Kachio Tasaka

Cisplatin (CDDP) specifically induces apoptosis via sequential activation of caspase-8, -3 and -6 in osteosarcoma

Purpose: Osteosarcoma is a common malignant tumor. The first choice of treatment plan for osteosarcoma is chemotherapy. In particular, preoperative chemotherapy is most important in clinical treatment in orthopedics. In these chemotherapies, multiple anticancer drugs such as Adriamycin (ADM), CDDP, cyclophosphamide (CPM), methotrexate (MTX) and vincristine (VCR) are commonly used in combination. Recently, anticancer drugs have been shown to trigger apoptosis in various cancer cells. However, many studies on this topic have been examined using leukemia cell lines, and many kinds of cancer cells established from solid tumor are resistant to the induction of apoptosis by anticancer drugs. So in this study, we examined the effects of the anticancer drugs ADM, CDDP, CPM, MTX and VCR on osteosarcoma cells in vitro. We also examined the signaling pathways of each anticancer drug by studying the induction of apoptosis and activation of caspases in the osteosarcoma cells. Methods: We examined the effects of the anticancer drugs ADM, CDDP, CPM, MTX and VCR, which are used clinically for the treatment of osteosarcoma, on cells of the human osteosarcoma (HOS) cell line. The cytotoxic effects of the anticancer drugs were evaluated using the MTT assay. We used both flow cytometry and activation of caspases to confirm the induction of apoptosis in the HOS cells. To dissect the pathway of the caspase cascade in apoptosis in HOS cells, we used the tetrapeptides YVAD-CHO, DMQD-CHO, VEID-CHO and IETD-CHO, which selectively inhibit caspase-1, -3, -6 and -8, respectively. Results: ADM, CDDP, CPM and VCR, but not MTX, induced death of HOS cells in a dose-dependent manner. CDDP at 10 μM, CPM at 7.5 μM, ADM at 20 μM and VCR at 150 μM caused 80% cell death of HOS cells after 12 h. However, the percentages of apoptotic cells were 5.6% (medium alone), 75.9% (CDDP), 20.0% (CPM), 22.2% (ADM), 20.5% (VCR) and 13.1% (MTX). In addition, direct measurement of caspase-3 activity revealed that CDDP but not the other drugs activated caspase-3 in HOS cells. These analyses revealed that only CDDP induced apoptosis of HOS cells via activation of caspases. Furthermore, DMQD-CHO, VEID-CHO and IETD-CHO inhibited CDDP-induced apoptosis of HOS cells, suggesting that caspase-3, -6 and -8 are involved in the signaling pathway of CDDP-induced apoptosis. In contrast, none of the caspase inhibitors inhibited cell death induced by the other anticancer drugs. Conclusions: This study demonstrates that CDDP specifically induces apoptosis via activation of caspases and the other anticancer drugs induce death of HOS cells via different signaling pathways. It also demonstrates that caspase-8 is a key molecule in the earliest stage of the signaling pathway of CDDP-induced apoptosis of HOS cells, and caspase-3 works downstream of caspase-8.

Cisplatin (CDDP) sensitizes human osteosarcoma cell to Fas/CD95‐mediated apoptosis by down‐regulating FLIP‐L expression

The mechanisms of escape from Fas/CD95‐mediated apoptosis induced by immunosurveillance(NK cells and T cells) in tumor cells are correlated to tumorigenicity. Human osteosarcoma cell MG‐63 constitutively expressed cell surface Fas antigen but was resistant to apoptosis by Fas stimulation. However, suboptimal dose of cisplatin(CDDP) could sensitize MG‐63 cells to Fas‐mediated apoptosis without up‐regulation of cell‐surface Fas antigen. Western blotting analysis showed that MG‐63 cells constitutively expressed FLICE inhibitory protein long form(FLIP‐L), which was a novel anti‐apoptotic protein and had a potency of tumorigenicity. CDDP down‐regulated FLIP‐L in a time‐dependent manner in MG‐63 cells but did not influence expression of other anti‐apoptotic molecules such as XIAP, c‐IAP‐1, c‐IAP‐2, FADD or pro‐caspase‐8. Moreover, antisense oligonucleotide to FLIP‐L confirmed that down‐regulation of FLIP‐L induced sensitization to Fas‐mediated apoptosis. These findings suggest that FLIP‐L contributes to resistance to Fas‐mediated apoptosis in MG‐63 cells, and sensitization to Fas‐mediated apoptosis by CDDP can be a new application of immune therapy. Int. J. Cancer 88:986–991, 2000.

Correlation of thrombospondin‐1 and transforming growth factor‐β expression with malignancy of glioma

The expression of thrombospondin‐1 (TSP‐1) and its role in gliomas have not been well examined. In the present study TSP‐1 expression in a panel of malignant glioma cell lines and the expression of TSP‐1 and transforming growth factor (TGF‐β) proteins in low‐grade and malignant glioma tissues were investigated. Reverse transcription‐polymerase chain reaction analysis showed that nine of nine malignant glioma cell lines expressed TSP‐1 mRNA, and seven of nine glioma lines expressed TSP‐2 mRNA. Production and secretion of TSP‐1 were examined in the T98G glioblastoma cell line by western blot analysis. Total TSP‐1 protein content in the supernatant was 10 times higher than that in the cell lysate. Secretion of TSP‐1 was examined in these glioma cell lines by western blot analysis. All glioma lines secreted significant levels of TSP‐1. Bioassay showed that all tumor lines had the capacity to activate latent TGF‐β. Localization of TSP‐1, TGF‐β1, ‐β2, and ‐β3 was examined immunohistochemically in surgically resected glioma tissues, including 11 glioblastomas, six anaplastic astrocytomas, and eight astrocytomas. Most glioblastomas expressed high levels of both TSP‐1 and TGF‐β. Anaplastic astrocytomas expressed moderate levels of TSP‐1 and TGF‐β. Most malignant gliomas expressed various levels of TGF‐β1, ‐β2, and ‐β3. The expression of both proteins, however, was weak in low‐grade gliomas. Normal brain tissues around the tumors were negatively or very weakly positively stained for TSP‐1 and TGF‐β. These results indicate that most malignant glioma cells express TSP‐1 in vitro and in vivo, and the expression of TSP‐1 and TGF‐βin vivo correlates with the histologic malignancy of glioma. Overexpression of both TSP‐1 and TGF‐β may increase the biologic malignancy of malignant gliomas, through generating the active form of TGF‐β in tumor tissues.

TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in mouse osteoblastic MC3T3-E1 cells

Tumour necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK), a member of the TNF family, is a multifunctional cytokine that regulates cell growth, migration, and survival principally through a TWEAK receptor, fibroblast growth factor-inducible 14 (Fn14). However, its physiological roles in bone are largely unknown. We herein report various effects of TWEAK on mouse osteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed Fn14 and produced RANTES (regulated upon activation, healthy T cell expressed and secreted) upon TWEAK stimulation through PI3K-Akt, but not nuclear factor-κB (NF-κB), pathway. In addition, TWEAK inhibited bone morphogenetic protein (BMP)-2-induced expression of osteoblast differentiation markers such as alkaline phosphatase through mitogen-activated protein kinase (MAPK) Erk pathway. Furthermore, TWEAK upregulated RANKL (receptor activation of NF-κB ligand) expression through MAPK Erk pathway in MC3T3-E1 cells. All these effects of TWEAK on MC3T3-E1 cells were abolished by mouse Fn14-Fc chimera. We also found significant TWEAK mRNA or protein expression in osteoblast – and osteoclast-lineage cell lines or the mouse bone tissue, respectively. Finally, we showed that human osteoblasts expressed Fn14 and induced RANTES and RANKL upon TWEAK stimulation. Collectively, TWEAK/Fn14 interaction regulates RANTES production, BMP-2-induced differentiation, and RANKL expression in MC3T3-E1 cells. TWEAK may thus be a novel cytokine that regulates several aspects of osteoblast function.

Involvement of mitochondrial permeability transition and caspase-9 activation in dimethyl sulfoxide-induced apoptosis of EL-4 lymphoma cells

We observed that dimethyl sulfoxide (DMSO) induced apoptotic changes in the EL-4 murine lymphoma cell line and that effect was dependent on the concentration and time period. Incubating cells over a period of 18 h, 2.5% DMSO was found to induce sub-G1 peak in DNA histograms analyzed by flowcytometer and nucleosomal ladder formation in DNA gel electrophoresis. We also found down-regulation of Bcl-2, collapse of mitochondrial membrane potential (delta psi m) occurred following DMSO treatment, and release of cytochrome c from the mitochondria to cytosol. These observations suggest that DMSO converted its pro-apoptotic signal at the mitochondria. In the involvement of caspases, caspase-9 and -3, but not caspase-8, were found to be activated responding to DMSO treatment. Inhibitory experiments demonstrated that caspase cascade of mitochondrial apoptotic pathway was indispensable for DMSO-induced apoptosis. In the caspase cascade, caspase-9 was an upstream initiator and its primary signal could be transduced and amplified by caspase-3, -6 and -7. Kinetic study of these data showed mitochondrial dysfunction and caspase activation occurred at 12 h and apoptotic change of nuclear DNA at 18 h, providing another support for the transduction of DMSO pro-apoptotic signal via the mitochondrial pathway.

Enhanced Expression of Fas-Associated Death Domain-Like IL-1-Converting Enzyme (FLICE)-Inhibitory Protein Induces Resistance to Fas-Mediated Apoptosis in Activated Mast Cells

Mast cells play a critical role in host immune responses and are implicated in the pathogenesis of allergic inflammation. Though mouse mast cell line MC/9 expresses cell surface Fas Ag and is sensitive to Fas-induced apoptosis, activated MC/9 cells are resistant to Fas-induced cell death by cross-linking of FcεRI or FcγR. Fas-associated death domain-like IL-1-converting enzyme (FLICE)-inhibitory protein (FLIP), a caspase-8 inhibitor that lacks the cisteine domain, is one of the negative regulators of receptor-mediated apoptosis. In this report, we show that activation of mast cells by cross-linking of FcεRI or FcγR can induce enhanced expression of FLIP and consequently a resistance to Fas-induced apoptosis, although the expression level of Fas Ag is not changed. Addition of antisense oligonucleotide for FLIP prevents resistance to Fas-induced apoptosis of activated mast cells, suggesting that endogenous FLIP inhibits Fas-mediated apoptosis in activated mast cells. Thus, the enhanced expression of FLIP in activated mast cells contributes to the resistance to Fas-induced apoptosis, which may result in the development and prolongation of allergic inflammation.

IFN-γ Induces the Apoptosis of WEHI 279 and Normal Pre-B Cell Lines by Expressing Direct Inhibitor of Apoptosis Protein Binding Protein with Low pI

Interferon-γ plays a crucial role in induction of Th1 response but is predominantly a negative regulator of B cell differentiation and Th2 response, so it is a key molecule in determining cellular or humoral immunity. In this study, we demonstrate that IFN-γ induces apoptosis in WEHI 279 mouse B cells and IL-7-dependent mouse pre-B cells by disrupting mitochondrial membrane potential and cytochrome c release via down-regulation of Bcl-2 and Bcl-xL. Furthermore, this apoptotic signal is promoted by the de novo synthesis of endogenous direct inhibitor of apoptosis protein binding protein with low pI (DIABLO) by IFN-γ and its release from mitochondria into the cytosol. Inhibition of DIABLO expression by antisense oligonucleotide is sufficient to decrease caspase activities and DNA fragmentation, but not cytochrome c release from mitochondria, suggesting that DIABLO plays a critical role in promoting apoptotic signals downstream of mitochondrial events. Thus, these findings demonstrate a signaling pathway during B cell apoptosis induced by IFN-γ and possible mechanisms by which B cell differentiation is negatively regulated by Th1-type cytokines.

Immunotoxicity of trichloroethylene: a study with MRL-lpr/lpr mice

In recent immunological studies, it has been suggested that trichloroethylene (TCE) participates in the onset of pneumatosis cystoides intestinalis (PCI) through a certain mechanism; however, the mechanism by which it develops remains unknown. Based on findings that secondary PCI is often linked with autoimmune disease, the possibility that some genetic or immunological mechanisms are involved in the development of PCI has been proposed. Pneumatosis cystoides intestinalis is not a type of disease where a dose–response relationship with TCE exposure can be recognized and it is difficult to reproduce its physiopathology through TCE exposure in ordinary experimental animals. In the present study, immunological changes caused by TCE exposure were investigated by employing MRL‐lpr/lpr mice that are genetically labile to autoimmune diseases. To observe changes in B cell functions, serum antibody titres were measured; and for the T cell function, T cell subsets were examined. The animals were exposed to TCE at dosages of 0, 500, 1000 and 2000 ppm through inhalation 4 h a day, 6 days a week, for 8 weeks. It was found that only IgG production capacity was suppressed and there were no changes in T cell subsets with TCE concentrations up to 1000 ppm. At a concentration of 2000 ppm, changes were noted in both T and B cell functions. Typical organs that are responsible for immunological functions were examined for their morphological changes under a light microscope: the spleen and liver exhibited dose–response changes at a concentration of 500 ppm or greater. The development of immunoblastoid cells at a concentration of 1000 ppm indicated a possibility that a change has occurred in the immunological system. These findings show that exposure to TCE at high concentrations affects the immune system, but the study failed to induce PCI in the experimental animals. Further studies on TCE exposure at lower concentrations for longer periods are needed. Copyright

Caspase-dependent and -independent apoptosis of mast cells induced by withdrawal of IL-3 is prevented by Toll-like receptor 4-mediated lipopolysaccharide stimulation.

IL‐3‐dependent mucosal‐like mast cells undergo apoptosis upon withdrawal of IL‐3. Generally, the apoptosis is mediated by the activation of caspases and inhibited by addition of the pan‐caspase inhibitors z‐VAD‐FMK or BOC‐D‐FMK. However, DNA fragmentation, a typical characteristic of apoptosis, is not inhibited by z‐VAD‐FMK or BOC‐D‐FMK in mast cell apoptosis. In this study, we demonstrate that the apoptosis of mast cells is mediated by both caspase‐dependent and ‐independent mechanisms. The caspase‐independent apoptosis is mediated by the translocation of endonuclease G from mitochondria into nuclei. Withdrawal of IL‐3 caused down‐regulation of Bcl‐xL, resulting in a drop in mitochondrial membrane transition potential followed by the release of cytochrome c and endonuclease G from mitochondria. However, stimulation of mast cells through Toll‐like receptor 4 (TLR4) by lipopolysaccharide prevented mast cell apoptosis by inducing expression of Bcl‐xL. Moreover, the activation of mast cells by LPS is enhanced in the presence of IFN‐γ, which up‐regulates the expression of cell surface TLR4. Taken together, these observations provide evidence that mast cells play importantroles not only in allergic reactions but also in innate immunity recognizing enterobacteria through TLR4, and are regulated differently from allergic inflammation by Th1 cytokines.

Down-regulation of Pim-1 and Bcl-2 is accompanied with apoptosis of interleukin-6-depleted mouse B-cell hybridoma 7TD1 cells.

Here we report, interleukin-6 (IL-6) dependent mouse B-cell hybridoma, 7TD1 cells underwent apoptotic cell death with the starvation of IL-6. First, 7TD1 cells cultured without IL-6 arrested at G0/G1 phase (maximum accumulation at 24 h ) of the cell cycle. After that, the parameters of apoptosis namely, decreased mitochondrial transmembrane potential (DeltaPsi(m)), activation of caspases, DNA fragmentation and morphological changes (condensed nucleus and formation of apoptotic bodies) were observed. As evidents by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses, down-regulation of Pim-1 (a serine/threonine kinase) and Bcl-2 was observed in the IL-6-depleted 7TD1 cells. There was no change in the expression of c-Myc, Bcl-xL and Mcl-1, even at 48 h of IL-6-depletion. Taken together, these results indicate that IL-6 withdrawn from the 7TD1 cells resulted in G0/G1 arrest and then caspase-dependent apoptosis via mitochondrial pathway by down-regulation of Pim-1 and Bcl-2, which may be essential for anti-apoptotic signals of IL-6.