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Helium–Neon Laser Irradiation Stimulates Cell Proliferation through Photostimulatory Effects in Mitochondria  Wan-Ping Hu, Jeh-Jeng Wang, Chia-Li Yu,

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Presentation on theme: "Helium–Neon Laser Irradiation Stimulates Cell Proliferation through Photostimulatory Effects in Mitochondria  Wan-Ping Hu, Jeh-Jeng Wang, Chia-Li Yu,"— Presentation transcript:

1 Helium–Neon Laser Irradiation Stimulates Cell Proliferation through Photostimulatory Effects in Mitochondria  Wan-Ping Hu, Jeh-Jeng Wang, Chia-Li Yu, Cheng-Che E. Lan, Gow-Shing Chen, Hsin-Su Yu  Journal of Investigative Dermatology  Volume 127, Issue 8, Pages (August 2007) DOI: /sj.jid Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Effect of He-Ne laser irradiation on A2058 cell proliferation. Cells in 24-well plates were irradiated in triplicate with 0, 0.5, 1.0, and 2.0J/cm2 He-Ne lasers. The number of visible cells was counted by trypsinization and assisted with trypan blue dye exclusion. *P<0.05 as compared with the control. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Immunocytochemical staining of p53, bcl-2, and ki-67 proteins of A2058 cells in response to different dosages of He-Ne laser irradiation. Percentage of A2058 cells with or without He-Ne laser irradiation in this study labeling for p53, bcl-2, and ki-67 at different levels, respectively. *P<0.05 as compared with the control. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Effect of He-Ne laser irradiation on the cytochrome c oxidase activity. The measurement of cytochrome c oxidase activity was determined following the experimental protocol described in the Materials and Methods section. The data are expressed as relative levels of cytochrome c oxidase activity compared with that of untreated control. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Effect of He-Ne laser irradiation on the mitochondrial membrane potential (ΔΨmt). (a) A2058 cells were treated with different dosages of He-Ne laser irradiation, then stained with DiOC6 dye and analyzed immediately by flow cytometry. The number M1 indicates the percentage with increased ΔΨmt. (b) As a control, ΔΨmt was measured in the presence of CCCP, an uncoupling agent that reduces ΔΨmt. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Effect of He-Ne laser irradiation on the intracellular ATP levels of the A2058 cells. (a) A2058 cells were irradiated with 0, 0.5, 1.0, and 2.0J/cm2 of an He-Ne laser. Relative ATP levels were calculated as the percentage of the dose 0J/cm2 level. (b) The relative ATP content of A2058 cells was obtained after 1.0J/cm2 He-Ne laser irradiation with or without 30μM CCCP. *P<0.05, **P<0.01 as compared with the control. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Effect of He-Ne laser irradiation on the intracellular cAMP levels of the A2058 cells. (a) The change of intracellular cAMP levels was determined using the experimental protocol. Relative cAMP levels were calculated as the percentage of the dose 0J/cm2 level. (b) The relative cAMP content of A2058 cells was obtained after 0 and 1.0J/cm2 He-Ne laser irradiation with or without ATP (1 × 10−4M). *P<0.05, **P<0.01 as compared with the control. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

8 Figure 7 cAMP analog significantly enhanced the phosphorylation of JNK. Cells were untreated or incubated with 0.3mM 8-bromo-cAMP for 60minutes before 1.0J/cm2 He-Ne laser irradiation, and JNK phosphorylation was detected. **P<0.01 as compared with the control. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

9 Figure 8 Immunoblot analysis showed the effect of He-Ne laser irradiation on phosphorylation of JNK. (a) After exposure to different dosages of laser, cell lysates were collected and immunoblotted with specific antibodies as indicated. For the internal control, the same amounts of protein extract were also probed with antibody against actin. (b) The pattern of JNK phosphorylation by A2058 cells showed a dose-responsive effect produced by He-Ne laser irradiation. *P<0.05, **P<0.01 as compared with the control. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

10 Figure 9 Effect of He-Ne laser irradiation on AP-1 activity. (a) A2058 cells were co-transfected with pAP1-luc and pSV-βgal reporter plasmids as described in the Materials and Methods section. The transfected cells were exposed to different dosages of an He-Ne laser and the cell lysates were collected. Luciferase activity was determined and then normalized with β-galactosidase activity. (b) The transfected A2058 cells were with or without 90μM SP before 1.0J/cm2 He-Ne laser treatment. *P<0.05, **P<0.01 as compared with the control. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

11 Figure 10 Effect of JNK inhibitor on IL-8 and TGF-β1 release after He-Ne laser treatment. A2058 cells were treated or untreated with 90μM SP before 1.0J/cm2 He-Ne laser treatment. The concentrations of growth factors in the culture supernatants derived from A2058 cells after 24hours irradiated were measured. **P<0.01 as compared with the control. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

12 Figure 11 A proposed mechanism for He-Ne laser-induced cell proliferation in the A2058 melanoma cell. He-Ne laser irradiation stimulates A2058 cell proliferation by the mitochondrial pathway and subsequent increases in the transcriptional activities of AP-1, which could be involved in increased expression of IL-8 and TGF-β1 leading to cell proliferation. The locations where various inhibitors (italicized) act are also shown. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

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