Banri Yamanoha

Leptosins isolated from marine fungus Leptoshaeria species inhibit DNA topoisomerases I and/or II and induce apoptosis by inactivation of Akt/protein kinase B

DNA topoisomerases (topo) I and II are molecular targets of several potent anticancer agents. Thus, inhibitors of these enzymes are potential candidates or model compounds for anticancer drugs. Leptosins (Leps) F and C, indole derivatives, were isolated from a marine fungus, Leptoshaeria sp. as cytotoxic substances. In vitro cytotoxic effects of Lep were measured using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide‐based viability assay. Lep F inhibited the activity of topos I and II, whereas Lep C inhibited topo I in vitro. Interestingly both of the compounds were found to be catalytic inhibitors of topo I, as evidenced by the lack of stabilization of reaction intermediate cleavable complex (CC), as camptothecin (CPT) does stabilize. Furthermore, Lep C inhibited the CC stabilization induced by CPT in vitro. In vivo band depletion analysis demonstrated that Lep C likewise appeared not to stabilize CC, and inhibited CC formation by CPT, indicating that Lep C is also a catalytic inhibitor of topo I in vivo. Cell cycle analysis of Lep C‐treated cells showed that Lep C appeared to inhibit the progress of cells from G1 to S phase. Lep C induced apoptosis in RPMI8402 cells, as revealed by the accumulation of cells in sub‐G1 phase, activation of caspase‐3 and the nucleosomal degradation of chromosomal DNA. Furthermore, Leps F and C inhibited the Akt pathway, as demonstrated by dose‐dependent and time‐dependent dephosphorylation of Akt (Ser473). Our study shows that Leps are a group of anticancer chemotherapeutic agents with single or dual catalytic inhibitory activities against topos I and II. (Cancer Sci 2005; 96: 816–824)

Isolation of a novel mouse gene, mSVS-1/SUSD2, reversing tumorigenic phenotypes of cancer cells in vitro

We report isolation of a novel tumor‐reversing gene, tentatively named SVS‐1, encoding a protein of 820 amino acids with localization on the plasma membrane as a type I transmembrane protein. The gene was found among those downregulated in the activated oncogene‐v‐K‐ras‐transformed NIH3T3 cells, Ki3T3, with tumorigenic phenotype. SVS‐1 protein harbors several functional domains inherent to adhesion molecules. Histochemical staining of mouse tissues using antibody raised against the protein showed the expression of the protein in restricted regions and cells, for example, strongly positive in apical membranes of epithelial cells in renal tubules and bronchial tubes. The protein inducibly expressed in human fibrosarcoma HT1080 cells and cervical carcinoma HeLa cells was found to be localized primarily on the plasma membrane, as stained with antibodies against FLAG tag in the N‐terminus and against the C‐terminal peptide of the protein. Expression of the protein in cells induced a variety of biological effects on cancer cells: detachment from the substratum and aggregation of cells and growth inhibition in HeLa cells, but no inhibition in non‐tumorigenic mouse NIH3T3 cells. Inhibition of clonogenicity, anchorage‐independent growth, migration and invasion through Matrigel was also observed. Taken together these results suggest that the SVS‐1 gene is a possible tumor‐reversing gene. (Cancer Sci 2007; 98: 900–908)

Down-regulation of Bcl-2-interacting protein BAG-1 confers resistance to anti-cancer drugs.

BAG-1 was originally identified as a binding partner of anti-apoptotic factor Bcl-2 [Takayama et al., Cell 80 (1995) 279-284]. Exogenous expression of BAG-1 was reported to confer cells resistance to several stresses [Chen et al., Oncogene 21 (2002) 7050]. We have obtained human cervical cancer HeLa cells with down-regulated BAG-1 levels by using a highly specific and efficient RNA interference approach. Surprisingly, cells with down-regulated BAG-1 exhibited significantly lower sensitivity against several anti-cancer drugs than parental cells expressing normal levels of the protein. Furthermore, growth rate of the cells was reduced when BAG-1 was down-regulated. Activity of ERK pathway appeared to be decreased in BAG-1 down-regulated cells, as shown by the reduced phosphorylation of ERK1/2 proteins. Taken together resistance against anti-cancer drugs acquired by BAG-1 down-regulated cells may well be accounted for by the retardation of cell cycle progression, implicating the importance of BAG-1 in cell growth regulation.

von Willebrand factor type D domain mutant of SVS‐1/SUSD2, vWDm, induces apoptosis in HeLa cells

SVS‐1/SUSD2 is a novel gene, which inhibits growth and reverses tumorigenic phenotypes of cancer cells in vitro. Here we report identification of a mutant of SVS‐1, designated SVS‐1‐vWDm, in which conserved amino acids GLLG at positions 591–594 in von Willebrand factor type D (vWD) domain are replaced by AAAA. As observed by laser confocal microscope, intracellular localization of the mutant protein has changed such that both the N‐terminus and the C‐terminus of SVS‐1‐vWDm were localized in the inner surface of the plasma membrane, whereas the N‐terminus of SVS‐1 was localized in the outer surface of the plasma membrane. Additionally, SVS‐1‐vWDm was processed much less efficiently and in a slightly different manner. In in vitro studies, adenovirus‐mediated transduction of the SVS‐1‐vWDmgene induced growth suppression of HeLa cells in a dose‐dependent manner, as the wild‐type gene and inhibition of anchorage‐independent growth. Of great interest is the finding that the mutant protein, vWDm, but not the wild‐type one induced apoptosis, as observed by nuclear as well as DNA fragmentation. Activation of caspase‐3 and ‐9, but not caspase‐8 or ‐12, was also demonstrated in vWDm‐expressing cells. An inhibition of Akt phosphorylation, a major survival signaling component, also occurred in vWDm‐expressing HeLa cells. Together these data suggest that vWDm induces apoptosis by inactivation of survival signaling component Akt and activation of caspase cascade (mitochondrial pathway) in HeLa cells. We propose SVS‐1‐vWDmas an alternative gene for use in developing new therapeutic strategies for the treatment of cancer. (Cancer Sci 2007; 98: 909–915)

532 nm low-power laser irradiation recovers γ-secretase inhibitor-mediated cell growth suppression and promotes cell proliferation via Akt signaling.

Background and Objective The γ-secretase inhibitor (GSI) has been shown to inhibit expression of amyloid beta (Aβ), but GSI also has a side effect of reducing cell survival. Since low-power laser irradiation (LLI) has been known to promote cell survival, we examined whether 532 nm LLI can rescue the GSI side effect or not. Study Design/Materials and Methods The human-derived glioblastoma cells (A-172) were cultured in 35 mm culture dishes or 96-well plate. The center of dish or selected wells was irradiated with 532 nm laser (Nd:YVO4, CW, 60 mW) for 20, 40 and 60 min, respectively. The irradiated cells were photographed at immediately after, 24 and 48 h later and counted. GSI was supplemented in medium 3 h before LLI. The MTT assay was also used to estimate viable cells at 48 h after irradiation. The expression of phosphorylated Akt (p-Akt) or phosphorylated PTEN (p-PTEN) was examined by immunofluorescent staining and measured by fluorescence intensity using the software (BZ-9000, KEYENCE, Japan). Results GSI application depressed cell proliferation as well as cell survival compared to control. GSI down-regulated Aβ but up-regulated p-PTEN and suppressed p-Akt. Application of 532 nm LLI in the presence of GSI significantly recovered the GSI-mediated effects, i.e., LLI could decrease elevated p-PTEN, while increased p-Akt expression with keeping Aβ suppression. The LLI effects had a dose-dependency. Conclusion We confirmed that GSI potently suppressed intracellular Aβ and decreased cell survival. We conclude that a combination of GSI application and 532 nm LLI can increase cell proliferation via Akt activation while keeping PTEN and Aβ suppressed.

A novel monoclonal antibody induces the differentiation of monocyte leukemic cells

A cell surface antigen (possibly a receptor) on human myeloid cell lines, that may play a crucial role in the differentiation of promonocytic cells, was detected by a murine monoclonal antibody (MAb 710F). When the myeloid cell lines, HL-60, THP-1 and U937, were cultured with the MAb 710F following pretreatment with phorbol 12-myristate 13-acetate (PMA), they displayed both cellular spreading and a strong adherence to the culture dish substratum. On transforming from their original round shape, the induced cells displayed the well-developed microvilli, spindles, or raffles that are characteristic of macrophages or dendrocytes. Similar morphological changes were not induced by treatment with either PMA or MAb 710F alone. By contrast, the lymphoid cell lines did not respond to these reagents. These results suggested that a signal for cellular adhesion and cytoskeletal reconstitution was triggered by the binding of MAb 710F to an antigen on the PMA-primed cells. Thus, the antigen 710F, which is preferentially expressed on peripheral blood monocytes, may represent a cell surface receptor closely associated with differentiation. The antigen/receptor 710F was shown to be a N-glycosylated protein with Mr. of 35-70k. This MAb may prove useful as a tool for both studies on the differentiation of myeloid lineage cells as well as on monocyte/macrophage adhesion function.

532 nm Low-Power Laser Irradiation Facilitates the Migration of GABAergic Neural Stem/Progenitor Cells in Mouse Neocortex.

Background and Objective Accumulating evidence has shown that low-power laser irradiation (LLI) affects cell proliferation and survival, but little is known about LLI effects on neural stem/progenitor cells (NSPCs). Here we investigate whether transcranial 532 nm LLI affects NSPCs in adult murine neocortex and in neurospheres from embryonic mice. Study Design/Materials and Methods We applied 532 nm LLI (Nd:YVO4, CW, 60 mW) on neocortical surface via cranium in adult mice and on cultured cells from embryonic mouse brains in vitro to investigate the proliferation and migration of NSPCs and Akt expression using immunohistochemical assays and Western blotting techniques. Results In vivo experiments demonstrated that 532 nm LLI significantly facilitated the migration of GABAergic NSPCs that were induced to proliferate in layer 1 by mild ischemia. In vitro experiments using GABAergic NSPCs derived from embryonic day 14 ganglionic eminence demonstrated that 532 nm LLI for 60 min promoted the migration of GAD67-immunopositive NSPCs with a significant increase of Akt expression. Meanwhile, the LLI induced proliferation, but not migration, of NSPCs that give rise to excitatory neurons. Conclusion It is concluded that 532 nm LLI promoted the migration of GABAergic NSPCs into deeper layers of the neocortex in vivo by elevating Akt expression.

Effect of hydrostatic pressure on the viability of non-adherent HL-60 cells

We investigated the effect of hydrostatic pressure on the viability of non-adherent HL-60 cell line derived from leukemic cells over a high pressure range. The HL-60 cells are resistant to pressures of up to 100 MPa under pressurization for 20 min at 25°C. However, cell viability decreased markedly between 100 and 200 MPa, and almost all cells died above 200 MPa. In the case of pressures up to 25 MPa at 25°C for four days, the viability of HL-60 cells was inhibited by increasing the pressure above 20 MPa. Although high viability was observed between 1.6 and 2.0 MPa for adherent astrocytes, viability did not change over pressures up to 2.0 MPa in the case of non-adherent HL-60 cells. It is thought that the response of cells to pressure varies among cell types.

Interactive effect of pressure and temperature on the preservation of rat primary-cultured astrocytes and human glioblastoma cell line A172

ABSTRACT Environmental factors such as temperature and pressure are important determinants of cell survival. Although the effect of temperature on cell preservation has been previously reported, the effects of pressure, an equally important thermodynamic parameter, have not been sufficiently investigated. In this study, we investigated the effect of temperature and pressure on cellular viability, morphology, adhesiveness, cell death, cell cycle and glucose metabolism in rat primary-cultured astrocytes and A172 human glioblastoma cell line subjected to 4-day preservation. It was revealed that under favorable preservation conditions (temperature: 15°C–20°C, pressure: 0.1–30 MPa) (1) cell morphology and adhesiveness of preserved cells were maintained similar to freshly isolated cells; (2) cell cycle was arrested; (3) glucose uptake and intra/extra-cellular pH decrease were suppressed. These results suggest that lowering temperature to 15°C–20°C or increasing pressure up to 30 MPa at temperatures of 20°C–25°C can reduce cellular metabolism and maintain cell-membrane fluidity, thus resulting in higher viability.

Glioblastoma growth inhibition by low-power laser irradiation

It has been reported that low-power laser irradiation (LLI) can modulate various biological processes including cell proliferation. Some reports suggest LLI interferes with cell cycle and inhibits cell proliferation, while others suggest LLI has a stimulatory effect. Mechanism underlying the effect of LLI remains unclear. We have reported that 808 nm (60 mW) LLI has a potential of suppressive effect on the cell proliferation of human-derived glioblastoma A-172. To reveal a wavelength dependency of such an effect, the present studies using 2 other kinds of diode laser generators (405 nm with 27 mW and 532 nm with 60 mW) were designed. The A-172 cells were cultivated in culture dishes or a 96-well plate. No irradiation was applied in control-group, whereas in experimental group, the center of dish or the selected well was irradiated for 20, 40 and 60 min, respectively. Cells were cultivated in CO2 incubator for 1 or 2 days. The dishes or wells were photographed at pre-, just post-irradiation, 24 and 48 hrs after irradiation with a digital camera. Cell countings were performed on the PC screen and the ratio of cell proliferation was measured. MTT colorimetric analysis was also applied at 48 hrs after irradiation and viable cells and cell proliferation were estimated. These analyses showed that 405 nm LLI provided a significant suppressive effect on the proliferation of A-172 cells (p < 0.05 or p < 0.01), and the effect of LLI had a dose-dependency, whereas 532 nm LLI showed a significant stimulatory effect on them (p < 0.05 or p < 0.01), and also had a dose-dependency. Taking the results of 808 nm (60 mW) application into consideration, it is concluded that LLI effects on A-172 cell proliferation have a wavelength dependency.