Mitsunobu R. Kano

Improvement of cancer-targeting therapy, using nanocarriers for intractable solid tumors by inhibition of TGF-β signaling

Transforming growth factor (TGF)-β plays a pivotal role in regulation of progression of cancer through effects on tumor microenvironment as well as on cancer cells. TGF-β inhibitors have recently been shown to prevent the growth and metastasis of certain cancers. However, there may be adverse effects caused by TGF-β signaling inhibition, including the induction of cancers by the repression of TGF-β-mediated growth inhibition. Here, we present an application of a short-acting, small-molecule TGF-β type I receptor (TβR-I) inhibitor at a low dose in treating several experimental intractable solid tumors, including pancreatic adenocarcinoma and diffuse-type gastric cancer, characterized by hypovascularity and thick fibrosis in tumor microenvironments. Low-dose TβR-I inhibitor altered neither TGF-β signaling in cancer cells nor the amount of fibrotic components. However, it decreased pericyte coverage of the endothelium without reducing endothelial area specifically in tumor neovasculature and promoted accumulation of macromolecules, including anticancer nanocarriers, in the tumors. Compared with the absence of TβR-I inhibitor, anticancer nanocarriers exhibited potent growth-inhibitory effects on these cancers in the presence of TβR-I inhibitor. The use of TβR-I inhibitor combined with nanocarriers may thus be of significant clinical and practical importance in treating intractable solid cancers.

VEGF-A and FGF-2 synergistically promote neoangiogenesis through enhancement of endogenous PDGF-B-PDGFRβ signaling

Combined stimulation with VEGF-A, FGF-2, or PDGF-BB has emerged as a potent strategy for therapeutic angiogenesis, although the mechanisms underlying the synergism of these factors are not well understood. In the present study, we investigated the mechanism of synergism between VEGF-A and FGF-2 by using Matrigel plug assay in vivo and embryonic stem cell (ESC)-derived VEGF receptor 2 (VEGFR2)-positive cells in vitro. Experiments in vitro revealed that, in addition to having direct mitogenic effects, these molecules enhance intercellular PDGF-B signaling in a cell-type specific manner: VEGF-A enhances endothelial PDGF-B expression, whereas FGF-2 enhances mural PDGF receptor β (PDGFRβ) expression. Co-stimulation with VEGF-A and FGF-2 caused significant mural cell recruitment in vitro and formation of functional neovasculature in vivo, compared with single-agent stimulation. These effects were abrogated not only by anti-PDGFRβ neutralizing antibody, but also by exogenous PDGF-BB, which could overwhelm the endogenous PDGF-BB distribution. These findings indicated the importance of preservation of the periendothelial PDGF-BB gradient. Thus, we demonstrated that the directional enhancement of endogenous PDGF-B–PDGFRβ signaling is indispensable for the synergistic effect of VEGF-A and FGF-2 on neoangiogenesis in adults. The findings provide insights into the mechanisms underlying the effects of co-stimulation by growth factors, which could lead to rational design of therapeutic angiogenic strategies.

Improving Drug Potency and Efficacy by Nanocarrier-Mediated Subcellular Targeting

Polymeric micelles containing a chemotherapeutic drug carry it adjacent to the DNA target in tumor cells, enhancing the drug potency. Special Delivery to the Nucleus Micelles are useful in the washing machine, where they self-assemble from soaps, trap grease inside, and carry it away. These spheres, formed by linear molecules with hydrophobic tails that cluster in the core and hydrophilic heads sticking out, can carry cargo other than dirt. Micelles self-assembled in the presence of a chemotherapeutic drug can ensnare and carry it to tumors, where they are ingested by cells. By creating micelles that disperse in specific environment within the late endosome and lysosome, a region of the cell near the nucleus, Murakami et al. force these soapy spheres to release their deadly cargo—in this case a platinum-based drug called DACHPt [(1,2-diaminocyclohexane) platinum(II)]—right in the neighborhood of its target: DNA. This direct assault on the genome proves to be an effective antitumor strategy: Tumor cells growing in mice succumb more readily to a micelle-delivered derivative of platinum than they do to free drug. The authors’ micelle carriers are carefully assembled from block copolymers with properties suited to their task. A poly(ethylene glycol) polymer is linked to a string of glutamic acids, with a boron dipyrromethene at each end. By attaching fluorescent tags of different colors to the ends, the authors endowed their micelles with the ability to signal to an observer whether they are intact. When all the poly(glutamic acid) segments were clustered in the core, their red fluorescence was quenched and only the green surface dye on the poly(ethylene glycol) was visible. Once the micelle encountered specific conditions in the late endosome and lysosome, the core dispersed, releasing the drug and dequenching the red dye. By taking advantage of these visible markers of the micelle state, the authors showed by time-lapse confocal laser scanning microscopy that the micelles were taken up into tumor cells by endocytosis and that they traveled to the late endosomal/lysosomal compartment, where the micelles dispersed and the drug was released. This color-coded behavior was apparent both in cultured tumor cells and in tumor cells growing subcutaneously in mice, which the authors monitored in the animals, also by confocal laser scanning microscopy. But does the direct delivery of DACHPt to the nuclear area improve its effectiveness? A comparison of free DACHPt to the micelle-carried drug shows that it can help with one serious problem of cancer therapeutics—tumors that become drug-resistant. After repeated exposure to DACHPt, tumor cells develop defensive proteins, such as metallothionein and methionine synthase, in their cytoplasm that inactivate the drug, protecting the tumor cell DNA from damage. Tumors that have become resistant to DACHPt grow well in the presence of the drug, but the micelle-delivered version effectively inhibited the tumors’ growth, most likely by bypassing the cells’ cytoplasmic defenses. Therefore, with appropriate chemical modifications, micelles can be used to carry medicinal cargo right where it is needed. Nanocarrier-mediated drug targeting is an emerging strategy for cancer therapy and is being used, for example, with chemotherapeutic agents for ovarian cancer. Nanocarriers are selectively accumulated in tumors as a result of their enhanced permeability and retention of macromolecules, thereby enhancing the antitumor activity of the nanocarrier-associated drugs. We investigated the real-time subcellular fate of polymeric micelles incorporating (1,2-diaminocyclohexane) platinum(II) (DACHPt/m), the parent complex of oxaliplatin, in tumor tissues by fluorescence-based assessment of their kinetic stability. These observations revealed that DACHPt/m was extravasated from blood vessels to the tumor tissue and dissociated inside each cell. Furthermore, DACHPt/m selectively dissociated within late endosomes, enhancing drug delivery to the nearby nucleus relative to free oxaliplatin, likely by circumvention of the cytoplasmic detoxification systems such as metallothionein and methionine synthase. Thus, these drug-loaded micelles exhibited higher antitumor activity than did oxaliplatin alone, even against oxaliplatin-resistant tumors. These findings suggest that nanocarriers targeting subcellular compartments may have considerable benefits in clinical applications.

Cilostazol Inhibits Oxidative Stress–Induced Premature Senescence Via Upregulation of Sirt1 in Human Endothelial Cells

Objective—Cilostazol, a selective inhibitor of PDE3, has a protective effect on endothelium after ischemic vascular damage, through production of nitric oxide (NO). The purpose of the present study was to clarify the molecular mechanisms underlying the preventive effect of treatment with cilostazol on oxidative stress–induced premature senescence in human endothelial cells. Methods and Results—Prematurely senescent human umbilical vein endothelial cells (HUVECs) were induced by treatment with hydrogen peroxide (H2O2) as judged by senescence-associated &bgr;-galactosidase assay (SA-&bgr;gal), cell morphological appearance, and plasminogen activator inhibitor-1 (PAI-1) expression. Treatment with H2O2 caused 93% of the cells to be SA-&bgr;gal positive, whereas 46% of cilostazol (100 &mgr;mol/L)-treated cells were positive. HUVECs treated with other cAMP-elevating agents and DETA-NO showed a reduction of SA-&bgr;gal–positive cells as well. Cilostazol increased phosphorylation of Akt at Ser473 and of endothelial nitric oxide synthase (eNOS) at Ser1177, with a dose-dependent increase in Sirt1 expression. Moreover, the effect of cilostazol on premature senescence was abrogated through inhibition of Sirt1. Conclusions—Our results indicated that cilostazol exerted protective effects against endothelial senescence and dysfunction, and enhancement of NO production is a key mediator in upregulation of Sirt1.

Inhibition of endogenous TGF-β signaling enhances lymphangiogenesis

Lymphangiogenesis is induced by various growth factors, including VEGF-C. Although TGF-beta plays crucial roles in angiogenesis, the roles of TGF-beta signaling in lymphangiogenesis are unknown. We show here that TGF-beta transduced signals in human dermal lymphatic microvascular endothelial cells (HDLECs) and inhibited the proliferation, cord formation, and migration toward VEGF-C of HDLECs. Expression of lymphatic endothelial cell (LEC) markers, including LYVE-1 and Prox1 in HDLECs, as well as early lymph vessel development in mouse embryonic stem cells in the presence of VEGF-A and C, were repressed by TGF-beta but were induced by TGF-beta type I receptor (TbetaR-I) inhibitor. Moreover, inhibition of endogenous TGF-beta signaling by TbetaR-I inhibitor accelerated lymphangiogenesis in a mouse model of chronic peritonitis. Lymphangiogenesis was also induced by TbetaR-I inhibitor in the presence of VEGF-C in pancreatic adenocarcinoma xenograft models inoculated in nude mice. These findings suggest that TGF-beta transduces signals in LECs and plays an important role in the regulation of lymphangiogenesis in vivo.

Autophagy Is Activated by TGF-β and Potentiates TGF-β–Mediated Growth Inhibition in Human Hepatocellular Carcinoma Cells

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that regulates cell growth, differentiation, and apoptosis of various types of cells. Autophagy is emerging as a critical response of normal and cancer cells to environmental changes, but the relationship between TGF-beta signaling and autophagy has been poorly understood. Here, we showed that TGF-beta activates autophagy in human hepatocellular carcinoma cell lines. TGF-beta induced accumulation of autophagosomes and conversion of microtubule-associated protein 1 light chain 3 and enhanced the degradation rate of long-lived proteins. TGF-beta increased the mRNA expression levels of BECLIN1, ATG5, ATG7, and death-associated protein kinase (DAPK). Knockdown of Smad2/3, Smad4, or DAPK, or inhibition of c-Jun NH(2)-terminal kinase, attenuated TGF-beta-induced autophagy, indicating the involvement of both Smad and non-Smad pathway(s). TGF-beta activated autophagy earlier than execution of apoptosis (6-12 versus 48 h), and reduction of autophagy genes by small interfering RNA attenuated TGF-beta-mediated growth inhibition and induction of proapoptotic genes Bim and Bmf, suggesting the contribution of autophagy pathway to the growth-inhibitory effect of TGF-beta. Additionally, TGF-beta also induced autophagy in some mammary carcinoma cells, including MDA-MB-231 cells. These findings show that TGF-beta signaling pathway activates autophagy in certain human cancer cells and that induction of autophagy is a novel aspect of biological functions of TGF-beta.

Inhibition of Cyclooxygenase-2 Suppresses Lymph Node Metastasis via Reduction of Lymphangiogenesis

Cyclooxygenase-2 (COX-2) inhibitor has been reported to suppress tumor progression. However, it is unclear whether this inhibitor can also prevent lymphatic metastasis. To determine the effects of COX-2 inhibitor on lymphatic metastasis, etodolac, a COX-2 inhibitor, was given p.o. to mice bearing orthotopic xenografts or with carcinomatous peritonitis induced with a highly metastatic human diffuse-type gastric carcinoma cell line, OCUM-2MLN. Tumor lymphangiogenesis was significantly decreased in etodolac-treated mice compared with control mice. Consistent with this decrease in lymphangiogenesis, the total weight of metastatic lymph nodes was less in etodolac-treated mice than in control mice. Immunohistochemical analysis revealed that the major source of vascular endothelial growth factor-C (VEGF-C) and VEGF-D was F4/80-positive macrophages in our models. The mRNA levels of VEGF-C in mouse macrophage-like RAW264.7 cells, as well as those in tumor tissues, were suppressed by etodolac. The growth of human dermal lymphatic microvascular endothelial cells was also suppressed by etodolac. Supporting these findings, etodolac also inhibited lymphangiogenesis in a model of chronic aseptic peritonitis, suggesting that COX-2 can enhance lymphangiogenesis in the absence of cancer cells. Our findings suggest that COX-2 inhibitor may be useful for prophylaxis of lymph node metastasis by reducing macrophage-mediated tumor lymphangiogenesis.

Induction of Endothelial Nitric Oxide Synthase, SIRT1, and Catalase by Statins Inhibits Endothelial Senescence Through the Akt Pathway

Objective—Statins (3-hydroxy-3-methylglutaryl–coenzyme A reductase inhibitors) have pleiotropic vascular protective effects besides cholesterol lowering. Recently, experimental and clinical studies have indicated that senescence of endothelial cells is involved in endothelial dysfunction and atherogenesis. Therefore, the present study was performed to determine whether statins would reduce endothelial senescence and to clarify the molecular mechanisms underlying the antisenescent property of statins. Methods and Results—Senescent human umbilical vein endothelial cells were induced by hydrogen peroxide (H2O2), as judged by senescence-associated &bgr;-galactosidase assay and cell morphological appearance. Atorvastatin, pravastatin, and pitavastatin inhibited the oxidative stress induced-endothelial senescence. These statins phosphorylated Akt at Ser473 and subsequently led to increased expression of endothelial nitric oxide synthase (eNOS), SIRT1, and catalase. Treatment with LY294002 or Akt short interfering RNA decreased the eNOS activation, SIRT1 expression, and antisenescent property of atorvastatin. Moreover, in streptozotocin-diabetic mice, administration of pitavastatin increased eNOS, SIRT1, and catalase expression and decreased endothelial senescence, but levels remained unaltered in Sirt1 knockout mice. Conclusion—Our results indicate that treatment with statins inhibits endothelial senescence and that enhancement of SIRT1 plays a critical role in prevention of endothelial senescence through the Akt pathway, a direct target of statins.

LPA4 regulates blood and lymphatic vessel formation during mouse embryogenesis

Lysophosphatidic acid (LPA) is a potent lipid mediator with a wide variety of biological actions mediated through G protein-coupled receptors (LPA(1-6)). LPA(4) has been identified as a G(13) protein-coupled receptor, but its physiological role is unknown. Here we show that a subset of LPA(4)-deficient embryos did not survive gestation and displayed hemorrhages and/or edema in many organs at multiple embryonic stages. The blood vessels of bleeding LPA(4)-deficient embryos were often dilated. The recruitment of mural cells, namely smooth muscle cells and pericytes, was impaired. Consistently, Matrigel plug assays showed decreased mural cell coverage of endothelial cells in the neovessels of LPA(4)-deficient adult mice. In situ hybridization detected Lpa4 mRNA in the endothelium of some vasculatures. Similarly, the lymphatic vessels of edematous embryos were dilated. These results suggest that LPA(4) regulates establishment of the structure and function of blood and lymphatic vessels during mouse embryogenesis. Considering the critical role of autotaxin (an enzyme involved in LPA production) and Gα(13) in vascular development, we suggest that LPA(4) provides a link between these 2 molecules.

Polyplex micelles prepared from ω-cholesteryl PEG-polycation block copolymers for systemic gene delivery.

Polyplex micelles formed with plasmid DNA (pDNA) and poly(ethylene glycol) (PEG)-block-poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} [PAsp(DET)] exhibit effective endosomal escaping properties based on di-protonation of diamine side chains with decreasing pH, which improves their transfection efficiency and thus are promising candidates for local in vivo gene transfer. Here, PEG-PAsp(DET) polyplex micelles were further improved as in vivo systemic vectors by introduction of cholesterol (Chole) into the ω-terminus of PEG-PAsp(DET) to obtain PEG-PAsp(DET)-Chole. Introduction of the cholesterol resulted in enhanced association of block copolymers with pDNA, which led to increased stability in proteinous medium and also in the blood stream after systemic injection compared to PEG-PAsp(DET) micelles. The synergistic effect between enhanced polymer association with pDNA and increased micelle stability of PEG-PAsp(DET)-Chole polyplex micelles led to high in vitro gene transfer even at relatively low concentrations, due to efficient cellular uptake and effective endosomal escape of block copolymers and pDNA. Finally, PEG-PAsp(DET)-Chole micelles achieved significant suppression of tumor growth following intravenous injection into mice bearing a subcutaneous pancreatic tumor using therapeutic pDNA encoding an anti-angiogenic protein. These results suggest that PEG-PAsp(DET)-Chole micelles can be effective systemic gene vectors for treatment of solid tumors.