Dynamin 2 along with microRNA-199a reciprocally regulate hypoxia-inducible factors and ovarian cancer metastasis.

Hypoxia-driven changes in the tumor microenvironment facilitate cancer metastasis. In the present study, we investigated the regulatory cross talk between endocytic pathway, hypoxia, and tumor metastasis. Dynamin 2 (DNM2), a GTPase, is a critical mediator of endocytosis. Hypoxia decreased the levels of DNM2. DNM2 promoter has multiple hypoxia-inducible factor (HIF)-binding sites and genetic deletion of them relieved hypoxia-induced transcriptional suppression. Interestingly, DNM2 reciprocally regulated HIF. Inhibition of DNM2 GTPase activity and dominant-negative mutant of DNM2 showed a functional role for DNM2 in regulating HIF. Furthermore, the opposite strand of DNM2 gene encodes miR-199a, which is similarly reduced in cancer cells under hypoxia. miR-199a targets the 3'-UTR of HIF-1α and HIF-2α. Decreased miR-199a expression in hypoxia increased HIF levels. Exogenous expression of miR-199a decreased HIF, cell migration, and metastasis of ovarian cancer cells. miR-199a-mediated changes in HIF levels affected expression of the matrix-remodeling enzyme, lysyloxidase (LOX). LOX levels negatively correlated with progression-free survival in ovarian cancer patients. These results demonstrate a regulatory relationship between DNM2, miR-199a, and HIF, with implications in cancer metastasis.

Dicer-substrate siRNA inhibits tumor necrosis factor alpha secretion in Kupffer cells in vitro: in vivo targeting of Kupffer cells by siRNA-liposomes.

Tumor necrosis factor alpha (TNF-α) plays a major role in the pathogenesis of many inflammatory diseases. Neutralizing TNF-α by antibodies or antisense oligodeoxynucleotides, alleviate disease symptoms. In this study, we introduce the new generation of gene-silencing molecules, namely the small interfering RNAs (siRNAs) to reduce TNF-α. Although siRNAs of 19-21bp are commonly used, it is reported that longer siRNAs have much higher efficacies. Here, we report the identification of a 27-mer Dicer-substrate siRNA (DsiRNA) against TNF-α mRNA. Primary cells of rat Kupffer cells were transfected with five 27-mer siRNA constructs (si27-1, si27-2 si27-3, si27-4 and si27-5) for 24h, following which, TNF-α secretion was induced by exposure to LPS (0.1µg/ml) for 2h. TNF-α released to the medium was measured by ELISA. Of the five si27 constructs, si27-3 had the highest inhibitory effect on TNF-α secretion. At 10nM, si27-3 inhibited TNF-α secretion by 80% compared to a 60% inhibition by a 21-mer (SSL3). Following encapsulation in anionic liposomes, si27-3 at 100µg/kg body weight, on two successive days by intravenous administration, inhibited the secretion of TNF-α by 50%. These data demonstrate the identification of a highly efficacious siRNA formulation, which can be used in the treatment of TNF-α mediated diseases.

Inhibition of ovarian cancer by RGD-P125A-endostatin-Fc fusion proteins.

Previous studies have shown that a single point mutation in endostatin at position 125 (P125A) can improve the biological activity of endostatin. Addition of an integrin-targeting moiety, R-G-D, resulted in better localization to tumor vasculature and improved the antiangiogenic activity of endostatin. Because endostatin has relatively shorter serum half-life, frequent dosing was required for inhibiting tumor growth. In our study, we have genetically fused RGD-P125A-endostatin to Fc of IgG4 isotype and evaluated its antiangiogenic and antitumor effects in athymic mice. Two genetic constructs were made, RGD-P125A-endostatin-Fc (RE-Fc) and P125A-endostatin-RGD-Fc (ER-Fc). Both constructs were cloned and expressed in mammalian cells. Purified fusion proteins inhibited endothelial cell migration and proliferation better than yeast-derived P125A-endostatin. Both RE-Fc and ER-Fc inhibited ovarian cancer growth and were found to be as effective as Bevacizumab treatment. Fusion protein showed marked increased half-life. Combination treatment with Bevacizumab and ER-Fc showed additive inhibition of ovarian cancer growth. These studies demonstrate that genetic fusion with human IgG4-Fc increases the half-life of P125A-endostatin and can be used along with Bevacizumab to improve antiangiogenic and antitumor activities.

Inhibition of tumor necrosis factor alpha secretion in rat Kupffer cells by siRNA: in vivo efficacy of siRNA-liposomes.

Tumor necrosis factor alpha (TNF-alpha), a pro-inflammatory cytokine, plays a key role in the pathogenesis of many inflammatory diseases, including alcoholic liver disease. In the liver, Kupffer cells are the primary source of the cytokine. Obliteration of Kupffer cells or neutralization of TNF-alpha by anti-TNF-alpha antibody or by an antisense oligonucleotide prevents ethanol-mediated liver damage. In this study, we report the identification of yet another highly efficacious gene-silencing molecule, the short interfering RNA (siRNA), SSL3, against TNF-alpha. The efficacies of various siRNA duplexes were tested against TNF-alpha mRNA in primary cultures of rat Kupffer cells. SSL3 (25 nM) inhibited lipopolysaccharide (LPS)-induced secretion of TNF-alpha by 55% (p<0.005) with a proportionate reduction in TNF-alpha mRNA; the inhibitory effect lasted for at least 96 h. Four nucleotide mismatches to SSL3 completely abolished the inhibitory effects of SSL3, suggesting the sequence specificity of the siRNA. Further, the in vivo efficacy of SSL3 was assessed following the i.v. administration of two doses (140 microg/kg body weight/day for 2 days) of liposome-encapsulated SSL3. The LPS-induced TNF-alpha secretion was inhibited by >60% (p<0.05) by SSL3 pre-treatment. These data demonstrate the identification of an siRNA against TNF-alpha, which, as a liposomal formulation, has therapeutic potential in the treatment of inflammatory diseases mediated by TNF-alpha.

Alteration of subcellular redox equilibrium and the consequent oxidative modification of nuclear factor kappaB are critical for anticancer cytotoxicity by emodin, a reactive oxygen species-producing agent.

We previously found that emodin produced reactive oxygen species (ROS) intracellularly. In various tumor cells at low doses it enhances the cytotoxicity of As(2)O(3), and at higher doses it renders cytotoxicity independently in vitro and in vivo. The effects involve redox-mediated inhibition of NF-kappaB activation. In this study, we focus on the mechanisms by which emodin inhibits NF-kappaB activation. Results in HeLa cells demonstrated that emodin at high doses or in combination with As(2)O(3), via generation of ROS especially in the nucleus, altered subcellular redox equilibrium and thus oxidized the redox-sensitive site on NF-kappaB and prevented its binding to the target DNA. In vivo study showed that tumors exposed to the arsenic/emodin cotreatment had dramatically smaller sizes and weaker antioxidant capacity, compared with arsenic alone. NF-kappaB binding and transactivation were inhibited in these tumors. These data help in the understanding of the mechanisms by which manipulation of cellular redox and NF-kappaB activation may enhance chemotherapy.

Identification of an ADAM17 cleavage region in human CD16 (FcγRIII) and the engineering of a non-cleavable version of the receptor in NK cells.

CD16a and CD16b are IgG Fc receptors expressed by human natural killer (NK) cells and neutrophils, respectively. Both CD16 isoforms undergo a rapid down-regulation in expression by ADAM17-mediated proteolytic cleavage upon cell activation by various stimuli. We examined soluble CD16 released from activated NK cells and neutrophils by mass spectrometric analysis, and identified three separate cleavage sites in close proximity at P1/P1' positions alanine195/valine196, valine196/serine197, and threonine198/isoleucine199, revealing a membrane proximal cleavage region in CD16. Substitution of the serine at position 197 in the middle of the cleavage region for a proline (S197P) effectively blocked CD16a and CD16b cleavage in cell-based assays. We also show that CD16a/S197P was resistant to cleavage when expressed in the human NK cell line NK92 and primary NK cells derived from genetically-engineered human induced pluripotent stem cells. CD16a is a potent activating receptor and despite blocking CD16a shedding, the S197P mutation did not disrupt IgG binding by the receptor or its activation of NK92 cells by antibody-treated tumor cells. Our findings provide further characterization of CD16 cleavage by ADAM17 and they demonstrate that a non-cleavable version of CD16a can be expressed in engineered NK cells.

Targeted delivery of an antibody-mutant human endostatin fusion protein results in enhanced antitumor efficacy.

The antiangiogenic protein endostatin showed considerable preclinical antitumor activity, but limited efficacy in phase I/II trials. Prior studies using an anti-HER2 antibody-murine endostatin fusion showed enhanced antitumor activity compared to anti-HER2 antibody or endostatin given alone, or in combination. We have generated two anti-HER2 human endostatin fusion proteins by fusing either wild-type or a mutant human endostatin (huEndo-P125A) to the 3' end of a humanized anti-HER2 IgG3 antibody. Antitumor efficacy was examined in murine and human breast tumor models. HuEndo-P125A antibody fusion protein (αHER2-huEndo-P125A) inhibited VEGF and bFGF induced endothelial cell proliferation, and tube formation in vitro, more efficiently than endostatin alone, wild-type endostatin fusion protein (αHER2-huEndo), or parental anti-HER2 antibody (αHER2 IgG3). Wild-type and mutant human endostatin was rapidly cleared from serum in mice (T½(2) = 2.0-2.1 hours), whereas αHER2-huEndo fusion proteins had a significantly prolonged half-life (T½(2) = 40.7-57.5 hours). Treatment of SK-BR-3 breast cancer xenografts with anti-HER2 IgG3-huEndo-P125A fusion resulted in greater inhibition of tumor growth and improved survival, compared to treatment with either αHER2 IgG3 (P = 0.025), human endostatin (P = 0.034), or anti-HER2 IgG3-huEndo (P = 0.016). αHER2-huEndo-P125A specifically inhibited tumors expressing HER2 in mice simultaneously implanted with murine mammary tumor EMT6 cells and with EMT6 engineered to express HER2 antigen (EMT6-HER2). Targeting of endostatin using antibody fusion proteins could improve antitumor activity of either anti-HER2 antibody and/or endostatin and provides a versatile approach that could be applied to other tumor targets with alternative antibody specificities.

Dicoumarol alters cellular redox state and inhibits nuclear factor kappa B to enhance arsenic trioxide-induced apoptosis.

The effects of a number of cytotoxic drugs are influenced by cellular reduction/oxidation (redox) state. In the present study, we attempt to explore if dicoumarol, an inhibitor of NADPH: quinone oxidoreductase (NQO1), alters the cellular redox state and how this alteration affects the redox-related apoptosis. Flow cytometry was used to assess the reactive oxygen species (ROS) level and apoptotic rates of HeLa cells treated with arsenic trioxide (As2O3) alone or in combination with natural anthraquinone emodin and dicoumarol or plus N-acetyl-cysteine. Western blot, immunofluorescence, electrophoretic mobility shift assay and luciferase assay were used to detect Nuclear Factor kappa B (NF-kappaB) activation. The results showed that dicoumarol synergized with emodin to sensitize HeLa cells to As2O3-induced apoptosis through raising the ROS level. More notably, this enhanced susceptibility was associated with a ROS-mediated inhibition of NF-kappaB activation in which the combinative treatment with dicoumarol prevented NF-kappaB from binding to target DNA. It was suggested that dicoumarol in combination with anthraquinones might be a novel strategy to expand the chemotherapeutic spectrum of As2O3 by means of interfering the cellular redox state.