Christina L. Rotoloni

Prevention of Barrett esophagus and esophageal adenocarcinoma by smoothened inhibitor in a rat model of gastroesophageal reflux disease.

Background:Activated hedgehog (Hh) pathway is associated with development of both Barrett esophagus (BE) and esophageal adenocarcinoma (EAC). We hypothesize that blockade of the Hh pathway with smoothened (Smo) inhibitor can prevent the development of BE/EAC in the Levrat model, in which induced gastroduodenoesophageal reflux (GDER) leads to esophageal carcinogenesis. Methods:GDER was induced in 6- to 8-week-old male Sprague-Dawley rats. The Smo inhibitor (10 mg/kg/d) was given orally on postoperative weeks 10 to 16, 18 to 22, and 24 to 28, and rats were killed on week 28. The primary outcome measure was the incidence of BE and EAC. To examine potential therapeutic effects of Smo inhibition on tumor tissue, semiquantitative immunohistochemistry for Ki-67 and caspase 3 was performed. In treated animals that developed cancer, gene expression was analyzed. Results:Thirty-eight of 48 controls and 32 of 46 treated animals survived to 28 weeks. messenger ribonucleic acid (mRNA) expression of Indian Hh, a ligand of transmembrane receptor patched 1, was 184× higher in BE and 99× higher in EAC compared with normal esophageal tissue (P = 0.0239 and P = 0.0004, respectively). Compared with controls, the incidence of BE and EAC was decreased in treated animals by 35.7% (relative risk reduction, 36%; P = 0.0015) and 36% (relative risk reduction, 62%; P = 0.0033), respectively. Compared with untreated EAC, Ki-67 was downregulated (P = 0.04) and cleaved caspase 3 was no different in treated EAC (P = 0.398). Of the 84 well-known genes involved in cancer drug resistance, 50 were dysregulated in treated EAC (P < 0.05 for each gene). Conclusions:Smo inhibitor prevents the development of BE and EAC in an in vivo model of GDER.

Smoothened Inhibition Leads to Decreased Proliferation and Induces Apoptosis in Esophageal Adenocarcinoma Cells

The Hedgehog (Hh) pathway is known to be active in Barretts carcinogenesis. Therefore, we evaluated the efficacy and underlying mechanisms of inhibition of cancer cell growth by the smoothened (Smo) antagonist BMS-833923 in esophageal adenocarcinoma (EAC) cell lines. Cell proliferation and apoptosis were evaluated by flow cytometry, Western blotting, immunofluorescence, and quantitative reverse transcription polymerase chain reactions. Results showed that the Smo antagonist led to reduced Hh pathway activity, resulting in decreased cell proliferation and induction of apoptosis via the intrinsic pathway in the esophageal cancer cells. In conclusion, the Smo antagonist may have application as an EAC chemotherapeutic agent.

Prognostic value and targeted inhibition of survivin expression in esophageal adenocarcinoma and cancer-adjacent squamous epithelium.

Background Survivin is an inhibitor of apoptosis and its over expression is associated with poor prognosis in several malignancies. While several studies have analyzed survivin expression in esophageal squamous cell carcinoma, few have focused on esophageal adenocarcinoma (EAC) and/or cancer-adjacent squamous epithelium (CASE). The purpose of this study was 1) to determine the degree of survivin up regulation in samples of EAC and CASE, 2) to evaluate if survivin expression in EAC and CASE correlates with recurrence and/or death, and 3) to examine the effect of survivin inhibition on apoptosis in EAC cells. Methods Fresh frozen samples of EAC and CASE from the same patient were used for qRT-PCR and Western blot analysis, and formalin-fixed, paraffin-embedded tissue was used for immunohistochemistry. EAC cell lines, OE19 and OE33, were transfected with small interfering RNAs (siRNAs) to knockdown survivin expression. This was confirmed by qRT-PCR for survivin expression and Western blot analysis of cleaved PARP, cleaved caspase 3 and survivin. Survivin expression data was correlated with clinical outcome. Results Survivin expression was significantly higher in EAC tumor samples compared to the CASE from the same patient. Patients with high expression of survivin in EAC tumor had an increased risk of death. Survivin expression was also noted in CASE and correlated with increased risk of distant recurrence. Cell line evaluation demonstrated that inhibition of survivin resulted in an increase in apoptosis. Conclusion Higher expression of survivin in tumor tissue was associated with increased risk of death; while survivin expression in CASE was a superior predictor of recurrence. Inhibition of survivin in EAC cell lines further showed increased apoptosis, supporting the potential benefits of therapeutic strategies targeted to this marker.

Preclinical Study of AUY922, a Novel Hsp90 Inhibitor, in the Treatment of Esophageal Adenocarcinoma.

Objective: To assess the efficacy of heat-shock protein 90 (Hsp90) inhibitor, NVP-AUY922-AG (AUY922), in the treatment of esophageal adenocarcinoma (EAC) in vitro and in vivo. Background: EAC is a leading cause of cancer death, and current treatment options are limited. Hsp90, a chaperone protein that regulates several oncoproteins, is upregulated in EAC, and may be a novel target for therapy. Methods: In vitro, EAC cell lines were utilized to evaluate AUY922, alone and in combination with 5-fluorouracil (5-FU) and cisplatin. BrdU ELISA and flow cytometry were used to assess proliferation and measure apoptosis, respectively. Western blot and RT-PCR were performed to quantitate Hsp90 pathway expression. In vivo, esophagojejunostomy was performed on rats and treatment animals received AUY922 32 to 40 weeks postoperatively. Drug efficacy was evaluated with magnetic resonance imaging (MRI), endoscopic biopsy, gross histological evaluation, and Hsp90 pathway expression. Results: In vitro, AUY922 demonstrated antiproliferative activity in both cell lines and showed enhanced efficacy with cisplatin and 5-FU. Western Blot and RT-PCR demonstrated downregulation of CDK1 and CDK4 and upregulation of Hsp72. In vivo, AUY922 showed decrease in tumor volume in 36.4% of rats (control = 9.4%), increase in 9.1% (control = 37.5%), and stable disease in 54.5% (control = 43.7%). Necropsy confirmed the presence of EAC in 50% of treatment animals and 75% of control animals. mRNA expression, pre- and posttreatment, demonstrated significant downregulation of MIF, Hsp70, Hsp90&bgr;, and CDK4, and upregulation of Hsp72. Conclusions: AUY922 exhibits antitumor efficacy in vitro and in vivo for EAC, suggesting the need for human clinical trials.

Abstract B05: MRI provides a more reliable method of esophageal tumor detection than endoscopy in a rat model

Background: The surgical rat model of end-to-side esophagojejunal anastomosis has proven utility in terms of its ability to replicate Barrett9s carcinogenesis by inducing gastroduodenoesophageal reflux (GDER). Previously, visual endoscopic analysis has been shown as a valuable tool for monitoring natural history of disease, with biopsy allowing for analysis of molecular markers while avoiding animal sacrifice. However, due to the inherent model limitation of the extraluminal presentation of the majority of tumors, as well as the small size of endoscopic biopsies, adenocarcinoma histology has only been validated post-mortem, and therefore treatment efficacy studies have been limited to chemoprevention. The current study aims to test and establish MRI and / or endoscopic visualization for diagnosis and monitoring of esophageal cancer in the rat model, in order to enhance utilization of this promising de novo model to study treatment response of novel agents in established esophageal cancer. Methods: Chronic bile and acid reflux was induced in 24 Sprague-Dawley rats through a modified Levrat9s model. At 40 weeks post-surgery, all animals underwent an endoscopy, T2-weighted turbo spin-echo (TSE) MRI scan and post-mortem histological analysis. A total of five esophageal surgeons and gastroenterologists participated in a blinded study and watched endoscopy videos to determine if esophageal tumor was present. Additionally, three radiology experts participated in a blinded study designed to assess correlated coronal and axial MRI scans and determine whether tumor was present. All videos and scans were studied in triplicate, and histology was used as the gold standard to assess accuracy of each participant9s analysis. Results: The accuracy of MRI and endoscopic analysis to correctly identify histology was 69.6% and 57.4%, respectively. The ROC curve analysis showed average MRI rating (AUC = 0.77, p-value = 0.032) was a better discriminator of tumor status in rats than average endoscopy rating (AUC = 0.595, p-value = 0.450). Additionally, false positive rates of MRI and endoscopy were 10.1% and 16.5%, respectively. Inter-observer variability was in substantial agreement for MRI participants (average Kappa = 0.768); whereas endoscopic analysis did not show greater agreement than that what would be expected by chance. All cases where MRI participants agreed on the presence of tumor, histology confirmed the positive findings. Conclusions: MRI is a more reliable diagnostic method than endoscopy for esophageal tumor in rats. Endoscopic examination remains a valuable tool in terms of its ability to biopsy specimens for analysis of biomarker correlates for therapeutic efficacy; however, MRI is a more dependable technique in terms of confirming tumor presence and disease progression for utilization in a treatment model. Citation Format: Juliann E. Kosovec, Yoshihiro Komatsu, Christina L. Rotoloni, Diane V. Thompson, Ali H. Zaidi, Blair A. Jobe. MRI provides a more reliable method of esophageal tumor detection than endoscopy in a rat model. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr B05.

Abstract A48: Enhanced efficacy of cisplatin and 5-fluorouracil combination with AUY-922 in esophageal adenocarcinoma cells

Background: Esophageal Adenocarcinoma (EAC) continues to rise in incidence, with prognosis remaining poor despite advances in multimodality therapy. Several novel target agents are now being explored as an option for treating EAC. One potential target is heat shock protein 90 (Hsp90), a chaperone protein that is involved in many diverse biological processes including cell signaling, proliferation, and survival. Many of the client proteins are known oncoproteins that allow Hsp90 to stabilize cancer cell growth by supporting proliferation and preventing apoptosis. The isoform, Hsp90β, is constituently expressed, while Hsp90α is inducible during times of stress, with expression increased 2-10 fold in cancers. Our hypothesis is that Hsp90 inhibition, in combination with standard chemotherapeutic drugs, will cause EAC cancer cells to be more susceptible to apoptosis and reduce the rate of proliferation. Methods: EAC cell lines, OE19 and OE33, were used to evaluate the effects of Hsp90 inhibitor, AUY-922, in cancer cell growth and apoptosis. ELISA of WST-1 and BrdU were used to determine the effective dosage and assess proliferation. Pathway inhibition was evaluated by Western Blot of Hsp90α and Hsp70. OE19, OE33, and patient samples of EAC tumor and gastroesophageal reflux tissue were used to assess the gene expression of Hsp90 and several client protein pathways by reverse transcription polymerase chain reaction (RT-PCR). Results: The ED50 of AUY-922 was determined to be 30ηM. A combination of chemotherapeutic drugs, cisplatin and 5-fluorouracil (5-Fu), along with AUY-922 showed significantly decreased proliferation compared to untreated and single agent treated cell lines. Western blot demonstrated that Hsp90 was inhibited by AUY-922 treatment, by a decrease in expression of Hsp90α, and an increase in the expression of Hsp70. RT-PCR in the cell line treatment groups showed an impact on many client oncoproteins involved in cancer cell survival and Hsp90 was shown to be upregulated in tumor samples when compared to normal GERD samples. Conclusion: The use of Hsp90 inhibitor, AUY-922, leads to reduced Hsp90 pathway expression, resulting in a degradation of many Hsp90 client proteins involved in cancer genesis. Cell proliferation was decreased with AUY-922 treatment, with the greatest demonstrated effect when used in combination with cisplatin and 5-Fu. Therefore, Hsp90 inhibition may have an application in multimodal EAC chemotherapy. Citation Format: Christina L. Rotoloni, Jaclyn M. LaRosa, Michael J. Porter, Lori A. Kelly, Katherine Nega, Emily Wolfson, Yoshihiro Komastu, Juliann E. Kosovec, Pashtoon M. Kasi, Toshitaka Hoppo, Ali H. Zaidi, Blair A. Jobe. Enhanced efficacy of cisplatin and 5-fluorouracil combination with AUY-922 in esophageal adenocarcinoma cells. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A48.

Prevention of Barrett’s esophagus (BE) and esophageal adenocarcinoma (EAC) in the levrat rat model of EAC by treatment with a smoothened (SMO) inhibitor.

P cancer is almost always fatal, in part because of its delayed diagnosis, poor prognosis, rapid progression and chemoresistance. Oncogenic proteins are stabilized by the heat shock protein 90 (Hsp90), making it a potential therapeutic target. We investigated the oxidative stress-mediated dysfunction of Hsp90 along with direct hindrance of its chaperonic activity by a carbazole alkaloid (CM-5), as a strategic therapeutic in pancreatic cancer. CM-5 exhibited anti-proliferative activity against several pancreatic cancer cells through apoptosis. It induced early accumulation of reactive oxygen species (ROS) leading to thiol oxidation, aggregation and dysfunction of Hsp90 in MIAPaCa-2 cell line. N-acetyl-L-cysteine prevented CM-5-induced ROS accumulation, aggregation of Hsp90, degradation of client proteins and cell death. CM-5 may possibly disrupted Hsp90Cdc37 chaperone complex in MIAPaCa-2 in addition to inducing ROS generation. Client proteins were restored by MG132, suggesting a possible role of ubiquitinylated protein degradation pathway. Surface plasmon resonance study gave the dissociation constant (KD) of Hsp90-CM-5 as 4.99 μM, which is in (a) good agreement with the dynamic simulation value (3.16 μM). CM-5 formed hydrogen bonds with Gln47 and Asn51 of Hsp90 and exhibited hydrophobic interactions with several amino acids in the Hsp90-Cdc37 interface. However, CM-5 did not impede the ATP binding pocket of Hsp90. CM-5 also reduced in vitro migration and tube formation in cancer cells. Further, it inhibited orthotopic pancreatic cancer in nude mice. Taken together, these results provide evidence for CM-5-induced ROS-mediated destabilization of Hsp90 chaperone activity resulting in Hsp90Cdc37 disruption leading to apoptosis, suggesting its potential as a specific target in pancreatic cancer. Chitra Mandal et al., J Cancer Sci Ther 2012, 4:10 http://dx.doi.org/10.4172/1948-5956.S1.0224129 Background: EAC from BE is increasing and the prognosis is poor. The benefit from prevention of BE and EAC is great. The Sonic Hedgehog (Shh) pathway, a mediator of foregut development, may play a role in development of BE/EAC. The Levrat model of gastroduodenojejunal reflux mimics progression from normal to BE to EAC. We studied the activity of a SMO inhibitor (BMS 833293) in prevention of BE/EAC in the Levrats rat model. METHODS Gastroduodenojejunal reflux was induced in 6-8 wk old male Sprague-Dawley rats, as approved by the Institutional Care and Use Committee. Esophagus was mobilized, preserving the vagus, and a loop of jejunum was identified. The gastroesophageal junction (GEJ) was divided and ligated, and an end-to-side anastomosis was constructed. The incidence of BE/EAC at 28 wks is 90%/70%. To attain a 42% reduction in incidence of EAC vs controls (70%) at 28 weeks, 32 rats/group (a = 0.05) were included. PK evaluation in operated rats yielded a dose of 10mg/kg. Oral dosing daily started at 10 wks post-op on weeks 10-16, 18-22 and 24-28. One pathologist (JD) evaluated all samples. RESULTS Surgical survival=90%. Dose schedule was daily; however, by 6 wks, rats lost 10-15% of their BW, necessitating an interrupted schedule. All animals have been sacrificed; however, data is available for a subset. Treatment toxicities included: wt loss at 6 weeks of 10-15%, porphyria and UGI bleeding. Specimens from 11 control and 14 treated animals were evaluated by JD. CONTROL BE=11/11 and EAC=4/11. TREATMENT BE=7/14 and EAC=0/14. Samples also showed ulcerative esophagitis in: 11/14 treated and 0/11 controls. Review of remaining samples is underway. CONCLUSIONS The Levrat model for evaluation of inhibition of BE/EAC by the smoothened inhibitor, BMS 833293, is feasible. Preliminary data suggest a treatment effect. Full data available at the meeting will include PD and Shh gene expression.M and invasiveness of cancer cells were shown to be associated with the suppressed ability to develop apoptosis in response to chemotherapy. However, the role of apoptotic (cytotoxic) DNA endonucleases, the key enzymes in inducing irreversible cell death, and their regulation in invasive cancer cells are not clearly understood. We have evaluated the expression of two most abundant endonucleases, DNase I and EndoG, in human breast and prostate cancer cell lines. This evaluation showed that invasive breast cancer cells (HCC1143, HCC1954 and HCC1395) and prostate cancer cells (PC3) have no detectable expression of DNase I, and significantly decreased expression of EndoG compared to normal or non-invasive cancer cells. The absence of EndoG in breast or prostate cancer cells negatively correlated with the sensitivity to anticancer drugs, such as cisplatin, etoposide, camptothecin, and docetaxel. The decrease of EndoG expression is caused by hypermethylation of EndoG gene, while suppression of DNA methylation activated the gene and made cells susceptible to chemotherapy drugs. The silencing of EndoG using specific siRNA decreased the chemoresistance of the cells, while overexpression of EndoG increased it. Finally, the expression of EndoG in orthotopic prostate PC3 cell xenografts in mice increased sensitivity of the tumors to docetaxel. Our findings suggest that the absence of EndoG in invasive breast or prostate cancer cells causes their decreased sensitivity to apoptosis induced by chemotherapy, while the delivery of EndoG gene restores the sensitivity and allows effective chemotherapy. Alexei G. Basnakian, J Cancer Sci Ther 2012, 4:10 http://dx.doi.org/10.4172/1948-5956.S1.023Backgrounds: Tuberculoma is a granulomatous inflammatory process mimicking a neoplasm, both clinically and radiologically. Although those with an infratentorial origin are rare, this disease is still a diagnostic challenge using conventional workup. However, this disease should not be overlooked because it is essentially curable with proper diagnosis and therapy, usually, a Mycobacterium Tuberculosis (MTB) DNA test is performed. Methods: We retrospectively analyzed the clinical presentations, CSF results, and images of 11 MTB DNA positive and clinically cured cases of infratentorial tuberculoma. Results: Infratentorial tuberculoma usually deteriorated before antituberculosis treatment (ATT). Magnetic resonance imaging showed space-occupying lesions without specific features, 4 within the cerebellum and 7 within the brainstem. Evidence of systemic tuberculosis was found in only 1 case. Clinical manifestations included various combinations of focal signs and symptoms in the brain stem and cerebellum. Cerebrospinal fluid (CSF) findings were also nonspecific. The diagnoses of these cases were based on the positive tests of a nested polymerase chain reaction (N-PCR) assay. Trial therapy with antituberculous drugs resulted in clinical improvement, as documented by MRI in all patients. Conclusions: Infratentorial tuberculoma should be suspected in patients with infratentorial space-occupying lesions who live in geographic areas where tuberculosis is endemic.M endogenous anti-cancer molecules which are released from extracellular matrix /Type IV collagen were identified as angioinhibitors of tumor growth. These endogenous angioinhibitory molecules bind to the cell surface integrins and transduce the signaling. Thus, cell surface integrins serve as transmembrane linkers between the extracellular matrix and cytoskeleton for outside-in signaling. Four of such endogenous molecules derived from the C-terminal non-collagenous domain of alpha1, 3 and 6 type IV collagen were identified as an inhibitor of angiogenesis, but their mediated angioinhibitory signaling are not known yet. Our findings suggests that these molecules interacting with different cell surface integrins and inhibiting angiogenesis by inhibiting different signaling that leading to inhibition of tumor angiogenesis and tumor growth both in-vitro and in-vivo.B cancers cells over express Tumor Associated Carbohydrate Antigens (TACA) but TACA as immunogens is restricted by a limited cooperation between TACA reactive B cell and T cells. To circumvent this draw back we have developed carbohydrate mimetic peptides (CMPs) with overlapping B and T cell epitopes to link TACA reactive humoral responses with anti-tumor cellular responses. Using molecular modeling we have designed a CMP reactive with anti-GD2 and anti-Lewis Y antibodies. CMP immunization leads to an inhibition of tumor cell growth in animal models, which proved dependent on cellular cytotoxicity in the context of Th1 responses induced by CMP vaccination and activation of NK cells. Combination with Cyclophosphamide and IL-12 augments survival in murine tumor models. We observe that the limited and targeted attack resembles more the selflimited disease in models of autoimmune inflammation in non-autoimmune prone individuals. Preclinical safety studies indicate that the CMP induces anti-tumor responses in the absence of autoimmunity. These findings encourage us to hypothesize about the possible cascade of immune events initiated by CMP immunization. Apparently, it can reshape cellular responses in vivo facilitating a multifaceted anti-tumor immune response. Such responses targeting TACA parallel those associated with immune surveillance mechanisms required for prevention of recurrence of breast cancer disease and blood borne dissemination of breast cancer cells. In a limited Phase I study CMP immunization is tolerable and induces an anti-CMP response that is cross-reactive with TACA expressing human tumor cells. Immune modulation based on CMPs provides a new intervention for enhancing antitumor immunotherapy. Thomas Kieber-Emmons, J Cancer Sci Ther 2012, 4:10 http://dx.doi.org/10.4172/1948-5956.S1.023M represent tandem iterations of 1-6 bp DNA constituting the hypervariable regions of the genome due to high mutation rates. Variations in the microsatellite regions referred to as microsatellite instability (MSI) resulting from insertions, deletions and single nucleotide transformation may alter the expression of microsatellite associated genes including those involved in DNA repair, tumor suppression and cell proliferation processes. Here, we present and critically analyze the information available on various aspects of MSI and its association with various organ-specific cancers with an assessment of using MSI as a prognostic marker for cancer.Background: Lung cancer remains the leading cause of cancer-related mortality worldwide. Early detection of lung cancer is problematic due to the lack of a marker with high diagnosis sensitivity and specificity. The purpose of this study was to develop techniques to identify the differential expression protein profiles between tumor and tumor free of lung cancer tissues. Methods: 2-dimensional differential ingel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to analyze four samples of lung cancer tissue (3 replicates each). Results: From optimized 2DE image, A total of 2561 spots were detected and 427 spots of these were differentially expressed (p<0.01). 40 spots were subjected to mass spectrometry including over expressed proteins and under expressed proteins. Some proteins were the products of oncogenes and others were involved in the regulation of cell cycle and signal transduction such as Annexin III, Selenium binding protein, glyceraldehydes-3-phosphate dehydrogenase, cathepsin D and catalase. Conclusion: These data are valuable for mass identification of differentially expressed proteins involved in lung carcinogenesis, establishing human lung cancer proteome database and screening molecular marker to further study human lung cancer. Using the DIGE approach, we were able to find many proteins that were expressed differently due to the disease state (tumor and tumor-free).R a wide variety of phytochemicals isolated from different plants such as flavones, polyphenols, thiols, and isothiocyanates, particularly sulforaphane (SFN) have garnered significant attention in cancer chemoprevention. While the efficacy of these phytochemicals for the prevention and treatment of chronic diseases including cancer is continuously being explored, controversy still surrounds their mechanisms of action. We and others have shown that the cytotoxic and apoptotic effects of SFN and another phytochemical withaferin A (WFA) isolated from the plant-Ashwagandha (ASH, Withania somnifera) maybe attributed largely to their oxidative stress-induced lipid peroxidation (LPO) in cancer as well as healthy normal cells, thereby limiting their clinical applications. Since extracts prepared from the whole plant or its part(s) are non-toxic to humans, we have devised a novel strategy to utilize it by fortifying with an optimized amount of the purified bioactive agent(s) for a selective chemosensitization of the tumor cells. Comparative studies conducted by us on normal lung epithelial and non-small cell lung cancer (NSCLC) cells with WFA and the crude ASH extract fortified with optimum amounts of WFA (FASH) demonstrated a significant attenuation in the cyotoxic and apoptotic effects of WFA in FASH-treated normal cells with little or no change in FASH-treated NSCLC cells. These effects were associated with a preferential stimulation of the antioxidant defense systems and modulation of several protective signaling proteins and their transcription factors in normal cells when compared to cancer cells. These studies suggest that enrichment of the plant extract with the purified bioactive compound (s) may be an excellent approach to selectively chemosensitize cancer cells. Rajendra Sharma et al., J Cancer Sci Ther 2012, 4:10 http://dx.doi.org/10.4172/1948-5956.S1.022C (CQ), N’-(7-chloroquinolin-4-yl)-N,N-diethyl-pentane-1,4-diamine, is widely used as an effective and safe agent to treat malaria, rheumatoid arthritis, lupus erythematosus and amoebic hepatitis. Although CQ was discovered 1934, it was ignored for a decade because it was considered too toxic to use in humans. CQ was “re-discovered” during World War II in the United States in the course of anti-malarial drug development. Since then, CQ remains the drug of choice for malarial treatment because it is effective and well-tolerated by humans. More recently, CQ has been “discovered” again as an enhancing agent in cancer therapies. We and others found that CQ can effectively sensitize cell killing by ionizing radiation and chemotherapeutics in a cancer-specific manner. We then created many CQ analogs and examined their efficacy and safety, alone or in combination with other cancer therapeutics. We found that combinations of Akt inhibitors and CQ/CQ derivatives greatly enhanced cellkilling effective. Importantly, this cell-killing effect by CQ/CQ derivatives is mostly cancer-specific. For example, a combination of an Akt 1⁄2 inhibitor with a CQ derivative could kill cancer cells 120-fold more effectively than non-cancer cells. We found that most of the CQ derivatives retained the lysosomotrophic property like CQ, although some showed only weak affinity to lysosome. We also found that some of the derivatives we created effectively induce cell cycle arrest and activate apoptosis in a cancerspecific manner. Taken together, CQ and its analogs can dramatically enhance the efficacy of conventional cancer therapies with minimum side effects. Hoyun Lee, J Cancer Sci Ther 2012, 4:10 http://dx.doi.org/10.4172/1948-5956.S1.023L or 511 constituting the extracellular matrix (ECM) of tumors and basement membranes (BM) of blood capillaries promotes the cellular migration of tumoral and endothelial cells, thus enhancing tumor growth, metastasis and angiogenesis. Cellular survival or migration promoting activities of laminin-511 have been attributed to the EGF-like domains that are released from the laminin-511 by MT1-MMP cleavage which activate EGFR signaling in target cancer cells. We have identified that cleavage of laminin-10 into 310 kDa fragment by MT1-MMP was prevented by human type IV collagen alpha 6 chain derived non-collagenous domain [Col4(α6)NC1]. The inhibitory effect of Col4(α6)NC1 on laminin alpha 5 chain cleavage was also exhibited by this domain on cell mediated laminin-10 cleavage. Further, Col4(α6)NC1 inhibited migration of cells on laminn-10 coated plates, phosphorylation of EGFR in LLC and breast cancer tumor cells. Thus, the present study elucidates the novel inhibitory mechanism(s) of MT1-MMP mediated laminin cleavage by Col4(α6)NC1, which prevents tumor progression Venugopal Gunda et al., J Cancer Sci Ther 2012, 4:10 http://dx.doi.org/10.4172/1948-5956.S1.022T transforming growth factor-beta (TGF-beta) receptor interacting protein TRIP-1 is a WD40 repeat-containing protein that has the ability to bind to the cytoplasmic domain of the TGF-beta type II receptor in a kinase-dependent manner. Evidences have demonstrated that the transforming growth factor beta (TGF-beta) signal pathway plays an important role in metastasis promotion in the later stages of cancer progression, although it mediates growth inhibition in early stages. As a modulator of the TGF-beta response, the potential role of TRIP-1 in cancer invasion and metastasis through the TGF-beta signaling pathway has not been elucidated. In this study, we firstly examine the protein-protein interactions between TRIP-1 and Ezrin (a key component in tumor metastasis) through GST pull-down technology and found that TRIP-1 can bind with Ezrin. Co-immunoprecipitation experiments further confirmed that the 567th threonine residue of Ezrin and the last 20 amino acids of TRIP-1 are necessary for the binding interaction between Ezrin and TRIP-1. Phospho-mimicking mutant ezrinT567D, but not the nonphosphorylatable mutant ezrinT567A, stimulated formation of protein complexes. Immunofluorescent localization experiments revealed co-localization of proteins at a cell membrane. Importantly, we found that knock-down of TRIP-1 by RNA interference resulted in a blockade to Ezrin-induced hepatocellular carcinoma cell adhesion and migration by wound healing assay. Our findings suggest TRIP-1 may act as a negative regulator to reduce tumor metastasis by blocking TGF-β-mediated phosphorylation of ezrin. Fei Yan, J Cancer Sci Ther 2012, 4:10 http://dx.doi.org/10.4172/1948-5956.S1.022A play a different role in tumer cell death and survival, the effection of autophagy in CD133+ stem cell like caner cells is still unclear. The purpose of this study was to investigate the change and effection of autophagy induced by chemotherapeutics and hypoxia in CD133+ cells. The CD133+ subpopulation of colon carcinoma cell line SW480 was purified with MACS, and enriched by serum-free medium culture system. In order to simulate the hypoxia microenvironment with 1% oxygen concentration, the anaerobic jar was used. Furthemore, the cells were treated with 1μg/mL 5-fluorouracil(5-FU). After treatment with that two factors for 24 to 48 hours, the emergence of a large number of autophagsomes was observed by TEM, the number of MDC positive utophagy vesica increased too, and the fluorescence intensity of MDC was significantly increased using FCM assay(P<0.05). In that process, the transformation of autophagy related protein LC3-I to LC3-II enhanced, the level of LC3-II significantly increased, but the transformation decreased following addition of 3-MA for 48 hours, and the level of LC3-II reduced. Simultaneously, the viability and cloning efficiency didn’t decline until addition of 3-MA (P<0.05). Immunohistochemical datas in 94 cases of colorectal cancer tissue demonstrated that both of HIF-2α and Beclin 1 had a positive relationship with classification and Duke’s staging in colorectal cancer (P<0.05), and the metastasis of lymphnode was positively correlative with Beclin 1 and negatively with HIF-2α (P<0.05). These results suggest that 5-FU and hypoxia microenvironment could induce the enhancement of autophagy and maintain cell viability in CD133+ cancer cells.H an RNA binding protein involved in the post-transcriptional regulation of a wide spectrum of mRNAs, has been demonstrated to be a determinant of carcinogenesis and tumor aggressiveness in several cancer types. In this study, we investigated the role of HuR in the apoptosis and in the chemoresistance induced by the widely used anticancer drug doxorubicin in human breast cancer cells (MCF-7). We challenged a small library of about 80 chemical compounds with an high content screening assay to quantitavely measure HuR translocation. We showed that HuR acts in the early phase of cell response to doxorubicin, being induced to translocate into the cytoplasm upon phosphorylation. Reducing HuR levels diminished the apoptotic response to doxorubicin. We identified a number of compounds that can inhibit HuR cytoplasmic accumulation and pointed to PKA, Rho kinase and PKCδ as potential HuR regulators. Among the hits rottlerin showed to be the most effective in blocking HuR nuclear export and in having correspondingly antagonistic effects with doxorubicin on cell toxicity. Co-immunoprecipitation of PKCδ and HuR upon doxorubicin confirmed the validity of HCS indications. In in vitro selected doxorubicin resistant MCF-7 cells (MCF-7/doxoR) overexpressing the multidrug resistance (MDR) related ABCG2 transporter, we observed a significant HuR downregulation that was paralleled by a corresponding downregulation of HuR targets as TOP2A and by loss of rottlerin toxicity. Restoration of HuR expression in these cells resensitized MCF-7/doxoR cells to doxorubicin, reactivating the apoptotic response. The present study shows that HuR is necessary to elicit the apoptotic cell response to doxorubicin, that restoration of HuR expression in resistant cells resensitizes them to the action of this drug. Moreover we suggest a novel mechanism of pharmacoresistance based on the interplay among the doxorubicin target TOP2A, its post-transcriptionally regulator HuR and the signaling control of PKCδ. Alessandro Provenzani, J Cancer Sci Ther 2012, 4:10 http://dx.doi.org/10.4172/1948-5956.S1.022A tremendous challenge in the treatment and therapy of cancers is due to their propensity to undergo metastasis. Until now there are no effective inhibitors of metastasisin clinical use, excepting perhaps angiogenesis inhibitors. The cystatins are small molecular weight protein inhibitors of cathepsins which have anti-metastatic effects. While certain cathepsins are generally elevated in different tumors, the cystatins are either unchanged or decreased in levels. The cystatins display in general metastasis suppressor actions. Melanoma and a number of other cancers (glioblastoma, breast, prostate and colon) are found to have decreased metastasis when cystatin C is overexpressed in each of the cancer cell types. We noted earlier dramatic inhibition of migration and invasion of melanoma cells overexpressing cystatin. An increase in apoptosis was also noted in a mouse model of metastasis. The cystatins are also known angiogenesis inhibitors which may play a role in reduced metastatic tumor growth. Decreases in melanoma cell signaling and cell migration on collagen are a major current focus. Migration related changes including rhoprotein and calcium regulation are the current approaches we have taken to look at cystatinmechanism of action. The anti-metastatic actions of a natural protein, cystatin, may help unlock future cancer treatment options.During the course of our study we found that the chemotherapeutic drug 5-fluoruracyl (5-FU) induces increased expression of miR-483-3p in HepG2 and Hep3B cells. 5-FU treatment does not affect the expression of miR-483 expression in all the HCC cell lines that we tested, suggesting a possible heterogeneity in cellular apoptosis resistance depending on the miR-483-3p induced expression. Although the IGF2 and miR-483 expressions are strictly correlated, we have demonstrated that the oncoprotein β-catenin cooperates with the USF1 to transcribe the miR-483 by a separate mechanism. In addition we found that also CMYC is involved in the regulation of miR-483 expression. In Hepatocarcinoma (HCC) IGF2/483 locus is often over-expressed by Loss of Imprinting (LOI) at the IGF2/H19 imprinted control region (ICR) and HepG2 and Hep3B HCC cell lines shown the entire IGF2/483 locus up-regulated. As a matter of fact we found that the treatment with the de-methylating agent 5-azacytidine, induces an important down-regulation of IGF2 and miR-483-3p genes.H is a known endogenous angioinhibitor molecule derived from alpha6 chain non-collagenous (NC1) domain of Type IV collagen, where as its angioinhibitory mechanism(s) is not yet known clearly. Here in the study, we have cloned hexastatin in pET22b (+) vector and expressed in E. Coli. Soluble and biologically active hexastatin was purified using different affinity and size exclusion chromatographies and studied its pro-apoptotic activities in-vitro using endothelial cells. Our preliminary in-vitro studies demonstrate that hexastatin inhibiting VEGF mediated endothelial cell proliferation, migration and tube formation. In addition, we have identified that hexastatin promoting endothelial cell apoptosis by activating FasL and its mediated downstream apoptotic cascade activation. Smita C. Pawar et al., J Cancer Sci Ther 2012, 4:10 http://dx.doi.org/10.4172/1948-5956.S1.022R of colorectal cancer (CRC) increases after exposure to ionizing radiation (IR). However, CRC risk prediction following heavy ion space radiation exposure is hindered due to scarcity of in vivo mechanistic understanding. Therefore, intestinal tumorigenesis and mechanisms of tumorigenesis were studied after 1.6 Gy of 56Fe radiation (energy: 1000 MeV/nucleon) exposure in APCMin/+ and wild type C57BL/6J mice (n=20; 6 to 8 wks old; female) and results were compared to equitoxic 2 Gy γ radiation. For tumorigenesis study, APCMin/+ mice were humanely sacrificed between 100 and 110 days after radiation exposure, intestine surgically removed, lumen washed and cut open along the length, tumors counted, and tissues preserved for molecular analysis. Because chronic oxidative stress is implicated in carcinogenesis, we also assessed reactive oxygen species, mitochondrial status, and antioxidant activity in wild type mouse intestine 1-year after radiation exposure. In APCMin/+ mice, relative to controls and γ-rays, intestinal tumor frequency was higher after 56Fe. Staining for phospho-histone H3 indicating proliferation was more and alcian blue staining indicating differentiation was less in 56Fe than γ tumors. Activation of β-catenin was more in 56Fe-irradiated normal and tumor tissues. When considered with higher levels of cyclin-D1, we concluded that relative to γ radiation high-energy 56Fe irradiation led to higher intestinal tumorigenesis, tumor proliferative index, and reduced tumor cell differentiation due to preferentially greater activation of β-catenin and its downstream effectors. In wild type mice, long-term functional dysregulation of mitochondria and increased NADPH oxidase activity are major contributing factors towards heavy ion radiation-induced persistent oxidative stress in IEC with potential for neoplastic transformation.Methods: We used high-field NMR, in parallel with GC-MS, and direct infusion nanospray FT-ICR-MS as well as stable isotoperesolved metabolomics to trace the fate of 13C and 15N from labeled glucose or glutamine in the P493 human B lymphoma under aerobic, hypoxic glucose-deprived conditions. Levels of specific enzymes, which are involved in glucose and glutamine metabolism, were determined by immunoblotting and LC-MRM-MS and compared with the metabolomic profiles.R to chemotherapy drugs results in poor response rates and treatment failure in more than 90% of patients with metastatic breast cancers. Understanding the mechanisms underlying such resistance is therefore crucial for the development of new, efficacious cancer drugs. Unfortunately, in spite of extensive inquiry in this field, little is known about the key molecules/ signaling pathways that regulate this phenomenon. We have discovered that microRNAs (miRNAs) may play critical roles in mediating drug sensitivity/resistance in breast cancers. We have identified miRNAs that are differentially expressed between chemo-resistant and sensitive HER2+ breast cancer cells. Specifically, through high-throughput miRNA inhibitor library screens, we have identified miRNAs that sensitize drug-resistant breast cancer cells to paclitaxel and trastuzumab (herceptin), a drug combination commonly used for the treatment of HER2+ metastatic breast cancers. Our studies reveal that cognate miRNA/s, which sensitize/de-sensitize resistant tumor cells to drug-induced cell death are differentially expressed in the Her2+ metastatic breast cancer paitents compared to normal matched controls. Importantly, using liposome-based or biocompatible nanoparticles-based approaches, we have demonstrated that candidate miRNAs can be systemically delivered to sensitize and therefore, eliminate chemoresistant metastatic breast cancers in tumor bearing mice. These findings suggest that certain miRNAs are selectively cytotoxic in a drug-specific manner and these miRNAs may serve as novel biomarkers and provide potent adjuvant therapeutic tools for the treatment of drug-resistant metastatic breast cancers.