Subcutaneous Xenografts In Nude Mice Research Articles

Novel acylureidoindolin-2-one derivatives as dual Aurora B/FLT3 inhibitors for the treatment of acute myeloid leukemia

A series of 6-acylureido derivatives containing a 3-(pyrrol-2-ylmethylidene)indolin-2-one scaffold were synthesized as potential dual Aurora B/FLT3 inhibitors by replacing the 6-arylureido moiety in 6-arylureidoindolin-2-one-based multi-kinase inhibitors. (Z)-N-(2-(pyrrolidin-1-yl)ethyl)-5-((6-(3-(2-fluoro-4-methoxybenzoyl)ureido)-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxamide (54) was identified as a dual Aurora B/FLT3 inhibitor (IC50 = 0.4 nM and 0.5 nM, respectively). Compound 54 also exhibited potent cytotoxicity with single-digit nanomolar IC50 values against the FLT3 mutant-associated human acute myeloid leukemia (AML) cell lines MV4-11 (FLT3-ITD) and MOLM-13 (FLT3-ITD). Compound 54 also specifically induced extrinsic apoptosis by inhibiting the phosphorylation of the Aurora B and FLT3 pathways in MOLM-13 cells. Compound 54 had a moderate pharmacokinetic profile. The mesylate salt of 54 efficiently inhibited tumor growth and reduced the mortality of BALB/c nude mice (subcutaneous xenograft model) that had been implanted with AML MOLM-13 cells. Compound 54 is more potent than sunitinib not only against FLT3-WT AML cells but also active against sunitinib-resistant FLT3-ITD AML cells. This study demonstrates the significance of dual Aurora B/FLT3 inhibitors for the development of potential agents to treat AML.

Targeting breast cancer-initiating/stem cells with melanoma differentiation-associated gene-7/interleukin-24

Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) displays a broad range of antitumor properties including cancer-specific induction of apoptosis, inhibition of tumor angiogenesis and modulation of antitumor immune responses. In our study, we elucidated the role of MDA-7/IL-24 in inhibiting growth of breast cancer-initiating/stem cells. Ad.mda-7 infection decreased proliferation of breast cancer-initiating/stem cells without affecting normal breast stem cells. Ad.mda-7 induced apoptosis and endoplasmic reticulum stress in breast cancer-initiating/stem cells similar to unsorted breast cancer cells and inhibited the self-renewal property of breast cancer-initiating/stem cells by suppressing Wnt/β-catenin signaling. Prevention of inhibition of Wnt signaling by LiCl increased cell survival upon Ad.mda-7 treatment, suggesting that Wnt signaling inhibition might play a key role in MDA-7/IL-24-mediated death of breast cancer-initiating/stem cells. In a nude mouse subcutaneous xenograft model, Ad.mda-7 injection profoundly inhibited growth of tumors generated from breast cancer-initiating/stem cells and also exerted a potent "bystander" activity inhibiting growth of distant uninjected tumors. Further studies revealed that tumor growth inhibition by Ad.mda-7 was associated with a decrease in proliferation and angiogenesis, two intrinsic features of MDA-7/IL-24, and a reduction in vivo in the percentage of breast cancer-initiating/stem cells. Our findings demonstrate that MDA-7/IL-24 is not only nontoxic to normal cells and normal stem cells but also can kill both unsorted cancer cells and enriched populations of cancer-initiating/stem cells, providing further documentation that MDA-7/IL-24 might be a safe and effective way to eradicate cancers and also potentially establish disease-free survival.

Increase of cyclooxygenase-2 inhibition with celecoxib combined with 5-FU enhances tumor cell apoptosis and antitumor efficacy in a subcutaneous implantation tumor model of human colon cancer

BackgroundThe purpose of this study was to investigate the anti-tumor effect and explore the mechanisms of celecoxib (a selective cyclooxygenase-2 inhibitor) combined with 5-fluorouracil (5-FU) on the treatment of human colorectal cancer in a BALB/C nude mouse subcutaneous xenograft model.MethodsEffects of celecoxib combined with 5-FU on the proliferation of xenograft carcinoma induced by HT-29 were investigated. The apoptotic cells were detected by electron microscope and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. Immunohistochemistry and Western blot were used to estimate the expression of cytochrome C, caspase-3 and caspase-9.ResultsCompared with the control group, treatment groups showed significant inhibition of tumor growth. More apoptotic cells existed after treatment with celecoxib combined with 5-FU. Cytochrome C, caspase-3 and caspase-9 were increased in treated groups, and more obviously in the drug combination group. Cyclooxygenase-2 (COX-2) were decreased after treatment with celecoxib only or combined with 5-FU. And the combined group showed a greater decrease.ConclusionsCelecoxib combined with 5-FU could inhibit the growth of tumors in vivo by inducing apoptosis and activation of the cytochrome C dependency apoptosis signal pathway. A decrease of COX-2 and an increase of cytochrome C, caspase-3 and caspase-9 may be involved in this process.

Microrna-149 Inhibits Proliferation and Invasion of Glioma Cells via Blockade of Akt1 Signaling

MicroRNAs (miRNAs) play important roles in the regulation of gene expressions. Aberrant expression of miRNAs is implicated in a variety of biological and pathological processes, including the tumorigenesis of glioma (GM). Though the molecular mechanisms of protein kinase B (AKT) survival signal have been comprehensively explored, the role of miR-149 in glioblastoma (GBM) and its regulation on AKT signaling have not yet been ascertained. The present study aimed to elucidate the role and molecular mechanisms of miR-149 in U251 GM cells. Using a gain-of-function approach, we investigated the effects of lentivirus-mediated overexpression of miR-149 on the expression of phosphated-AKT1 (p-AKT1), proliferating cell nuclear antigen (PCNA), matrix metallopeptidase-2 (MMP-2) and CyclinD1 in U251 cells and nude mice subcutaneous xenograft tumors by Real-time PCR, Western blot and immunohistochemical assays. Proliferative activities indicated by MTT assay, invasive potential by Transwell and cycle distribution by flow cytometry were carried out for functional analysis of U251 cells after infection with miR-149 mimic. As a consequence, miR-149 inhibited the expression of p-AKT1, PCNA, CyclinD1 and MMP-2, reduced the proliferative activities and invasive potential, and induced cycle arrest in G0/G1 phase in U251 cells. In conclusion, our findings show that miR-149 as tumor suppressor may be involved in the proliferation and invasion of GM cells via blockade of the AKT1 signaling, and be considered as a candidate target for the treatment of cancer.

Mer or Axl receptor tyrosine kinase inhibition promotes apoptosis, blocks growth and enhances chemosensitivity of human non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is a prevalent and devastating disease that claims more lives than breast, prostate, colon, and pancreatic cancers combined. Current research suggests that standard chemotherapy regimens have been optimized to maximal efficiency. Promising new treatment strategies involve novel agents targeting molecular aberrations present in subsets of NSCLC. We evaluated 88 human NSCLC tumors of diverse histology and identified Mer and Axl as receptor tyrosine kinases (RTKs) overexpressed in 69% and 93%, respectively, of tumors relative to surrounding normal lung tissue. Mer and Axl were also frequently overexpressed and activated in NSCLC cell lines. Ligand-dependent Mer or Axl activation stimulated MAPK, AKT, and FAK signaling pathways indicating roles for these RTKs in multiple oncogenic processes. In addition, we identified a novel pro-survival pathway—involving AKT, CREB, Bcl-xL, survivin, and Bcl-2—downstream of Mer, which is differentially modulated by Axl signaling. We demonstrated that shRNA knockdown of Mer or Axl significantly reduced NSCLC colony formation and growth of subcutaneous xenografts in nude mice. Mer or Axl knockdown also improved in vitro NSCLC sensitivity to chemotherapeutic agents by promoting apoptosis. When comparing the effects of Mer and Axl knockdown, Mer inhibition exhibited more complete blockade of tumor growth while Axl knockdown more robustly improved chemosensitivity. These results indicate that Mer and Axl play complementary and overlapping roles in NSCLC and suggest that treatment strategies targeting both RTKs may be more effective than singly-targeted agents. Our findings validate Mer and Axl as potential therapeutic targets in NSCLC and provide justification for development of novel therapeutic compounds that selectively inhibit Mer and/or Axl.

Abstract 1448: Whole genome RNAi screen identifies proteasome inhibition as a strategy for NSCLC radiosensitization

Abstract Background: Each year, approximately 60,000 patients in the US are diagnosed with locally advanced non-small cell lung carcinoma (NSCLC). The majority is treated with a combination of radiation therapy (RT), chemotherapy +/− surgery. Despite best available therapy, over a third of patients fail locoregionally. The addition of concurrent chemotherapy to RT adds only about a 5% absolute survival benefit. Novel radiosensitizers may improve the therapeutic index of RT. Materials and Methods: We performed whole genome pooled RNAi screens using the Hannon-Elledge library of 74,705 distinct shRNAs directed against over 18,000 genes, in two NSCLC cell lines A549 and NCI-H460. After stable transduction, we passaged the lines for 12 population doublings, and extracted DNA both before and after passaging to compare the representation of each shRNA at each time point. We defined top hits as those genes for which multiple shRNAs decreased in representation by at least 50% during passaging, signifying silencings that were detrimental to proliferation. Results: The top hit of our screen was PSMA1, a subunit of the 20S proteasome; out of six shRNAs, between four and six scored in the two lines in screens performed in triplicate. Bortezomib, an inhibitor of the 20S proteasome, has shown activity against NSCLC in preclinical studies and Phase I/II clinical trials. We observed that bortezomib results in synergistic cytotoxicity in cells treated with fractionated ionizing radiation (IR) in vitro. 10 nM bortezomib resulted in 46% clonogenic survival of unirradiated A549 cells, but only 29% survival of cells treated with 1 Gy x 3 daily fractions compared to cells treated with IR alone. Similar results were observed with MG-132 and with NCI-H460. In soft agar anchorage-independent growth assays, bortezomib resulted in less than 1% colony formation following 1 Gy x 3 daily fractions compared to cells treated with IR alone. In vivo experiments are underway with subcutaneous xenografts in nude mice treated with concurrent CT-guided conformal RT. A retrospective analysis of 443 patients with profiled lung adenocarcinomas showed worse overall survival among patients with tumors showing high proteasome gene expression (median OS 4.2 vs. 6.2 y, 5-year OS 47.5 vs. 61.0%, log rank p = 0.04). Conclusions: Proteasome inhibition emerged as the top drug target of an RNAi screen designed to identify targets with sustained activity over multiple cell generations. Its synergistic impact on NSCLC viability when coupled with fractionated RT should be further explored in preclinical studies and early clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1448. doi:1538-7445.AM2012-1448

Inactivation of Transcription Factor Pit-1 to Target Tumoral Somatolactotroph Cells

The treatment of growth hormone (GH)- and prolactin (PRL)-secreting tumors resistant to current therapeutic molecules (somatostatin and dopamine analogues) remains challenging. To target these tumors specifically, we chose to inactivate a gene coding for a crucial factor in cell proliferation and hormonal regulation, specifically expressed in pituitary, by using a dominant-negative form of this gene involved in human pituitary deficiencies: transcription factor Pit-1 (POU1F1) mutated on arginine 271 to tryptophan (R271W). After lentiviral transfer, the effect of R271W was studied in vitro on human tumoral somatotroph and lactotroph cells and on the murine mammosomatotroph cell line GH4C1 and in vivo on GH4C1 subcutaneous xenografts in nude mice. R271W induced a decrease in GH and PRL hypersecretion by controlling the transcription of the corresponding hormones. This mutant decreased cell viability by an apoptotic mechanism and in vivo blocked the tumoral growth and GH secretion of xenografts obtained after transplantation of GH4C1 expressing mutant R271W. The strategy of using a dominant-negative form of a main factor controlling cell proliferation and hormonal secretion, and exclusively expressed in pituitary, seems promising for the gene therapy of human pituitary tumors and may be translated to other types of tumors maintaining some differentiation features.

The effects of cyclooxygenase-2 gene silencing by siRNA on cell proliferation, cell apoptosis, cell cycle and tumorigenicity of Capan-2 human pancreatic cancer cells

The prognosis of pancreatic cancer is still very poor. No specific effective gene therapy for pancreatic cancer has been found. As a key enzyme of the metabolic process of arachidonic acid, cyclooxygenase-2 (COX-2) has been found to be closely related to the tumorigenesis of epithelial cancers. However, the antitumor effect of small interfering RNA (siRNA) targeting COX-2 in pancreatic cancer has not yet been verified. Therefore, the aim of this study was to investigate the effects of COX-2 gene silencing by siRNA on cell proliferation, cell apoptosis, cell cycle and tumorigenicity of pancreatic cancer cells. COX-2 mRNA was detected by RT-PCR and real-time PCR. COX-2 protein was detected by Western blotting. The cell proliferation was measured by cell counting using microscopy. The cell apoptosis and cell cycle were measured by flow cytometry. The tumorigenicity of Capan-2 pancreatic cancer cells transfected with COX-2 siRNA was evaluated using a nude mouse xenograft model. The expression of COX-2 mRNA as well as COX-2 protein were downregulated after COX-2 siRNA transfection. COX-2 siRNA could inhibit the growth of Capan-2 cells significantly by decreasing the cell proliferation, increasing cell apoptosis and regulating cell cycle as well. In vivo experiments demonstrated that the mean volume and weight of subcutaneous xenografts in nude mice derived from Capan-2 cells transfected with COX-2 siRNA were significantly decreased. COX-2 siRNA could inhibit the growth of Capan-2 pancreatic cancer cells and also decrease the tumorigenicity of Capan-2 cells, implicating a new potential therapeutic target in pancreatic cancer.

Delta-like ligand 4-notch blockade and tumor radiation response.

The microenvironment plays an important role in regulating tumor response to radiotherapy. Ionizing radiation can disrupt tumor vasculature, and Notch pathway inhibition can interfere with functional angiogenesis. We explored the potential cooperativity between Notch inhibition and ionizing radiation in delaying tumor growth. Human colorectal carcinoma LS174T cells, which express the Notch ligand delta-like ligand 4 (DLL4), and human head and neck cancer FaDu cells, which do not, were grown as subcutaneous xenografts in nude mice. The mice were treated with dibenzazepine (DBZ), a γ-secretase inhibitor that blocks all Notch signaling, or a DLL4-specific blocking monoclonal antibody, alone or in combination with ionizing radiation (n = 5-10 mice per group), and response was assessed by tumor growth delay. Microbubble contrast Doppler ultrasound was used to measure tumor blood flow. Tumor Notch activity was monitored by in vivo bioluminescence from a Notch luciferase reporter. Vessel density was assessed using Chalkley vessel counting. All statistical tests were two-sided. In LS174T xenografts, the average time for tumor volumes to reach four times the starting volume was longer for mice treated with the DLL4 monoclonal antibody than for mice treated with DBZ (16.4 vs 9.5 days, difference = 6.9 days, 95% confidence interval [CI] = 3.7 to 10.1 days, P < .001). Both Notch inhibitors suppressed tumor Notch activity within 24 hours of administration compared with vehicle (change in luciferase activity, vehicle vs DBZ: 103% vs 28%, difference = 75%, 95% CI = 39% to 109%, P = .002; vehicle vs DLL4 antibody: 172% vs 26%, difference = 146%, 95% CI = 86% to 205%, P < .001). Administration of the DLL4 antibody or DBZ after ionizing radiation resulted in a supra-additive growth delay compared with vehicle (vehicle vs DLL4 antibody + ionizing radiation: 6.8 vs 44.3 days, difference = 37.5 days, 95% CI = 32 to 43 days, P < .001; vehicle vs DBZ + ionizing radiation: 7.1 vs 24.4 days, difference = 17.3 days, 95% CI = 15.9 to 18.6 days, P < .001). Treatment of mice with the DLL4 antibody alone or in combination with ionizing radiation increased tumor vessel density but reduced tumor blood flow. Combination therapy with DLL4 antibody and ionizing radiation resulted in extensive tumor necrosis in LS174T xenografts and enhanced tumor growth delay in FaDu xenografts. The combination of specific DLL4-Notch blockade and ionizing radiation impairs tumor growth by promoting nonfunctional tumor angiogenesis and extensive tumor necrosis, independent of tumor DLL4 expression.

RETRACTED: RNA Interference Targeting Slug Increases Cholangiocarcinoma Cell Sensitivity to Cisplatin via Upregulating PUMA

Slug is an E-cadherin repressor and a suppressor of PUMA (p53 upregulated modulator of apoptosis) and it has recently been demonstrated that Slug plays an important role in controlling apoptosis. In this study, we examined whether Slug’s ability to silence expression suppresses the growth of cholangiocarcinoma cells and/or sensitizes cholangiocarcinoma cells to chemotherapeutic agents through induction of apoptosis. We targeted the Slug gene using siRNA (Slug siRNA) via full Slug cDNA plasmid (Slug cDNA) transfection of cholangiocarcinoma cells. Slug siRNA, cisplatin, or Slug siRNA in combination with cisplatin, were used to treat cholangiocarcinoma cells in vitro. Western blot was used to detect the expression of Slug, PUMA, and E-cadherin protein. TUNEL, Annexin V Staining, and cell cycle analysis were used to detect apoptosis. A nude mice subcutaneous xenograft model of QBC939 cells was used to assess the effect of Slug silencing and/or cisplatin on tumor growth. Immunohistochemical staining was used to analyze the expression of Slug and PUMA. TUNEL was used to detect apoptosis in vivo. The results showed that PUMA and E-cadherin expression in cholangiocarcinoma cells is Slug dependent. We demonstrated that Slug silencing and cisplatin both promote apoptosis by upregulation of PUMA, not by upregulation of E-cadherin. Slug silencing significantly sensitized cholangiocarcinoma cells to cisplatin through upregulation of PUMA. Finally, we showed that Slug silencing suppressed the growth of QBC939 xenograft tumors and sensitized the tumor cells to cisplatin through PUMA upregulation and induction of apoptosis. Our findings indicate that Slug is an important modulator of the therapeutic response of cholangiocarcinoma cells and is potentially useful as a sensitizer in cholangiocarcinoma therapy. One of the mechanisms is the regulation of PUMA by Slug.

Stemness markers characterize IGR-CaP1, a new cell line derived from primary epithelial prostate cancer

Deciphering molecular pathways involved in the early steps of prostate oncogenesis requires both in vitro and in vivo models derived from human primary tumors. However the few recognized models of human prostate epithelial cancer originate from metastases. To date, very few models are proposed from primary tumors and immortalizing normal human prostate cells does not recapitulate the natural history of the disease. By culturing human prostate primary tumor cells onto human epithelial extra-cellular matrix, we successfully selected a new prostate cancer cell line, IGR-CaP1, and clonally-derived subclones. IGR-CaP1 cells, that harbor a tetraploid karyotype, high telomerase activity and mutated TP53, rapidly induced subcutaneous xenografts in nude mice. Furthermore, IGR-CaP1 cell lines, all exhibiting negativity for the androgen receptor and PSA, express the specific prostate markers alpha-methylacyl-CoA racemase and a low level of the prostate-specific membrane antigen PSMA, along with the prostate basal epithelial markers CK5 and CK14. More importantly, these clones express high CD44, CD133, and CXCR4 levels associated with high expression of α2β1-integrin and Oct4 which are reported to be prostate cancer stemness markers. RT-PCR data also revealed high activation of the Sonic Hedgehog signalling pathway in these cells. Additionally, the IGR-CaP1 cells possess a 3D sphere-forming ability and a renewal capacity by maintaining their CSC potential after xenografting in mice. As a result, the hormone-independent IGR-CaP1 cellular clones exhibit the original features of both basal prostate tissue and cancer stemness. Tumorigenic IGR-CaP1 clones constitute invaluable human models for studying prostate cancer progression and drug assessment in vitro as well as in animals specifically for developing new therapeutic approaches targeting prostate cancer stem cells.

Abstract 4167: Establishment and characterization of a nude mouse model directly derived from human primary colon cancer

Abstract The human tumor models established by inoculation of transformed tumorigenic tumor cell lines in immunodeficient mice have long been used in drug R&amp;D for the evaluation of anti-cancer therapies; however, the prediction for clinical effectiveness is not satisfactory by far. One of the obvious gaps is due to the adaptation of the tumor lines in culture environment and/or in vivo passages that make them step away from the original clinical tumor, e.g. in the reproducibility of the development and malignant behaviors, the specific gene expression profiles and the responsiveness to anti-cancer drug treatment, etc. We took a strategy of directly inoculation of the tumor tissues collected from Chinese colon cancer patients to establish human primary colon cancer model in nude mice. We successfully kept the tumor survived in vivo, but within limited passages to minimize the unnecessary adaptive change. One of the primary colon cancer models established with subcutaneous inoculation (HPTCSC-10-3) was further characterized. The histopathological analysis of the tumors from each passage showed the characters of a moderately differentiated colon epithelial adenocarcinoma with gland-like columnar epithelial lining mixed with mucin secretion, identical to the original tumor. The tumor growth rate following subcutaneous xenograft in nude mice was sensitive to the treatment of multiple drugs: 69%, 50% and 77% inhibition in response to the treatment for 3 weeks with CPT-11 (10 mg/kg, i.v., twice weekly), Docetaxel (2 mg/kg, i.v., twice weekly) and Sutent (40 mg/kg, p.o., daily), respectively. A 90.1% inhibition of tumor growth was reached by CPT-11 treatment at same dosing regimen following orthotopic xenograft in cecum. The primary cells of HPTCSC-10-3 tumor demonstrated to be more sensitive to Dox treatment compared to HCT-116 (IC50: 456 vs. 650 ng/ml) in MTT assay. Gene analysis of the tumor tissues using limited panel RT-PCR revealed that the expression of quit a few genes, such as Erk-1 and MMP-2, were increased in a passage-dependent pattern, indicating the adaptation activities following in vivo passage. The data demonstrated that a novel human colon cancer model was successfully established. The preliminary characterization indicated that this model was highly representative of a clinical colon cancer when used within 7 passages, providing a useful set of tools for investigation of anticancer intervention by subcutaneous or othortopic xenograft in nude mice, or assays in the cell culture. The collection of such human primary tumor models will greatly facilitate the translational studies of anti-cancer compounds in Chinese colon cancer patients. 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 4167.

1438 THE CANCER DRUG ENIGMOL ALTERS SPHINGOLIPID METABOLISM AND REDUCES MACROPHAGE PRESENCE IN PROSTATE TUMORS

You have accessJournal of UrologyProstate Cancer: Basic Research VIII1 Apr 20101438 THE CANCER DRUG ENIGMOL ALTERS SPHINGOLIPID METABOLISM AND REDUCES MACROPHAGE PRESENCE IN PROSTATE TUMORS Suzanne Mays, Harsha Ramaraju, Anatoliy Buushnev, Adeboye Osunkoya, Elayne Wang, Dennis Liotta, Alfred Merrill, and John Petros Suzanne MaysSuzanne Mays Atlanta, GA More articles by this author , Harsha RamarajuHarsha Ramaraju Ann Arbor, MI More articles by this author , Anatoliy BuushnevAnatoliy Buushnev Atlanta, GA More articles by this author , Adeboye OsunkoyaAdeboye Osunkoya Atlanta, GA More articles by this author , Elayne WangElayne Wang Atlanta, GA More articles by this author , Dennis LiottaDennis Liotta Atlanta, GA More articles by this author , Alfred MerrillAlfred Merrill Atlanta, GA More articles by this author , and John PetrosJohn Petros Atlanta, GA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1131AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Enigmol (2-amino-3,5-dihydroxyoctadecane[2S,3S,5S]) is an orally active anti-cancer drug designed by our group specifically to disrupt sphingolipid metabolism. We have demonstrated that this drug is as effective as standard therapies for prostate cancer (androgen deprivation or taxane chemotherapy) in preclinical models. The objective of these studies was to investigate Enigmol's ability to alter sphingolipids in prostate cancer xenografts and to identify molecular pathways that may be responsible for anti-tumor effects. METHODS LNCaP or PC-3 tumors were grown as subcutaneous xenografts in nude mice. Mice with established tumors received daily gavage of vehicle or Enigmol (10 or 50mg/kg/day for 12-26 days). Tumors from 4 mice representing the median tumor volumes of each group were excised for analysis. Sphingolipids were extracted and quantified by liquid chromatography and tandem mass spectrometry (LC-MS/MS). In PC-3 tumors, whole genome microarray was used to identify transcriptional changes. Macrophages were detected by immunohistochemistry (IHC) using the macrophage marker F4/80. RESULTS In both LNCaP and PC-3 tumors,Enigmol treatment significantly inhibited tumor growth. Enigmol accumulated in tumors in a dose-dependent manner. Comparing tumors in which similar concentrations of Enigmol were detected,sphingosine- and sphinganine 1-phospate were decreased by 1.5 and 1.8-fold, respectively, in LNCaP tumors (Enigmol treated vs controls) but unchanged in PC-3 tumors. Sphingosine (So) and sphinganine (Sa) were decreased nearly 3-fold in LNCaP tumors but were unchanged in PC-3 tumors; however, higher Enigmol concentrations in PC-3 tumors resulted in decreased Sa and So. Microarray analysis of PC-3 tumors treated with vehicle or Enigmol revealed a large set of genes related to immune response that were down regulated at least 1.5-fold upon Enigmol treatment, and IHC indicated a decreased presence of macrophages in tumor interiors. CONCLUSIONS Enigmol, an effective oral agent in in vivo models of human prostate cancer, alters sphingolipid metabolism in prostate tumors. Gene expression analysis indicated transcriptional down-regulation of many genes relating to immunity, and examination of tumors revealed decreased macrophage infiltration in treated tumors compared to controls. Enigmol likely acts at least in part by altering ratios of key sphingolipids in tumors with concomitant modification of components of the immune response. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e554 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Suzanne Mays Atlanta, GA More articles by this author Harsha Ramaraju Ann Arbor, MI More articles by this author Anatoliy Buushnev Atlanta, GA More articles by this author Adeboye Osunkoya Atlanta, GA More articles by this author Elayne Wang Atlanta, GA More articles by this author Dennis Liotta Atlanta, GA More articles by this author Alfred Merrill Atlanta, GA More articles by this author John Petros Atlanta, GA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Toxicological Study and Efficacy of Blank and Paclitaxel-Loaded Lipid Nanocapsules After i.v. Administration in Mice

Lipid nanocapsules (LNCs) are solvent-free drug nanocarriers permitting entrapment of paclitaxel and increasing its antitumoural effect in animal models after i.v. injection. The tolerance and efficacy of LNCs after repeated dose i.v. administration were assessed in mice. The maximum tolerated dose (MTD) and 50 percent lethal dose (LD50) were studied. Paclitaxel-loaded LNC formulation was given i.v. at the dose of 12 mg/kg per day for 5 consecutive days in comparison with blank LNCs and saline. Histological examination, complete blood counts and biochemical quantification were performed after a recovery of 7 days. Growth of NCI-H460 subcutaneous xenografts in nude mice receiving one of the aforementioned schedules was assessed. MTD and LD50 were determined by Irwin test. No mortality was observed in repeated injections studies. Histological studies revealed no lesions and no accumulation of lipids. Blood studies were normal. The tumoural growth was significantly reduced in the group treated by paclitaxel-loaded LNCs. The MTDs/LD50s of Taxol, paclitaxel-loaded LNCs and blank LNCs were 12/19.5, 96/216 and above 288/288 mg/kg, respectively. This study demonstrates that a five-day i.v. injection schedule of paclitaxel-loaded LNC dispersions induces no histological or biochemical abnormalities in mice and improves paclitaxel efficacy and therapeutic index in comparison with Taxol.

The Role of microRNA Genes in Breast Cancer Progression and Metastasis.

Abstract MicroRNAs influence tumor progression and metastasis via action as metastasis promoting genes (for example mir-373 and mir-520) or metastasis suppressor genes (for example mir-335). To identify additional microRNAs capable of driving breast cancer metastasis in vivo, we employed a functional genomics screen using the '4T1' syngeneic (Balb/c) mouse model of breast cancer progression. Approximately 400 microRNA sequences were subcloned into the pMSCV_puro retroviral expression vector and the resultant microRNA expression library transduced into fluorescent non-metastatic 67NR-pMSCV_mCherry and mildy-metastatic 66cl4-pMSCV_mCherry cells and selected with puromycin. The transduced cell lines were then implanted into the mammary fat pads of 20 syngeneic Balb/c mice per cell line. Following establishment of primary tumors, the primary tumors and secondary organs (lung, liver, spine, femurs), including any overt metastatic deposits, were dissected under a fluorescent stereomicroscope and the microRNA inserts amplified from the integrated retroviral genome by PCR.In an alternative approach, we have studied two populations of MDA-MB-231 human breast cancer cells that exhibit different behavior in vitro and in vivo to elucidate processes involved in cellular invasion and metastasis. A late-passage MDA-MB-231 (231variant) population formed invasive clusters when embedded in Matrigel, whereas early-passage (P3) MDA-MB-231 (231) cells did not. Moreover, 231v cells were more motile than 231 cells and showed enhanced tumorigenicity when grown as subcutaneous xenografts in nude mice, whereas proliferation and apoptotic response were indistinguishable between the two lines in vitro. Molecular characterization of the two cell populations cultured in 3D by array-based gene expression profiling revealed elevated expression of NF kappa B target genes in 231v cells (IL1A, IL1B, IL6, PTX3, NR4A2, HMOX1, FLIP). MicroRNA array analysis of 3D cultures also revealed marked induction (approximately 8-fold) of the related NF kappa B target microRNAs, mir-146a and mir-146b (Figure 1). Further evaluation of a panel of human breast cancer cell lines revealed that mir-146a and mir-146b were dramatically elevated in metastatic human breast cancer cell lines that display both epithelial-to-mesenchymal transition and high NF kappa B activity (MDA-MB-231, Hs578T, MDA-MB-436), compared to non-metastatic lines. Experiments to elucidate the function of mir-146a and mir-146b during breast cancer progression are underway. Figure 1: TaqMan qPCR analysis of mir-146a and mir-146b expression in 231 and 231v cells. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6142.

Hypoxia-inducible Factor-1-mediated Regulation of Semaphorin 4D Affects Tumor Growth and Vascularity

Tumor progression and metastasis depend on the ability of cancer cells to initiate angiogenesis to ensure delivery of oxygen, nutrients, and growth factors to tumor cells and provide access to the systemic circulation. Hypoxia-inducible factor-1 (HIF-1) can activate expression of a broad range of genes that mediate many of the adaptive responses to decreased oxygen concentration, such as enhanced glucose uptake and formation of new blood vessels. Acting through Plexin-B1 on endothelial cells, Semaphorin 4D (Sema4D) has been shown to promote angiogenesis and enhance invasive growth and proliferation in some tumors. Here we show that the gene for Sema4D, the product of which is elevated in head and neck squamous cell carcinoma (HNSCC) cells, contains upstream hypoxia response elements (HRE) and is strongly induced in hypoxia in a HIF-1-dependent manner. Knocking down Sema4D expression with short hairpin (sh) RNA reduces in vitro endothelial cell migration and growth and vascularity of HNSCC xenografts expressing a degradation resistant HIF-1alpha subunit. We also demonstrate a correlation between HIF-1 activity and Sema4D expression in HNSCC specimens. These findings indicate that Sema4D is induced by hypoxia in a HIF-1-dependent manner and influences endothelial cell migration and tumor vascularity. Expression of Sema4D may be a strategy by which carcinomas promote angiogenesis and therefore could represent a therapeutic target for these malignancies.

Insulin-like growth factor-I receptor blockade reduces the invasiveness of gastrointestinal cancers via blocking production of matrilysin

Insulin-like growth factor-I receptor (IGF-IR) signaling is required for carcinogenicity and proliferation of gastrointestinal (GI) cancers. We have previously shown significant therapeutic activity for recombinant adenoviruses expressing dominant-negative insulin-like growth factor-I receptor (IGF-IR/dn), including suppression of tumor invasion. In this study, we sought to evaluate the mechanism of inhibition of invasion and the relationship between IGF-IR and matrix metalloproteinase (MMP) activity in GI carcinomas. We analyzed the role of IGF-IR on invasion in three GI cancer cell lines, colorectal adenocarcinoma, HT29; pancreatic adenocarcinoma, BxPC3 and gastric adenocarcinoma, MKN45, using a modified Boyden chamber method and subcutaneous xenografts in nude mice. The impact of IGF-IR signaling on the expression of MMPs and the effects of blockade of matrilysin or IGF-IR on invasiveness were assessed using recombinant adenoviruses, a tyrosine kinase inhibitor NVP-AEW541 and antisense matrilysin. Invasive subcutaneous tumors expressed several MMPs. IGF-IR/dn reduced the expression of these MMPs but especially matrilysin (MMP-7). Insulin-like growth factor (IGF) stimulated secretion of matrilysin and IGF-IR/dn blocked IGF-mediated matrilysin induction in three GI cancers. Both IGF-IR/dn and inhibition of matrilysin reduced in vitro invasion to the same degree. NVP-AEW541 also reduced cancer cell invasion both in vitro and in murine xenograft tumors via suppression of matrilysin. Thus, blockade of IGF-IR is involved in the suppression of cancer cell invasion through downregulation of matrilysin. Strategies of targeting IGF-IR may have significant therapeutic utility to prevent invasion and progression of human GI carcinomas.

Synergistic use of epidermal growth factor and 5-fluorouracil for the treatment of human colorectal cancer in BALB/C nude mice subcutaneous xenograft model

To investigate the synergistic effect of epidermal growth factor(EGF) and 5-fluorouracil for the treatment of human colorectal cancer in BALB/C nude mice subcutaneous xenografts model. Human colorectal cancer Caco-2 cells were transplanted into the subcutaneous tissue of nude mice. The tumor growth was followed up every 4 days after treatment, and estimated tumor weight, tumor growth curve, and results from histologic examination were used to evaluate the effects of EGF on the growth of tumors. Proliferation of the tumor cells was estimated by PCNA labeling index. The combined use of EGF and 5-fluorouracil significantly inhibited the growth of colorectal cancer xenografts in nude mice with the inhibitory rate of 57.05% which was higher than 5-fluorouracil did (40.97%)(P<0.05). No pathologic changes were observed in organs. PCNA labeling index was elevated in combined group which implied more tumor cells reentry cell cycle. Epidermal growth factor, which may recruit colorectal cancer cells into activated phases of the cell cycle, can enhance the sensitivity of colorectal cancer cell Caco-2 to 5-fluorouracil.

Effects of the EGFR Inhibitor Erlotinib on Tumor Microenvironment: Implications for Therapy

Purpose/Objective(s): A recently published randomized study showed that combining an EGFR inhibitor with radiation increases survival and local control in patients with locally advanced head and neck cancer. We were interested in understanding the effects of EGFR inhibition on the tumor microenvironment in a head and neck cancer xenograft model, as this could provide insight into its effectiveness in combination with radiation and chemotherapy. Materials/Methods: We used SQ20B head and neck squamous cell carcinoma cells either in vitro or grown as subcutaneous xenografts in nude mice. Both in vitro (Western blotting, radiation cell survival assays) and in vivo studies (tumor regrowth assay, EF5 staining) were conducted. Results: We saw a minimal effect of erlotinib oninvitro radiosensitization. However, mice with SQ20B xenografts treated with the drug had prolonged time to tumor regrowth after 6 Gy of radiation compared to mice given radiation only. Because the in vivo effects appeared greater than the in vitro effects, we investigated the effects of erlotinib on the tumor microenvironment. Erlotinib decreased both vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1a expression in vitro and in tumor xenografts grown in vivo. We measured vascular permeability within the xenografts using Evan’s blue dye and found a 75% reduction in permeability in the tumors of mice treated with the drug. Consistent with these results, tomato lectin staining of the tumor vasculature showed that erlotinib treatment led to a dramatic change in vessel morphology. Using Power Doppler we found that there was a greater than 3-fold increase in tumor perfusion within 48 hours of starting erlotinib treatment. This increased vascular perfusion was evident even at 96 hours, in spite of the fact that the tumors showed a 50% decrease in size by this time. Using the hypoxia marker EF5 we found that the pattern of hypoxia was heterogeneous in these tumors but that erlotinib treatment led to improved oxygenation. Conclusions: Erlotinib decreases HIF-1a/VEGF expression in SQ20B xenografts but paradoxically increases vascular perfusion. A potential explanation for this is the idea of ‘‘vascular normalization’’ as proposed by Jain (Science 2005;307:58). This could lead to increased delivery of chemotherapy to tumors in patients responding to erlotinib. Improved vascular perfusion may also lead to increased oxygenation that could play a role in increased in vivo radiosensitization.

Ligand-Targeted Liposomal Therapies of Neuroblastoma

The central problem in cancer chemotherapy is the severe toxic side effects of anticancer drugs on healthy tissues. The use of liposomes as drug delivery vehicles for antitumour therapeutics has great potential to revolutionise the future of cancer therapy. As tumour architecture causes liposomes to preferentially accumulate at the tumour site, their use as drug carriers results in the localization of a greater amount of the loaded drug at the tumour site, thus improving cancer therapy and reducing the harmful non-specific side effects of chemotherapeutics. In addition, targeting of liposomal anticancer drugs to antigens expressed or over-expressed on tumour cells provides a very efficient system for increasing the therapeutic indices of the drugs. Animal models allow detailed examination of molecular and physiological basis of diseases and offer a frontline testing system for studying the involvement of specific genes and the efficacy of novel therapeutic approaches. Until recently, the most resorted experimental model of paediatric Neuroblastoma (NB) tumour is the subcutaneous xenograft in nude mice. However, the main disadvantage of this animal model is that it does not reflect the metastatic potential of NB cells, ultimately responsible for poor patient survival. A more realistic view of the clinical potential of targeted therapies could be obtained if a tumour model were available that better reflects the growth of advanced NB in children (i.e. large adrenal gland tumours and multiple small metastatic lesions). All current data support this concept and recommend that orthotopic implantation of tumour cells in recipient animals is mandatory for studies of tumour progression, angiogenesis, invasion, and metastasis. This review will focus on the description of the most clinically relevant animal models established to test the efficacy of targeted liposomal anti-tumour formulations for the treatment of Neuroblastoma.