title: Nox4 inhibition enhances the cytotoxicity of cisplatin in human renal cancer cells.
abstract: OBJECTIVE
  Kidney cancer is notoriously chemo-resistant and abundantly expresses the Nox4 NADPH oxidase. To determine if Nox4 superoxide generation contributes to drug resistance, we assayed in vitro drug cytotoxicity following Nox4 shRNA silencing in human renal cancer cells.
  
  
  METHODS
  Human conventional kidney cell lines, 786-0 and RCC4 expressing Nox4-specific shRNA or a non-targeting, control shRNA were grown in serial dilutions of cisplatin, vincristine, doxorubicin, or etoposide. Cell viability curves were generated and the concentration required to kill 50% of the cells (IC50) calculated for each drug. Apopotosis was estimated by TUNEL assay. Quantitative RT-PCR and Western blots were used to confirm Nox4 silencing and evaluate expression of apoptotic pathway proteins.
  
  
  RESULTS
  Silencing significantly lowered the IC50 for cisplatin, vincristine and etoposide, and promoted drug-induced apoptosis by TUNEL assay. Improved sensitivity to cisplatin was reproduced by Nox inhibiton with diphenyliodonium, whereas induction of intracellular superoxide by dithiothreitol superoxide enhanced chemo-resistance. RT-PCR and Western blot revealed decreased expression of anti-apoptotic Bcl-XL and Bcl-2 and increased expression of pro-apoptotic Bax following Nox4 knockdown.
  
  
  CONCLUSIONS
  Nox4 contributes to RCC chemo-resistance through modulation of pro-apoptotic and anti-apoptotic signaling, suggesting that Nox4 inhibition might enhance the efficacy of conventional cytotoxic drugs against RCC.
authors.affiliation: Department of Urology, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: Guimin
authors.initials: G
authors.lastname: Chang
authors.forename: Li
authors.initials: L
authors.lastname: Chen
authors.forename: Hui-Min
authors.initials: HM
authors.lastname: Lin
authors.forename: Yan
authors.initials: Y
authors.lastname: Lin
authors.forename: Jodi K
authors.initials: JK
authors.lastname: Maranchie
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002945:Cisplatin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics; D020868:Gene Silencing; D006801:Humans; D007680:Kidney Neoplasms / Q000235:genetics / Q000378:metabolism; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D017382:Reactive Oxygen Species; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D002945:Cisplatin