediatric tumors are scarce. By immunohistochemistry, HIF-1α expression was readily detectable in 18/28 primary Ewing′s sarcoma family tumors (ESFT), a group of highly malignant bone-associated tumors in children and young adults, hence we studied the effect of hypoxia on ESFT cell lines in-vitro. Intriguingly, we found that EWS-FLI1 protein expression, which characterizes ESFT, is transiently up-regulated by hypoxia in a HIF-1α-dependent manner. Hypoxia modulated the EWS-FLI1 transcriptional signature relative to normoxic conditions. Both synergistic as well as antagonistic transcriptional effects of EWS-FLI1 and of hypoxia were observed. Consistent with alterations in the expression of metastasis related genes, hypoxia stimulated the invasiveness and soft-agar colony formation of ESFT cells in-vitro. Our data represents the first transcriptome analysis of hypoxic ESFT cells and identifies hypoxia as an important microenvironmental factor modulating EWS-FLI1 expression and target gene activity that may impact the malignant properties of ESFT.

This study was funded by: “European Embryonal Tumor Pipeline” 6th framework program of the European Commission (STREP “E.E.T. Pipeline” contract LSHC-CT-2006-037260) and grant 12675 from the Austrian National Bank Jubilaeumsfonds.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 444.
Abstract 445: Significance of carbonic anhydrase-9 in gastric carcinoma
Authors: Yukihiro Kato , Yashiro Masakazu , Kashiwagi Shinichiro , Fuyuhiro Yuhiko , Noda Satoru , Doi Yosuke , Kubo Naoshi , Tanaka Hiroaki , Muguruma Kazuya , Inoue Toru , Maeda Kiyoshi , Sawada Tetsuji , Ohira Masaichi , Hirakawa Kosei
Published: February 2011

[purpose] Gastric carcinoma is one of the most frequent causes of cancer death. Various kinds of solid tumor including gastric cancer have heterogeneously a hypoxic environment. In gastric cancers, the hypoxia reported to develop their malignant potential. Cancer cell under hypoxic condition show resistance against various types of therapies. The poor outcome is, however, not only due to the resistance to treatment but also to the more aggressive behavior of cancer cells. Hypoxia selects tumor cells with a reduced apoptotic potential and angiogenic phenotype, which invade the surrounding tissue and easily metastasize due to the activation of matrix metalloproteinases. CA-9 (originally named MN) is a member of the α-family of carbonic anhydrases of zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to carbonic acid. The purpose of our study was to clarify significance of the hypoxia in gastric carcinoma using of CA-9.

[materials and methods] A total of 265 patients who had undergone a resection of the primary tumor and were confirmed histologically to have sporadic gastric cancer, were enrolled in this study. Tumor specimens were fixed in 10% formaldehyde solution and embedded in paraffin. The expression of CA-9 was immunohistochemically determined using the antibodies against CA-9 proteins. CA-9 immunostaining was scored as 0 for 0%, 1+ for 1-20%, 2+ for 21-50%, and 3+ for >50%, taking into account the percentage of cells showing membrane in all pieces of specimen from each patient mounted in one slide. Correlation of CA-9 expression and clinic-pathologic features was statistically analyzed using the Chi-square test or Fisher's exact test.

[results] The CA-9 expression was positive in 88 (33%) of 265 gastric carcinomas. The CA-9 expression level was significantly high in type 4 tumors (60%, p<0.001) and diffuse type carcinoma (41%, p<0.001) and significantly correlated with invasion depth (p<0.001), lymph node metastasis (p<0.001) and lymphatic invasion (p=0.002). In addition, the CA-9 positive example was poor prognosis in comparison with a negative example significantly, (P <0.001) while multivariate analysis did not show significant difference.

[conclusion] Hypoxia might be associated with aggressive tumor phenotypes of gastric carcinomas. The hypoxic marker, CA-9, may be a useful prognostic indicator.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 445.
Abstract 4452: Induction of cell and autophagy by the anticancer redox agent mitoquinone is regulated by Nrf2
Authors: Rao V. Ashutosh , Klein Sarah , Mizuno Naoko , Zielonka Jacek , Kalyanaraman Balarama , Shacter Emily
Published: February 2011

Mitoquinone (MitoQ) is a synthetically-modified, redox-active ubiquinone compound that accumulates predominantly in mitochondria. We evaluated the potential use of MitoQ as an anti-cancer agent and found that it is 30-fold more cytotoxic to breast cancer cells than to healthy mammary cells. MitoQ caused irreversible inhibition of the clonogenic growth of breast cancer cells by a combination of autophagic and apoptotic cell death mechanisms, with autophagy playing the major role. None of the cytotoxic effects of MitoQ were seen with the parent ubiquinone coenzyme Q10. Growth inhibition from MitoQ was associated with G1/S cell cycle arrest and phosphorylation of the checkpoint kinases Chk1 and Chk2. A possible role for oxidative stress in MitoQ activity is supported by observed increases in products of hydroethidine oxidation (2-hydroxyethidium, ethidium and diethidium) at cytotoxic concentrations of MitoQ. We will present data showing that Nrf2 and Keap1 regulate the response to the oxidative perturbations caused by MitoQ.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4452.
Abstract 4457: Omega-3 polyunsaturated fatty acids induce apoptosis cell death and inhibit LPS-induced invasion in PC3 cells
Authors: Song Kyoung-Sub , Shin Soyeon , Jing Kaipeng , Kim Nayeong , Jeong Soyeon , Kim Jong-Seok , Heo Jun-Young , Park Ji-Hoon , Seo Kang-Sik , Kwon Hyun-Joo , Kang Jing X. , Park Jong-Il , Park Seung-kiel , Kweon Gi-Ryang , Yoon Wan-Hee , Hwang Byung-Doo , Lim Kyu
Published: February 2011

Dietary fat as a potential risk factor for cancer has been the focus of many epidemiologic and basic researchers, but the findings have been inconclusive. Toll-like receptor 4 (TLR4)-mediated signaling has been implicated in tumor cell invasion, survival, and metastasis in a variety of cancers. In this study, we have investigated anticancer action of ω3-PUFAs and effect of ω3-PUFAs on LPS-mediated TLR4 signaling in prostate cancer. DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid) treatment resulted in a dose-dependent reduction of cell viability with apoptosis of PC3 cells as confirmed by TUNEL assay. In contrast, AA (arachidonic acid), a ω6-PUFA, exhibited no significant effect. Moreover, invasiveness of PC3 cells was inhibited in a dose-dependent manner by treatment of DHA, while AA showed no significant effect. In zymography, MMP-2 activity was inhibited by DHA treatment and MMP-9 and MMP-2 promoter activities were also inhibited. DHA inhibited Cox-2 and VEGF promoter activities and NF-kB reporter activity was also decreased. The expression of TLR4 was confirmed by RT-PCR in PC3 cells and DHA dramatically inhibited the LPS-induced invasion of PC3 cells. In in vivo experiments, when mouse prostate cancer cells (RM1) were injected into the tail vein of Fat1 mice (Fat1 transgenic mice express a Caenorhabditis elegans ω3-desaturase converting ω6- to ω3-PUFAs endogenously.) and WT (wild type mice), lung metastasis was significantly inhibited in Fat1 transgenic mice compared to WT mice. In conclusion, the present study suggests that ω3-PUFAs may inhibit prostate cancer cell growth, and invasion through decrease of MMPs, Cox-2, VEGF and NF-kB reporter activities. Moreover LPS-mediated invasiveness was inhibited by DHA. These findings provide important preclinical evidence and molecular insight for utilization of ω-3 PUFAs for the chemoprevention and treatment of human prostate cancer. [This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Infection Signaling Network Research Center (R13-2007-020-01000-0) at Chungnam National University].

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4457.
Abstract 4460: Mechanism of action of small molecule CXCR4 antagonists in human B-lymphoma cell lines
Authors: Hu Yanping , Nguyen Tri-Hung , Swistak Mark , Gale Matt , Lonning Scott , Kaplan Johanne , Siders William
Published: February 2011

CXCR4 is a chemokine receptor frequently over-expressed on hematological cancers and solid tumors. CXCR4 and its ligand SDF1α (stromal derived factor-1α or CXCL12) play an important role in cancer metastasis, regulation of stem cell trafficking and neovascularization. We have observed previously that treatment with the small molecule CXCR4 antagonists Plerixafor (AMD3100, Mozobil) and AMD3465 in combination with Campath or Rituxan had a synergistic effect resulting in a prolongation of survival greater than that obtained with either agent alone in disseminated human B-lymphoma tumor models. We next explored the mechanism of action of CXCR4 antagonists in vitro using the human B-lymphoma cell lines Ramos, Raji, B104, BL31, IM9 or Wien133 which display similarly strong levels of cell surface CXCR4 expression. All cell lines responded to SDF1α but, in spite of comparable levels of CXCR4 expression, showed varying degrees of calcium influx and cell migration when stimulated. There was a good correlation between calcium influx and migratory responses for a given cell line. Addition of the CXCR4 antagonists Plerixafor or AMD3465 inhibited both responses. The compounds did not show any direct cytotoxicity and failed to inhibit spontaneous proliferation of Ramos, Raji and B104 human B-lymphoma cell lines. However, they did inhibit SDF1α-induced proliferation of these cell lines in agreement with their ability to block CXCR4. Moreover, CXCR4 antagonists inhibited SDF1α-induced phosphorylation of multiple proteins involved in cell migration, proliferation and survival signal pathways as well as some transcription factors, including Akt, ERK1/2, JNK, p70S6, p90RSK, GSK-3a/b, Tyk2, p38, MEK1, c-Jun, STAT3 and CREB.

These results suggest that the CXCR4/SDF1α axis plays an important role in the migration, proliferation and survival of human B-lymphoma cells and that disruption of these functions with CXCR4 antagonists may contribute to their observed ability to enhance the anti-tumor activity of Campath and Rituxan in human B-lymphoma models.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4460.
Abstract 4475: Discovery and characterization of PI3K isoform-selective inhibitors
Authors: Virone-Oddos Angela , Halley Franck , Certal Victor , Carry Jean-Christophe , Thompson Fabienne , Schio Laurent , Debussche Laurent , Bonnevaux Helene , Nicolas Jean-Paul , Delorme Cécile , Karlsson Andreas , Morales Renaud , Abecassis Pierre-Yves , Lejeune Pascale , Benard Tsiala , Michot Nadine , Foster Paul , Bussenius Joerg , Jaeger Chris , Trowe Torsten , Raeber Oliver , Wang Jing , Nguyen Lam , Aoyama Ron , Rice Ken , Matthews David , Lengauer Christoph
Published: February 2011

Abnormal PI3K pathway activation plays a major role in cancer, as a result of either RTK activation or somatic mutations of major components of the pathway, including activating point mutations and amplification of the PIK3CA gene as well as loss of negative regulatory proteins such as PTEN. Most of the ATP-competitive PI3K inhibitors currently in clinical development inhibit all class I PI3K isoforms: however, several recent reports support the development of isoform-specific inhibitors. In particular, while PI3Kα specific inhibitors are predicted to inhibit growth of tumors with PIK3CA mutations, PTEN-deficient tumors have been shown to depend on PI3Kβ. In addition, isoform specific PI3K inhibitors may exhibit better safety profiles compared to pan-selective PI3K inhibitors, and thus may be easier to combine with other targeted or cytotoxic therapies. Here we report the discovery of ATP-competitive inhibitors with selectivity for PI3Kα, PI3Kα/mTOR, or PI3Kβ, which were identified and optimized by means of high-throughput screening and medicinal chemistry. These inhibitors exhibit biochemical IC50 values below 100 nM and good selectivity over other PI3K isoforms and a diverse panel of protein kinases. Cellular assays demonstrate that PI3Kα or PI3Kα/mTOR compounds inhibit phosphorylation of targets downstream of PI3K (Akt) and mTOR (S6 and p70S6 kinase) in the PI3Kα-activated MCF7 cell line, and that effects on PI3K pathway readouts are less pronounced in PC3 cells lacking PTEN. In contrast, PI3Kβ compounds potently inhibit phosphorylation of AKT (cellular IC50s < 200 nM) in the PTEN-deficient PC3 tumor cell line, and the most PI3Kβ-selective inhibitors are inactive on AKT phosphorylation in PI3Kα-activated H460 cells. In vivo pharmacodynamic analyses following oral administration of these isoform-selective inhibitors to mice bearing xenografted tumors demonstrate dose-dependent inhibition of phosphorylation of PI3K and mTOR effectors at well-tolerated doses.

These results strongly argue for the development of isoform-selective PI3K inhibitors for the treatment of cancer patients harboring tumors with PTEN- deficient or PI3Kα-activated specific genotypes.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4475.
Abstract 4477: Dual-targeting of AMPK and PI3K/mTOR in a panel of breast cancer cell lines
Authors: Elizalde Violeta Serra , Russillo Michelangelo , Scaltriti Maurizio , Rodriguez Olga , Guzman Marta , Cosimo Serena Di , Baselga José
Published: February 2011

Several PI3K/AKT/mTOR pathway inhibitors are presently in early clinical trials for the treatment of cancer. Due to their mechanism of action, many of these compounds markedly increase the blood glucose levels. Metformin is an AMPK activator widely used in clinical practice to contrast hyperglycemia and is also known to reduce cancer risk as well as to improve cancer prognosis. The aim of this study is to explore the in vitro anti-tumor effect of metformin alone and in combination with the PI3K/mTOR inhibitor NVP-BEZ235 in a panel of breast cancer cell lines.

Breast cancer cells (BT474, SKBR3, MCF7, MDA-468, MDA-231) were exposed to escalating doses of BEZ235 as single agent and in combination with metformin. Levels of phosphorylated and total AMPK, MAPK, EGFR, HER2 and S6 ribosomal protein were evaluated by western blot. Metformin induced dose-dependent growth inhibition. The combined treatment of NVP-BEZ235 plus metformin resulted in an inhibitory effect on cell proliferation greater than with either treatment alone in EGFR and HER2 positive breast cancer cell lines. At the IC50 of 20 mM we found that metformin activates AMPK reducing mTORC1 activity and, in turn, decreasing the levels of p-S6 ribosomal protein. Metformin treatment was also associated with reduced receptor tyrosine kinases (RTKs) expression (EGFR and HER2) and decreased p-MAPK. The dual PI3K/mTOR inhibitor NVP-BEZ235 potently decreases p-AKT and p-S6. As described for other mTOR inhibitors, NVP-BEZ235 increases MAPK phosphorylation by transactivation of several RTKs of the HER family. Treatment with metformin downregulates these RTKs preventing NVP-BEZ235-dependent MAPK pathway transactivation, providing a possible explanation for the potent anti-proliferative activity of the combination in HER2 positive breast cancer cell lines. The combination of metformin and NVP-BEZ235 significantly inhibits the growth of several breast cancer cell lines. We propose that inhibiting the PI3K/Akt/mTOR pathway in combination with metformin treatment would result in superior antitumor activity. Further studies are warranted to determine the applicability of this approach in the clinic.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4477.
Abstract 4486: OSI-027, a selective dual mTORC1/TORC2 kinase inhibitor displays broad spectrum anti-tumor activity in preclinical models of human cancer
Authors: Gokhale Prafulla C. , Bhagwat Shripad V. , Crew Andrew P. , Cooke Andrew , Mantis Christine , Workman Jennifer E. , Landfair Darla , Bittner Mark , Epstein David M. , Pachter Jonathan A. , Wild Robert
Published: February 2011

The mammalian target of rapamycin (mTOR) protein kinase plays a central role in regulating cell proliferation and cell survival. The PI3K/AKT signaling pathway that activates mTOR is frequently altered in many human cancers. mTOR exists as multi-protein complexes with distinct signaling functions. Complexes containing mTOR and RAPTOR (mTORC1) phosphorylate S6K and 4E-BP1 and are rapamycin sensitive. Whereas, complexes that contain mTOR and RICTOR (mTORC2) phosphorylate AKT and 4E-BP1 and are insensitive to rapamycin treatment. OSI-027 is a selective small molecule dual inhibitor of both mTORC1 and mTORC2 kinase activity with biochemical IC50 values of 22 nM and 65 nM, respectively. In the MDA-MB 231 breast carcinoma xenograft model, a single 65 mg/kg oral dose of OSI-027 resulted in significant inhibition of 4E-BP1 phosphorylation (>70%) up to 16 h in tumor tissue. At 24 h post-dose, 59% inhibition of phospho-4E-BP1 was observed with a corresponding plasma OSI-027 concentration of 2.2 μM. Consistent with its ability to inhibit mTORC2 in vitro, a single dose of 65 mg/kg also resulted in significant inhibition of phospho-AKT (Ser473) for up to 8 hours with slow recovery thereafter. Such extended target suppression at 65 mg/kg was associated with significant efficacy corresponding to 100% tumor growth inhibition (TGI) with 19% regression in the MDA-MB 231 xenograft model when dosed for 14 consecutive days. In comparison, a lower dose of 25 mg/kg resulted in only 39% TGI which corresponded with a shorter duration of pharmacodynamic effects in vivo. In particular, at the 25 mg/kg dose, inhibition of 4E-BP1 phosphorylation was observed only up to 8 h in tumor tissue with return to predose levels by 16 h which corresponded with plasma drug concentrations of <0.1 µM. These data suggest that maximal anti-tumor efficacy may require plasma levels sufficient to chronically suppress phospho-4E-BP1 in tumor tissue. In addition to significant efficacy in MDA-MB 231 breast carcinoma model, OSI-027 demonstrated robust anti-tumor activity in several different human xenograft models representing different histologies, including colon carcinomas, non-small cell lung carcinomas and ovarian carcinomas. These preclinical studies indicate that OSI-027, a dual mTORC1/2 kinase inhibitor, may show broad spectrum anti-tumor activity, including superior efficacy compared to mTORC1-selective rapalogs against some human cancers. OSI-027 is currently being evaluated in Phase I clinical studies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4486.
Abstract 4489: The activity of novel, potent, selective PI3K/mTOR pathway inhibitors in B-cell malignancies
Authors: Jueliger Simone , Maharaj Lenushka , Iyengar Sunil , Ghazaly Essam , Cutillas Pedro , Auer Rebecca , Rommel Christian , Li Liansheng , Gribben John , Vanhaesebroeck Bart , Joel Simon P.
Published: February 2011

Background: Activation of the PI3K pathway is implicated in a number of tumour types, including haematological malignancies. Agents that target this pathway at different levels (e.g. RTK, PI3K, mTOR etc) have shown promising activity in preclinical models and in early phase trials. We have therefore investigated the activity of novel, potent, pan- and isoform-selective PI3K pathway inhibitors in lymphoma and myeloma models. Methods: Compounds studied included those with selectivity for mTOR (INK128, mTOR IC50 1.6 nM), PI3Kδ/γ (INK713, INK1048, IC50s <10 nM for δ/γ isoforms, >50 nM for others) and PI3Kδ/γ/β (INK1138, IC50s <10 nM for each isoform). IC87114 (δ- selective), GDC-0941 (α/δ selective), rapamycin and doxorubicin were included as comparators. Agents were studied in a panel of lymphoma and myeloma cell lines, in normal PBMCs and in primary tumours cultured with stromal cells. Effects on cell viability and proliferation were assessed using the Guava Viacount assay, on apoptosis induction by annexin V labelling, and on cell cycle distribution by flow cytometry. PI3K pathway activity and inhibition was determined by Western blotting as well as mass spectrometry. Results: In cell lines, inhibition of mTOR by INK128 resulted in potent inhibition of cell proliferation, in many at concentrations as low as 10 nM. Selective PI3K inhibitors were less potent anti-proliferative agents, although activity was seen in cells with activation of the PI3K pathway as determined by western blot analysis. For all compounds tested, the major impact was on cell proliferation rather than cell viability. PI3K inhibitors showed little effect on cell viability at concentrations < 1µM. The effect of these compounds in primary mantle cell lymphoma, follicular lymphoma, chronic lymphocytic leukaemia and myeloma samples was variable, both between and within tumour types, possibly due to differences in basal PI3K pathway activity. Effects in normal PBMCs were markedly different to those seen in tumour cells, with a concentration-dependent increase in cell number with either mTOR or PI3K inhibition. Conclusion: Selective PI3K pathway inhibition, particularly at the level of mTOR, results in cytostatic, and at higher concentrations cytotoxic, responses in haematological malignancies. The variable activity of these compounds between cell lines and primary samples suggests it will be important to characterise the activity of the PI3K pathway in individual tumours to identify patients likely to benefit from such agents.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4489.
Abstract 4502: Synthesis and evaluation of curcumin-like compounds as inhibitors of the JAK/STAT pathway in cancer
Authors: Abdelhamid Dalia , Corcoran Mike , Etter Jonathan P. , Hu Sheng , Pandit Bulbul , Li Chenglong , Li Pui-Kai , Fuchs James R.
Published: February 2011

Curcumin is an attractive lead compound for drug discovery and development. It displays a range of remarkable and diverse biological activities due to its interaction with numerous biological targets including the receptor tyrosine kinase Janus Kinase 2 (JAK2) which transduces cytokine and growth factor mediated signals by activating signal transducer and activator of transcription 3 (STAT3). The significant role of JAK-STAT pathway activation in the development, progression, and survival of cancer has been widely recognized since the 1980s. Based on our interest in developing more potent curcumin analogs which act as inhibitors of the JAK-STAT pathway we synthesized a series of monoketone curcumin analogs that bear a strong structural resemblance to AG490 and WP1066; known synthetic small molecule inhibitors of JAK2. A thorough investigation of the SAR of these compounds was pursued to elucidate which parts of the molecules are essential for the anti-proliferative activity. Thus hybrid molecules containing structural motifs found in all three classes of compounds were synthesized and screened using breast, prostate and colon cancer cell lines. Enzyme-linked immunosorbent assay (ELISA) measuring the relative amount of STAT3 phosphorylation was utilized to determine whether the anti-proliferative activity of these compounds was directly related to inhibition of the JAK-STAT pathway, and therefore whether these three classes of compounds exhibit similar cytotoxic mechanisms of action. Our results showed that although the monoketone curcumin analogs displayed potent cytotoxicity; they failed to inhibit STAT3 phosphorylation. The WP1066 analogs also showed potent cytotoxicity correlating directly to STAT3 inhibition. The pyridine and the halogen at the C3 position of the pyridine ring are essential for activity and appear to have a synergistic effect. Longer aliphatic chains at the benzylic position enhanced the cytotoxic activity and aromatic substitution usually led to a decrease in activity. Different structural modifications of the AG490 analogs by protection of the catechol moiety and/or changing the relative position of the substituents did not result in remarkable change in cytotoxic activity. We concluded that although the monoketone curcumin analogs bear remarkable structural resemblance to WP1066 and AG490, they do not inhibit the JAK-STAT pathway and may have a different anti-proliferative mechanism of action.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4502.
Abstract 4516: A new class of MDM2 inhibitors cause growth inhibition and stabilize wt p53 in lymphoma cells but do not interfere with MDM2 E3 ligase activity
Authors: Sosin Angela M. , Burger Angelika M. , Yang Dajun , Mohammad Ramzi M. , Al-Katib Ayad
Published: February 2011

The p53 tumor suppressor protein function is impaired in over 50% of all cancers via inactivating mutations. However, the frequency of p53 mutations is much lower in hematological malignancies. Most lymphomas have wild-type (wt) p53, mutant (mt) p53 is found in less than 30% of all cases. The ubiquitin E3 ligase MDM2 plays a central role in cancer development and progression and is typically overexpressed in lymphomas. MDM2 expression destabilizes functional wt p53 by causing its polyubiquitination and subsequent degradation in the proteasome. Small-molecule inhibitors (SMI) that can block the MDM2-p53 protein-protein interaction and restore p53 function are a promising therapeutic strategy for the treatment of cancers retaining wt p53 that is degraded by overexpression of MDM2. By utilizing two wt p53 and two mt p53 lymphoma cell lines whose p53 status was confirmed by DNA sequencing, we studied the growth inhibitory potential of two different classes of MDM2 SMIs: Nutlin-3, a cis-imidazoline, and MI-219, a new spiro-oxindole MDM2 SMI. Both trypan blue exclusion and MTT assays demonstrated IC50s between 2 and 4 µM in wt p53 cells in vitro. Mutant p53 cells displayed IC50s greater than 10 µM. Growth inhibition was time- and dose-dependent. Additional flow cytometric analyses empolying Annexin V and propidium iodide staining illustrated increasing apoptosis with cells decreasing in G2/M and S phase that also showed time- and dose-dependency. The MDM2 SMI MI-219 showed a much more prominent cell cycle arrest compared to Nutlin-3. Western blotting demonstrated a stabilization of wt p53 and proapoptotic proteins such as p21, Bcl-2, and Bax. In order to determine whether MDM2 SMIs affect the E3 ligase RING-finger domain of MDM2, in vitro autoubiquitination assays were performed using recombinant MDM2 with various concentration of drug. MDM2 autoubiquitination was not affected, even at a concentration as high as 50 µM. These results show that MDM2 autoubiquitination and thus, its degradation remains unaffected in the presence of MDM2 inhibitors that interfere with the p53-MDM2 binding site. This feature of MDM2 SMI is attractive since inhibition of MDM2 autoubiquitination would lead to its stabilization and could negate effects on p53. Our data suggest that MI-219 would be a useful treatment for lymphomas expressing wt p53 and that this should be explored in further preclinical and future clinical studies.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4516.
Abstract 4531: Anticancer potential of curcurbitacin IIa trough STAT3/JAK2-independent, survivin and PARP mediated apoptosis and disruption of actin cytoskeleton
Authors: Lu Qun , Boykin Christi , Zhang Gen , Chen Yan-Hua , Zhang Ren-wei , Fan XE , Yang Weimin
Published: February 2011

The medicinal plant Hemsleya amalils Diels has been used as a homeopathic treatment for various diseases in China for centuries and has recently been indicated as being effective in inhibiting cancer cell growth. To investigate the molecular mechanisms underlying the anti-cancer activity of Hemsleya family, we purified the active component curcurbitacin IIa. Curcurbitacin IIa inhibited cell growth, induced apoptosis in several cancer cell lines, and reduced tumor growth in vivo. Immunofluorescent light microscopy as well as time-lapse imaging demonstrated the remarkable clustering of filamentous actin and the increases in G2/M populations in cell cycle, indicating the disruption of mitosis as the potential mechanism of inducing apoptosis by curcurbitacin IIa. Flowcytometry confirmed increased cell death while western blots showed reduced phospho-histone H3 immunoreactivity, consistent with reduced mitosis. Cleavage of Poly (ADP-ribose) polymerase or PARP, immediate upstream of DNA breakdown as a result of caspase activation, was increased dramatically while AKT phosphorylation and the Inhibitor of Apoptosis Protein survivin expression were reduced. Prostate cancer cells stably transfected with oncoprotein δ-catenin in which survivin expression was increased showed reduced efficacy of curcurbitacin IIa to induce cell death. Combined with lack of its effects on STAT3/JAK2 phosphorylation, theses studies highlight curcurbitacin IIa as a new class of anti-cancer drugs in suppression of cancer cell expansion by disrupting the actin cytoskeleton and directing the cell to undergo apoptosis through inhibition of surviving, AKT, and PARP. Supported by Department of Defense (PC040569) and National Cancer Institute (CA111891).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4531.