Mutsuhiko Minami

Evodiamine, a constituent of Evodiae Fructus, induces anti-proliferating effects in tumor cells

We found that evodiamine, a major alkaloidal component of Evodiae Fructus (Goshuyu in Japan), inhibited proliferation of several tumor cell lines, but had less effect on human peripheral blood mononuclear cells (PBMC). We used human cervical cancer cells, HeLa, as a model to elucidate the molecular mechanisms of evodiamine‐induced tumor cell death. The results showed that evodiamine induced oligonucleosomal fragmentation of DNA in HeLa cells and increased the activity of caspase‐3, but not that of caspase‐1, in vitro. Both evodiamine‐induced DNA fragmentation and caspase‐3 activity were effectively inhibited by a caspase‐3 inhibitor, z‐DEVD‐fmk (z‐Asp‐Glu‐Val‐Asp‐fmk). In addition, evodiamine increased the expression of the apoptosis inducer Bax, but decreased the expression of the apoptosis suppressor Bcl‐2 in mitochondria. Taken together, our data indicated that evodiamine alters the balance of Bcl‐2 and Bax gene expression and induces apoptosis through the caspase pathway in HeLa cells. (Cancer Sci 2003; 94: 92–98)

P53-mediated cell cycle arrest and apoptosis through a caspase-3- independent, but caspase-9-dependent pathway in oridonin-treated MCF-7 human breast cancer cells.

AbstractAim:To study the caspase-3-independent mechanisms in oridonin-induced MCF-7 human breast cancer cell apoptosis in vitro.Methods:The viability of oridonin-treated MCF-7 cells was measured by MTT (thiazole blue) assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. The apoptotic ratio was determined by lactate dehydrogenase assay. Cell cycle alternation and mitochondrial membrane potential were measured by flow cytometric analysis. Bax, Bcl-2, caspase-3, caspase-9, heat shock protein (Hsp)90, p53, p-p53, p21, Poly (ADP-ribose) polymerase (PARP), and the inhibitor of caspase-activated DNase (ICAD) protein expressions were detected by Western blot analysis.Results:Oridonin inhibited cell growth in a time- and dose-dependent manner. Cell cycle was altered through the upregulation of p53 and p21 protein expressions. Pancaspase inhibitor Z-VAD-fmk and calpain inhibitor II both decreased cell death ratio. Nucleosomal DNA fragmentation and the downregulation of ΔΨmit were detected in oridonin-induced MCF-7 cell apoptosis, which was involved in a postmitochondrial caspase-9-dependent pathway. Decreased Bcl-2 and Hsp90 expression levels and increased Bax and p21 expression levels were positively correlated with elevated levels of phosphorylated p53 phosphorylation. Moreover, PARP was partially cleaved by calpain rather than by capase-3.Conclusion:DNA damage provoked alternations in the mitochondrial and caspase-9 pathways as well as p53-mediated cell cycle arrest, but was not related to caspase-3 activity in oridonin-induced MCF-7 cells.

MHC class I-mediated exogenous antigen presentation by exosomes secreted from immature and mature bone marrow derived dendritic cells

Exosomes are 50-90 nm vesicles with antigen presenting ability carrying major histocompatibility complex (MHC) class I, class II, abundant co-stimulatory molecules and some tetraspan proteins. Although dendritic cells (DCs) are one of the professional antigen presenting cells capable of presenting exogenous antigens in MHC class I-mediated antigen specific manner (cross-presentation), the cross-presentation ability by exosomes from immature or mature DCs are unknown. Here we show that exosomes released from ovalbumin (OVA) protein-pulsed bone marrow derived dendritic cells (BM-DCs) weakly present the peptide determinants to OVA specific MHC class I-restricted CD8(+) T cell hybridomas. The exosomes secreted by OVA(257-264) peptide- or OVA protein-pulsed mature BM-DCs activated OVA specific MHC class I-restricted T cell hybridomas more efficiently than those from immature BM-DCs. Transporters associated with antigen processing (TAP) deficient mice-derived BM-DCs were also used to examine whether functional TAP activity was required for cross-presentation by exosomes. The exosomes obtained from OVA(257-264) peptide-pulsed BM-DCs derived from TAP(-/-) mice showed a significant antigen presenting ability to OVA specific MHC class I-restricted T cell hybridomas. Altogether, our data indicate that BM-DCs secrete exosomes with weak cross-presentation ability.

Requirement of CD80 and CD86 molecules for antigen presentation by eosinophils.

The authors analysed the antigen‐presenting ability of eosinophils purified from peritoneal exudate cells of interleukin‐5 (IL‐5) transgenic mice. The granulocyte‐macrophage colony‐stimulating factor (GM‐CSF)‐treated eosinophils induced proliferative responses of primed lymph node T cells and thymus T cells to staphylococcal enterotoxin B (SEB), while untreated eosinophils induced little or no response. GM‐CSF‐treated eosinophils also induced proliferation of ovalbumin (OVA)‐primed lymph node T cells to OVA. Although untreated eosinophils expressed no MHC class II molecule on the surface the eosinophils could be induced to express major histocompatibility complex (MHC) class II molecules when treated with GM‐CSF. In the present study, anti‐I‐Ak monoclonal antibodies (MoAbs) strongly inhibited proliferation of thymus T cells and proliferation of OVA‐primed lymph node T cells in response to OVA, but weakly inhibited proliferation of primed T cells in response to SEB. Furthermore, CD80 (B7‐1) and CD86 (B7‐2) were expressed on the surfaces of untreated eosinophils. The expression of those two molecules on the eosinophils was increased by incubation with GM‐CSF. Moreover, anti‐CD80 or anti‐CD86 MoAbs blocked proliferative responses of primed lymph node T cells and thymus T cells to SEB, and also blocked responses of primed lymph node T cells to OVA. Thus, CD80 and CD86 play an important role in stimulation of T cells by eosinophils.

Activation and suppression of renin-angiotensin system in human dendritic cells.

We previously identified the gene expression of renin-angiotensin system in human monocyte-derived dendritic cells (DCs). This study was conducted to examine the mechanisms by which angiotensin II and captopril, the inhibitor of the angiotensin-converting enzyme (ACE), affect human DCs. In DCs, lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-alpha), interleukin-(IL)-1alpha, IL-10, IL-12, and IL-18 was significantly inhibited by captopril. In contrast, angiotensin II treatment resulted in a significant increase in TNF-alpha and IL-6 protein biosynthesis by DCs. In addition, we have studied the global expression of 2400 genes in DCs from two donors. Here, we demonstrated the specific down-regulation of the ACE gene expression in captopril-treated DCs. Our finding indicates the possible activation of NF-kappaB through the up-regulation of expressions of MEFV gene (encoding PYRIN protein) and heterogeneous nuclear ribonucleoprotein R in DCs. This is the first study on the modulation of cytokine and gene expression by angiotensin II and captopril in DCs.

The delivery of an antigen from the endocytic compartment into the cytosol for cross-presentation is restricted to early immature dendritic cells

Dendritic cells (DCs) are the only antigen‐presenting cell population having a cross‐presentation capacity. For cross‐presentation, however, the intracellular antigen‐processing pathway and its regulatory mechanism have not been defined. Here we report the differences in cross‐presentation ability among murine bone marrow‐derived immature DC, early immature day8‐DC and late immature day10‐DC, and fully mature day10 + lipopolysaccharide DC. Day8‐DCs and day10‐DCs show an immature phenotypic profile but are different in morphology. Day8‐DCs can internalize an abundant volume of exogenous soluble ovalbumin (OVA) and result in cross‐presentation. In contrast, day10‐DCs are not able to cross‐present, although they maintain efficient macropinocytosis. Exogenously internalized OVA antigens are stored in the endocytic compartments. The endocytic compartments are temporarily maintained at mildly acidic pH in day8‐DCs and are rapidly acidified in day10‐DCs after uptake of antigens. We show that OVA antigens accumulated in the endocytic compartments move into the cytosol in day8‐DCs but do not in day10‐DCs. NH4Cl‐treatment, which neutralizes the acidic endocytic compartments and/or delays endosomal maturation, restores day10‐DCs for transport the stored OVA antigens from the endocytic compartments into the cytosol. Diphenyleneiodonium chloride‐treatment, which acidifies the endocytic compartments, decreases an amount of transported OVA antigen into the cytosol in day8‐DCs. These data indicate that only the early immature stage of DC interferes with endosomal maturation, even after uptake of exogenous antigens, and then transports the antigens into the cytosol.

Immune Response Induced by Airway Sensitization after Influenza A Virus Infection Depends on Timing of Antigen Exposure in Mice

ABSTRACT To study which phase of viral infection promotes antigen sensitization via the airway and which type of antigen-presenting cells contributes to antigen sensitization, BALB/c mice were sensitized by inhalation of ovalbumin (OA) during the acute phase or the recovery phase of influenza A virus infection, and then 3 weeks later animals were challenged with OA. The numbers of eosinophils and lymphocytes, the amounts of interleukin-4 (IL-4) and IL-5 in the bronchoalveolar lavage fluid, and the serum levels of OA-specific immunoglobulin G1 (IgG1) and IgE increased in mice sensitized during the acute phase (acute phase group), while a high level of gamma interferon production was detected in those sensitized during the recovery phase (recovery phase group). In the acute phase group, both major histocompatibility complex class II molecules and CD11c were strongly stained on the bronchial epithelium; in the recovery phase group, however, neither molecule was detected. OA-capturing dendritic cells (DCs) migrated to the regional lymph nodes, and a small number of OA-capturing macrophages were also observed in the lymph nodes of the acute phase group. In the recovery group, however, no OA-capturing DCs were detected in either the lungs or the lymph nodes, while OA-capturing macrophages were observed in the lymph nodes. These results indicate that the timing of antigen sensitization after viral infection determines the type of immune response.

Impaired function of dendritic cells in alymphoplasia (aly/aly) mice for expansion of CD25+CD4+ regulatory T cells

Alymphoplasia (aly/aly) mice are from a naturally occurring strain with a mutation in nuclear factor-kappa B inducing kinase (NIK). The NIK mutation causes disruption of the architecture of the thymus and spleen and aly/aly mice show decreased numbers of CD25+CD4+T cells in the spleen. For the expansion of CD25+CD4+T cells, interactions between dendritic cells (DCs) and CD25+CD4+ regulatory T cells are necessary. We investigated the ability of DCs to induce expansion of CD25+CD4+T cells. We found that DCs are reduced in the spleen of aly/aly mice, and showed low expressions of CD80, CD86 and MHC class II molecules on the surface. DCs from aly/aly mice showed decreased ability to present ovalbumin (OVA) to T cells from OVA specific TCR transgenic mice, and a decreased ability for alloantigen presentation. Further, DCs showed a decreased ability to induce expansion of CD25+CD4+T cells in vitro. Our results suggested that the impairment of DCs in aly/aly mice is responsible, at least in part, for the decreased numbers of CD25+CD4+T cells in the periphery of aly/aly mice.

Dracorhodin perchlorate induces apoptosis in HL-60 cells.

Dracorhodin perchlorate, an anthocyanin red pigment, induces human premyelocytic leukemia HL-60 cell death through apoptotic pathway. Caspase -1, -3, -8, -9, and -10 inhibitors partially reversed the cell death induced by dracorhodin perchlorate. Caspase-3 and -8 were activated followed to the degradation of caspase-3 substrates, inhibitor of caspase-activated DNase (ICAD) and poly-(ADP-ribose) polymerase (PARP). Dracorhodin perchlorate up-regulated the expression ratio of mitochondrial proteins, Bax/Bcl-XL. The cell death was accompanied with phosphorylation of ERK, JNK and p38 MAPK and partially reduced by MEK inhibitor (PD98059), JNK MAPK inhibitor (SP600125) and p38 MAPK inhibitor (SB 203580). Taken together, dracorhodin perchlorate-induced apoptosis in HL-60 cells via up-regulation of Bax, activation of caspases and ERK/p38/JNK MAPKs.

Experimental gene therapy against subcutaneously implanted glioma with a herpes simplex virus-defective vector expressing interferon-γ

We investigated the feasibility of local treatment or tumor vaccination with a herpes simplex virus (HSV) type 1-defective vector. The vector was engineered to express murine interferon-γ (IFN-γ) for experimental gene therapy against mouse glioma Rous sarcoma virus (RSV). The murine IFN-γ gene was driven by the cytomegalovirus promoter. The helper virus (tsk) was thermosensitive; consequently, this vector could only proliferate at 31°C. A high level of murine IFN-γ expression was confirmed in vitro and in vivo by immunohistochemistry using anti-mouse IFN-γ monoclonal antibody. This engineered vector (dvHSV/MuIFN-γ) inhibited the proliferation of mouse glioma RSV cells in vitro, and an intratumoral (i.t.) local injection of the vector caused i.t. necrosis in vivo. The immunological effect of dvHSV/MuIFN-γ was also examined in a mouse glioma RSV cell implantation model. A subcutaneous (s.c.) implant of 1 × 106 mouse glioma RSV cells after treatment with dvHSV/MuIFN-γ was rejected. However, the implant after treatment with an engineered HSV-defective vector containing an antisense nucleotide sequence of the murine IFN-γ gene was not rejected. In addition, in another group of mice in which RSV cells treated with dvHSV/MuIFN-γ were implanted into a femoral (s.c.) region and nontreated RSV cells were implanted into a contralateral femoral (s.c.) region, the implanted RSV cells were rejected. The rejection of the implanted mouse glioma RSV was blocked by anti-asialo GM1, which was known to inhibit natural killer cell activity. These results revealed that the HSV-defective vector could realize a high efficiency of transfection to glioma cells through short-time treatment, and that the IFN-γ gene transferred to the cells had the effect of tumor vaccination, which was suggested be related to natural killer cells. In conclusion, dvHSV/MuIFN-γ may be useful for the gene therapy of malignant glioma through either i.t. local injection or a practical tumor vaccination with ex vivo gene transfer.