Direct Growth-inhibitory Effect Research Articles

Nucleos(t)ide analogs for hepatitis B virus infection differentially regulate the growth factor signaling in hepatocytes.

Recent clinical studies have suggested that the risk of developing HCC might be lower in patients with chronic hepatitis B receiving tenofovir disoproxil fumarate than in patients receiving entecavir, although there is no difference in biochemical and virological remission between the 2 drugs. The effects of nucleoside analogs (NsAs; lamivudine and entecavir) or nucleotide analogs (NtAs; adefovir disoproxil, tenofovir disoproxil fumarate, and tenofovir alafenamide) on cell growth and the expression of growth signaling molecules in hepatoma cell lines and PXB cells were investigated in vitro. The tumor inhibitory effects of NsAs or NtAs were evaluated using a mouse xenograft model, and protein phosphorylation profiles were investigated. The binding of NsAs or NtAs to the insulin receptor (INSR) was investigated by thermal shift assays. NtAs, but not NsAs, showed direct growth inhibitory effects on hepatoma cell lines in vitro and a mouse model in vivo. A phosphoprotein array revealed that INSR signaling was impaired and the levels of phosphorylated (p)-INSRβ and downstream molecules phosphorylated (p)-IRS1, p-AKT, p-Gab1, and p-SHP2 were substantially reduced by NtAs. In addition, p-epidermal growth factor receptor and p-AKT levels were substantially reduced by NtAs. Similar findings were also found in PXB cells and nontumor lesions of liver tissues from patients with chronic hepatitis B. Prodrug NtAs, but not their metabolites (adefovir, adefovir monophosphate, adefovir diphosphate, tenofovir, tenofovir monophosphate, and tenofovir diphosphate), had such effects. A thermal shift assay showed the binding of NtAs to INSRβ. NtAs (adefovir disoproxil, tenofovir disoproxil fumarate, and tenofovir alafenamide), which are adenine derivative acyclic nucleotide analogs, potentially bind to the ATP-binding site of growth factor receptors and inhibit their autophosphorylation, which might reduce the risk of HCC in patients with chronic hepatitis B.

A Seedling Growth Inhibition Assay to Measure Phytocytokine Activity.

The study of immunomodulatory peptides, both of exogenous and endogenous origin, attracted increasing attention over the last years. Numerous methods are widely used to study the sensitivity of plants to peptide elicitation, ranging from measuring early to late induced responses. Seedling growth inhibition is a prominent and easy-to-measure output induced by prolonged peptide treatment. Here, we describe a robust Arabidopsis thaliana seedling growth inhibition experiment that can be used to measure the direct growth-inhibitory effect of peptides, exemplified by RAPID ALKALINIZATION FACTOR 23 (RALF23) treatment. We also show how the assay can be used to assess the modulatory effect of peptide co-treatment on microbe-associated molecular pattern (MAMP)-triggered seedling growth inhibition, exemplified by GOLVEN 2 (GLV2)`s effect on flagellin (flg22)-induced seedling growth inhibition.

A Novel Fc-Optimized Antibody Drug Conjugate Targeting CD7 As a Therapeutic Strategy in T-Cell Acute Lymphoblastic Leukemia

Despite progress in improving treatment regimens for patients with T-cell acute lymphoblastic leukemia (T-ALL), the therapeutic options are still limited, and especially antibody-based immunotherapy is not established. The CD7 antigen represents a promising target structure in T-ALL since it is strongly expressed in different T-ALL subtypes including early T-cell precursor (ETP)-ALL. Therefore, different approaches are currently pursued for targeting CD7, including CAR-T cell therapy. Due to its high internalization capacity CD7 also represents an ideal target structure for antibody drug conjugates (ADC). Here, a novel antibody engineering approach for CD7-targeting was evaluated in vitro and in xenograft mouse models of T-ALL. A CD7 antibody was optimized for its ability to trigger antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) by introducing two amino acid substitutions (S239D/I332E; DE-variant) in the Fc-domain. In addition, the Fc-engineered antibody was conjugated to the microtubule-disrupting agent monomethyl auristatin E (MMAE) via an enzymatic-cleavable linker (mc-vc-PABC). The Fc-optimized ADC, CD7-DE-vcMMAE, as well as the unconjugated antibody, CD7-DE, showed improved binding to activating Fcγ receptors (FcγRIIa, FcγRIIIa) compared to a CD7 antibody lacking the DE-modification. This resulted in potent ADCC- and ADCP-activity against different T-ALL cell lines mediated by NK-cells or macrophages as effector cells (EC 50 ADCC, 1-7 pM). In contrast to the CD7-DE antibody, CD7-DE-vcMMAE showed strong cytotoxic effects independently of immune effector cell engagement by releasing its cytotoxic compound into T-ALL cells in an antigen-restricted manner, thereby inducing G2/M cell cycle arrest and apoptosis. CD7-DE-vcMMAE was active at subnanomolar concentrations demonstrating dose-dependent cytotoxic effects in six T-ALL cell lines (IC 50 0.2 - 1 nM). The extent of maximum growth inhibition ranged between 54-98 % and correlated with CD7 antigen density. Yet, although CD7-DE-vcMMAE did not kill CD7-negative cells directly, the linker design facilitated bystander killing activity. Thus, in co-culture experiments using CEM cells and CEM-CD7-knockout cells mimicking CD7-antigen escape, the ADC demonstrated significant killing of neighbouring CD7-negative cells, thereby extending its mode of action. The antitumor activity of the CD7-ADC was further investigated in xenograft mouse models of T-ALL. In a first model, CEM cells were injected subcutaneously into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and CD7-DE-vcMMAE treatment was evaluated in comparison to the unconjugated antibody CD7-DE. Treatment with CD7-DE-vcMMAE led to a significantly reduced tumor growth in comparison to CD7-DE or untreated animals (p<0.05; see panel A). A preclinical phase II-like patient-derived xenograft (PDX) study employing eight randomly selected T-ALL-PDX samples from pediatric and adult patients was conducted. PDX-cells were injected intravenously into NSG mice and treatment was started when the leukemia load reached 1 % human blasts in the peripheral blood, reflecting an overt leukemia situation. Animals receiving therapy with CD7-DE-vcMMAE showed significant prolongation of median survival in comparison to animals treated with a similarly designed control ADC targeting an irrelevant antigen (control-DE-vcMMAE) or which were left untreated (median survival time; CD7-DE-vc-MMAE: 82 days; untreated/control-DE-vcMMAE: 60 days; p<0.05; see panel B). Importantly, no leukemic blasts were found in the peripheral blood, spleen or bone marrow in animals treated with CD7-DE-vcMMAE and surviving the experimental period of 150 days. Together, the novel ADC CD7-DE-vcMMAE showed a unique set of Fc effector functions, potent direct growth inhibitory effects, bystander killing activity and efficacy in xenograft models of T-ALL. These results exhibit CD7-DE-vcMMAE as a promising therapeutic strategy and form the basis for new approaches in the treatment of T-ALL.

Abstract LB170: HER2-targeted interferon-beta-1a mutein, a potent immunocytokine for the treatment of HER2-positive cancers

Abstract Background: Interferon beta (IFN-β), a promising potent cytokine, has been attracting attention for treatment of cancer. The pleiotropic antitumor effects of IFN-β have been studied, with specific reference to their direct role on cancer cells and indirect action through the immune effector cells. However, its systemic toxicities and poor biophysical properties prevent IFN-β from being widely used for cancer therapy. Since the potential of cytokine therapies are impaired due to dose-limiting systemic toxicities, novel treatment methods should be developed to safely deliver effective drug quantities at the tumor sites. Like antibody-drug conjugates (ADCs), immunocytokine can be attempted to induce cytokine's organ-targeting and alleviate its systemic side effects. Here, we designed recombinant IFN-β mutein immunocytokines that comprise a HER2-targeting antibody and IFN-β mutein, and evaluate the antitumor properties against HER2-positive cancers.Method: A panel of human gastric cancer cell lines was treated with trastuzumab-IFN-β mutein to evaluate direct antitumor effect. In addition, to test the immune cell-mediated antitumor effect, cancer cells were co-cultured with effector cells (e.g. PBMC) in the absence or drug. The antitumor efficacy of trastuzumab-IFN-β mutein in vivo was tested in HER2-positive cancer xenograft models using nude mice or humanized mice. Result: Trastuzumab-IFN-β mutein directly inhibited the growth of HER2-positive gastric cancer cell lines and was more effective than trastuzumab or IFN-β mutein alone. Trastuzumab-IFN-β mutein also displayed enhanced immune cell-mediated cytotoxicity. Collectively, trastuzumab-IFN-β mutein may have indirect immune cell-mediated antitumor effects and direct cell growth inhibitory effects. Moreover, trastuzumab-IFN-β mutein significantly suppress tumor growth in HER2-positive cancer xenograft models. Tumor-infiltration of lymphocytes was enhanced by trastuzumab-IFN-β mutein, implying that the tumor-targeting IFN-β may have an antitumor effect through increased immune response. Conclusion: We have characterized and evaluated the antitumor properties of trastuzumab-IFN-β mutein in HER2-expressing cancers. Since IFN-β activates antitumor immune responses, it is expected to be administered in combination with immunotherapeutic drugs. Therefore, the study suggests that trastuzumab-IFN-β mutein is a promising candidate for the treatment of HER2-positive carcinoma. Citation Format: Chan Gyu Lee, Tae Eun Kim, Sungyoul Hong, Jongwan Chu, Ju Eun Kang, Hae Min Jeong, Young Kee Shin. HER2-targeted interferon-beta-1a mutein, a potent immunocytokine for the treatment of HER2-positive cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB170.

Role of epidermal growth factor receptor inhibitor-induced interferon pathway signaling in the head and neck squamous cell carcinoma therapeutic response

BackgroundEpidermal growth factor receptor (EGFR) is frequently amplified or overexpressed in head and neck squamous cell carcinoma (HNSCC) and is a clinically validated target for the therapeutic antibody, cetuximab, in the management of this cancer. The degree of response to EGFR inhibitors measured by tumor shrinkage varies widely among HNSCC patients, and the biological mechanisms that underlie therapeutic heterogeneity amongst HNSCC patients remain ill-defined.MethodsEGFR-dependent human and murine HNSCC cell lines were treated with the EGFR/ERBB inhibitors, gefitinib and AZD8931, and submitted to RNAseq, GSEA, and qRT-PCR. Conditioned media was analyzed by ELISA and Luminex assays. Murine HNSCC tumors were stained for T cell markers by immunofluorescence. Primary HSNCC patient specimens treated with single agent cetuximab were stained with Vectra multispectral immunofluorescence.ResultsThe transcriptional reprogramming response to EGFR/ERBB-specific TKIs was measured in a panel of EGFR-dependent human HNSCC cell lines and interferon (IFN) α and γ responses identified as top-ranked TKI-induced pathways. Despite similar drug sensitivity, responses among 7 cell lines varied quantitatively and qualitatively, especially regarding the induced chemokine and cytokine profiles. Of note, the anti-tumorigenic chemokine, CXCL10, and the pro-tumorigenic factor, IL6, exhibited wide-ranging and non-overlapping induction. Similarly, AZD8931 exerted potent growth inhibition, IFNα/IFNγ pathway activation, and CXCL10 induction in murine B4B8 HNSCC cells. AZD8931 treatment of immune-competent mice bearing orthotopic B4B8 tumors increased CD8 + T cell content and the therapeutic response was abrogated in nu/nu mice relative to BALB/c mice. Finally, Vectra 3.0 analysis of HNSCC patient tumors prior to and after 3–4 weeks of single agent cetuximab treatment revealed increased CD8 + T cell content in specimens from patients exhibiting a therapeutic response relative to non-responders.ConclusionsThe findings reveal heterogeneous, tumor cell-intrinsic, EGFR/ERBB inhibitor-induced IFN pathway activation in HNSCC and suggest that individual tumor responses to oncogene-targeted agents are a sum of direct growth inhibitory effects and variably-induced participation of host immune cells.

Antibody-Based Targeting of Interferon-Beta-1a Mutein in HER2-Positive Cancer Enhances Antitumor Effects Through Immune Responses and Direct Cell Killing.

Type I interferon (IFN) has been approved as an anticancer agent to treat some malignancies. However, IFNs have a short in vivo half-life, systemic toxicity, and poor biophysical properties, which prevent it from being widely used for cancer therapy. This study aimed to construct recombinant IFN-β-1a mutein immunocytokines that comprise a human epidermal growth factor receptor 2 (HER2)-targeting antibody and IFN-β muteins with an additional glycosylation, which can overcome the limitation of the cytokine itself. Hence, the molecular design aims to 1) enhance productivity and biophysical properties by adding secondary glycosylation in IFN-β, 2) increase the therapeutic index of IFN-β therapy by preferential retention at the tumor by possessing high affinity for HER2-expressing cancer cells, and 3) improve the pharmacokinetics and, thus, the convenience of IFN-β administration. The yield of trastuzumab-IFN-β mutein was higher than that of trastuzumab-wild-type IFN-β in the mammalian cell culture system. Trastuzumab-IFN-β mutein showed similar IFN activity and HER2-targeting ability equivalent to that of IFN-β mutein and trastuzumab, respectively. Trastuzumab-IFN-β mutein directly inhibited the growth of HER2-positive gastric cancer cell lines and was more effective than trastuzumab or IFN-β mutein alone. Trastuzumab-IFN-β mutein and IFN-β mutein displayed enhanced immune cell-mediated cytotoxicity. Collectively, trastuzumab-IFN-β mutein may have indirect immune cell-mediated antitumor effects and direct cell growth inhibitory effects. Tumor-targeting effect of trastuzumab-IFN-β mutein was analyzed using in vivo fluorescence imaging. The accumulation of trastuzumab-IFN-β mutein was observed in HER2-positive tumors rather than other tissues except the liver. To evaluate the both direct tumor growth inhibition effect and indirect immune cell-mediated antitumor effect, we tested the effect of trastuzumab-IFN-β mutein in HER2-positive cancer xenograft models using nude mice or humanized mice. Trastuzumab-IFN-β mutein could significantly enhance tumor regression when compared with trastuzumab or IFN-β mutein. In addition, an increase in tumor-infiltrating lymphocytes was observed in the trastuzumab-IFN-β mutein-treated group, implying that the tumor-targeting IFN-β may have an enhanced antitumor effect through increased immune response. Therefore, targeting IFN-β with an anti-HER2 monoclonal antibody makes the immunocytokine more potent than either agent alone. These novel findings suggest that trastuzumab-IFN-β mutein merits clinical evaluation as a new candidate of anticancer therapeutics.

Synthesis, Stability and Direct Antiproliferative Effect of New Cysteine Modified GnRH Analogs

It is demonstrated that gonadotropin-releasing hormone (GnRH) analogs can directly inhibit the proliferation of reproductive tissue cancer cells, but the poor pharmacokinetic properties still restrict their application in treating hormone-dependent diseases. Modifications in position 6 and 10 of natural GnRH can improve the metabolic stability. In order to study the effect of incorporation Cys6 substitution with C-terminal Pro9-NHEt modification and dimerization of linear peptides on metabolic stability and antiproliferative activity of GnRH analogs, two new GnRH analogs [l-Cys6, desGly10, Pro9-NHEt]-GnRH (1) and [d-Cys6, desGly10, Pro9-NHEt]-GnRH (2), and their corresponding dimer derivatives ([l-Cys6, desGly10, Pro9-NHEt]-GnRH)2 (3) and ([d-Cys6, desGly10, Pro9-NHEt]-GnRH)2 (4) were synthesized. Incubation of these analogs with human serum was carried out to evaluate their metabolic stability, and direct growth inhibitory effect of the two dimer derivatives 3 and 4 on MCF-7 human breast cancer cell line was examined by MTT assay. The metabolic stability of dimer derivatives 3 and 4 was remarkably improved in comparison with natural GnRH. The d-Cys6 substituted dimer derivative 4 exhibited higher inhibitory effect (29.6–39.7% growth reduction) on cell growth than its corresponding counterpart 3 (21.8–26.2% growth reduction) at concentration range of 50, 100 and 200 μΜ. The cell growth inhibition of leuprolide was 16.4–27.2% at the tested concentrations. The dimer derivative 4 was the most stable and active GnRH analog in this study and has the potential for future preclinical investigations as promising antitumor drug candidate.

Direct Growth Inhibitory Effect of Platelet Activating Factor C-16 and Its Structural Analogs on Mycobacteria.

Mycobacterium tuberculosis, the causative agent of tuberculosis, is one of the leading causes of human deaths due to a single infectious agent. M. tuberculosis infection of the host initiates a local inflammatory response, resulting in the production of a range of inflammatory factors at the site of infection. These inflammatory factors may come in direct contact with M. tuberculosis and immune cells to activate different signaling pathways. One such factor produced in excess during inflammation is a phospholipid compound, Platelet Activating Factor C-16 (PAF C-16). In this study, PAF C-16 was shown to have a direct inhibitory effect on the growth of Mycobacterium bovis BCG (M. bovis BCG) and Mycobacterium smegmatis (M. smegmatis) in a dose- and time-dependent manner. Use of a range of PAF C-16 structural analogs, including the precursor form Lyso-PAF, revealed that small modifications in the structure of PAF C-16 did not alter its mycobacterial growth inhibitory properties. Subsequent experiments suggested that the attachment of aliphatic carbon tail via ether bond to the glycerol backbone of PAF C-16 was likely to play a vital role in its growth inhibition ability against mycobacteria. Fluorescence microscopy and flow cytometry using Propidium iodide (PI) indicated that PAF C-16 treatment had a damaging effect on the cell membrane of M. bovis BCG and M. smegmatis. Furthermore, the growth inhibitory effect of PAF C-16 was partially mitigated by treatment with membrane-stabilizing agents, α-tocopherol and Tween-80, which further suggests that the growth inhibitory effect of PAF C-16 was mediated through bacterial cell membrane damage.

In vitro effect of chlorambucil on human glioma cell lines (SF767 and U87-MG), and human microvascular endothelial cell (HMVEC) and endothelial progenitor cells (ECFCs), in the context of plasma chlorambucil concentrations in tumor-bearing dogs.

The objective of this study was to investigate a possible mechanism of action of metronomic chlorambucil on glioma by studying the in vitro cytotoxicity and anti-angiogenic effects on glioma and endothelial cells, respectively. The in vitro LD50 and IC50 of chlorambucil were determined using human SF767 and U87-MG glioma cell lines, human microvascular endothelial cells (HMVECs) and human endothelial colony forming cells (ECFCs). Results were analyzed in the context of chlorambucil concentrations measured in the plasma of tumor-bearing dogs receiving 4 mg m-2 metronomic chlorambucil. The LD50 and IC50 of chlorambucil were 270 μM and 114 μM for SF767, and 390 μM and 96 μM for U87-MG, respectively. The IC50 of chlorambucil was 0.53 μM and 145 μM for the HMVECs and ECFCs, respectively. In pharmacokinetic studies, the mean plasma Cmax of chlorambucil was 0.06 μM. Results suggest that metronomic chlorambucil in dogs does not achieve plasma concentrations high enough to cause direct cytotoxic or growth inhibitory effects on either glioma or endothelial cells.

Prostaglandin E2 Receptor Antagonist with Antimicrobial Activity against Methicillin-Resistant Staphylococcus aureus.

Polymicrobial intra-abdominal infections (IAI) involving Candida albicans and Staphylococcus aureus are associated with severe morbidity and mortality (∼80%). Our laboratory discovered that the immunomodulatory eicosanoid prostaglandin E2 (PGE2) plays a key role in the lethal inflammatory response during polymicrobial IAI using a mouse model of infection. In studies designed to uncover key PGE2 biosynthesis/signaling components involved in the response, selective eicosanoid enzyme inhibitors and receptor antagonists were selected and prescreened for antimicrobial activity against C. albicans or S. aureus Unexpectedly, we found that the EP4 receptor antagonist L-161,982 had direct growth-inhibitory effects on S. aureusin vitro at the physiological concentration required to block the PGE2 interaction with EP4 This antimicrobial activity was observed with methicillin-sensitive S. aureus and methicillin-resistant S. aureus (MRSA) strains, with the MIC and minimum bactericidal concentration values for planktonic cells being 50 μg/ml and 100 μg/ml, respectively. In addition, L-161,982 inhibited S. aureus biofilm formation and had activity against preformed mature biofilms. More importantly, treatment of mice with L-161,982 following intraperitoneal inoculation with a lethal dose of MRSA significantly reduced the bioburden and enhanced survival. Furthermore, L-161,982 protected mice against the synergistic lethality induced by coinfection with C. albicans and S. aureus The antimicrobial activity of L-161,982 is independent of EP4 receptor inhibitory activity; an alternative EP4 receptor antagonist exerted no antimicrobial or protective effects. Taken together, these findings demonstrate that L-161,982 has potent antimicrobial activity against MRSA and may represent a significant therapeutic alternative in improving the prognosis of mono- or polymicrobial infections involving MRSA.

Direct growth-inhibitory effects of prostaglandin E2 in pancreatic cancer cells in vitro through an EP4/PKA-mediated mechanism

There is strong evidence linking inflammation and the development of pancreatic ductal adenocarcinoma. Cyclooxygenase-2 (COX-2) and COX-2-derived PGE2 are overexpressed in human and murine pancreatic ductal adenocarcinoma. Several studies have demonstrated an important role of COX-2-derived PGE2 in tumor-stroma interactions; however, the direct growth effects of prostaglandin E2 (PGE2) on pancreatic ductal adenocarcinoma cells is less well defined. Our aim was to investigate the effects of PGE2 on pancreatic ductal adenocarcinoma cell growth and to characterize the underlying mechanisms. Human pancreatic ductal adenocarcinoma cell lines, Panc-1 and MIA PaCa-2, were treated with PGE2 in varying doses (0-10μM). Effects on the phosphorylation of ERK1/2 were evaluated by Western blot. Colony formation was observed for cells treated with PGE2 for 11days. DNA synthesis was determined by (3H)-thymidine incorporation assay. Gene expression of E-type prostaglandin (EP)2/EP4 receptors and their correlation with survival in patients with pancreatic ductal adenocarcinoma were assessed using the RNA-Seq data set from The Cancer Genome Atlas Research Network. PGE2 decreased the size and number of colonies in Panc-1 but not MIA PaCa-2cells. In the Panc-1cells, PGE2 activated PKA/CREB and decreased phosphorylation of ERK1/2, which was reversed by an EP4 receptor antagonist, while an EP2 receptor antagonist had no effect. In contrast, in MIA PaCa-2cells, PGE2 had no effect on ERK1/2 phosphorylation. Treatment of both Panc-1 and MIA PaCa-2cells with forskolin/IBMX decreased ERK1/2 phosphorylation. Finally, PGE2 decreased DNA synthesis only in Panc-1cells, which was reversed by an EP4 receptor antagonist. In human pancreatic ductal adenocarcinoma, high EP2 and low EP4 gene expression was correlated to worse median overall survival (15.6 vs 20.8months, log-rank P=.017). Our study provides evidence that PGE2 can inhibit directly pancreatic ductal adenocarcinoma cell growth through an EP4-mediated mechanism. Together with our gene expression and survival analysis, this observation suggests a protective role of EP4 receptors in human pancreatic ductal adenocarcinoma that expresses E-type prostaglandin receptors.

In vitro effect of molluscan hemocyanins on CAL-29 and T-24 bladder cancer cell lines

The aim of this study was to investigate the anti-tumor effects of molluscan hemocyanins (Hcs) isolated from the marine snail Rapana venosa (RvH) and the garden snail Helix lucorum (HlH) on human bladder cancer cell lines. The antitumor effect of the native molecules of the above-mentioned Hcs and their subunits were examined in comparison to keyhole limpet hemocyanin (KLH), which is the most thoroughly studied Hc. The experiments were conducted using 2 human bladder cancer cell lines: CAL-29 and T-24. Doxorubicin hydrochloride (DOX) and mitomycin-C (MIT-C), which are routinely used in clinical practice to treat bladder cancer, were used for comparison. The viability of the 2 bladder cancer cell lines, used at a concentration of 20,000 cells/well, was measured by WST-1 assay at 24, 48 and 72 h after treatment with the above-mentioned Hcs and their isoforms at a concentration ranging from 0.8 to 500 μg/ml. A direct growth inhibitory effect on the tumor cells was observed mainly after treatment with the native molecule of HlH and the structural subunit, RvH1, at a concentration of 500 μg/ml. The native molecule of RvH exhibited an efficacy similar to that of KLH. However, the observed growth inhibitory effect of HlH was superior to that observed for KLH and RvH, when used at the same concentration. These findings demonstrate the antitumor effect of other Hcs, apart from KLH. Our data suggest that the native molecule of HlH and the subunit, RvH1, are alternative candidates for the treatment of human superficial bladder cancer.

Maternal corticosterone regulates nutrient allocation to fetal growth in mice

Stresses during pregnancy that increase maternal glucocorticoids reduce birth weight in several species. However, the role of natural glucocorticoids in the mother in fetal acquisition of nutrients for growth remains unknown. This study aimed to determine whether fetal growth was reduced as a consequence of altered amino acid supply when mice were given corticosterone in their drinking water for 5 day periods in mid to late pregnancy (day, D, 11-16 or D14-19). Compared to controls drinking tap water, fetal weight was always reduced by corticosterone. At D16, corticosterone had no effect on materno-fetal transfer of [(14)C]methylaminoisobutyric acid (MeAIB), although placental MeAIB accumulation and expression of the Slc38a1 and Slc38a2 transporters were increased. However, at D19, 3 days after treatment ended, materno-fetal transfer of MeAIB was increased by 37% (P < 0.04). During treatment at D19, placental accumulation and materno-fetal transfer of MeAIB were reduced by 40% (P < 0.01), although expression of Slc38a1 was again elevated. Permanent reductions in placental vascularity occurred during the earlier but not the later period of treatment. Placental Hsd11b2 expression, which regulates feto-placental glucocorticoid bioavailability, was also affected by treatment at D19 only. Maternal corticosterone concentrations inversely correlated with materno-fetal MeAIB clearance and fetal weight at D19 but not D16. On D19, weight gain of the maternal carcass was normal during corticosterone treatment but reduced in those mice treated from D11 to D16, in which corticosterone levels were lowest. Maternal corticosterone is, therefore, a physiological regulator of the amino acid supply for fetal growth via actions on placental phenotype.

Antitumor Activity of BIBF 1120, a Triple Angiokinase Inhibitor, and Use of VEGFR2+pTyr+ Peripheral Blood Leukocytes as a Pharmacodynamic Biomarker In Vivo

BIBF 1120 is a potent, orally available triple angiokinase inhibitor that inhibits VEGF receptors (VEGFR) 1, 2, and 3, fibroblast growth factor receptors, and platelet-derived growth factor receptors. This study examined the antitumor effects of BIBF 1120 on hepatocellular carcinoma (HCC) and attempted to identify a pharmacodynamic biomarker for use in early clinical trials. We evaluated the antitumor and antiangiogenic effects of BIBF 1120 against HCC cell line both in vitro and in vivo. For the pharmacodynamic study, the phosphorylation levels of VEGFR2 in VEGF-stimulated peripheral blood leukocytes (PBL) were evaluated in mice inoculated with HCC cells and treated with BIBF 1120. BIBF 1120 (0.01 μmol/L) clearly inhibited the VEGFR2 signaling in vitro. The direct growth inhibitory effects of BIBF 1120 on four HCC cell lines were relatively mild in vitro (IC(50) values: 2-5 μmol/L); however, the oral administration of BIBF 1120 (50 or 100 mg/kg/d) significantly inhibited the tumor growth and angiogenesis in a HepG2 xenograft model. A flow cytometric analysis revealed that BIBF 1120 significantly decreased the phosphotyrosine (pTyr) levels of VEGFR2(+)CD45(dim) PBLs and the percentage of VEGFR2(+)pTyr(+) PBLs in vivo; the latter parameter seemed to be a more feasible pharmacodynamic biomarker. We found that BIBF 1120 exhibited potent antitumor and antiangiogenic activity against HCC and identified VEGFR2(+)pTyr(+) PBLs as a feasible and noninvasive pharmacodynamic biomarker in vivo.

Intracellular uptake and associated toxicity of silver nanoparticles in Caenorhabditis elegans

Silver nanoparticles (AgNPs) are frequently used as antimicrobials. While the mechanism(s) by which AgNPs are toxic are unclear, their increasing use raises the concern that release into the environment could lead to environmental toxicity. We characterized the physicochemical behavior, uptake, toxicity (growth inhibition), and mechanism of toxicity of three AgNPs with different sizes and polyvinylpyrrolidone (PVP) or citrate coatings to the nematode Caenorhabditis elegans. We used wild-type (N2) C. elegans and strains expected to be sensitive to oxidative stress ( nth-1, sod-2 and mev-1), genotoxins ( xpa-1 and nth-1), and metals ( mtl-2). Using traditional and novel analytical methods, we observed significant aggregation and extra-organismal dissolution of silver, organismal uptake and, in one case, transgenerational transfer of AgNPs. We also observed growth inhibition by all tested AgNPs at concentrations in the low mg/L levels. A metallothionein-deficient ( mtl-2) strain was the only mutant tested that exhibited consistently greater AgNP sensitivity than wild-type. Although all tested AgNPs were internalized (passed cell membranes) in C. elegans, at least part of the toxicity observed was mediated by ionic silver. Finally, we describe a modified growth assay that permits differentiation between direct growth-inhibitory effects and indirect inhibition mediated by toxicity to the food source.

Combination of interferon-alpha and 5-fluorouracil inhibits endothelial cell growth directly and by regulation of angiogenic factors released by tumor cells

BackgroundThe combination therapy of interferon (IFN)-alpha and 5-fluorouracil (5-FU) improved the prognosis of the patients with hepatocellular carcinoma (HCC). To determine the molecular mechanisms of the anti-tumor and anti-angiogenic effects, we examined the direct anti-proliferative effects on human umbilical vein endothelial cells (HUVEC) and indirect effects by regulating secretion of angiogenic factors from HCC cells.MethodsThe direct effects on HUVEC were examined by TUNEL, Annexin-V assays and cell cycles analysis. For analysis of the indirect effects, the apoptosis induced by the conditioned medium from HCC cell treated by IFN-alpha/5-FU and expression of angiogenic factors was examined.ResultsIFN-alpha and 5-FU alone had anti-proliferative properties on HUVEC and their combination significantly inhibited the growth (compared with control, 5-FU or IFN alone). TUNEL and Annexin-V assays showed no apoptosis. Cell cycle analysis revealed that IFN-alpha and 5-FU delayed cell cycle progression in HUVEC with S-phase accumulation. The conditioned medium from HuH-7 cells after treatment with IFN/5-FU significantly inhibited HUVEC growth and induced apoptosis, and contained high levels of angiopoietin (Ang)-1 and low levels of vascular endothelial growth factor (VEGF) and Ang-2. Knockdown of Ang-1 in HuH-7 cells abrogated the anti-proliferative effects on HUVEC while knockdown of Ang-2 partially rescue the cells.ConclusionThese results suggested that IFN-alpha and 5-FU had direct growth inhibitory effects on endothelial cells, as well as anti-angiogenic effects through regulation of angiogenic factors released from HCC cells. Modulation of VEGF and Angs secretion by IFN-alpha and 5-FU may contribute to their anti-angiogenic and anti-tumor effects on HCC.

Growth-inhibitory effect of adiponectin via adiponectin receptor 1 on human breast cancer cells through inhibition of S-phase entry without inducing apoptosis

Adiponectin is one of the most important adipocytokines secreted from adipose tissue. In addition to its effects on glucose and fatty acid metabolism, it has been reported that adiponectin has a direct growth-inhibitory effect on breast cancer cells. However, it still remains to be established how adiponectin affects cell cycle and apoptosis and whether or not its inhibitory effect is mediated through adiponectin receptors. Here, we demonstrated that adiponectin treatment resulted in a significant dose-dependent growth inhibition of both MDA-MB-231 and T47D cells. In both cell lines, the G0/G1 population significantly increased after adiponectin treatment, but apoptosis was not induced. High expression of mRNA and protein of adiponectin receptor 1 was observed, but expression of adiponectin receptor 2 was very low in both cell lines. Treatment with small interference RNA against adiponectin receptor 1 significantly reduced the growth inhibition induced by adiponectin in both cell lines. Taken together, adiponectin decreases breast cancer cell proliferation by inhibiting the entry into S-phase without inducing apoptosis, and this inhibitory effect is mediated through adiponectin receptor 1.

Adipokines as endocrine, paracrine, and autocrine factors in breast cancer risk and progression

Adipokines (leptin, adiponectin, and hepatocyte growth factor (HGF)) secreted from adipose tissue have come to be recognized for their contribution to the mechanisms by which obesity and related metabolic disorders influence breast cancer risk. In this review, we discuss the direct and indirect effects of these protein factors on the biological and clinical aspects of breast cancer biology, and emphasize their distinctive modes of action through endocrine-, paracrine-, and autocrine-mediated pathways. The stimulatory effects of leptin on breast cancer growth were considered to occur primarily via activation of the estrogen receptor; however, new evidence suggests that leptin may be acting on downstream cell signaling pathways in both estrogen-dependent and -independent cell types. Another secretory adipokine, HGF, may act largely not only to promote tumor cell invasion, but also to enhance tumor growth indirectly by stimulating angiogenesis. In contrast, adiponectin, an endogenous insulin sensitizer, exerts a direct growth-inhibitory effect on tumor cells by downregulating cell proliferation and upregulating apoptosis, and also inhibits tumor-related angiogenesis.

Molecular targets for apigenin-induced cell cycle arrest and apoptosis in prostate cancer cell xenograft

Apigenin (4',5,7-trihydroxyflavone) is a promising chemopreventive agent abundantly present in fruits and vegetables that has been shown to promote cell cycle arrest and apoptosis in various malignant cell lines. To determine whether pharmacologic intervention with apigenin has a direct growth inhibitory effect on human prostate tumors implanted in athymic nude mice, we examined cell cycle regulatory molecules as precise molecular targets of apigenin action. Apigenin feeding by gavage to these mice at doses of 20 and 50 microg/mouse/d in 0.2 mL of a vehicle containing 0.5% methyl cellulose and 0.025% Tween 20 resulted in significant decreases in tumor volume and mass of androgen-sensitive 22Rv1 and androgen-insensitive PC-3-implanted cells. Oral intake of apigenin resulted in dose-dependent (a) increase in the protein expression of WAF1/p21, KIP1/p27, INK4a/p16, and INK4c/p18; (b) down-modulation of the protein expression of cyclins D1, D2, and E; and cyclin-dependent kinases (cdk), cdk2, cdk4, and cdk6; (c) decrease in retinoblastoma phosphorylation at serine 780; (d) increase in the binding of cyclin D1 toward WAF1/p21 and KIP1/p27; and (e) decrease in the binding of cyclin E toward cdk2 in both types of tumors. In addition, apigenin feeding resulted in stabilization of p53 by phosphorylation at serine 15 in 22Rv1 tumors, which seems to exhibit p53-dependent growth inhibitory responses. Apigenin intake by these mice also resulted in induction of apoptosis, which positively correlated with serum and tumor apigenin levels. Taken together, this is the first systematic in vivo study showing the involvement of cell cycle regulatory proteins as potential molecular targets of apigenin.

Interferon-γ induces regression of epithelial cell carcinoma: critical roles of IRF-1 and ICSBP transcription factors

We have developed an epithelial cell carcinoma model for studying efficacy of IFNgamma gene therapy and have identified components of IFNgamma-signaling pathway responsible for its direct anti-tumor actions. The tumor results from ectopic expression of SV40 Large T-Antigen (SV40 T-Ag) oncogene in lens of transgenic mouse (alphaT3) and complete regression of the tumor is induced by targeting expression of IFNgamma into malignant lens cells. Inflammatory cells are absent in lens of alphaT3 or DT (co-expressing IFNgamma and SV40-T-Antigen) mice and the transformed lens cells are non-immunogenic, suggesting non-involvement of immunologic cells. We show that IFNgamma has direct growth-inhibitory effects on tumor cells, induces death of tumor cells by apoptosis and that these effects are mediated by two transcription factors, IRF-1 (interferon-regulatory factor-1) and ICSBP (interferon-consensus sequence-binding protein) induced by IFNgamma. Furthermore, stable transfection with ICSBP or IRF-1 construct inhibits lens carcinoma cell growth by upregulating Caspase-1, p21(WAF1) and p27 expression. In contrast, tumor progression in alphaT3 lens correlates with inhibition of IRF-1 and ICSBP expression. Our results suggest that IFNgamma gene therapy maybe effective in malignant diseases for which DNA tumor viruses are etiologic agents and that antitumor actions of IRF-1/ICSBP can be exploited therapeutically to circumvent adverse clinical effects associated with IFN therapy.