Carcinoma Xenografts In Mice Research Articles

TLR9 Agonists Oppositely Modulate DNA Repair Genes in Tumor versus Immune Cells and Enhance Chemotherapy Effects

Synthetic oligodeoxynucleotides expressing CpG motifs (CpG-ODN) are a Toll-like receptor 9 (TLR9) agonist that can enhance the antitumor activity of DNA-damaging chemotherapy and radiation therapy in preclinical mouse models. We hypothesized that the success of these combinations is related to the ability of CpG-ODN to modulate genes involved in DNA repair. We conducted an in silico analysis of genes implicated in DNA repair in data sets obtained from murine colon carcinoma cells in mice injected intratumorally with CpG-ODN and from splenocytes in mice treated intraperitoneally with CpG-ODN. CpG-ODN treatment caused downregulation of DNA repair genes in tumors. Microarray analyses of human IGROV-1 ovarian carcinoma xenografts in mice treated intraperitoneally with CpG-ODN confirmed in silico findings. When combined with the DNA-damaging drug cisplatin, CpG-ODN significantly increased the life span of mice compared with individual treatments. In contrast, CpG-ODN led to an upregulation of genes involved in DNA repair in immune cells. Cisplatin-treated patients with ovarian carcinoma as well as anthracycline-treated patients with breast cancer who are classified as "CpG-like" for the level of expression of CpG-ODN modulated DNA repair genes have a better outcome than patients classified as "CpG-untreated-like," indicating the relevance of these genes in the tumor cell response to DNA-damaging drugs. Taken together, the findings provide evidence that the tumor microenvironment can sensitize cancer cells to DNA-damaging chemotherapy, thereby expanding the benefits of CpG-ODN therapy beyond induction of a strong immune response.

Image-Guided Tumor-Selective Radioiodine Therapy of Liver Cancer After Systemic Nonviral Delivery of the Sodium Iodide Symporter Gene

We reported the induction of tumor-selective iodide uptake and therapeutic efficacy of (131)I in a hepatocellular carcinoma (HCC) xenograft mouse model, using novel polyplexes based on linear polyethylenimine (LPEI), shielded by polyethylene glycol (PEG), and coupled with the epidermal growth factor receptor-specific peptide GE11 (LPEI-PEG-GE11). The aim of the current study in the same HCC model was to evaluate the potential of biodegradable nanoparticle vectors based on pseudodendritic oligoamines (G2-HD-OEI) for systemic sodium iodide symporter (NIS) gene delivery and to compare efficiency and tumor specificity with LPEI-PEG-GE11. Transfection of HCC cells with NIS cDNA, using G2-HD-OEI, resulted in a 44-fold increase in iodide uptake in vitro as compared with a 22-fold increase using LPEI-PEG-GE11. After intravenous application of G2-HD-OEI/NIS HCC tumors accumulated 6-11% ID/g (123)I (percentage of the injected dose per gram tumor tissue) with an effective half-life of 10 hr (tumor-absorbed dose, 281 mGy/MBq) as measured by (123)I scintigraphic gamma camera or single-photon emission computed tomography computed tomography (SPECT CT) imaging, as compared with 6.5-9% ID/g with an effective half-life of only 6 hr (tumor-absorbed dose, 47 mGy/MBq) for LPEI-PEG-GE11. After only two cycles of G2-HD-OEI/NIS/(131)I application, a significant delay in tumor growth was observed with markedly improved survival. A similar degree of therapeutic efficacy had been observed after four cycles of LPEI-PEG-GE11/(131)I. These results clearly demonstrate that biodegradable nanoparticles based on OEI-grafted oligoamines show increased efficiency for systemic NIS gene transfer in an HCC model with similar tumor selectivity as compared with LPEI-PEG-GE11, and therefore represent a promising strategy for NIS-mediated radioiodine therapy of HCC.

MiR-143 Overexpression Impairs Growth of Human Colon Carcinoma Xenografts in Mice with Induction of Apoptosis and Inhibition of Proliferation

BackgroundMicroRNAs (miRNAs) are aberrantly expressed in human cancer and involved in the (dys)regulation of cell survival, proliferation, differentiation and death. Specifically, miRNA-143 (miR-143) is down-regulated in human colon cancer. In the present study, we evaluated the role of miR-143 overexpression on the growth of human colon carcinoma cells xenografted in nude mice (immunodeficient mouse strain: N: NIH(s) II-nu/nu).Methodology/Principal FindingsHCT116 cells with stable miR-143 overexpression (Over-143) and control (Empty) cells were subcutaneously injected into the flanks of nude mice, and tumor growth was evaluated over time. Tumors arose ∼ 14 days after tumor cell implantation, and the experiment was ended at 40 days after implantation. miR-143 was confirmed to be significantly overexpressed in Over-143 versus Empty xenografts, by TaqMan® Real-time PCR (p<0.05). Importantly, Over-143 xenografts displayed slower tumor growth compared to Empty xenografts from 23 until 40 days in vivo (p<0.05), with final volumes of 928±338 and 2512±387 mm3, respectively. Evaluation of apoptotic proteins showed that Over-143 versus Empty xenografts displayed reduced Bcl-2 levels, and increased caspase-3 activation and PARP cleavage (p<0.05). In addition, the incidence of apoptotic tumor cells, assessed by TUNEL, was increased in Over-143 versus Empty xenografts (p<0.01). Finally, Over-143 versus Empty xenografts displayed significantly reduced NF-κB activation and ERK5 levels and activation (p<0.05), as well as reduced proliferative index, evaluated by Ki-67 immunohistochemistry (p<0.01).ConclusionsOur results suggest that reduced tumor volume in Over-143 versus Empty xenografts may result from increased apoptosis and decreased proliferation induced by miR-143. This reinforces the relevance of miR-143 in colon cancer, indicating an important role in the control of in vivo tumor progression, and suggesting that miR-143 may constitute a putative novel therapeutic tool for colon cancer treatment that warrants further investigation.

Stimulated γδ T Cells Increase the In Vivo Efficacy of Trastuzumab in HER-2+ Breast Cancer

One fourth of women with HER-2(+) metastatic breast carcinoma are treated with a combination regimen with trastuzumab, but the frequent resistance to this Ab requires definition of new means to improve its bioactivity. The mechanisms of action of trastuzumab involve several pathways including Ab-dependent cellular cytotoxicity. Because human γδ T lymphocytes mediate Ab-dependent cellular cytotoxicity and can be activated further by phosphoantigens, these cells are prone to improve the efficacy of Abs, as recently demonstrated for CD20(+) B cell lymphomas. Whether this concept applies as well with carcinomas remained to be demonstrated in vivo, however. In this study, we asked whether a combination of trastuzumab and phosphoantigen-stimulated γδ lymphocytes increases the efficacy of trastuzumab against HER-2(+) breast carcinoma cell lines in vivo. We report that repeated infusions of this combination had a better efficacy than that of trastuzumab alone against HER-2(+) mammary carcinoma xenografts in mice. In these models, reduction of tumor growth was observed together with trastuzumab opsonization of HER-2(+) cells and tumor infiltration by γδ lymphocytes. In addition in humans, the mammary carcinomas of 27 of 30 patients showed significant γδ T cell infiltrates. Altogether, these findings indicate that combination of trastuzumab and stimulated γδ cells represents a new strategy to improve the efficacy of Herceptin (trastuzumab) in HER-2(+) breast cancer.

Metabolomics study of alcohol-induced liver injury and hepatocellular carcinoma xenografts in mice

Alcohol abuse is one of the major causes of liver injury and a promoter for hepatocellular carcinoma (HCC). To understand the disease-associated metabolic changes, we investigated and compared the profiles of metabolites in nude mice with alcohol-induced liver injury or bearing a HCC xenograft (HCCX). Alcohol-induced liver injury was achieved by daily administration of grain liquor, and HCC xenografts were generated by subcutaneous inoculation of HepG2 cells in nude mice. Metabolites in serum samples were profiled by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS). The acquired data was analyzed by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) to identify potential disease-specific biomarkers. Results showed that the phosphatidylcholine (PC) levels were significantly higher in both liver injury and HCCX mice compared with the control. Interestingly, lysophosphatidylcholines (LPCs) that contain saturated or monounsaturated fatty acids were reduced in both liver injury and HCCX mice, but polyunsaturated fatty acids LPCs were elevated in liver injury mice only. These data delineated the disease-related metabolic alterations of LPCs in liver injury and HCC, suggesting that the LPC profile in serum may be biomarkers for these two common liver diseases.

Induction ofIn VitroandIn VivoNK Cell Cytotoxicity Using High-Avidity Immunoligands Targeting Prostate-Specific Membrane Antigen in Prostate Carcinoma

Cancer that might develop as host natural killer (NK) cells fail to detect ligands for their activating NK receptors. Immunoligands represent promising immunotherapeutic tools to overcome this deficit. These are fusion proteins containing a single-chain antibody fragment (scFv) to target an available tumor antigen and ULBP2 to activate host NK cells by targeting the activatory receptor NKG2D. Prostate-specific membrane antigen (PSMA) is an integral non-shed type 2 membrane protein that is highly and specifically expressed on prostate epithelial cells and strongly upregulated in prostate cancer. Here, we compare the impact of various anti-PSMA immunoligand formats on the therapeutic efficacy against prostate carcinoma cells by activating NK cells via NKG2D. Shortening of the linker separating the heavy and light chain antibody domain leads to the formation of dimers, trimers, and higher molecular mass oligomers. NK cells are most efficiently activated by multimeric immunoligands, thus showing an altered cytokine release pattern. The high avidity format is also superior in in vitro NK-mediated tumor cell targeting as shown in cytotoxicity assays. Finally, the efficacy of a multimeric immunoligand is shown in a prostate carcinoma mouse xenograft model showing a strong activity against advanced established tumors.

Cyclooxygenase‐2 inhibition causes antiangiogenic effects on tumor endothelial and vascular progenitor cells

Tumor angiogenesis is necessary for solid tumor progression and metastasis. Cyclooxygenase (COX)-2 is known to play an important role in cancer growth and invasion, and it activates the signaling pathways controlling cell proliferation, migration, apoptosis, and angiogenesis. COX-2 is reported to be expressed in many cancer cells. Several studies have reported successful treatment of cancer cells with COX-2 inhibitors (COX-2is). However, the effect of COX-2 inhibition on the tumor endothelium remains to be elucidated. Our study shows that COX-2 is expressed in the vasculature of surgically resected human tumors. To investigate the effects of COX-2 inhibition on the tumor endothelium in vitro, we isolated tumor endothelial cells (TECs) from human melanoma and oral carcinoma xenografts in mice, in which we confirmed that tumor growth was suppressed by inhibiting angiogenesis with the COX-2is NS398. COX-2 mRNA was upregulated in TECs compared to normal endothelial cells (NECs). Cell migration and proliferation were suppressed by NS398 in TECs but not in NECs. The effects of NS398 in vivo were consistent with the in vitro results. The number of CD133+ /vascular endothelial growth factor receptor-2+ cells in circulation was significantly suppressed by COX-2 inhibition. In addition, the number of progenitor marker-positive cells decreased in the tumor blood vessels after COX-2i treatment, which suggests that the homing of progenitor cells into the tumor was also blocked. We conclude that NS398 specifically targets both TECs and vascular progenitor cells without affecting NECs.

A nanoemulsion formulation of dacarbazine reduces tumor size in a xenograft mouse epidermoid carcinoma model compared to dacarbazine suspension

Dacarbazine (DAC) is an anticancer drug that has been used to treat various types of cancers. The aim of the current study was to test whether there is an increased efficacy of DAC as a nanoemulsion on reducing tumor size in an epidermoid carcinoma xenograft mouse model. Tumors were induced in 5-week-old nude mice by subcutaneous injection. The mice were untreated or treated with a suspension of DAC (0.1 mg/kg), a nanoemulsion of DAC (0.1 mg/kg), or Nano-Control (same composition as the suspension and nanoemulsion but no DAC), every 2 days by either intramuscular injection (IM) or topical application. After 40 days, the final tumor size of mice receiving the nanoemulsion of DAC IM (0.83 ± 0.55 mm 3) was significantly reduced compared to the suspension of DAC IM (4.75 ± 0.49 mm 3), Nano-Control IM (7.63 ± 0.91 mm 3), and untreated (10.46 ± 0.06 mm 3). The final tumor size of mice receiving the nanoemulsion of DAC topically (3.33 ± 0.63 mm3) was also significantly reduced compared to the suspension of DAC topically (7.64 ± 0.68 mm 3). This increased efficacy maybe partially attributed to: 1) the reduced particle size of the nanoemulsion in comparison with suspension (111 versus > 6000 nm), 2) reduction in zeta potential of the nanoemulsion compared to suspension (−3.2 versus −89.1 mV), 3) production of a stable water dispersion relative to unstable suspension, 4) decreased polydispersity index of the nanoemulsion compared to suspension, and 5) greater stability of drug with the nanoemulsion in comparison with the suspension. From the Clinical Editor In this clinically relevant study, the anti-tumor efficacy of dacarbazine was found to be significantly increased as a nanoemulsion in epidermoid carcinoma xenograft mice, both with IM and topical administration.

Effects of endostatin on the growth and lymphangiogenesis of Lewis lung carcinoma xenograft in mice

to investigate the effects of endostar, a recombined humanized endostatin, on the growth, lymphangiogenesis and lymphatic metastasis of Lewis lung carcinoma xenograft in mice. lewis lung carcinoma (LLC) xenograft were established in C57 mice by intravenous transplantation of 1 × 10(6) cells. Then tumor-bearing mice were assigned into two groups: control group received caudal vein injections of 0.2 ml of 0.9% sodium chloride for 15 days, and treatment group received 500 µg endostar daily. Six weeks after LLC cell injection mice were sacrificed, and then tumor numbers and size were recorded. The expression of vascular endothelial growth factor-c (VEGF-C) and microlymphatic vessel density (MLVD) were observed by immunohistochemical staining. tumor number and size of control group were significantly higher than those of treatment group. The microlymphatic vessel density (MLVD) was 5.7 ± 1.6 in the treatment group, which was markedly lower than in the control mice (7.8 ± 1.6). Two lymph node metastases were observed in treatment group, and eight in control group. Lymphatic metastases were more frequent in control group than in treatment group. Expression of VEGF-C in control group was significantly higher than that in treatment group. endostar significantly inhibits the growth, lymphangiogenesis and lymphatic metastasis of Lewis lung carcinoma xenografts, and the inhibitory effect is due to its ability to regulate the expression of VEGF-C of tumors in part.

A Novel p53-Based Strategy for Protection of Bone Marrow and Gastrointestinal (GI) Track From Total Body Irradiation (TBI)-Induced Damage.

AbstractAbstract 1454 Purpose:Recognizing p53 activation as the primary mechanism that underlies radiation-induced damage to bone marrow and GI track, we aim to explore the use of low dose arsenic-induced transient p53 inhibition as a novel strategy for radiotherapy protection. Methods:Expanding from our recent finding that low-dose of arsenic can suppress radiation-induced p53 activation and subsequent apoptosis, we used a mouse model to test the potential of arsenic in protection of bone marrow and GI track against TBI induced damage. Arsenic pretreatment was carried out by feeding sex-matched mice (4-6 weeks of age) with water with or without 1.0 mg/L sodium arsenic for three days. Mice were randomized into following groups; control; arsenic only; irradiation only; arsenic and irradiation. Mice were then irradiated with 2Gy TBI daily for 7 days. Radiation-induced bone marrow and GI track damages were analyzed histologically with H&E staining 4 weeks after last treatment. Animal body weight was monitored as a measure of toxicity. To test the prediction that arsenic would not provide protection for malignant cells because of their defect in p53, a lung carcinoma cell line NCI-H358 was used to generate a mouse xenograft model. Treatments were initiated three weeks after implantation when the lung carcinoma cells developed into readily visible tumor with an average volume of approximately 100 mm3. Tumor volumes were measured periodically. Tumor volume was calculated using the equation: (volume = length × width × depth × 0.5236 mm3). Two independent experiments were done and the tumor volumes are means ± SE from total of 10 mice per group. [Display omitted] Results:Consistent with published results, mice treated with irradiation significantly lost their body weight. TBI-induced body weight loss was effectively prevented by arsenic pretreatment (Fig. 1A). In line with the data of body weight change, radiation treatments were associated with severe damages to small intestine and bone marrow cells, and remarkably, such damages were significantly reduced by low-dose arsenic pretreatment (Fig. 1B). The results together demonstrate the potential of low-dose arsenic to effectively protect bone marrow and GI track to radiation-induced damage. [Display omitted] In tumor xenograft models, the tumor volume of the control group continued to increase with time. Arsenic treatment did not have any detectable effect on growth of the implanted tumors. As expected, irradiation with 2Gy of TBI daily for 7 days resulted in marked tumor regression. Arsenic pretreatment showed little effect on irradiation-induced tumor suppression (Fig. 2), indicating that low-dose arsenic pretreatment does not detectably affect the efficacy of radiation, at least in the human lung carcinoma xenograft mouse model. Collectively, the results demonstrate that a brief treatment with low-dose arsenic is associated with a marked protection of bone marrow and GI track without compromising the ability of irradiation to kill carcinoma cells. Conclusion:The animal studies have provided proof-of-principle for the use of low-dose arsenic in radiotherapy protection of bone marrow and GI track, serving as strong rationales to initiate clinical trials. Disclosures:No relevant conflicts of interest to declare.

Measurement of reoxygenation during fractionated radiotherapy in head and neck squamous cell carcinoma xenografts

Hypoxic tissues lack adequate oxygenation and it has been long established that tumours commonly exhibit hypoxia and that hypoxia is a factor contributing towards resistance to radiotherapy. To develop computer models and make predictions about the affects of tumour hypoxia on treatment outcome, quantitative tumour oxygenation and reoxygenation data from in vivo systems is required. The aim of this study was to investigate the timing and degree of reoxygenation during radiotherapy in a human head and neck squamous cell carcinoma xenograft mouse model (FaDu). Mice were immobilised using a novel restraining system and exposed unanaesthetised in 3 or 5 Gy fractions, up to a maximum of 40 Gy. Partial pressures of oxygen (pO2) measurements were recorded at six time points throughout the 2 week course of radiotherapy, using a fibre optic system. Tumours receiving 0-30 Gy did not exhibit an increase in pO2. However, the mean pO2 after 2 weeks of accelerated fractionated radiotherapy (40 Gy) was significantly increased (P<0.01) compared to the mean pO2 of tumours not receiving the full schedule (0-30 Gy). These results lead to the conclusion of an average reoxygenation onset time of 2 weeks in this group of xenografts. A relatively large range of pO2 values measured at each dose point in the study indicate a large inter-tumour variation in oxygenation among the tumours. Data from this experimental work will be used to define the range of reoxygenation onset times implemented in a Monte Carlo computer model, simulating hypoxic head and neck cancer growth and radiotherapy.

Inhibition of Mac-1 (CD11b/CD18) enhances tumor response to radiation by reducing myeloid cell recruitment

Despite recent advances in radiotherapy, loco-regional failures are still the leading cause of death in many cancer patients. We have previously reported that bone marrow-derived CD11b(+) myeloid cells are recruited to tumors grown in irradiated tissues, thereby restoring the vasculature and tumor growth. In this study, we examined whether neutralizing CD11b monoclonal antibodies could inhibit the recruitment of myeloid cells into irradiated tumors and inhibit their regrowth. We observed a significant enhancement of antitumor response to radiation in squamous cell carcinoma xenografts in mice when CD11b antibodies are administered systemically. Histological examination of tumors revealed that CD11b antibodies reduced infiltration of myeloid cells expressing S100A8 and matrix metalloproteinase-9. CD11b antibodies further inhibited bone marrow-derived cell adhesion and transmigration to C166 endothelial cell monolayers and chemotactic stimuli, respectively, to levels comparable to those from CD11b knockout or CD18 hypomorphic mice. Given the clinical availability of humanized CD18 antibodies, we tested two murine tumor models in CD18 hypomorphic or CD11b knockout mice and found that tumors were more sensitive to irradiation when grown in CD18 hypomorphic mice but not in CD11b knockout mice. When CD18 hypomorphism was partially rescued by reconstitution with the wild-type bone marrow, the resistance of the tumors to irradiation was restored. Our study thus supports the rationale of using clinically available Mac-1 (CD11b/CD18) antibodies as an adjuvant therapy to radiotherapy.

Abstract 5154: Human umbilical cord matrix stem cells transfected with the interferon-β gene markedly attenuated the growth of bronchioloalveolar carcinoma xenografts in mice

Abstract Mesenchymal stem cells derived from the human umbilical cord matrix (UCMSC) have great potential for therapeutic use in multiple diseases including cancer. UCMSC can be used as carriers for targeted delivery of genes or therapeutics and for local production of biologic agents. The therapeutic strategy using UCMSC as cellular vehicles for targeted gene delivery can avoid the problem of short life or excessive toxicity of a particular gene product in vivo. Interferon-β (IFN-β) has demonstrated a potent anti-tumor effect on many tumor cell lines in vitro. However, in vivo inhibition of tumor cell growth by IFN-β required blood concentrations much higher than the maximally tolerated dose. Therefore, clinical therapy by systemic administration of IFN-β was not possible. The aim of this study was to determine the anti-cancer effect of IFN-β gene transfected-UCMSC (IFN-UCMSC) on bronchioloalveolar carcinoma (BAC) cell lines in vitro and in vivo. The conditioned medium derived from IFN-UCMSC significantly attenuated cell growth of H358 and SW1573 BAC cell lines in vitro compared to the medium derived from non-transfected UCMSC. The percentage of dead cells, as measured by the trypan blue exclusion test, was significantly higher in the cells treated with the medium derived from IFN-UCMSC than UCMSC alone. The co-culture of IFN-UCMSC with cancer cells inhibited cell growth significantly through stimulation of apoptosis. Finally, systemic administration of IFN-UCMSC (0.3 million cells/injection, 5 day interval for four times) markedly attenuated tumor multiplicity and size of orthotopic H358 BAC xenografts in SCID mouse lungs. Histochemical analysis of the tumors indicated tumor-targeted localization of UCMSC and significant reduction of tumor cell proliferation index as measured by Ki-67 staining. These results clearly indicated that IFN-β production by IFN-UCMSC attenuated growth of BAC cells by inducing programmed cell death both in vitro and in vivo. This research clearly demonstrated that IFN-UCMSC are a powerful anti-cancer therapeutic tool as delivery vehicles for IFN-β. This work was supported by the KSU Terry C. Johnson Center for Basic Cancer Research, KSU Provost's Fund, KSU CVM Dean's Fund, NIH P20 RR017686 and a Joan's Legacy Research Grant. 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 5154.

Abstract 594: RNAi-mediated therapeutic knockdown of HER family members in vivo through polyethylenimine (PEI)/siRNA complexes and establishment of oligomaltose-modified PEI (OM-PEIs) as novel siRNA delivery platform

Abstract The therapeutic application of RNA interference (RNAi) for the knockdown of tumor-relevant genes critically relies on the efficient delivery of small interfering RNAs (siRNAs) or shRNA-encoding DNA plasmids in vivo. Previously, we have established polyethylenimine (PEI)/siRNA complexes as a highly efficient and ease-to-use system for gene targeting and a universally applicable platform for therapeutic RNAi. Here, we analyse the anti-tumor effects of the therapeutic knockdown of various members of the HER family, i.e. HER1 (c-erbB1, EGFR), HER2 (c-erbB2/neu) and HER3 (c-erbB3) in s.c. ovarian carcinoma xenografts in mice. While little effects are seen upon HER1 knockdown, the treatment of the mice with PEI/complexed siRNAs targeting HER2 or HER3 lead to a significant reduction in tumor growth. Notably, additive anti-tumor effects are observed upon double knockdown of HER2 and HER3. The biocompatibility of this PEI-based siRNA delivery platform for in vivo RNAi can be further increased by modifications of the polyethylenimines. To this end, we systematically analyse physicochemical and biological properties of DNA and siRNA complexes prepared from a set of maltose-, maltotriose- or maltoheptaose-modified hyperbranched PEIs (termed (oligo-)maltose-modified PEIs; OM-PEIs). While (oligo-)maltose grafting generally leads to reduced in vitro DNA transfection efficacies, this effect is less profound in OM-PEI/siRNA complexes for the induction of RNAi. This is particularly true for NH2-monosubstituted OM-PEIs. Independent of the maltose architecture, DNA and siRNA complexes based on maltose-grafted PEI show considerably lower toxicity as compared to PEI complexes. More importantly, pharmacokinetics in mice are markedly altered in OM-PEI/siRNA complexes. While their halflives in the blood circulation upon i.v. injection are generally increased as compared to PEI/siRNA complexes, the tissue distribution of radioactively labelled (OM-)PEI complexed siRNA molecules critically relies on the degree and pattern of (oligo-)maltose grafting. Marked differences in siRNA tissue distribution profiles are observed between different OM-PEIs. Enhanced tissue siRNA levels upon treatment of mice with OM-PEI/siRNA complexes as compared to PEI-based complexes are observed. Taken together, we (i) demonstrate the therapeutic anti-tumor effects of PEI/siRNA-mediated (double) knockdown of members of the HER family and (ii) establish OM-PEI complexes as novel promising tools for the in vivo application of siRNAs. 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 594.

Abstract 352: Preclinical evaluation of LYS6K1, a novel, highly selective, orally bioavailable inhibitor of p70 S6 kinase currently in phase I clinical trials for cancer

Abstract PI3K/AKT/mTOR/S6K signaling pathway (AKT pathway) controls cell survival, cell-cycle progression, cell growth and metabolism through a cascade phosphorylation of a number of key substrates. This pathway is regulated by three well characterized tumor suppressors; pten, tsc2, and lkb1. Deletion of these genes results in activation of the AKT pathway and proliferative disorders. Similarly, activating mutations of the receptor tyrosine kinases or PI3 Kinase result in the activation of the pathway. Therefore, multiple nodes of the pathway have become drug targets. As part of a comprehensive drug discovery platform aimed at targeting the PI3K pathway, we have developed a potent small molecule inhibitor of p70 S6 kinase that is a downstream effector in this pathway. LYS6K1 is a potent, highly selective ATP competitive inhibitor against p70 S6 kinase with an IC50 of 0.002 uM. In vitro, LYS6K1 inhibits the phosphorylation of S6 ribosomal protein in HCT116 colon cancer cells with an IC50 of 0.2 uM and similar activity is seen in a broad range of other cell lines. In vivo, LYS6K1.tosylate demonstrates potent phospho-S6 inhibition in nude mice bearing HCT116 colon carcinoma cells, with an ED50 value of 2.2 mg/kg and a ED90 value of 10 mg/kg 4 hours after a single oral dose. In these studies, LYS6K1.tosylate did not show statistically significant elevation of phospho-AKT, in spite of potent p70 S6 kinase inhibition. Proportional dose, exposure, and pharmacodynamic relationships were observed for LYS6K1 in dose- and time- dependent studies. In vivo, LYS6K1.tosylate effectively inhibits the growth of HCT116 colon carcinoma xenografts in mice and the growth of U87MG glioblastoma tumor as a single agent at 2.5 mg/kg given twice daily. Overall, LYS6K1.tosylate has high permeability that resulted in good oral absorption and PK properties. Based on these pre-clinical observations, LYS6K1.tosylate has advanced to Phase I studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 352.

Antitumor 1-nitroacridine derivative C-1748, induces apoptosis, necrosis or senescence in human colon carcinoma HCT8 and HT29 cells

C-1748 is a DNA-binding agent with potent antitumor activity, especially towards prostate and colon carcinoma xenografts in mice. Here, we elucidated the nature of cellular response of human colon carcinoma HCT8 and HT29 cells to C-1748 treatment, at biologically relevant concentrations (EC 90 and their multiplicity). Cell cycle analysis showed gradual increase in HCT8 cells with sub-G1 DNA content (25% after 72 h) considered as apoptotic. Hypodiploid cell population increased up to 60% upon treatment with 4× EC 90 concentration of the drug. Compared with HCT8 cells, the fraction of sub-G1 HT29 cells did not exceed 14%, even following 4-fold dose escalation. Morphological changes and biochemical markers such as: phosphatydylserine externalization, apoptotic DNA breaks, mitochondrial dysfunction and caspase activation confirmed the presence of considerable amount of apoptotic HCT8 cells but only a low amount of apoptotic HT29 cells. Next, we demonstrated that HCT8 cells surviving after exposure to C-1748 were in the state of senescence, based on altered cell morphology and expression of a pH 6-dependent β-galactosidase. On the contrary, no β-galactosidase staining was observed in HT29 cells after C-1748 treatment. Moreover, prolonged drug incubation (up to 168 h) resulted in massive detachment of cells from culture plates, which together with Annexin V/PI results, indicated that necrosis was the main response of HT29 cells to C-1748 treatment. We also determined the ability of C-1748 to induce reactive oxygen species (ROS) in colon cancer cells and demonstrated, that generation of ROS was not essential for C-1748-induced apoptosis and cytotoxic activity of this drug.

N3-o-toluyl-fluorouracil inhibits human hepatocellular carcinoma cell growth via sustained release of 5-FU

PurposeN3-o-toluyl-fluorouracil (TFU), the prodrug of 5-fluorouracil (5-FU), is the metabolite of N1-acetyl-N3-o-toluyl-fluorouracil (atofluding). In the present study, we aimed to evaluate the efficacy of TFU on the inhibition of human hepatocellular carcinoma cells via sustained release of 5-FU. The metabolism of TFU underlying the inhibitory effect was also analyzed.MethodsIn vitro assays, inhibition of cell growth by TFU was evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method. The levels of TFU and 5-FU in the cell culture supernatant fluid were measured by high-performance liquid chromatography (HPLC). In vivo assays, the efficacy of TFU was evaluated in a human hepatocellular carcinoma xenograft mice model after 3 weeks of oral administration. The distributions of TFU and 5-FU in plasma and homogenate tissues including liver, lung and tumor were determined by HPLC.ResultsN3-o-toluyl-fluorouracil weakly inhibited the proliferation of SMMC-7721 and PLC/PRF/5 cells in the absence of liver microsomal enzymes. In contrast, the inhibition rates were significantly increased in the presence of these enzymes. HPLC results revealed that TFU was metabolized slowly by liver microsomal enzymes and therefore the concentration of 5-FU was gradually increased with a longer retention time in cell culture supernatant fluid. The efficacy of TFU was confirmed in SMMC-7721 xenografts in Balb/c athymic (nu+/nu+) mice model. TFU treatment induced inhibition of SMMC-7721 growth with few side effects. HPLC results showed that high levels of TFU were still in liver 48 h after the end of oral administration, implying that TFU preferentially accumulated in liver with slow conversion to 5-FU by enzymes. This led to a long-lasting concentration of 5-FU in plasma. Further, a high level of 5-FU was found in tumors with a relatively low level in lungs. These results suggest that the metabolite of TFU was preferentially converted or taken up by tumor cells. The distributions of 5-FU may contribute to its high anti-tumor activity and low adverse reactions in vivo.ConclusionThese results demonstrate that TFU is a promising prodrug of 5-FU for cancer treatment via sustained release of 5-FU in liver.

Carbogen breathing increases prostate cancer oxygenation: a translational MRI study in murine xenografts and humans

Hypoxia has been associated with poor local tumour control and relapse in many cancer sites, including carcinoma of the prostate. This translational study tests whether breathing carbogen gas improves the oxygenation of human prostate carcinoma xenografts in mice and in human patients with prostate cancer. A total of 23 DU145 tumour-bearing mice, 17 PC3 tumour-bearing mice and 17 human patients with prostate cancer were investigated. Intrinsic susceptibility-weighted MRI was performed before and during a period of carbogen gas breathing. Quantitative R2* pixel maps were produced for each tumour and at each time point and changes in R2* induced by carbogen were determined. There was a mean reduction in R2* of 6.4% (P=0.003) for DU145 xenografts and 5.8% (P=0.007) for PC3 xenografts. In all, 14 human subjects were evaluable; 64% had reductions in tumour R2* during carbogen inhalation with a mean reduction of 21.6% (P=0.0005). Decreases in prostate tumour R2* in both animal models and human patients as a result of carbogen inhalation suggests the presence of significant hypoxia. The finding that carbogen gas breathing improves prostate tumour oxygenation provides a rationale for testing the radiosensitising effects of combining carbogen gas breathing with radiotherapy in prostate cancer patients.

The Inhibition of the Highly Expressed Mir-221 and Mir-222 Impairs the Growth of Prostate Carcinoma Xenografts in Mice

BackgroundMiR-221 and miR-222 are two highly homologous microRNAs whose upregulation has been recently described in several types of human tumors, for some of which their oncogenic role was explained by the discovery of their target p27, a key cell cycle regulator. We previously showed this regulatory relationship in prostate carcinoma cell lines in vitro, underlying the role of miR-221/222 as inducers of proliferation and tumorigenicity.Methodology/Principal FindingsHere we describe a number of in vivo approaches confirming our previous data. The ectopic overexpression of miR-221 is able, per se, to confer a high growth advantage to LNCaP-derived tumors in SCID mice. Consistently, the anti-miR-221/222 antagomir treatment of established subcutaneous tumors derived from the highly aggressive PC3 cell line, naturally expressing high levels of miR-221/222, reduces tumor growth by increasing intratumoral p27 amount; this effect is long lasting, as it is detectable as long as 25 days after the treatment. Furthermore, we provide evidence in favour of a clinical relevance of the role of miR-221/222 in prostate carcinoma, by showing their general upregulation in patient-derived primary cell lines, where we find a significant inverse correlation with p27 expression.Conclusions/SignificanceThese findings suggest that modulating miR-221/222 levels may have a therapeutic potential in prostate carcinoma.

Tumor targeting of baculovirus displaying a lymphatic homing peptide

Tumor-associated cells and vasculature express attractive molecular markers for site-specific vector targeting. To attain tumor-selective tropism, we recently developed a baculovirus vector displaying the lymphatic homing peptide LyP-1, originally identified by ex vivo/in vivo screening of phage display libraries, on the viral envelope by fusion to the transmembrane anchor of vesicular stomatitis virus G-protein. In the present study, we explored the specificity and kinetics of viral binding and internalization as well as in vivo tumor homing of the LyP-1 displaying virus to elucidate the applicability of baculovirus for targeted therapies. We demonstrated that the LyP-1 peptide contributes to saturable binding of baculovirus in human MDA-MB-435 and HepG2 carcinoma cells and escalates the kinetics of viral internalization leading to earlier nuclear accumulation and enhanced transgene expression. The LyP-1 displaying virus also showed stronger competitiveness against transduction with wild-type baculovirus, suggesting involvement of a specific receptor in cellular attachment and entry. Following intravenous injections, the modified virus accumulated within the human MDA-MB-435 and MDA-MB-231 carcinoma xenografts in mice with higher specificity and efficiency than the control virus. Targeting of the modified virus was more specific in the MDA-MB-435 than in the MDA-MB-231 xenografts as demonstrated by higher tumor accumulation and lower distribution in nontarget organs. No apparent cytotoxicity was associated with the surface modification. This first demonstration of in vivo tumor targeting of a systemically administered, tropism-modified baculoviral vector highlights the potential of baculovirus-mediated targeted therapies.