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

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.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).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.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

Immunofluorescent staining of Akt and PTEN.

<p>A: The LLI effects on immunofluorescence staining of p-PTEN and p-Akt in cultures cells (bottom). Corresponding phase contrast micrographs are shown on top of each fluorescent image. It should be noted that tumor cells normally proliferate in high proportions and p-Akt is often highly expressed in cancer cells of different natures, whereas our result of control group shows low level of p-Akt expression due to haze reduction. Cal.: 100 µm. B: Average fluorescence intensity for p-PTEN or p-Akt normalized to the control value. Asterisks: one-way ANOVA, * p<0.05, ** p<0.01.</p

Effects of LLI on the number of A-172 cells.

<p>A: Sample images of A-172 cells under light microscope. The number of cells increased 48 h post-LLI (right column) after 20, 40, 60 min LLI compared to pre-LLI (left column). Cal.: 100 µm. B: Proliferation ratio (the ratio of cell number at 24 or 48 hours following LLI and cell number before LLI) was normalized to control (no LLI) (n = 12 for each group). C: A summary of colorimetric analysis by MTT staining performed at 48 h after LLI (each group: n = 16). The optical density of each group was normalized to the value of control group (no LLI) at 48 h after initial condition. Asterisks: one-way ANOVA, * p<0.05, ** p<0.01.</p

Effects of LLI, DAPT and combined application of both on cell proliferation.

<p>Proliferation ratios for each group at 24 h and 48 h after LLI for control (white bars), 60 min LLI (light grey bars), DAPT (grey bars) or the combination of LLI and DAPT (dark grey bars) are shown. Asterisks: one-way ANOVA, * p<0.05, ** p<0.01.</p

Effects of LLI on the fluorescence intensities of p-Akt, p-PTEN and Aβ.

<p>A, B: Schematic diagrams of Notch and APP signaling pathways. GS can cleave APP and Notch, making Aβ and ICN, respectively (A). GSI inhibits Aβ expression but also inhibits ICN expression (B), a side-effect against cell survival through PTEN activation (dashed box in B). C: LLI effects on immunofluorescence staining of p-Akt, p-PTEN and Aβ in cells pretreated with GSI. Cal.: 100 µm. D: Average fluorescence intensity for p-PTEN (green bars), p-Akt (pink bars) and Aβ (blue bars) was normalized to control. Asterisks: one-way ANOVA, ** p<0.01.</p

Effects of LLI on ATP level in cell lysates.

<p>The ATP/ADP ratio measured using a luminescence-based assay for control and for 20, 40 and 60 min LLI. The luminescent densities for ATP and ADP were measured 48 h after LLI treatment. The ATP/ADP ratio was calculated and averaged (n = 5). Asterisks: one-way ANOVA, * p<0.05.</p

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

<div><p>Background and Objective</p><p>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.</p><p>Study Design/Materials and Methods</p><p>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.</p><p>Results</p><p>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.</p><p>Conclusion</p><p>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.</p></div