Altered binding avidities and improved growth inhibitory effects of novel anti-HER3 mAb against human cancers in the presence of HER1-or HER2-targeted drugs.

HER1-and HER2-targeted drugs are effective in cancer therapy, especially against lung, breast and colon malignancies; however, resistance of cancer cells to HER1-and HER2-targeted therapies is becoming a serious problem. The avidity/affinity constant (KA) and growth inhibitory effect of anti-HER3 rat monoclonal antibodies (mAb, Ab1∼Ab6) in the presence of therapeutic mAb or low-molecular-weight inhibitors against HER family proteins were analyzed by flow cytometry-based Scatchard plots (Splot) and cell proliferation assay. The KA of Ab3 and Ab6, but not Ab1 or Ab4, split into dual (high and low) modes of KA, and Ab6 exhibited greater anti-proliferative effects against LS-174T colon cancer cells in the presence of Pertuzumab (anti-HER2 mAb). A high KA by Ab6 and Ab6-mediated increased growth inhibition were observed against NCI-H1838 lung or BT474 breast cancer cells, respectively, in the presence of Panitumumab (anti-HER1 mAb) or Perutuzumab. A high KA by Ab6 and Ab6-mediated increased anti-proliferative effects against NCI-H1838 or BT474 were also respectively observed in the presence of Erlotinib (HER1 inhibitor) or Lapatinib (HER1/HER2 inhibitor). In HER1-knockout (KO) NCI-H1838, the reactivity and KA of Ab4 increased compared with in parent NCI-H1838. In HER1-KO or HER3-KO SW1116 colon cancer cells, dual modes of KA with Pertuzumab were noted, and the combination Ab6 and Pertuzumab promoted growth inhibition of HER1-KO, but not of parent SW1116.

Serum levels of galectin-3 in idiopathic inflammatory myopathies: a potential biomarker of disease activity.

OBJECTIVES: Galectin-3 is involved in various biological activities, including immune activations and fibrosis. Idiopathic inflammatory myopathies (IIMs) are autoimmune diseases of unknown aetiology, often complicated by interstitial lung disease (ILD). The aim of this study was to evaluate the expression of galectin-3 in sera and tissues of patients with IIM and assess the associations of galectin-3 with patient characteristics and disease activity. RESULTS: Serum galectin-3 levels were significantly higher in IIM patients than in healthy controls. The serum galectin-3 levels positively correlated with serum levels of inflammatory markers and proinflammatory cytokines/chemokines and the Myositis Intention-to-Treat Activity Index. Stratification analysis revealed that patients with IIM-associated ILD (IIM-ILD) had significantly higher levels of serum galectin-3 than those without IIM-ILD. In addition, patients with acute/subacute interstitial pneumonia had significantly higher levels of serum galectin-3 than those with chronic interstitial pneumonia. Furthermore, serum galectin-3 levels in IIM-ILD patients correlated with the radiological assessments of parenchymal lung involvement and treatment response. Immunohistochemical analysis revealed that galectin-3 was expressed in inflammatory cells of myositis and dermatitis sections, whereas in ILD sections, galectin-3 was expressed in interstitial fibrosis and inflammatory cells. CONCLUSION: Galectin-3 may be involved in the pathogenesis of inflammatory and fibrotic conditions in IIM and can serve as a potential biomarker of disease activity, especially in patients with IIM-ILD.

Pr1E11, a novel anti-TROP-2 antibody isolated by adenovirus-based antibody screening, recognizes a unique epitope.

TROP-2 is a type Ⅰ transmembrane glycoprotein that is highly expressed in various epithelial cancer cells, and its increased expression correlates with poor prognosis. Although several anti-TROP-2 antibodies have been described, they were found unsuitable for antitumor therapy use in vivo as naked antibodies. In this study, we established a novel anti-TROP-2 antibody, designated Pr1E11, from mice immunized with primary prostate cancer cells. Antibody screening was based on the infection activity of Adv-LacZ-FZ33, which displays an immunoglobulin G binding domain in the adenoviral fiber protein. We found that Pr1E11 specifically binds to TROP-2 with high affinity and recognizes diverse epithelial cancer cell lines and primary pancreatic cancer tissues. Epitope analysis using TROP-2 deletion mutants revealed that binding site of Pr1E11 is a cysteine-rich domain, a unique epitope compared with other available anti-TROP-2 antibodies. In addition, Pr1E11 exhibited low internalization activity, which may make it suitable for naked antibody therapeutics. Our results suggest that Pr1E11 may stimulate different biological activities from other anti-TROP-2 antibodies based on its unique binding epitope, and is a potential candidate for naked antibody therapeutics for various epithelial cancer treatments.

The usefulness of three-dimensional cell culture in induction of cancer stem cells from esophageal squamous cell carcinoma cell lines.

In recent years, research on resistance to chemotherapy and radiotherapy in cancer treatment has come under the spotlight, and researchers have also begun investigating the relationship between resistance and cancer stem cells. Cancer stem cells are assumed to be present in esophageal cancer, but experimental methods for identification and culture of these cells have not yet been established. To solve this problem, we created spheroids using a NanoCulture® Plate (NCP) for 3-dimensional (3-D) cell culture, which was designed as a means for experimentally reproducing the 3-D structures found in the body. We investigated the potential for induction of cancer stem cells from esophageal cancer cells. Using flow cytometry we analyzed the expression of surface antigen markers CD44, CD133, CD338 (ABCG2), CD318 (CDCP1), and CD326 (EpCAM), which are known cancer stem cell markers. None of these surface antigen markers showed enhanced expression in 3-D cultured cells. We then analyzed aldehyde dehydrogenase (ALDH) enzymatic activity using the ALDEFLUOR reagent, which can identify immature cells such as stem cells and precursor cells. 3-D-cultured cells were strongly positive for ALDH enzyme activity. We also analyzed the expression of the stem cell-related genes Sox-2, Nanog, Oct3/4, and Lin28 using RT-PCR. Expression of Sox-2, Nanog, and Lin28 was enhanced. Analysis of expression of the hypoxic surface antigen marker carbonic anhydrase-9 (CA-9), which is an indicator of cancer stem cell induction and maintenance, revealed that CA-9 expression was enhanced, suggesting that hypoxia had been induced. Comparison of cancer drug resistance using cisplatin and doxorubicin in 3-D-cultured esophageal cancer cells showed that cancer drug resistance had increased. These results indicate that 3-D culture of esophageal squamous cell carcinoma lines is a useful method for inducing cancer stem cells.

Novel [ 111 In]In-BnDTPA-EphA2-230-1 Antibody for Single-Photon Emission Computed Tomography Imaging Tracer Targeting of EphA2.

Erythropoietin-producing hepatocellular receptor A2 (EphA2) is overexpressed in cancer cells and causes abnormal cell proliferation. Therefore, it has attracted attention as a target for diagnostic agents. In this study, the EphA2-230-1 monoclonal antibody (EphA2-230-1) was labeled with [111In]In and evaluated as an imaging tracer for single-photon emission computed tomography (SPECT) of EphA2. EphA2-230-1 was conjugated with 2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (p-SCN-BnDTPA) and then labeled with [111In]In. [111In]In-BnDTPA-EphA2-230-1 was evaluated in cell-binding, biodistribution, and SPECT/computed tomography (CT) studies. The cellular uptake ratio of [111In]In-BnDTPA-EphA2-230-1 was 14.0 ± 2.1%/mg protein at 4 h in the cell-binding study. In the biodistribution study, a high uptake of [111In]In-BnDTPA-EphA2-230-1 was observed in tumor tissue (14.6 ± 3.2% injected dose/g at 72 h). The superior accumulation of [111In]In-BnDTPA-EphA2-230-1 in tumors was also confirmed using SPECT/CT. Therefore, [111In]In-BnDTPA-EphA2-230-1 has potential as a SPECT imaging tracer for EphA2.

Circulating tumor necrosis factor-related biomarkers predict kidney function decline in Japanese patients with diabetes: An observational cohort study.

AIMS: Tumor necrosis factor (TNF) receptors (TNFRs: TNFR1 and, TNFR2) are reportedly associated with chronic kidney disease (CKD) progression chiefly in Caucasian patients with diabetes. We assessed the prognostic value of TNF-related biomarkers for CKD progression in Japanese patients with diabetes. METHODS: We estimated TNF-related biomarkers using an enzyme-linked immunosorbent assay in 640 patients with diabetes. Cox proportional hazards analysis was performed to estimate hazard ratios (HRs) per one standard deviation (SD) increase in a log-transformed biomarker. The kidney and the composite outcome were defined as a 30% reduction in estimated glomerular filtration rate (eGFR) from baseline, and kidney outcome plus death before kidney outcome, respectively. RESULTS: During the median follow-up of 5.4 years, 75 (11.7%) patients reached the kidney outcome and 37 (5.8%) died before reaching the kidney outcome. Each SD increase in baseline circulating TNFR1, TNFR2, and ephrin type-A receptor 2 (EphA2) was associated with a higher risk of the kidney outcome independently from baseline eGFR and urine albumin-to-creatinine ratio. However, circulating osteoprotegerin was associated with the composite outcome only. CONCLUSIONS: Elevated TNFR1, TNFR2, and EphA2 were associated with both kidney and composite outcomes in Japanese patients with diabetes.

Heme oxygenase-1 (HO-1) is constitutively up-regulated in top alpinists.

Alpinists who challenge Mt. Everest need adaptation to hypoxia before the attack of Mt. Everest. Although this adaptation is important for the success of climbing Mt. Everest, the molecular mechanism on the adaptation to hypoxia is not well understood. In order to clarify this mechanism, we investigated hypoxia-induced gene expressions specific for top alpinists using microarray analyses. We report here that heme oxygenase-1 (HO-1) is significantly higher in the blood of top alpinist compared with non-alpinists. Although HO-1 expression of non-alpinists is also up-regulated in response to hypoxia, HO-1 level of the top alpinists are constitutively higher than that of non-alpinists. Serial examinations of HO-1 in one top alpinist revealed that the higher expression of HO-1 is maintained in high-level several months after the attack of top mountains. Taken together with the biochemical function of HO-1 that catalyzes heme into CO and bilirubin, HO-1 expression may improve the circulation and compensate with oxidative tissue damages induced by hypoxia. These data also suggest that peripheral blood has the memory on hypoxia independent of antigens by maintaining the high-level of HO-1 expression in top alpinists, which merits the rapid adaptation to hypoxia for 8000m climbing.

Development of an In-House EphA2 ELISA for Human Serum and Measurement of Circulating Levels of EphA2 in Hypertensive Patients with Renal Dysfunction.

Identifying novel biomarkers of kidney function in patients with chronic kidney disease (CKD) has strong clinical value as current measures have limitations. This study aims to develop and validate a sensitive and specific ephrin type-A receptor 2 (EphA2) enzyme-linked immunosorbent assay (ELISA) for human serum, and determine whether its results correlate with traditional renal measures in patients with hypertension. The novel ELISA of the current study was validated and used to measure circulating EphA2 levels in 80 hypertensive patients with and without kidney function decline (eGFR less than 60 mL/min/1.73 m2). Validation of the EphA2 ELISA showed good recovery (87%) and linearity (103%) and no cross-reactivity with other Eph receptors. Patients with kidney function decline had lower diastolic blood pressure, and higher UPCR and EphA2 than those without kidney function decline. The association of age and eGFR with EphA2 was maintained in the stepwise multiple regression analysis. In a multivariate logistic model, EphA2 was associated with a lower eGFR (<60 mL/min/1.73 m2) after adjustment for age, sex, and UPCR. High circulating EphA2 levels have potential application as a clinical biomarker for the presence of CKD in patients with hypertension.

Neural cell adhesion molecule 2 as a target molecule for prostate and breast cancer gene therapy.

In adenovirus-derived gene therapy, one of the problems is the difficulty in specific targeting. We have recently demonstrated that monoclonal antibody (mAb) libraries screened by fiber-modified adenovirus vector (Adv-FZ33), which is capable of binding to immunoglobulin-G (IgG), provide a powerful approach for the identification of suitable target antigens for prostate cancer therapy. Hybridoma libraries from mice immunized with androgen-dependent prostate cancer cell line LNCaP were screened and mAb were selected. Through this screening, we obtained one mAb, designated LNI-29, that recognizes a glycoprotein with an apparent molecular mass of 100 kD. It was identified as neural cell adhesion molecule 2 (NCAM2). Some prostate and breast cancer cell lines highly expressed NCAM2 whereas normal prostate cell lines expressed NCAM2 at low levels. In contrast to the low efficiency of gene transduction by Adv-FZ33 with a control antibody, LNI-29-mediated Adv-FZ33 infection induces high rates of gene delivery in NCAM2-positive cancers. NCAM2-mediated therapeutic gene transduction of uracil phosphoribosyltransferase (UPRT) had a highly effective cytotoxic effect on NCAM2-positive cancer cells, whereas it had less of an effect in cases with a control antibody. In conclusion, NCAM2 should be a novel gene therapy target for the treatment of prostate and breast cancer.

NK Cells Can Preferentially Target Prostate Cancer Stem-like Cells via the TRAIL/DR5 Signaling Pathway.

BACKGROUND: The occurrence of androgen-dependent prostate cancer mainly depends on prostate cancer stem cells. To reduce the risk of androgen-dependent prostate cancer, the direct elimination of prostate cancer stem cells is important, but an elimination strategy has not yet been established. A previous study showed that natural killer (NK) cells can preferentially target cancer stem cells in several solid tumors except prostate cancer. In this context, this study was undertaken to investigate if NK cells can selectively attack androgen-dependent prostate cancer stem cells. METHODS: Prostate cancer stem-like cells were separated from an androgen-dependent prostate cancer cell line (LNCaP) using a three-dimensional culture system. LNCaP stem-like cells or LNCaP cells were co-cultured with human NK cells (KHYG-1) for 24-72 h, and cell viability was determined using the WST-8 method. The expression of each protein in the cell membrane was evaluated through FACS analysis, and mRNA levels were determined using real-time PCR. RESULTS: KHYG-1 cells had more potent cytotoxicity against LNCaP stem-like cells than LNCaP cells, and the potency of the cytotoxicity was strongly related to the TRAIL/DR5 cell death pathway. CONCLUSION: NK cells can preferentially target prostate cancer stem-like cells via the TRAIL/DR5 pathway.

Habitual cigarette smoking attenuates shear-mediated dilation in the brachial artery but not in the carotid artery in young adults.

In the present study, we hypothesized that habitual cigarette smoking attenuates endothelial function in the cerebral circulation as well as that of the peripheral circulation in young adults. To test this hypothesis, we measured cerebrovascular and peripheral flow-mediated dilation (FMD) in young smokers and nonsmokers in the present study. Ten healthy nonsmokers and 10 smokers participated in the study. We measured blood velocity and diameter in the brachial artery and internal carotid artery (ICA) using Doppler ultrasound. We identified shear-mediated dilation in the brachial artery and ICA by the percentage change in peak diameter during hyperemia stimulation (reactive hyperemia and hypercapnia). We measured the baseline diameter and the shear rate area under the curve from the onset of hyperemia to peak dilation in the brachial artery and ICA, finding the measurements of the smokers and those of the nonsmokers did not differ (p > .05). In contrast to brachial FMD (5.07 ± 1.79% vs. 7.92 ± 3.01%; smokers vs. nonsmokers, p = .019), FMD in the ICA was not attenuated in the smokers compared with that of the nonsmokers (5.46 ± 2.32% vs. 4.57 ± 2.70%; p = .442). These findings indicate that in young healthy smokers, cerebral endothelial function was preserved, and the response of cerebral endothelial function to smoking was different from that of peripheral vasculature.

Emerging concepts in biomarker discovery; the US-Japan Workshop on Immunological Molecular Markers in Oncology.

Supported by the Office of International Affairs, National Cancer Institute (NCI), the "US-Japan Workshop on Immunological Biomarkers in Oncology" was held in March 2009. The workshop was related to a task force launched by the International Society for the Biological Therapy of Cancer (iSBTc) and the United States Food and Drug Administration (FDA) to identify strategies for biomarker discovery and validation in the field of biotherapy. The effort will culminate on October 28th 2009 in the "iSBTc-FDA-NCI Workshop on Prognostic and Predictive Immunologic Biomarkers in Cancer", which will be held in Washington DC in association with the Annual Meeting. The purposes of the US-Japan workshop were a) to discuss novel approaches to enhance the discovery of predictive and/or prognostic markers in cancer immunotherapy; b) to define the state of the science in biomarker discovery and validation. The participation of Japanese and US scientists provided the opportunity to identify shared or discordant themes across the distinct immune genetic background and the diverse prevalence of disease between the two Nations. Converging concepts were identified: enhanced knowledge of interferon-related pathways was found to be central to the understanding of immune-mediated tissue-specific destruction (TSD) of which tumor rejection is a representative facet. Although the expression of interferon-stimulated genes (ISGs) likely mediates the inflammatory process leading to tumor rejection, it is insufficient by itself and the associated mechanisms need to be identified. It is likely that adaptive immune responses play a broader role in tumor rejection than those strictly related to their antigen-specificity; likely, their primary role is to trigger an acute and tissue-specific inflammatory response at the tumor site that leads to rejection upon recruitment of additional innate and adaptive immune mechanisms. Other candidate systemic and/or tissue-specific biomarkers were recognized that might be added to the list of known entities applicable in immunotherapy trials. The need for a systematic approach to biomarker discovery that takes advantage of powerful high-throughput technologies was recognized; it was clear from the current state of the science that immunotherapy is still in a discovery phase and only a few of the current biomarkers warrant extensive validation. It was, finally, clear that, while current technologies have almost limitless potential, inadequate study design, limited standardization and cross-validation among laboratories and suboptimal comparability of data remain major road blocks. The institution of an interactive consortium for high throughput molecular monitoring of clinical trials with voluntary participation might provide cost-effective solutions.

Advanced microscopic evaluation of parallel type I and type II cell deaths induced by multi-functionalized gold nanocages in breast cancer.

Despite aggressive surgical resections and combinatorial chemoradiations, certain highly malignant populations of tumor cells resurrect and metastasize. Mixed-grade cancer cells fail to respond to standard-of-care therapies by developing intrinsic chemoresistance and subsequently result in tumor relapse. Macroautophagy is a membrane trafficking process that underlies drug resistance and tumorigenesis in most breast cancers. Manipulating cellular homeostasis by a combinatorial nanotherapeutic model, one can evaluate the crosstalk between type I and type II cell death and decipher the fate of cancer therapy. Here, we present a multi-strategic approach in cancer targeting to mitigate the autophagic flux with subcellular toxicity via lysosome permeation, accompanied by mitochondrial perturbation and apoptosis. In this way, a nanoformulation is developed with a unique blend of a lysosomotropic agent, an immunomodulating sulfated-polysaccharide, an adjuvant chemotherapeutic agent, and a monoclonal antibody as a broad-spectrum complex for combinatorial nanotherapy of all breast cancers. To the best of our knowledge, this manuscript illustrates for the first time the applications of advanced microscopic techniques such as electron tomography, three-dimensional rendering and segmentation of subcellular interactions, and fate of the multifunctional therapeutic gold nanocages specifically targeted toward breast cancer cells.

Combination Effect of Bowman-Birk Inhibitor and α-Tocopheryl Succinate on Prostate Cancer Stem-Like Cells.

The reoccurrence of androgen-dependent prostate cancer after anti-androgen therapy mainly depends on prostate cancer stem-like cells. To reduce the risk, it is important to delete the cancer stem-like cells. Furthermore, to induce differentiation of cancer stem-like cells is critical to abrogate stemness of the cells. Therefore, we tried to investigate a possibility on the establishment of a new effective therapy to eradicate the cancer stem-like cells via the induction of differentiation in this study. Prostate cancer stem-like cells from an androgen-dependent prostate cancer cell line (LNCaP cell) had severe resistance against an anti-androgen therapeutic agent. We selected Bowman-Birk inhibitor (BBI) from soybeans reported as a chemopreventive agent in prostate cancer to differentiate the caner stem-like cells and α-tocopheryl succinate (TOS) known as a mitocan to induce effectively cytotoxic effect against the cancer stem-like cells. In fact, only TOS treatment had cytotoxic effect against the cancer stem-like cells, but the addition of BBI treatment to the cells treated with TOS reinforced TOS-mediated cytotoxicity in the cancer stem-like cells. This reinforcement coincided with the combination-enhanced apoptosis in the stem-like cells. Also, we confirmed caspase9-caspase3 cascade mainly contributed to the enhancement of the cytotoxicity in the stem-like cells caused by the combination, indicating that the reinforcement of BBI on TOS-mediated apoptosis via mitochondria related to the enhancing cytotoxic effect of the combination on the prostate cancer stem-like cells. Overall, it seems that the combination is an effective new approach to reduce the reoccurrence of prostate cancer targeting prostate cancer stem cells.

Selective gene transfer into neurons via Na,K-ATPase beta1. Targeting gene transfer with monoclonal antibody and adenovirus vector.

BACKGROUND: Neuron-selective gene transfer is an attractive therapeutic strategy for neurological disorders. However, optimal targets and gene delivery systems remain to be determined. METHODS: Following immunization of mice with PC12 cells, hybridomas were screened by beta-Gal reporter gene assay using FZ33 fiber-modified adenovirus vectors. Subsequently, the efficacy and specificity of monoclonal antibody (mAb)-mediated gene transfer via FZ33 and FdZ adenovirus vectors were evaluated by flow cytometry, chemiluminescent beta-Gal reporter gene assay, and immunocytochemistry. Finally, the antigen recognized by the mAb was identified by mass spectrometry and transfection analysis. RESULTS: A hybridoma clone 6E3 producing monoclonal antibody, mAb6E3, was screened. Flow cytometry, chemiluminescent beta-Gal reporter gene assay, and immunocytochemistry with mAb6E3 and the fiber mutant adenovirus demonstrated efficient gene transfer into the PC12 cells. Treatment of neuron-glia cocultures with mAb6E3 and FdZ adenovirus resulted in neuron-selective gene transfer. Immunohistochemical images of rat spinal cord tissue showed that mAb6E3 reacts specifically with neurons. Finally, Na,K-ATPase beta1 was identified as the antigen of mAb6E3. CONCLUSIONS: Hybridoma screening using FZ33 fiber-modified adenovirus vectors serves as an efficient approach to detect antigens in mAb-targeted gene transfer. Neuronal tropism in the central nervous system through mAb6E3 represents an important initial step towards neuron-selective gene transfer in the treatment of local neurological disorders, such as spinal cord injury.

Metalloprotease inhibitors block release of soluble CD27 and enhance the immune stimulatory activity of chronic lymphocytic leukemia cells.

OBJECTIVE: Chronic lymphocytic leukemia (CLL) B cells from most patients express both membrane-bound CD27 (mCD27) and soluble CD27 (sCD27). Expression of sCD27 inhibits CD27-dependent T-cell or CLL-cell activation mediated by its ligand, CD70. In this study, we evaluated whether protease inhibitors could inhibit the release of sCD27 from CLL cells and enhance T-cell activation mediated by CD27-CD70 interaction. METHODS: CLL cells exposed to hydroxamic acid-based matrix metalloprotease (MMP) inhibitors were evaluated for the release of sCD27 by sandwich enzyme-linked immunosorbent assay and immunoprecipitation. We examined for phenotypic changes in CLL cells treated with MMP inhibitors by flow cytometry and T-cell activation by CLL cells was assessed by [(3)H] thymidine incorporation assay and the production of interferon-gamma. RESULTS: Treatment of CLL cells with MMP inhibitors blocked the release of sCD27 to the culture supernatant. In contrast, a non-hydroxamic acid control compound or inhibitors of other proteases, including serine, cysteine, and aspartyl proteases, were ineffective. Furthermore, CLL cells treated with MMP inhibitors expressed significantly higher levels of accessory molecules, such as CD54, CD80, and CD95. Consistent with such changes, we found that CLL cells treated with MMP inhibitors, but not control treated cells, could stimulate allogeneic and autologous T cells in mixed lymphocyte reactions. CONCLUSION: These data reveal that metalloprotease inhibitors can block production of sCD27, which can interfere with mCD27-CD70 interactions that induce expression of immune costimulatory molecules on CLL B cells. Conceivably, treatment of CLL cells with metalloprotease inhibitors may enhance their potential for stimulating cellular immune recognition of leukemia-associated antigens.

Wnt3/RhoA/ROCK signaling pathway is involved in adhesion-mediated drug resistance of multiple myeloma in an autocrine mechanism.

Adhesion of myeloma cells to bone marrow stromal cells is now considered to play a critical role in chemoresistance. However, little is known about the molecular mechanism governing cell adhesion-mediated drug resistance (CAM-DR) of myeloma cells. In this study, we focused our interests on the implication of the Wnt signal in CAM-DR. We first screened the expression of Wnt family in myeloma cell lines and found that Wnt3 was overexpressed in all the myeloma cells examined. KMS-5 and ARH77, which highly expressed Wnt3 protein, tightly adhered to human bone marrow stromal cells, and accumulation of beta-catenin and GTP-bounded RhoA was observed in these myeloma cell lines. Conversely, RPMI8226 and MM1S, which modestly expressed Wnt3 protein, rather weakly adhered to human bone marrow stromal. We then examined the relevance of Wnt3 expression to adhesive property to stromal cells and to CAM-DR of myeloma cells. KMS-5 and ARH-77 exhibited apparent CAM-DR against doxorubicin. This CAM-DR was significantly reduced by anti-integrin beta(1) antibody, anti-integrin alpha(6) antibody and a Wnt-receptor competitor, secreted Frizzled-related protein-1, and Rho kinase inhibitor Y27632, but not by the specific inhibitor of canonical signaling (Dickkopf-1), indicating that Wnt-mediated CAM-DR that is dependent on integrin alpha(6)/beta(1) (VLA-6)-mediated attachment to stromal cells is induced by the Wnt/RhoA/Rho kinase pathway signal. This CAM-DR was also significantly reduced by Wnt3 small interfering RNA transfer to KMS-5. These results indicate that Wnt3 contributes to VLA-6-mediated CAM-DR via the Wnt/RhoA/ROCK pathway of myeloma cells in an autocrine manner. Thus, the Wnt3 signaling pathway could be a promising molecular target to overcome CAM-DR of myeloma cells.

An Agonistic Antibody to EPHA2 Exhibits Antitumor Effects on Human Melanoma Cells.

BACKGROUND/AIM: EPH receptor A2 (EPHA2) is highly expressed in aggressive types of human cancer, and is expected to be an excellent target molecule for antibody treatments. In this study, we investigated the therapeutic potential of antibody to EPHA2 against melanoma in vitro. MATERIALS AND METHODS: We generated three monoclonal antibodies (mAbs) to EPHA2 and examined cell-surface expression by flow cytometry. To investigate the ability to inhibit tumor cell migration therapy with mAbs to EPHA2, we performed a wound scratch assay and invasion assay. We investigated the therapeutic effects of immunotoxins consisting of toxin-conjugated EPHA2 mAbs. RESULTS: All human melanoma cell lines studied expressed EPHA2. Like natural ligand ephrin-A1, one of EPHA2 mAbs, SHM16, inhibited metastatic behavior of cells, such as migration and invasion. In addition, drastic growth inhibition and cytotoxicity were found using immunotoxin-conjugated SHM16. CONCLUSION: These observations indicate a promising role for EPHA2 as a target in antibody treatments for melanoma, and demonstrate the potential therapeutic effects of an agonistic antibody to EPHA2.

Cancer-targeting gene therapy using tropism-modified adenovirus.

Gene therapy has the potential to provide highly selective, curative cancer treatments without inducing systemic toxicity. Adenoviral vectors have been extensively used for cancer gene therapy because of their relatively high efficacy of gene transfer. However, gene transduction to cancer cells is limited by the necessity of using adenoviral type 5 vectors. This is because these vectors have a low transduction efficiency due to weak expression of the adenovirus receptor, coxsackie-adenovirus receptor (CAR), on cancer cells. Moreover, there may be side-effects to the treatment as normal cells also express CAR. In order to eradicate cancer cells without side-effects, the development of a targeting-vector is therefore crucial. In this review, the recent targeting strategies of adenoviral vectors for cancer gene therapy are summarized.

Carcinoembryonic antigen-targeted selective gene therapy for gastric cancer through FZ33 fiber-modified adenovirus vectors.

PURPOSE: A major problem when using the adenoviral vectors for gene therapy applications is thought to be related to low transduction efficiency in cancer cells or to side effects in normal cells. There is an urgent requirement to improve the specificity of gene delivery in the context of cancer gene therapy. EXPERIMENTAL DESIGN: We constructed a genetically modified adenovirus incorporating an IgG Fc-binding motif from the Staphylococcus protein A, Z33, within the HI loop (Adv-FZ33). A remarkable degree of targeted gene delivery to gastric cancer cells was obtained with Adv-FZ33 with the fully human anti-carcinoembryonic antigen (CEA) monoclonal antibody, C2-45. RESULTS: In vitro LacZ or EGFP gene expression after Adv-FZ33 infection via C2-45 was 20 times higher than control monoclonal antibody in MKN-45 at 1,000 viral particles/cell. We generated Ax3CAUP-FZ33 (UP-FZ33), which is an Adv-FZ33 derivative vector expressing a therapeutic gene (i.e., Escherichia coli uracil phosphoribosyltransferase), which converts 5-fluorouracil (5-FU) directly to 5-fluoro-UMP. UP-FZ33 with C2-45 enhanced the cytotoxicity of 5-FU by 10.5-fold in terms of IC(50) against MKN-45 compared with control IgG4. In a nude mouse peritoneal dissemination model, tumor growth in mice treated with UP-FZ33/C2-45/5-FU was significantly suppressed, and tumor volumes were less than one-fourth of those of the control IgG4 group (P < 0.05). The median survival time of the UP-FZ33/C2-45/5-FU group was significantly longer than those treated with PBS or 5-FU only (P < 0.01). CONCLUSIONS: These data suggest that CEA-targeted FZ33 mutant adenovirus-mediated gene delivery offers a strong and selective therapeutic modality against CEA-producing cancers.