Akemi Hayakawa

Seaweed Prevents Breast Cancer

To investigate the chemopreventive effects of seaweed on breast cancer, we have been studying the relationship between iodine and breast cancer. We found earlier that the seaweed, wakame, showed a suppressive effect on the proliferation of DMBA (dimethylbenz(a)anthracene)‐induced rat mammary tumors, possibly via apoptosis induction. In the present study, powdered mekabu was placed in distilled water, and left to stand for 24 h at 4°C. The filtered supernatant was used as mekabu solution. It showed an extremely strong suppressive effect on rat mammary carcinogenesis when used in daily drinking water, without toxicity. In vitro, mekabu solution strongly induced apoptosis in 3 kinds of human breast cancer cells. These effects were stronger than those of a chemothera‐peutic agent widely used to treat human breast cancer. Furthermore, no apoptosis induction was observed in normal human mammary cells. In Japan, mekabu is widely consumed as a safe, inexpensive food. Our results suggest that mekabu has potential for chemoprevention of human breast

Gene therapy for hepatocellular carcinoma using sonoporation enhanced by contrast agents

We examined whether sonoporation enhanced by a contrast agent (BR14) was effective in gene therapy for hepatocelluar carcinoma (HCC). Human hepatic cancer cells (SK-Hep1) and plasmid cDNAs expressing green fluorescent protein (GFP), interferonβ (IFNβ), and LacZ were used. In vitro, SK-Hep1 cell suspensions with DNA and BR14 were sonoporated. Expressions of every plasmid cDNA and the antitumor effect of IFNβ were analyzed. In vivo, GFP and IFNβ genes with BR14 were directly injected into subcutaneous tumors using SK-Hep1 in nude mice, and transcutaneous sonoporation of the tumors was performed. GFP gene transfections and tumor diameters after IFNβ gene transfection were examined. In vitro, no SK-Hep1 cells were transfected without sonication, whereas transfections were successful after sonication with BR14. Antitumor effect of IFNβ gene transfection by ultrasound (US) and with BR14 was revealed. In vivo, the SK-Hep1 cells expressed GFP, and the IFNβ gene transfection by US with BR14 reduced tumor size significantly. In conclusion, gene therapy with sonoporation enhanced by a contrast agent may become a new treatment option for HCC.

Paeoniflorin induces apoptosis of lymphocytes through a redox‐linked mechanism

Paeoniflorin (PF), isolated from paeony root, has been used as a herbal medicine for more than 1,200 years in China, Korea, and Japan for its anti‐allergic, anti‐inflamatory, and immunoregulatory effects. In this study, we found that PF induces apoptosis in both murine T‐lineage cells and human T‐cell leukemia Jurkat cells. This apoptosis was mediated through the reduction of mitochondrial membrane potential, activation of caspase, and fragmentation of DNA. Interestingly, PF induced generation of reactive oxygen species (ROS) and a reducing agent, dithiothreitol (DTT), and a ROS scavenger, N‐acetyl cysteine (NAC), successfully attenuated the PF‐induced apoptosis. Additionally, PF induced the phosphorylation of three mitogen‐activated protein (MAP) family kinases, extracellular signal‐regulated kinase, c‐Jun amino‐terminal kinase (JNK), and p38 MAP kinase. Curcumin, an anti‐oxidant and JNK inhibitor, inhibited PF‐induced apoptosis, suggesting the possible involvement of curcumin‐sensitive JNK or other redox‐sensitive elements in PF‐induced apoptosis. These results partially explain the action mechanism of PF‐containing paeony root as a herbal medicine.

Changes in intracellular Ca2+ concentrations related to PDT-induced apoptosis in photosensitized human cancer cells

For photodynamic therapy (PDT), human squamous cell carcinoma cells (HSC-2) were treated with 3 microg/ml of photofrin 24 h prior to irradiating the cultures with the excimer dye laser at a dose of 2 J/cm2. Extensive DNA fragmentation was recognized within 2 h of PDT. The proportion of cells with DNA fragmentation on flow cytometric analysis was significantly increased to 44 and 78% at 1 and 2 h after PDT, respectively, compared to control groups. Confocal laser scanning microscopy revealed a slight change in the intracellular Ca2+ concentration occurring at 30 min after PDT and a subsequent marked increase in the Ca2+ concentration 1-2 h after PDT, but no change was observed in the cells exposed to laser irradiation alone and to photofrin alone and in the cells immediately after PDT. These findings suggest that an increase in the intracellular Ca2+ concentration may play an important role in the induction of PDT-induced apoptosis.

Cepharanthine Activates Caspases and Induces Apoptosis in Jurkat and K562 Human Leukemia Cell Lines

Cepharanthine (CEP) is a known membrane stabilizer that has been widely used in Japan for the treatment of several disorders such as anticancer therapy‐provoked leukopenia. We here report that apoptosis was induced by low concentrations (1–5 μM) of CEP in a human leukemia T cell line, Jurkat, and by slightly higher concentrations (5–10 μM) in a human chronic myelogenous leukemia (CML) cell line K562, which expresses a p210 antiapoptotic Bcr‐Abl fusion protein. Induction of apoptosis was confirmed in both Jurkat and K562 cells by DNA fragmentation and typical apoptotic nuclear change, which were preceded by disruption of mitochondrial membrane potential and were induced through a Fas‐independent pathway. CEP treatment induced activation of caspase‐9 and ‐3 accompanied by cleavage of PARP, Bid, lamin B1, and DFF45/ICAD in both Jurkat and K562 cells, whereas caspase‐8 activation and Akt cleavage were observed only in Jurkat cells. The CEP‐induced apoptosis was completely blocked by zVAD‐fmk, a broad caspase inhibitor. Interestingly, CEP treatment induced remarkable degradation of the Bcr‐Abl protein in K562 cells, and this degradation was prevented partially by zVAD‐fmk. When used in combination with a nontoxic concentration of herbimycin A, lower concentrations (2–5 μM) of CEP induced obvious apoptosis in K562 cells with rapid degradation or decrease in the amount of Bcr‐Abl and Akt proteins. Our results suggest that CEP, which does not have bone marrow toxicity, may possess therapeutic potential against human leukemias, including CML, which is resistant to anticancer drugs and radiotherapy. J. Cell. Biochem. 82: 200–214, 2001.

Combination effect of anti-Fas antibody and chemotherapeutic drugs in ovarian cancer cells in vitro.

To clarify the significance of the Fas antigen (Ag) in gynecologic tumors, its expression in gynecologic cancer cell lines was examined. The Fas Ag was expressed in 6 of 15 cell lines. Five of 8 ovarian cancer cell lines but none of 4 choriocarcinoma cell lines expressed the Fas Ag. In drug-resistant cell lines derived from one of the Fas-positive cells, its expression was not lost after development of resistance to cisplatin, SN-38 or etoposide, but its expression was absent in the cell line resistant to Adriamycin. The effect of the anti-Fas antibody (Ab) was then studied. Apoptosis was induced in 7 of 9 Fas-positive cell lines, whereas the remaining two cell lines were unaffected. Furthermore, the combination effect of the anti-Fas Ab and drugs was examined in an ovarian cancer cell line and its drug-resistant variants, and a synergistic effect was observed. These results suggest important roles of the Fas Ag in ovarian cancer and the potential for overcoming drug resistance by a combination of the anti-Fas Ab and various drugs.

Intracellular signaling in the induction of apoptosis in a human breast cancer cell line by water extract of Mekabu

BackgroundWe previously reported that water extract of Mekabu, a kind of seaweed, induced apoptosis in a human breast cancer cell line. In the present study we investigated intracellular signaling in apoptosis, with a focus on caspases.MethodsMekabu extract, obtained with ultrapure water, was used to induce apoptosis in a human breast cancer cell line, MDA-MB231, and DNA fractionation was investigated by flow cytometry and electrophoresis. In addition, using the caspase detection kit Caspa Tag, activation of caspases 3, 6, 8, and 9 was observed under a fluorescence microscope. Furthermore, using antibodies to caspases 3, 8, 9, and Bid, we conducted a protein analysis by Western blotting to determine the activation of these substances.ResultsObvious ladder formation demonstrating DNA fractionation was seen, confirming that Mekabu extract induced apoptosis. In the fluorescence microscope observations, activation of caspases 3, 6, and 8, but not caspase 9, was seen. Activated caspases 3 and 8 were detected in the Western blotting analysis, but no proteins of activated caspase 9 or Bid were detected.ConclusionMekabu extract activates caspases 3, 6, and 8 and contributes to intracellular signaling to induce apoptosis in a human breast cancer cell line. This signaling is not via the mitochondria.

Activation of caspase-8 is critical for sensitivity to cytotoxic anti-Fas antibody-induced apoptosis in human ovarian cancer cells

Two ovarian cancer cell lines named NOS4 and SKOV-3 have been shown to have different sensitivities to a cytotoxic anti-Fas antibody, CH-11. Although both cell lines express Fas molecules on the cell surfaces at the same intensities, apoptosis is induced by CH-11 in NOS4 cells but not in SKOV-3 cells. In this study, the different apoptosis-sensitivities of these cells were assessed. Both cell lines express almost the same levels of FADD, RIP, c-FLIP, FAP-1, Bax, Bcl-2 and Bcl-XL. Evidence of caspase-8, caspase-9 and caspase-3 activation and of cleavage of PARP and Bid was obtained in NOS4 cells but not in SKOV-3 cells. When triggered by FasL protein, DNA fragmentation and caspase-8 activation were observed in SKOV-3 cells, though they were not as clear as in NOS4 cells. All the anti-Fas antibody-mediated signals for apoptosis induction in NOS4 cells were completely blocked by a caspase-8-specific inhibitor, Z-IETD-FMK. These results indicate that the different sensitivities to the anti-Fas antibody are solely dependent on the activation of caspase-8, which could be influenced by yet unknown qualitative or quantitative abnormalities in molecules involved in DISC formation.

Fas Antigen Expression of Hepatocytes and Its Modification by Immunosuppressants

Active cell death induced by ligation of the Fasantigen (Fas-Ag) with its antibody, Fas ligand (Fas-L),has been known to play a major role in cell killing viaapoptosis by cytotoxic T lymphocytes (CTL). Thus, in liver transplantation, Fas-Agexpression of hepatocytes and its modification byimmunosuppressive agents such as FK 506 or CsA cantheoretically influence allograft survival. Mousehepatocytes (BALB/c) were isolated and cultured with orwithout FK 506 or CsA, and Fas-Ag expression wasdetermined by flow cytometry. Fas-Ag expression in thecontrol was 17.2 ± 2.5% after 24 hr of culture.When FK 506 or CsA was added, Fas-Ag expression withFK 506 at a concentration of 0.01-0.1 μg/ml wassignificantly lower than that with CsA (P < 0.05).When the cells were incubated with apoptosis-inducing anti-Fas-Ag monoclonal antibody, agarose gelelectrophoresis of the control cells yielded a typicalpattern of DNA fragmentations. The cells with FK 506 at0.01 μg/ml yielded the least DNA fragmentation. These findings suggested that in the in vivosetting, the hepatocytes of the allograft would have alower chance of being attacked by CTL in the hosttreated with FK 506.

Murine lymph node‐derived stromal cells effectively support survival but induce no activation/proliferation of peripheral resting T cells in vitro

Little is known about the homeostatic mechanisms by which the levels of peripheral lymphocytes are maintained. The survival of naïve T cells in vivo must be maintained by some factors that have not been characterized in an in vitro culture system. In this study, we established a culture system of stromal cells derived from murine lymph nodes and investigated the action of the stromal cells in supporting the survival of resting T cells in vitro. Most of the T cells cocultured with the stromal cells did not die, and the supernatant of cultured stromal cells increase the viability of T cells. This T‐cell survival‐supporting activity was maintained for more than 7 days. Although interleukin (IL)‐4, IL‐6, IL‐7, and interferon‐β also rescued peripheral T cells from spontaneous cell death, medium‐soluble and heat‐sensitive factor(s) derived from the stromal cells supported the survival of T cells more effectively and for a longer time than did these cytokines. T cells maintained in the culture system with the stromal cells appeared to remain in a resting G0/G1 state and did not show remarkable DNA synthesis. From these results, it is presumed that some soluble factor(s) other than the tested cytokines that have been identified as supporting T‐cell survival are produced from lymph node stromal cells. These factor(s) play an important role in maintenance of resting T cells.