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.

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 (Aß), 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 Aß 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 Aß suppression. The LLI effects had a dose-dependency. CONCLUSION: We confirmed that GSI potently suppressed intracellular Aß 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 Aß suppressed.

Low-power 808-nm laser irradiation inhibits cell proliferation of a human-derived glioblastoma cell line in vitro.

It has been reported that low-power laser irradiation (LLI) can modulate various biological processes including cell proliferation. Some reports suggest that LLI interferes with the cell cycle and inhibits cell proliferation, while others suggest that LLI has a stimulatory effect. Mechanisms underlying the effects of LLI remain unclear. Since the effects of LLI on cancer cells are not well understood, with the aim of developing an LLI therapy for malignant glioblastoma, we investigated the effects of LLI on the cell proliferation of the human-derived glioblastoma cell line A-172. Glioblastoma cell cultures were irradiated with a diode laser at a wavelength of 808 nm and the effects on cell viability and proliferation were examined. Cell counting at 24 and 48 h after irradiation showed that LLI (at 18, 36 and 54 J/cm(2)) suppressed proliferation of A-172 cells in a fluence-dependent manner (irradiation for 20, 40 and 60 min). A reduction in the number of viable cells was also demonstrated by a fluorescent marker for viable cells, calcein acetoxymethylester (calcein-AM). The reduction in cell viability was not associated with morphological changes in the cells or with necrotic cell death as demonstrated by propidium iodide staining. LLI also had little effect on cell proliferation as shown by 5-bromo-2'-deoxyuridine staining. We discuss possible mechanisms underlying the suppressive effect of 808-nm LLI on the viability of human-derived glioblastoma A-172 cells.

Immunocytochemical studies on the effect of 405-nm low-power laser irradiation on human-derived A-172 glioblastoma cells.

The application of low-power laser irradiation (LLI) affects the cell cycle and cell proliferation in various kinds of cells. LLI at a wavelength of 808 nm and a power of 30 mW has been found to significantly decrease the proliferation rate of cells of the human-derived glioblastoma cell line A-172. To determine if this effect of LLI is specific to 808-nm LLI, the present study was designed to reveal the effects of 405-nm LLI under the same experimental conditions. A-172 glioblastoma cells were cultured in 96-well plates according to the conventional protocol. Two different schedules of 405-nm LLI (27 mW) were tested: longer periods of 20, 40 and 60 min and shorter periods of 1, 2, 3, 5, 10 and 15 min. Cells on a digital image displayed on a computer monitor were counted and the proliferation ratio was determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) staining. Annexin-V-FLUOS staining and acridine-orange/ethidium-bromide staining were in an immunocytochemical assay to determine if cells were viable or dead (due to apoptosis or necrosis). Cell counting and MTT staining showed that longer 405-nm LLI significantly suppressed the proliferation of A-172 cells at 48 h after LLI (p < 0.05 or p < 0.01) and that the effect of LLI tended to be dose-dependent with morphological changes including cell death. At 90 min after LLI, shorter 405-nm LLI caused necrotic as well as apoptotic cell death, and these effects depended on irradiation time, power and energy density. Detailed analysis revealed that this lethal effect occurred after LLI and was not sustainable. It is concluded that 405-nm LLI has a lethal effect on human-derived glioblastoma A-172 cells, that is different from the suppressive effect without morphological changes induced by 808-nm LLI.

von Willebrand factor type D domain mutant of SVS-1/SUSD2, vWD(m), 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-vWD(m), 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-vWD(m) 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-vWD(m) was processed much less efficiently and in a slightly different manner. In in vitro studies, adenovirus-mediated transduction of the SVS-1-vWD(m)gene 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, vWD(m), 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 vWD(m)-expressing cells. An inhibition of Akt phosphorylation, a major survival signaling component, also occurred in vWD(m)-expressing HeLa cells. Together these data suggest that vWD(m) induces apoptosis by inactivation of survival signaling component Akt and activation of caspase cascade (mitochondrial pathway) in HeLa cells. We propose SVS-1-vWD(m)as an alternative gene for use in developing new therapeutic strategies for the treatment of cancer.

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.

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 G(1) to S phase. Lep C induced apoptosis in RPMI8402 cells, as revealed by the accumulation of cells in sub-G(1) 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.

Daily variation in cellular content of UV-absorbing compounds mycosporine-like amino acids in the marine dinoflagellate Scrippsiella sweeneyae.

Sudden exposure experiments to high PAR (photosynthetically available radiation) or high PAR+UVR (ultraviolet radiation) were conducted for the marine dinoflagellate Scrippsiella sweeneyae acclimated to either low PAR or high PAR to determine the induction of cellular mycosporine-like amino acid (MAA) in relation to photosynthesis status. When the exposure to high PAR (30.8 Wm(-2)) was provided at different time in the light period for S. sweeneyae acclimated to low PAR (7.7 Wm(-2)) which suppressed photosynthesis, S. sweeneyae could enhance the induction of MAA but it only occurred in the first half of the light period. When UVR exposure was provided for the culture acclimated to high PAR which enhanced photosynthesis, cellular MAA content did not increase during the entire light period, but displayed daily variation similar to the control for two and half days. Daily variation of cellular MAA content did not synchronized with that of cell volume and cellular chlorophyll a content. The individual MAAs also revealed similar daily variations with different phase, which increased for a few hours in the beginning of the light period, except for cellular palythine content. Thus the total cellular MAA content revealed daily variation with changing the relative composition within a few hours. As one of the biological protective strategies against harmful UVR in sunlight, the daily vertical migration in the bloom forming dinoflagellates might be accompanied by the daily variation of cellular MAA content for a photosynthesis at daytime.

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.