title: Downregulation of ROCK2 through nanocomplex sensitizes the cytotoxic effect of temozolomide in U251 glioma cells.
abstract: OBJECTIVE
  Rho-associated coiled-coil kinase 2 (ROCK2) is an attractive therapeutic target because it is overexpressed in many malignancies, including glioma. Therefore, we designed the current study to determine whether the downregulation of ROCK2 would sensitize the cytotoxic effect of temozolomide (TMZ) in U251 cells.
  
  
  METHODS
  Glycol-polyethyleneimine (PEG-PEI) was used to deliver siROCK2 to U251 cells, and the physical characteristics of the PEG-PEI/siROCK2 complex (referred to as the siROCK2 complex) were investigated. The transfection efficiency and cell uptake were determined by flow cytometry (FCM) and confocal laser microscopy (CLSM), respectively. U251 cells were then treated with 100 μM TMZ, siROCK2 complexes or their combination. The apoptosis rate and cell migration were measured by FCM and wound-healing assay, respectively. The levels of Bax, Bcl-2, cleaved caspase-3, MMP-2, and MMP-9 were detected to analyze the degrees of apoptosis and migration.
  
  
  RESULTS
  Our results revealed that the characteristics of the siROCK2 complexes depended closely on the N/P ratios. PEG-PEI served as a good vector for siROCK2 and exhibited low cytotoxicity toward U251 cells. The CLSM assay showed that the siROCK2 complexes were successfully uptaken and that both the protein and mRNA levels of ROCK2 were significantly suppressed. Furthermore, the combination treatment induced a higher apoptosis rate and markedly increased the gap distance of U251 cells in the wound-healing assay. Levels of the proapoptotic proteins Bax and cleaved caspase-3 were significantly increased, whereas levels of the antiapoptotic protein Bcl-2 and the migration-related proteins MMP-2 and MMP-9 were significantly reduced by the combination treatment compared with either treatment alone.
  
  
  CONCLUSIONS
  In conclusion, our results demonstrate that the combination of TMZ and siROCK2 effectively induces apoptosis and inhibits the migration of U251 cells. Therefore, the combination of TMZ and siROCK2 complex is a potential therapeutic approach for human glioma.
authors.affiliation: Department of Neurology, Sun Yat-sen Memorial Hospital,Sun Yat-sen University, Guangzhou, China.
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Wen
authors.affiliation: State Key Laboratory of Oncology in Southern China, Sun Yat-sen University Cancer Center, Guangzhou, China.
authors.forename: Amin
authors.initials: A
authors.lastname: Huang
authors.affiliation: Department of Neurology, Sun Yat-sen Memorial Hospital,Sun Yat-sen University, Guangzhou, China.
authors.forename: Zhonglin
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Department of Neurology, the Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China.
authors.forename: Yunyun
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Neurology, Sun Yat-sen Memorial Hospital,Sun Yat-sen University, Guangzhou, China.
authors.forename: Jingyang
authors.initials: J
authors.lastname: Hu
authors.affiliation: Department of Neurology, Sun Yat-sen Memorial Hospital,Sun Yat-sen University, Guangzhou, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.affiliation: Center of Biomedical Engineering, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China.
authors.forename: Xintao
authors.initials: X
authors.lastname: Shuai
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D058726:Nanoconjugates / Q000737:chemistry; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D011092:Polyethylene Glycols / Q000737:chemistry; D011094:Polyethyleneimine / Q000031:analogs & derivatives / Q000737:chemistry; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D000077204:Temozolomide; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism
chemical_list: D018906:Antineoplastic Agents, Alkylating; D058726:Nanoconjugates; D019253:Proto-Oncogene Proteins c-bcl-2; D034741:RNA, Small Interfering; C544016:poly(ethylene glycol)-co-poly(ethyleneimine); D011092:Polyethylene Glycols; D003606:Dacarbazine; D011094:Polyethyleneimine; C515852:ROCK2 protein, human; D054460:rho-Associated Kinases; D053148:Caspase 3; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D000077204:Temozolomide