bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5380: Disulfiram sensitised cytotoxicity of gemcitabine in human glioblastoma cell lines
Authors: Brown Sarah , Liu Peng , Goktug Tuge , Darling John , Wang Weiguang
Published: February 2011

The blood and brain barrier (BBB) and chemoresistance are the major obstacles for the success of glioblastoma multiforme (GBM) chemotherapy. It has been shown that high nuclear factor kappa B (NFκB) activity is highly related to chemoresistance. Gemcitabine (dFdC) can easily cross the BBB and gets into GBM tissues. Due to the chemoresistance of GBM cells to dFdC induced cytotoxicity, this drug is currently only used as a radiosensitiser for GBM treatment. Disulfiram (DS) is an anti-alcoholism medicine. Our previous study demonstrates that DS strongly inhibits NFκB activity and sensitises cancer cell lines to several anticancer drugs including dFdC. DS can freely pass through BBB. Therefore, we tested the chemosensitising effect of DS on dFdC in three GBM cell lines (U251MG, U87MG and U373MG). DS was cytotoxic to GBM cell lines. Copper (Cu) was essential for DS induced cytotoxicity in GBM cell lines. Cu or DS alone was not cytotoxic to GBM cell lines in vitro. Whereas high cytotoxicity (IC50 = 150 - 250 nM) was demonstrated when DS was dosed in combination with physiological concentration of Cu (1 µM). We also tested the chemosensitising effect of DS/Cu complex on dFdC in GBM cell lines. The GBM cell lines were highly resistant to dFdC induced cell death. DS significantly sensitised the cytotoxicity of dFdC in GBM cell lines (∼ 6 - 23-fold; Table 1). CI-isobologram analysis indicated that the combination effect between DS/Cu and dFdC was highly synergistic (Table 1). Flow cytometric data demonstrated that DS/Cu enhanced dFdC induced apoptosis. The molecular chemosensitising mechanisms of DS/Cu to dFdC in GBM cells are still not full elucidated. Our preliminary data indicated that DS/Cu simultaneously inhibited NFκB pathway and triggered reactive oxygen species (ROS)-JNK pathway.Cytotoxicity of dFdC alone or dFdC plus DS/Cu in GBM cell lines U251MGU87MGU373MGdFdC alone100.4 (22.3)>40078.6 (5.6)dFdC + DS/Cu (1:10 ratio)17.5 (5.1)17.1 (3.0)6.8 (1.8)CI0.390.470.82

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 5380.
Abstract 5381: Curcumin induces chemo/radio-sensitization in ovarian cancer cells and curcumin nanoparticles inhibit ovarian cancer cell growth
Authors: Maher Diane , Yallapu Murali M. , Sundram Vasudha , Bell Maria C. , Jaggi Meena , Chauhan Subhash C.
Published: February 2011

Background: Chemo/radio-resistance is a major obstacle in treating advanced ovarian cancer. The efficacy of current treatments may be improved by increasing the sensitivity of cancer cells to chemo/radiation therapies. Curcumin is a naturally occurring compound with anti-cancer activity in multiple cancers; however, its chemo/radio-sensitizing potential is not well studied in ovarian cancer. Herein, we demonstrate the chemo/radio-sensitizing potential of curcumin in a cisplatin resistant ovarian cancer cell line model. Additionally, due to suboptimal pharmacokinetics and low uptake of curcumin in tumors in vivo, we developed a curcumin nanoparticle formulation to improve its therapeutic efficacy.

Methods: Cisplatin resistant A2780CP ovarian cancer cells were pre-treated with curcumin followed by exposure to cisplatin or radiation and the effect on cell growth was determined by MTS and colony formation assays. The effect of curcumin pre-treatment on the expression of apoptosis related proteins and β-catenin was determined by Western blotting or Flow Cytometry. A luciferase reporter assay was used to determine the effect of curcumin on β-catenin transcription activity. The poly(lactic acid-co-glycolic acid) (PLGA) nanoparticle formulation of curcumin (Nano-CUR) was developed by a modified nano-precipitation method and physico-chemical characterization was performed by transmission electron microscopy and dynamic light scattering methods.

Results: Curcumin pre-treatment considerably reduced the dose of cisplatin and radiation required to inhibit the growth of cisplatin resistant ovarian cancer cells. During the 6 hrs pre-treatment, curcumin down regulated the expression of Bcl-XL and Mcl-1 pro-survival proteins. Curcumin pre-treatment followed by exposure to low doses of cisplatin increased apoptosis as indicated by annexin V staining and cleavage of caspase 9 and Poly (ADP-ribose) polymerase (PARP). Additionally, curcumin pre-treatment lowered β-catenin expression and transcriptional activity. Physico-chemical characterization of Nano-CUR indicated an average particle size of ∼70 nm, steady release of curcumin over a period of 18 days and antibody conjugation characteristics. The Nano-CUR formulation also effectively inhibited the growth of ovarian cancer cells.

Conclusion: Curcumin pre-treatment enhances chemo/radio-sensitization in A2780CP ovarian cancer cells through multiple molecular mechanisms. Therefore, curcumin pre-treatment may effectively improve ovarian cancer therapeutics. A targeted PLGA nanoparticle formulation of curcumin is feasible and may improve the in vivo therapeutic efficacy of curcumin.

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 5381.
Abstract 5390: QBI-139, a human RNase variant in a phase I trial, works in combination
Authors: Strong Laura E. , Kink John A. , Mei Baigen , Shahan Mark N. , Raines Ronald T.
Published: February 2011

The EVade™ Ribonucleases (RNases) are a new class of anti-cancer drugs that target the RNA in cancer cells. These EVade™ RNases are variants of native mammalian RNases that have been converted to anti-cancer agents by diminishing their binding to their natural inhibitor protein.

One of these EVade™ RNases, called QBI-139, has advanced to human clinical trials. QBI-139 shares 95% sequence identity with the wild type human RNase 1. QBI-139 has shown tumor growth inhibition against a variety of cancers, including: colon, non-small cell lung, ovarian, pancreatic and prostate cancers. QBI-139 is currently in a Phase I clinical trial in patients with solid tumors. A frog RNase called Ranpirnase (Alfacell) that shares only 20% sequence identity with human RNase 1 has been in clinical trials and responses have been seen in esophageal, non-small cell lung and breast cancers as well as unresectable mesothelioma.

The next stage of clinical development of QBI-139 will require selection of a specific indication as well as an existing treatment regimen to be used in combination with QBI-139. The Phase I trial may provide insights about sensitive tumor types. However, mouse models of cancer will be used to select the combinations for development.

QBI-139 has shown efficacy against non-small cell lung cancer (A549 cell line) in xenograft models. Cisplatin is a standard of care treatment for non-small cell lung cancer in patients. The ability of QBI-139 and cisplatin to work together to cause tumor growth inhibition was determined in a non-small cell lung cancer xenograft model (A549 cell line). Cisplatin (3 mg/kg) and QBI-139 (15 mg/kg) were used as controls. The combination of cisplatin and QBI-139 (3 and 15 mg/kg, respectively) was also tested. Cisplatin treatment resulted in tumor growth inhibition (TGI) of 60% while QBI-139 caused 76% TGI. The combination of the two drugs resulted in a TGI of 96%, strongly supporting the use of QBI-139 in combination with cisplatin for the treatment of lung cancer. Even the combination of the two drugs exhibited minimal impact on the health of the animals.

EVade™ Ribonucleases are novel agents that provide a new way to target the RNA in cancer cells and are an innovative new tool for oncologists. QBI-139 has a variety of benefits, including: a novel mechanism of action with clinical support, broad in vivo anti-tumor activity alone and in combination with standard of care therapies and a strong tolerability profile.

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 5390.
Abstract 540: Lactate as a mediator of mesenchymal stem cell interactions within the tumor microenvironment
Authors: Rattigan Yanique I. , Martin Alister F. , Glod John , Banerjee Debabrata
Published: February 2011

Mesenchymal stem cells (MSCs) are multipotent stromal precursor cells that have the capacity to migrate, proliferate, differentiate and contribute in vivo to the hypoxic tumor stroma. Cancer cells primarily depend on the anaerobic breakdown of glucose to generate ATP, even in the presence of oxygen. A consequence of this altered metabolic phenotype of cancer cells is the accumulation and secretion of lactate. Recent reports suggest that lactate produced by cancer cells may be taken up by stromal cells and recycled to regenerate pyruvate. Interestingly, stromal cells can efflux pyruvate to serve as a power supply to sustain the metabolic needs of cancer cells. Here we provide evidence that MDA-MB-231 and MCF-7 breast cancer cell lines secrete enhanced levels of lactate (2-fold) under hypoxia (1.5% O2) as compared to normoxia (21% O2). Analysis of MSC migration, showed that 15mM lactate enhanced MSC migration over 3-fold compared to control medium. Lactate stimulation of MSCs induced expression of a functional monocarboxylate transporter MCT-1, one of the proteins involved in lactate transport, at both mRNA and protein levels. Furthermore, activation of Stat3 signaling leading to enhanced MSC migration and NF-[[Unable to Display Character: ĸ]]B regulated survival pathways following lactate stimulation was observed suggesting that lactate plays a role in the metabolic fate of cancer cells through the recruitment of MSCs. Delineating the interaction between MSCs, lactate and the tumor microenvironment will be fundamental in understanding tumor cell metabolism and provide knowledge for the development of novel anticancer therapies.

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 540.
Abstract 5405: PPARγ-agonist can render bladder tumor sensitive to EGFR inhibition
Authors: Mansure Jose J. , Szymanski Konrad , Nassim Roland , Rocha Joice , Aldousari Saad , Chevalier Simone , Kassouf Wassim
Published: February 2011

Abstract

Purpose: The two signalling molecules that are extremely attractive for targeted therapy are the epidermal growth factor receptor (EGFR) and the peroxisome proliferator-activated receptor γ (PPARγ). We evaluate the integration of combined drugs against these targets in the management of bladder cancer therapy.

Methods: The effect of EGFR inhibitor (Gefitinib) and PPARγ-agonist DIM-C, on cell growth, were screened in a panel of 9 human urothelial carcinoma cell lines derived from well-differentiated superficial bladder tumors as well as from high-grade invasive tumors. Cell proliferation was determined with an MTT assay after incubation with varying concentrations of the targeted agents (10-3 to 102 mM) for 72 h. Antiproliferative effects of combined therapy (Gefitinib and DIM-C) compared to each drug alone were monitored in vitro, by MTT assays. Levels of expression of EGFR and PPARγ were evaluated by Western Blot analysis at baseline and after pre-treatment with EGFR inhibitor (Gefitinib). Immunofluorescence was then used to determine PPARγ nuclear accumulation mediated by Gefitinib. In vivo, the effect of Gefitinib, DIM-C alone and combined were determined in nude mice bearing human KU-7 subcutaneous xenografts.

Results: Several bladder cancer cell lines were demonstrated to be relatively resistant to Gefitinib. Induction of PPARγ expression was observed in response to different concentrations of Gefitinib for 24 h. Moreover, nuclear accumulation of PPARγ was observed following its upregulation. MTT assay shows that PPARγ agonist significantly sensitized bladder cancer cell lines that were resistant to EGFR inhibition in a schedule-specific manner. These findings were confirmed in vivo where combination therapy maximally inhibited tumor growth in contrast to each treatment alone when compared to control (p<0.04).

Conclusions: Preliminary results suggest that PPARγ-agonist DIM-C can render bladder tumor sensitive to EGFR inhibition and combination efficacy might be achieved in a schedule-specific manner.

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 5405.
Abstract 5407: Inhibitory Raf-1 phosphorylations at Ser-43 and Ser-259 are induced in vitamin K1 enhancement of Sorafenib-mediated HCC cell growth inhibition
Authors: Wang Ziqiu , Wang Meifang , Carr Brian I.
Published: February 2011

Sorafenib, a multi-kinase inhibitor, is an FDA-approved agent for treatment of human hepatocellular carcinoma (HCC). However, tumor shrinkage is minor. K vitamins can activate protein kinase A (PKA), which in turn can mediate inhibitory Raf phosphorylation. We developed a new strategy to combine naturally occurring K vitamins with Sorafenib to treat HCC, and found that K vitamins were able to enhance the inhibition of HCC cell growth in vitro and in vivo by Sorafenib. To explore the mechanisms involved, we examined the role of Raf-1 kinase, because it has been reported that Sorafenib inhibits tumor cell growth as a direct inhibitor of Raf, whereas vitamin K2 can induce PKA activity, which is a Raf-1 upstream kinase. We found that while lower doses of vitamin K1 (10 μM) or Sorafenib (2.5 μM) alone slightly induced Raf-1 phosphorylation at both Ser-43 and Ser-259 in Hep3B cells, combination vitamin K1 plus Sorafenib treatment resulted in strong Raf-1 phosphorylation at these two Raf-inhibitory serine residues. Raf-1 protein kinase activity assay showed that lower concentrations of vitamin K1 or Sorafenib alone had little, if any effects on Raf-1 activity, combination vitamin K1 plus Sorafenib at the same low concentrations significantly inhibited Raf-1 activity, judged by strong inhibition of MEK, and of ERK phosphorylation, its down stream targets. Since Raf-1 phosphorylation at Ser-43 and Ser-259 can be regulated by either PKA or Akt kinase, we examined the effects of both vitamin K1 and Sorafenib on their phosphorylation. Although both vitamin K1 and Sorafenib alone induced PKA phosphorylation, no synergistic phosphorylation effects on PKA were found using the combination. However, vitamin K1 enhanced Sorafenib-induced Akt phosphorylation, which was associated with enhanced c-Met tyrosine phosphorylation at Tyr-1349, a known phospho-Akt regulator. Our data suggest that enhanced inhibitory Raf-1 phosphorylation at Ser-43 and Ser-259 may play a central role in vitamin K1 plus Sorafenib synergy in inhibiting HCC cell growth.

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 5407.
Abstract 5421: Targeting glucose and glutamine for the metabolic management of systemic metastatic cancer
Authors: Shelton Laura M. , Huysentruyt Leanne C. , Seyfried Thomas N.
Published: February 2011

Metastatic cancer is a major cause of morbidity and mortality. Current therapeutic options consist of chemotherapy, radiation, or targeted therapies. However, these therapies are often toxic, effective over a small range of cancer types, or result in drug resistance. Therefore, a more global, less toxic strategy for the management of metastatic cancer is required. Though most cancers display increased glucose metabolism, glutamine is also a major energy substrate for many cancers. We evaluated the anti-metastatic potential of 6-diazo-5-oxo-L-norleucine (DON), a glutamine analog, using the new VM mouse model of systemic metastasis. In addition we evaluated the combined inhibition of glucose and glutamine metabolism by treating mice with both DON, which targets glutamine, and calorie restriction (CR), which reduces circulating glucose levels. We found that primary tumor growth was about 20-fold less in DON treated mice than in untreated control mice. We also found that DON treatment administered alone or in combination with CR inhibited metastasis to liver, lung, and kidney as detected by bioluminescence imaging and histology. Although DON treatment alone did not reduce the incidence of tumor metastasis to spleen compared to the controls (68% controls, 50% DON treatment), DON administered together with CR significantly reduced the incidence of metastasis to the spleen to 27%, indicating a diet/drug synergy. Furthermore, DON toxicity was less in the DON + CR group than in the DON alone group. Less DON was needed to achieve the same level of tumor growth inhibition when administered with CR than administered alone. Finally, survival was greater in the DON + CR group than in the DON group alone further illustrating drug/diet synergy. Our findings provide proof of concept that metabolic therapies targeting both glucose and glutamine metabolism can manage systemic metastatic cancer. (Supported by Boston College REG).

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 5421.
Abstract 5423: Effects of atorvastatin, celecoxib and tipifarnib alone or in combination on the growth of pancreatic cancer in Panc-1 cells cultured in vitro or Panc-1 tumors grown in immunodeficient mice
Authors: Zheng Xi , Cui Xiao-Xing , Gao Zhi , Shi Yi , Rabson Arnold , Conney Allan H.
Published: February 2011

Ras mutation and overexpression of cyclooxygenase-2 (Cox-2) are present in the majority of pancreas cancer patients. An approach for inhibiting the progression and growth of pancreas cancer is the use of agents that inhibit the function of both Ras and COX-2. In the present study, we assessed the effects of atorvastatin, celecoxib and tipifarnib alone or in combination on the growth of human pancreatic cancer Panc-1 cells cultured in vitro or grown as xenograft tumors in severe combined immunodeficient (SCID) mice. In the in vitro studies, we found that treatment of Panc-1 cells with a combination of atorvastatin, celecoxib and tipifarnib had a stronger inhibitory effect on proliferation and a stronger stimulatory effect on apoptosis than each drug alone or for any combination of two drugs. We also found that treatment of Panc-1 cells with a combination of the 3 drugs decreased the level of phosphorylated Erk1/2 and Atk to a much greater extent than the individual compounds alone or any combination of two compounds. In animal experiment, we found that daily i.p injections of a combination of atorvastatin, celecoxib and tipifarnib had a stronger inhibitory effect on the growth of Panc-1 tumors in SCID mice than each drug alone or for any combination of two drugs. The results from our study indicate that a combination of atorvastatin, celecoxib and tipifarnib may be an effective strategy for the treatment of pancreatic cancer.

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 5423.
Abstract 5445: Suberoylanilide hydroxamic acid induces Epstein-Barr virus lytic cycle and enhanced apoptosis in nasopharyngeal carcinoma
Authors: Hui Kwai Fung , Tsao George Sai-Wah , Chiang Alan Kowk-Shing
Published: February 2011

Nasopharyngeal carcinoma (NPC) has uniquely high prevalence in Southern Chinese populations and a predilection to affect young adult males. Epstein-Barr virus (EBV) is harbored in every tumour cell of NPC but it persists in tightly latent form expressing few viral proteins and evading the immune system. Induction of EBV lytic cycle will lead to expression of a much larger number of viral proteins which may serve as potential therapeutic targets against the cancer. We reported that suberoylanilide hydroxamic acid (SAHA), a FDA-approved histone deacetylase inhibitor, could strongly induce viral lytic cycle in EBV-positive gastric carcinoma cells. Here, we demonstrated that SAHA (at 5-10 μM) could potently induce EBV lytic cycle in a panel of EBV-positive NPC cell lines, as evidenced by increased viral DNA replication, expression of immediate early (Zta and Rta), early (BMRF1) and late (VCA-p40 and gp350/220) viral lytic proteins and production of infectious viral particles. Immunofluorescent staining demonstrated expression of viral lytic proteins in more than 40% of the NPC cells. Enhanced killing of EBV-positive NPC cells when compared with that of EBV-negative counterparts was observed and cleaved form of PARP was predominantly expressed in EBV-positive than EBV-negative NPC cells, following treatment with SAHA for 24, 48 and 72 hours. The kinetics of expression of cleaved PARP correlated with that of Zta viral protein in EBV-positive NPC cells. In conclusion, SAHA is found to be a potent EBV lytic cycle inducing agent and mediates enhanced apoptosis of NPC cells. Further investigation into SAHA's development as a novel virus-targeted therapeutic drug in NPC is warranted.

This work is funded by EBV research and NPC AoE grants of AKSC.

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 5445.
Abstract 5447: A novel inhibitor of DNA methylation
Authors: Malik Mohammad T. , Rinaldo Francesca M. , Mitra Shams , Casson Lavona K. , Zacharias Wolfgang , Waigel Sabine , Arumugam Vennila , Hasan Nazarul , Hu Chuan , Bo Xu , Hammond Gerald B. , Bates Paula J.
Published: February 2011

We previously reported the discovery of XB05, a synthetic small molecule with antiproliferative activity. XB05 was found to be a strong inhibitor of DNMT1 activity in cell-free and cell-based assays. However, screening in the NCI 60 tumor cell lines and analysis using COMPARE indicated a novel mechanism of action for XB05, different from standard demethylating agents such as 5-azacytidine (azaC) and decitabine. Here, we report on new research to further characterize XB05 activity. We have examined the effects of XB05 on clonogenicity, promoter methylation, gene expression, and DNMT1 protein. XB05 was found to inhibit colony formation of HCT116 colon carcinoma cells in a dose-dependent manner with a sub-micromolar IC50. Western blots of nuclear extracts from HCT116 cells treated for 72 h with 100 nM XB05 or 10 µM azaC (as positive control) showed greatly decreased levels of DNMT1, suggesting that binding to XB05 can induce degradation of DNMT1 (previous research indicates direct drug-enzyme binding). Analysis of CDKN2A promoter methylation by bisulfite modification and sequencing demonstrated heavy methylation in untreated HCT116 cells. This was reversed by treatment of with 100 nM XB05 or 10 µM azaC, resulting in re-expression of the gene (which encodes tumor suppressor, p16), as shown by qRT-PCR. Experiments to assess promoter methylation using multiplex arrays and to examine gene expression changes using DNA microarrays were recently completed and data analysis is ongoing. To evaluate in vivo activity, we treated mice bearing subcutaneous HCT116 xenografts by IP injection of XB05 with multiple doses of up to 10 mg/kg. There was no evidence of toxicity as judged by body weights and gross necropsies. The tumors of mice treated with XB05, although not significantly smaller than in control animals, were characterized by extensive necrosis in the center of the tumor, leading to a “hollow” tumor in the majority (12/20) of mice. No similar necrosis was observed in vehicle-treated or azaC-treated mice, suggesting that this effect is treatment related. Because induced necrosis is seen with vascular disrupting agents, we also examined the effect of XB05 on endothelial cells in an in vitro assay, and observed reproducible inhibition of endothelial tube formation at 400 nM or higher. In summary, our new data confirm that XB05 is a potent inhibitor of DNA methylation in cultured cancer cells, and provide the first evidence of in vivo activity, which may involve effects on the tumor vasculature, as well as the tumor itself.

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 5447.
Abstract 5448: The tricyclic anti-depressant amitriptyline inhibits D-cyclin transactivation and induces myeloma cell apoptosis by inhibiting histone deacetylases
Authors: Mao Xinliang , Wang Wenjie , Cao Biyin , Li Zhihua , Hurren Rose , Trudel Suzanne , Schimmer Aaron D.
Published: February 2011

D-cyclins are universally dysregulated in multiple myeloma and contribute to the pathogenesis and chemoresistance of this disease. To identify novel pharmaceutical inhibitors of cyclin D transactivation, we compiled and screened a library of on-patent and off-patent drugs using NIH 3T3 cells stably expressing the Cyclin D2 promoter-driving a luciferase reporter gene. From this screen we identified the tricyclic antidepressant amitriptyline as a novel inhibitor of Cyclin D2 transactivation. In secondary assays, Amitriptyline reduced expression of cyclins D1, D2, and D3 in myeloma cell lines at low micromolar concentrations across a variety of transforming events including c-maf over-expression (JJN3, KMS11, My5 cells), FGFR3 translocation (KMS11, OPM2 cells), and cyclin D1 translocation (KMS12 cells). Consistent with its effects on D-cyclin expression, amitriptyline arrested cells in the G0/G1 phase of the cell cycle at concentrations associated with its ability to decrease D-cyclins. Decreased cyclin D expression and G1 arrest can induce apoptosis. Therefore we tested the effects of amitriptyline on cell viability. Myeloma cell lines were treated with increasing concentrations of Amit and cell viability was measured by the MTS assay. Amitriptyline reduced the viability of myeloma cells with an IC50 < 20 uM within 72hr. In addition, the combination of amitriptyline and dexamethasone synergistically induced cell death. Cell death and apoptosis were confirmed by Annexin V and PI staining and were associated with activation of Caspase-3 and Poly(ADP-ribose)Polymerase (PARP) as detected by immunoblotting. To understand the mechanism by which amitryptiline inhibits D-cyclin transactivation and induces cell death, we examined the effects of this compound on HDAC levels and activity. By immunoblotting, treatment with amitriptyline increased levels of acetyl-Histone 3 in myeloma cell lines. Furthermore amitriptyline decreased levels of HDACs 3, 6, 7 and 8 in examined cell lines with a concentration consistent with the cell death and cyclin D2 inhibition.

Thus, in summary, amitriptyline inhibits D-cyclin transactivation in myeloma cells that is associated with reductions in HDAC levels. Given the preclinical activity of amitriptyline in myeloma, this work highlights a potential therapeutic strategy for this disease.

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 5448.
Abstract 5452: Cancer-associated metabolite 2-hydroxyglutarate accumulates in AML with IDH1/2 mutations
Authors: Fantin Valeria R. , Gross Stefan , Cairns Rob A. , Minden Mark D. , Driggers Edward M. , Jang Hyun Gyung , Sasaki Masato , Jin Shengfang , Schenkein David P. , Su Shinsan M. , Dang Lenny , Mak Tak W.
Published: February 2011

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2), are present in most gliomas and secondary glioblastomas, but are rare in other neoplasms. IDH1/2 mutations are heterozygous, and affect a single arginine residue. Recently, IDH1 mutations were identified in 8% of acute myelogenous leukemia (AML) patients. Our previous study revealed that IDH1 mutations cause a gain of function, resulting in the production and accumulation of 2-hydroxyglutarate (2-HG). Genotyping of 145 AML biopsies identified 11 IDH1 R132 mutant samples. Liquid chromatography-mass spectrometry metabolite screening revealed increased 2-HG levels in IDH1 R132 mutant cells and sera, and uncovered two IDH2 R172K mutations. IDH1/2 mutations were associated with normal karyotypes. Recombinant IDH1 R132C and IDH2 R172K proteins catalyze the novel NADPH -dependent reduction of alpha-ketoglutarate (a-KG) to 2-HG. The IDH1 R132C mutation commonly found in AML reduces the affinity for isocitrate, and increases the affinity for NADPH and a-KG. This prevents the oxidative decarboxylation of isocitrate to a-KG, and facilitates the conversion of a-KG to 2-HG. IDH1/2 mutations confer an enzymatic gain of function that dramatically increases 2-HG in AML. This provides an explanation for the heterozygous acquisition of these mutations during tumorigenesis. 2-HG is a tractable metabolic biomarker of mutant IDH1/2 enzyme activity.

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 5452.
Abstract 5466: nNOS, a novel target for melanoma prevention and therapy
Authors: Yang Sun , Yang Zhen , Meyskens Frank L.
Published: February 2011

It is well recognized that nitric oxide (NO) is involved in tumor progression, including human melanoma. Our data showed that NO stress by NO donor DETA treatment remarkably stimulated the proliferation of normal primary melanocytes with foci formed in culture and the metastatic capacity of human melanoma cells detected by matrigel invasion assays was also significantly induced with DETA incubation. Our previous mechanistic studies have revealed that NO-induced melanoma progression is mediated by Apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE/Ref-1), which plays an important role in melanomagenesis and development, in a feed-forward loop manner. Both our in vitro cell culture (Immunoblotting) and in vivo human biopsy (Immunohistochemistry) studies have demonstrated the predominant elevations of nNOS expression levels in melanoma cells. Notably, induction of nNOS was evident with UVB radiation and growth factor bFGF treatments. Knockdown of nNOS with siRNA efficiently reduced the DETA-induced melanoma proliferation and invasion potential, and was associated with reductions of many genes that related to proliferation, anti-apoptosis and invasion, such as c-Jun, Bcl-2, and MMP-1. A novel synthesized nNOS inhibitor JI-11 was utilized to inhibit nNOS activity and our data showed that co-treatment of JI-11 at 1uM concentration significantly attenuated the alterations induced by UVB radiation and DETA treatments. MTS analysis also revealed that pretreatment of JI-11 sensitized melanoma cells to chemo-drug Cisplatin. We hypothesize that targeting nNOS/NO to prevent and treat melanoma is innovative and potentially of great benefit.

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 5466.
Abstract 5467: Breflate upregulates TRAIL gene transcription in Bax-null cells and induces TRAIL-mediated apoptosis independently of p53 in TRAIL-sensitive tumor lines
Authors: Allen Joshua E. , Patel Luv , Krigsfeld Gabriel , Mayes Patrick A. , Wu Gen Sheng , El-Deiry Wafik S.
Published: February 2011

TNF-related apoptosis-inducing ligand (TRAIL) has been hailed as a promising cancer drug target due to its apoptosis-inducing capabilities which are aimed selectively at cancer cells. Methods of inducing endogenous TRAIL as well as introducing exogenous recombinant human TRAIL (rhTRAIL) are being explored in preclinical studies and in the case of the latter, clinical trials. While the gene encoding for DR5, a TRAIL death receptor, has been known to be regulated by p53, we have recently shown that TRAIL is regulated by p53 at base pairs 504-1102 of its promoter (Kuribayashi et al., Cancer Biology and Therapy. 7:2034-2038, 2008). p53 is mutated in over half of all cancers; therefore, a method of upregulating endogenous TRAIL via a mechanism independent of p53 holds great potential therapeutic value for cancer. We searched for small molecule p53-independent upregulators of TRAIL using a luciferase reporter assay which utilized a plasmid with the gene encoding for firefly luciferase under the transcriptional control of the first 504 base pairs of the human TRAIL promoter. Screening with the NCI Diversity Set II library yielded several hits with >2-fold TRAIL induction which were repeated and further analyzed for use as a drug and to gain mechanistic insight. Interestingly, one hit from this screen was breflate, the prodrug of Brefeldin A (BFA). BFA inhibits retrograde protein transport from the Golgi apparatus to the endoplasmic reticulum and is currently being investigated as a cancer therapeutic. Shao et al. (Exp. Cell Res. 227:190-196, 1996) have previously reported that BFA is a potent inducer of apoptosis in some cancer cell lines independently of p53, though the mechanistic relationship was not clear. Breflate clearly increased intracellular levels of TRAIL in HCT116 Bax−/− cells as determined by Western blot analysis, and in addition to the original reporter assay this is being corroborated by reverse transcriptase polymerase chain reaction (RT-PCR). Moreover, sub-G1 analysis found that breflate induced levels of apoptosis >2-fold relative to control levels at 10μM after 24 hours in HCT116 p53−/− cells. Further mechanistic and ongoing in vivo studies are being conducted with breflate alone and in combination with chemotherapy or targeted cancer therapy.

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 5467.
Abstract 5473: Antiproliferative and antimetastasic activity of a novel vasopressin V2 receptor peptide agonist in a breast cancer model
Authors: Ripoll Giselle V. , Garona Juan , Iannucci Nancy , Orlando Ulises , Cascone Osvaldo , Podestá Ernesto , Gomez Daniel E. , Alonso Daniel F.
Published: February 2011

Desmopressin (1-deamino-8-D-arginine vasopressin) is a synthetic analog of vasopressin with antimetastatic properties. The molecule is a well known and safe hemostatic compound that acts as a selective agonist for the vasopressin V2 membrane receptor, which is expressed in endothelium and also in some tumor cells. Previous data revealed that perioperative administration of desmopressin can minimize the spread and survival of residual cancer cells. This cyclic nonapeptide was substituted in positions 4 and 5 searching for compounds with improved biological activity and half life. Such positions belong to the disulphide loop (between positions 1-6), which have a major interaction with its receptor. In this work, we evaluated the in vitro biological activity of a novel analog designated VQ (1-deamino-4-valine-5-glutamine-8-D-arginine vasopressin) in MCF7 human mammary carcinoma cells, as well as its in vivo antitumor action in combination with chemotherapy using the syngeneic F3II mammary carcinoma model in Balb/c mice. Because V2 receptor signaling has been associated with cytostatic effects, we have examined peptide ability to modify in vitro cell proliferation of MCF7 cells expressing V2 receptor. Incubation with VQ analog (100-1500 nM) during 72 h significantly reduced cell proliferation (p<0.001) showing a better performance than desmopressin at higher doses of 500-1500 nM (p<0.05). Inhibition of cell proliferation was associated with an increase of intracellular cAMP levels and this was correlated with PKA activation in MCF7 cells. A similar antiproliferative profile was obtained with loop tetrapeptides (aminoacid positions 2-5) at low doses (<500 nM), although cytostatic activity of tetrapeptides was lost at high concentrations. Three weekly i.v. doses of VQ (0.3 µg/kg/dose) reduced by 40% the subcutaneous tumor volume in mice implanted with F3II mammary carcinoma cells, while VQ treatment plus weekly i.p. cycles of carmustine (20 mg/kg/cycle) was even more effective (p<0.001). Furthermore, VQ treatment, either in combination or not with carmustine cycles, prevented lung metastatic progression in tumor-bearing mice. VQ also inhibited F3II lung colonization in the experimental metastasis assay. Our results indicate that the novel cyclic peptide analog VQ can exert a remarkable antitumor and antimetastatic effect in a preclinical model of breast carcinoma. Loop conformation of the peptide seems to be essential for interaction with V2 receptor and for the biological activity of the compound.

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 5473.
Abstract 5479: Glycogen synthase kinase-3 (GSK3) differently targets in MLL leukemia cells
Authors: Hur Eun-Hye , Kim Dae-Young , Kang Mun-Jung , Lee Je-Whan
Published: February 2011

Propose: We evaluated the anti-leukemic effect of GSK3 inhibitor for specific type of MLL-tranlocated cell line.

Materials and methods:

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GSK3 inhibitor: LiCl, SB216763, GSK3-IX

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Cell line: MV4-11 [human lymphoblast, t(4:11)]

MOLM-13 [human acute myeloid leukemia, ins(9:11)(q23;p22p23)]

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Cell proliferation assay: XTT assay, Clonogenic assay

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GSK3i activity & detection of histone acetylation and H3K4 methylation: Immunoblotting

Summary:

MLL gene encodes an enzyme which methylates histone H3 DNA-binding protein and positively regulates the expression of target genes in normal tissue. MLL rearrangement which is found in acute myeloid leukemia (AML), especially therapy-related AML, encodes fusion proteins that have lost H3 lys4-specific (H3K4) methyltransferase activity, and causes a transformation of hematopoietic cells into leukemia stem cells.

GSK3, a serine/threonine kinase, plays a key role in glycogen metabolism and is constitutively expressed in resting cell. Although it has been thought that the inhibition of GSK3 causes apoptosis in neurons and other tissues, a paradoxical and unexpected cell death effect of GSK3 inhibition were reported recently in leukemic cell line with MLL translocation, especially t(4;11).

Regarding the mechanism of the cytotoxic effect of GSK3 inhibitor, various molecules associated with the signaling pathway around MLL gene have been investigated. Acetylation of H3 peptides stimulates the activity of SET domain in MLL. MLL gene is cleaved at 2 conserved sites by taspase-1; N-terminal 320 kD fragment (N320) and a C-terminal 180 kD fragment (C180). RNA interference-mediated knockdown of taspase-1 resulted in an unprocessed MLL, and finally a loss of proper HOX gene expression. However, the exact mechanism has been still unknown and it remains to be uncertain whether GSK3 inhibitor shows cell death effect on various leukemic cell lines of MLL translocation with other partner gene.

We intended to determine whether the inhibition of GSK3 induced cell death in different types of leukemia cell lines with MLL translocation. We examined the apoptotic effects of GSK3 inhibitors on MV4-11 and MOLM-13 cell line. LiCl and GSK3-IX induced cell death, H3K4 methylation and histone acetylation in dose-dependent manners on MV4-11 but did not induce on MOLM-13. However, SB216763 did not induced cell death both cell lines.

Conclusion: Cell death effects with the inhibition of GSK3 may be different among leukemic cell line with MLL translocation according to the partner gene of MLL translocation.

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 5479.
Abstract 5483: Targeted intracellular photodynamic therapy (TIP) reduces tumor mass and prolongs survival in a mouse model of cancer
Authors: Luboshitz Galia , Raisa Laptev , Arad-Yellin Rina , Green Bernard G. , Firer Michael A.
Published: February 2011

Targeted Intracellular Photodynamic Therapy (TIP) is a novel advance on traditional photodynamic therapy (PDT) in which photosensitizer activation is afforded by chemiluminescence rather than by laser beam. This advance allows PDT access to target cells essentially anywhere in the body. TIP also has advantages over bioluminescence as there is no need for transfection of cells with enzymes such as luciferase. It can therefore be more easily applied both in vitro and in vivo and has potential as a new targeted cancer therapeutic modality that could be applied to a wide variety of cancers. We have previously described in vitro the efficacy and specificity of TIP toward murine leukemic cells (Brit. J. Cancer, 95; 189-196, 2006). We now report that TIP is effective in vivo using a murine model of cancer.

To target the photosensitizer, Protoporphyrin IX was conjugated to Transferrin (Trnsf-PrP). Balb/c mice were injected subcutaneously with the mouse hybridomas U13.6 or U7.6, both of which overexpress the Transferrin receptor CD71. The use of Trnsf-PrP resulted in rapid uptake of photosensitizer into cells and conjugate was retained for over 48hrs. Animals were treated with TIP by injecting Trnsf-PrP and the chemiluminescent agent Luminol (LM) under reduced light conditions. Experiments were performed whereby different parameters of the system were varied, including: time of treatment initiation after tumor cell inoculation; dose and number of Trnsf-PrP and luminol treatments; and time intervals between treatments. Initial experiments in which TIP was performed at the time of tumor inoculation showed that this procedure completely abolished tumor growth. In subsequent experiments where tumors were palpable before treatment began, various treatment protocols were tested. TIP reduced tumor mass by over 40% and increased survival by 50%. These results demonstrate that TIP is active against cancer cells in vivo and encourages the further development of the modality as a targeted cancer therapy.

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 5483.
Abstract 5486: Developing homing aptamers for targeted therapy in neuroblastoma
Authors: Barman Jharna , Beckstead Wesley , Song Young , Wei Jun , Metaferia Belhu , Khan Javed
Published: February 2011

Neuroblastoma is the most common extracranial solid tumour in children. It accounts for 7-10% of malignancies in patients younger than 15 years and 15% of childhood oncology deaths. Each year, there are about 650 new cases of neuroblastoma occurring in the US. The survival rate is 70 - 95 % for low stage (stage 1, 2, 3) tumors. Unfortunately more than 50% of the patients present with advanced stage and the overall survival rate is less than 40% (less than 30% of the children over 1 year old) despite current aggressive multimodal therapies. We have reached the maximal tolerated dosage for most of the chemotherapeutic agents currently used to treat neuroblastoma. Therefore, better targeted therapeutic agents are required to reduce the toxicity related to the therapies. To identify specific targeting agents for neuroblastoma cells, we have undertaken nucleic acid based approach, known as aptamers.

Aptamers are short single stranded nucleic acid ligands which can bind specifically to target protein or small molecules because of their unique 3D structures. Low or no immunogenicity together with high specificity makes aptamers potential candidates for therapeutic agents. In our study we have applied Systematic Evolution of Ligands by EXponential Enrichment (SELEX) to isolate RNA aptamers that target neuroblastoma. A modified RNA pool containing a theoretical 109 unique molecules was generated from a synthetic combinatorial library of nucleic acids, by using 2′-F pyrimidine and 2′-OH purine nucleotides in order to increase the stability toward nucleases.

We performed 13 rounds of selection against one of the aggressive stage 4 neuroblastoma cell lines, SK-NA-S. After preliminary analysis we have identified 15 modified RNA sequences and performed binding affinity studies by labeling those sequences with 33P. The apparent dissociation constant (Kd) values are in nanomolar range.

The aptamers with the lowest Kd values are chosen for further study in order to visualize the site of binding as well as to identify the target protein they are binding to. Once a neuroblastoma specific aptamer is verified, it will be evaluated for targeted therapeutics in neroblastoma by conjugating it with the polymerized liposomal nanoparticles containing small molecule 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 5486.