Hirokazu Nagawa

Vaccination with autologous endothelium inhibits angiogenesis and metastasis of colon cancer through autoimmunity

Overcoming immune tolerance of tumor angiogenesis should be useful for adjuvant therapy of cancer. We hypothesized that vaccination with autologous endothelium would induce an autoimmune response targeting tumor angiogenesis. To test this concept, we immunized BALB/c mice with a vaccine of glutaraldehyde‐fixed murine hepatic sinusoidal endothelial cells (HSEs) in a lung metastasis model of Colon‐26 cancer. Vaccination with autologous HSEs induced both preventive and therapeutic anti‐tumor immunity that significantly inhibited the development of metastases. ELISA revealed an immunoglobulin response involving IgM and IgG subclasses. These antibodies had a strong affinity for antigens of both murine and human endothelium, and lyzed endothelial cells in the CDC assay. Flow‐cytometry and chromium‐release cytotoxicity assay revealed a specific CTL response against endothelial cells, which were lyzed in an effector: target ratio‐dependent manner. Neither antibodies nor CTLs reacted with Colon26. The effect of autologous HSEs was more pronounced than that of xenogeneic human umbilical vein endothelial cells (HU‐VECs), which were tested in the same experimental setting. Our results suggest that vaccination with autologous endothelium can overcome peripheral tolerance of self‐angiogenic antigens and therefore should be useful for adjuvant immunotherapy of cancer. (Cancer Sci 2004; 95: 85–90)

Inhibition of autophagy potentiates sulforaphane-induced apoptosis in human colon cancer cells.

BackgroundSulforaphane (SUL), an isothiocyanate naturally present in widely consumed vegetables, particularly broccoli, has recently attracted attention due to its inhibitory effects on tumor cell growth by inducing apoptosis. We investigated the ability of SUL to induce autophagy in human colon cancer cells and whether inhibition of autophagy could potentiate the proapoptotic effect of SUL.MethodsThe proliferation of cells treated with SUL was assessed by MTS assay and colony-forming assay. Apoptosis and caspases activity were investigated by flow cytometry. The formation of acidic vesicular organelles (AVOs) was detected in acridine-orange-stained cells by flow cytometry. Western blotting was used for the detection of light chain 3 (LC3). Localizations of LC3 and cytochrome c were analyzed by immunocytochemistry.ResultsThe proapoptotic effect was observed by treatment of cells with relatively high concentrations of SUL for long periods of time. After 16xa0h of treatment, evident formation of AVOs and recruitment of LC3 to autophagosomes, features of autophagy, were observed. Treatment of cells with a specific autophagy inhibitor (3-methyladenine) potentiated the proapoptotic effect of SUL, which was dependent on the activation of caspases and the release of cytochrome c to the cytosol.ConclusionThe present results demonstrate induction of autophagy in colon cancer cells as a protective reaction against the proapoptotic effect of SUL, and consequently, the potentiation of the proapoptotic effect by autophagy inhibition. These findings provide a premise for use of autophagy inhibitors in combination with chemotherapeutic agents for treatment of colorectal cancer.

Anti-angiogenic property of zoledronic acid by inhibition of endothelial progenitor cell differentiation.

BACKGROUNDnZoledronic acid (ZOL) is clinically available for the treatment of skeletal complications. In preclinical studies, strong anti-cancer activities against breast cancer, prostate cancer, and leukemia were reported. It also inhibited the proliferation of cultured human endothelial cells, suggestive of an anti-angiogenic activity. Since ZOL has the tendency to accumulate in bone, we investigated the effect of ZOL on endothelial progenitor cells (EPCs), which originate from the bone marrow, and play important roles in angiogenesis.nnnMATERIALS AND METHODSnHuman peripheral blood mononuclear cells were cultured for 7 d to differentiate into EPCs. Cells were treated without/with ZOL or with geranylgeraniol (GGOH). Their endothelial phenotype was confirmed by the expression of CD144 and vascular endothelial growth factor receptor 2 and the tube-like formation ability on Matrigel (Becton Dickinson, Bedford, MA). Annexin V/propidium iodide staining was used to analyze apoptosis.nnnRESULTSnZOL treatment, even at low doses, from d 2 to 7 of culture resulted in impaired EPC differentiation and could be restored by co-treatment with GGOH. On the other hand, treatment of putative EPCs with ZOL at concentrations higher than 10 mum resulted in induction of apoptosis.nnnCONCLUSIONnZOL dose-dependently inhibited the differentiation of EPCs, the effect being observed even at low drug levels. At high concentrations, ZOL also induced the apoptotic death of putative EPCs. Since GGOH restored the inhibitory effect of ZOL on EPCs differentiation, the effect of ZOL appears to be dependent on the inhibition of prenylation of small-G-proteins. From these findings, we conclude that ZOL could be a potential anticancer agent by inhibiting angiogenesis.

Sulforaphane induces inhibition of human umbilical vein endothelial cells proliferation by apoptosis

Sulforaphane (SUL), one of the isothiocyanates (ITCs), has recently been focused due to its inhibitory effects on tumor cell growth in vitro and in vivo, which is dependent on the direct effect on cancer cells. In the present study, we aimed to investigate the potential anti-angiogenic effect of SUL and its mechanism of action. Using the human umbilical vein endothelial cells (HUVECs) as a model of angiogenesis, we investigated the effect of SUL on the various steps of angiogenesis, including the proliferation of endothelial cells, tubular formation, and matrix metalloproteinase (MMP) production. Sulforaphane induced a dose-dependent decrease in the proliferative activity of endothelial cells, which was dependent on cell apoptosis. Also SUL inhibited tube formation on matrigel, but did not affect MMP production. The present results demonstrate the anti-angiogenic activity of SUL and its potential use as an anti-cancer drug is suggested.

The inhibition of autophagy potentiates anti-angiogenic effects of sulforaphane by inducing apoptosis

BackgroundSulforaphane (SUL), a kind of isothiocyanate, has recently been focused due to its strong pro-apoptotic effect on cancer cells as well as tumor vascular endothelial cells (ECs). And recently, we demonstrated the induction of autophagy by colon cancer cells as a protective mechanism against SUL. In the present study, we aimed to investigate the possible role of autophagy induction by ECs as a defense mechanism against SUL.MethodsHuman umbilical vein endothelial cells (HUVECs) were used as the in vitro model of angiogenic ECs. The induction of autophagy was evaluated by the detection of acidic vesicular organelles (AVOs) by flow-cytometry, after the staining with acridine orange, as well as the detection of light chain 3(LC3) by Western blot. Finally, the functional implication of autophagy inhibition and SUL treatment in ECs was investigated by their ability to form vascular-like structures on Matrigel.ResultsTreatment of HUVECs with relatively low concentrations of SUL for 16xa0h resulted in the evident formation of AVOs and the recruitment of LC3 to autophagosomes, the pathognomonic features of autophagy. Co-treatment of cells with the specific autophagy inhibitor (3-methyladenine) potentiated the proapoptotic effect of SUL. And inhibition of autophagy potentiated the inhibitory effect of SUL on the ability of ECs to form capillary-like structures.ConclusionSimilar to cancer cells, ECs induced autophagy in response to the pro-apoptotic agent, SUL, and the inhibition of autophagy potentiated the pro-apoptotic effect. These findings open premises for the use of autophagy inhibitors in combination with anti–angiogenic agents.

Sulforaphane Stimulates Activation of Proapoptotic Protein Bax Leading to Apoptosis of Endothelial Progenitor Cells

Sulforaphane (SUL) is an isothiocyanate naturally present in widely consumed vegetables, particularly in broccoli. SUL has recently been focused as a result of its inhibitory effects on tumor cell growth in vitro and in vivo. We used endothelial progenitor cells (EPCs) as an in vitro model to investigate the effect of SUL on the various steps of vasculogenesis and angiogenesis. Peripheral blood mononuclear cells from blood of normal human volunteers were plated on fibronectin-coated 100 mm dishes and incubated for 7xa0days. The viability of EPCs, treated with SUL at different doses, was assessed by MTS assay. Cell apoptosis was analyzed by flow cytometry. To determine the relative contributions of caspase-8 and caspase-9 pathways to SUL-induced apoptosis, the effect of caspase inhibitors was determined. The expression of apoptosis-related proteins (Bax, Bcl-2) was investigated by Western blot test. Finally, the effect of SUL on the ability of EPCs to form vascular-like structures on Matrigel was investigated. We clearly demonstrated that SUL induced the dose-dependent inhibition of EPCs’ viability by induction of apoptosis. All caspases (caspase-3, −8, and −9) were activated during apoptosis induction by SUL, but the effect of caspase-9 was more prominent than that of caspase-8. Also, the expression of Bax was upregulated by SUL treatment. In addition to apoptosis induction, SUL dose-dependently inhibited the tube-like formation by EPCs on Matrigel. The present results demonstrate the antivasculogenic/antiangiogenic activity of SUL in vitro and open premise for the use of SUL as a multipotent anticancer agent that targets both cancer cells and the angiogenic endothelium.

Early-outgrowth of endothelial progenitor cells can function as antigen-presenting cells

Endothelial progenitor cells (EPCs) have been recently found to exist circulating in peripheral blood of adults, and home to sites of neovascularization in peripheral tissues. They can also be differentiated from peripheral blood mononuclear cells (PBMNCs). In tumor tissues, EPCs are found in highly vascularized lesions. Few reports exist in the literature concerning the characteristics of EPCs, especially related to their surface antigen expressions, except for endothelial markers. Here, we aimed to investigate the surface expression of differentiation markers, and the functional activities of early-outgrowth of EPCs (EO-EPCs), especially focusing on their antigen-presenting ability. EO-EPCs were generated from PBMNCs, by culture in the presence of angiogenic factors. These EO-EPCs had the morphological and functional features of endothelial cells and, additionally, they shared antigen-presenting ability. They induced the proliferation of allogeneic lymphocytes in a mixed-lymphocyte reaction, and could generate cytotoxic lymphocytes, with the ability to lyze tumor cells in an antigen-specific manner. The antigen-presenting ability of EO-EPCs, however, was weaker than that of monocyte-derived dendritic cells, but stronger than peripheral blood monocytes. Since EO-EPCs play an important role in the development of tumor angiogenesis, targeting EPCs would be an effective anti-angiogenic strategy. Alternatively, due to their antigen-presenting ability, EO-EPCs can be used as the effectors of anti-tumor immunotherapy. Since they share endothelial antigens, the activation of a cellular immunity against angiogenic vessels can be expected. In conclusion, EO-EPCs should be an interesting alternative for the development of new therapeutic strategies to combat cancer, either as the effectors or as the targets of cancer immunotherapy.