title: 5-Aminolevulinic acid-mediated sonodynamic therapy induces anti-tumor effects in malignant melanoma via p53-miR-34a-Sirt1 axis.
abstract: BACKGROUND
  Malignant melanoma is a very refractory skin tumor due to its high metastasis, poor prognosis, and insensitivity to chemotherapy. Sonodynamic therapy has recently evolved as a potential method to treat cancers. In this study, 5-aminolevulinic acid-mediated sonodynamic therapy (ALA-SDT) was used to treat malignant melanoma in vivo.
  
  
  OBJECTIVE
  To investigate whether ALA-SDT induces anti-tumor effects in malignant melanoma and to see if miRNAs are involved in this process.
  
  
  METHODS
  Tumor transplantation experiments in BALB/c nude mice were used to assess anti-tumor effects after ALA-SDT treatment. Cell apoptosis was evaluated by TUNEL assays and cell proliferation was measured using immunohistochemisty with anti-PCNA antibody. Microarray analysis was performed to measure miRNAs expressions. Endogenous miR-34a and its upstream and downstream genes were assayed by real-time PCR. Western blottings were used to determine these protein expressions. Intracellular ROS levels were detected by measuring the fluorescence intensity of DCF.
  
  
  RESULTS
  Tumor transplantation experiments revealed that ALA-SDT could inhibit mouse melanoma cell proliferation and tumor growth. Compared with the control group, TUNEL assays revealed that apoptosis was increased and proliferation was inhibited in the SDT group. Real-time PCR analysis showed 14-fold increase of miR-34a expression in the SDT group compared to the control group. In addition, ALA-SDT significantly increased intracellular ROS levels in vitro, which were almost inhibited by the ROS scavenger NAC. Also, the mRNA, total protein, and acetylation levels of p53 were increased, whereas some downstream anti-apoptotic or pro-proliferative factors of miR-34a such as BCL2, CCND1, CDK6, and SIRT1 were decreased in the SDT group compared with the control, ALA alone, and ultrasound alone groups. When miR-34a was inhibited in vitro, the protein expressions of BCL2, CCND1, CDK6, and SIRT1 recovered. By targeting SIRT1, which inhibits p53 acetylation, miR-34a promoted the transcriptional activity of p53, and finally led to increased expression of miR-34a itself. Therefore, the p53, miR-34a, and SIRT1 constituted a positive feedback loop.
  
  
  CONCLUSIONS
  ALA-SDT showed synergistic anti-tumor effects in malignant melanoma by constituting a positive feedback loop of p53-miR-34a-Sirt1 axis.
authors.affiliation: Laboratory of Sono- and Photo-theranostic Technologies, Harbin Institute of Technology, Harbin 150080, China.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Hu
authors.affiliation: Department of Anatomy, Harbin Medical University, Harbin 150081, China.
authors.forename: Haixia
authors.initials: H
authors.lastname: Fan
authors.affiliation: Department of Biochemistry and Molecular Biology, Harbin Medical University, Harbin 150086, China.
authors.forename: Guixiang
authors.initials: G
authors.lastname: Lv
authors.affiliation: Laboratory of Sono- and Photo-theranostic Technologies, Harbin Institute of Technology, Harbin 150080, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhou
authors.affiliation: Laboratory of Sono- and Photo-theranostic Technologies, Harbin Institute of Technology, Harbin 150080, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Yang
authors.affiliation: Department of Anatomy, Harbin Medical University, Harbin 150081, China. Electronic address: jhzheng@ems.hrbmu.edu.cn.
authors.forename: Jinhua
authors.initials: J
authors.lastname: Zheng
authors.affiliation: Laboratory of Sono- and Photo-theranostic Technologies, Harbin Institute of Technology, Harbin 150080, China; Department of Mathematics and Materials Research Institute, Pennsylvania State University, University Park, PA 16802, USA. Electronic address: dzk@psu.edu.
authors.forename: Wenwu
authors.initials: W
authors.lastname: Cao
mesh_terms: D000622:Aminolevulinic Acid / Q000627:therapeutic use*; D000818:Animals; D045744:Cell Line, Tumor; D054459:Elasticity Imaging Techniques; D008546:Melanoma, Experimental / Q000188:drug therapy* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D035683:MicroRNAs / Q000378:metabolism*; D023421:Models, Animal; D010778:Photochemotherapy / Q000379:methods; D017319:Photosensitizing Agents / Q000627:therapeutic use*; D060888:Real-Time Polymerase Chain Reaction; D056564:Sirtuin 1 / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D014464:Ultrasonic Therapy
chemical_list: C551848:MIRN34a microRNA, mouse; D035683:MicroRNAs; D017319:Photosensitizing Agents; D016159:Tumor Suppressor Protein p53; D000622:Aminolevulinic Acid; C470545:Sirt1 protein, mouse; D056564:Sirtuin 1
keywords: 5-Aminolevulinic acid; Anti-tumor effects; Malignant melanoma; Positive feedback loop; Sonodynamic therapy; microRNA