title: Oroxylin A promotes PTEN-mediated negative regulation of MDM2 transcription via SIRT3-mediated deacetylation to stabilize p53 and inhibit glycolysis in wt-p53 cancer cells.
abstract: BACKGROUND
  p53 plays important roles in regulating the metabolic reprogramming of cancer, such as aerobic glycolysis. Oroxylin A is a natural active flavonoid with strong anticancer effects both in vitro and in vivo.
  
  
  METHODS
  wt-p53 (MCF-7 and HCT116 cells) cancer cells and p53-null H1299 cancer cells were used. The glucose uptake and lactate production were analyzed using Lactic Acid production Detection kit and the Amplex Red Glucose Assay Kit. Then, the protein levels and RNA levels of p53, mouse double minute 2 (MDM2), and p53-targeted glycolytic enzymes were quantified using Western blotting and quantitative polymerase chain reaction (PCR), respectively. Immunoprecipitation were performed to assess the binding between p53, MDM2, and sirtuin-3 (SIRT3), and the deacetylation of phosphatase and tensin homolog (PTEN). Reporter assays were performed to assess the transcriptional activity of PTEN. In vivo, effects of oroxylin A was investigated in nude mice xenograft tumor-inoculated MCF-7 or HCT116 cells.
  
  
  RESULTS
  Here, we analyzed the underlying mechanisms that oroxylin A regulated p53 level and glycolytic metabolism in wt-p53 cancer cells, and found that oroxylin A inhibited glycolysis through upregulating p53 level. Oroxylin A did not directly affect the transcription of wt-p53, but suppressed the MDM2-mediated degradation of p53 via downregulating MDM2 transcription in wt-p53 cancer cells. In further studies, we found that oroxylin A induced a reduction in MDM2 transcription by promoting the lipid phosphatase activity of phosphatase and tensin homolog, which was upregulated via sirtuin3-mediated deacetylation. In vivo, oroxylin A inhibited the tumor growth of nude mice-inoculated MCF-7 or HCT116 cells. The expression of MDM2 protein in tumor tissue was downregulated by oroxylin A as well.
  
  
  CONCLUSIONS
  These results provide a p53-independent mechanism of MDM2 transcription and reveal the potential of oroxylin A on glycolytic regulation in both wt-p53 and mut-p53 cancer cells. The studies have important implications for the investigation on anticancer effects of oroxylin A, and provide the academic basis for the clinical trial of oroxylin A in cancer patients.
authors.affiliation: State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, The People's Republic of China. cpuzhaokai@163.com.
authors.forename: Kai
authors.initials: K
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, The People's Republic of China. zyxspain@hotmail.com.
authors.forename: Yuxin
authors.initials: Y
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, The People's Republic of China. qiaochen1987@163.com.
authors.forename: Chen
authors.initials: C
authors.lastname: Qiao
authors.affiliation: State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, The People's Republic of China. 1179720746@qq.com.
authors.forename: Ting
authors.initials: T
authors.lastname: Ni
authors.affiliation: Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, The People's Republic of China. zhiyuli@cpu.edu.cn.
authors.forename: Zhiyu
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Chemistry and Biochemistry, Florida International University, Miami, FL, 33199, USA. wangx@fiu.edu.
authors.forename: Xiaotang
authors.initials: X
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, The People's Republic of China. anticancer_drug@163.com.
authors.forename: Qinglong
authors.initials: Q
authors.lastname: Guo
authors.affiliation: State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, The People's Republic of China. luna555@163.com.
authors.forename: Na
authors.initials: N
authors.lastname: Lu
authors.affiliation: State Key Laboratory of Natural Medicines, Jiangsu Key Laboratory of Carcinogenesis and Intervention, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, The People's Republic of China. wlbiws_1986@aliyun.com.
authors.forename: Libin
authors.initials: L
authors.lastname: Wei
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D005419:Flavonoids / Q000494:pharmacology*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects* / Q000502:physiology; D006019:Glycolysis / Q000187:drug effects; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D008819:Mice, Nude; D018613:Microscopy, Confocal; D009369:Neoplasms / Q000235:genetics / Q000378:metabolism*; D051059:PTEN Phosphohydrolase / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D034741:RNA, Small Interfering; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D056566:Sirtuin 3 / Q000378:metabolism; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*; D023041:Xenograft Model Antitumor Assays
chemical_list: D000970:Antineoplastic Agents; D005419:Flavonoids; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; C080669:5,7-dihydroxy-6-methoxy-2-phenylchromen-4-one; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human; C466093:SIRT3 protein, human; D056566:Sirtuin 3