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
Authors: Chen Qiao
Kai Zhao
Zhiyu Li
Ting Ni
Xiaotang Wang
Yuxin Zhou
Libin Wei
Qinglong Guo
Na Lu
Journal: Journal of hematology & oncology (2015)
Discipline: CLINICAL MEDICINE
Keywords: p53; Oroxylin A; glycolysis; MDM2; cancer cells
Affiliations: 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. ., Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing, 210009, The People's Republic of China. ., Department of Chemistry and Biochemistry, Florida International University, Miami, FL, 33199, USA. .

Abstract:  Introduction: 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. 

Retracted: No

PubPeer comments:
 Expression of deepest concern for this article: Figure 2A and 2D: duplication of western blot data. Figure 3A and 3B: duplication of western blot data. Figure 3D and 3E: duplication of western blot data, please compare the background noise.    My eyes are crying and stop here. , 
 #1 Correction published [30 December 2019] :  The original article [1] contains several errors:  1) During WB experiment, authors conducted multiple batches of samples together, and thus confused the actin for different batches of samples. When organising the results, authors misused the incorrect WB band for actin of MCF7 cells in Fig. 2a as that of HCT116 cells in Fig. 2d. Authors would like to correct the actin protein band in Fig. 2d and correct version is shown ahead.  2) During WB experiment, authors conducted the samples for HCT116 cells in Fig. 3a and the samples for HCT116 cells in Fig. 3b together in the same gel. When organising the results, authors made a mistake with the actin protein band and erroneously used the actin protein band repetitively. The actin for Fig. 3b is correct. Authors would like to correct the actin protein band in Fig. 3a, and correct version is shown ahead.  3) The resolution and quality for some WB bands in Fig. 3d and e are low, resulting in potential misunderstanding. Authors would like to replace the low quality WB bands with a higher quality version shown ahead.  4) To avoid confusion, authors would like to clarify that actin bands shown in Figs. 3b and 4a are from the same gel in a one experiment. Thus, the actin protein bands for them are same. Fig. 2 (hhttps: media.springernature.com lw685 springer-static image art%3A10.1186%2Fs13045-019-0792-8 MediaObjects 13045_2019_792_Fig1_HTML.png)  Oroxylin A downregulates the protein and mRNA expression of p53-related glycolytic pathway components. (a) MCF-7 and HCT116 cells were treated with oroxylin A (100 and 200 u03bcM) for 48 h. Western blot assays were performed for the p53-targeted gene products p53, TIGAR, PGM, and GLUT4. (d) MCF-7 and HCT116 were transfected with siRNA targeting wt-p53 or with a non-targeting control siRNA, then incubated with 200 u03bcM oroxylin A for 48 h. Western blot assays were performed for the p53-targeted gene products TIGAR, PGM, and GLUT4. All the Western Blot bands were quantified. Bars, SD;*p 0.05 or **p 0.01 versus non-treated control Fig. 3   Oroxylin A enhances p53 expression through post-transcriptional regulation. (a) Cells were treated with oroxylin A (100 and 200 u03bcM) for 48 h. Western blot assays were performed for MDM2. (b) Effect of oroxylin A on p53 expression after co-treatment with CHX. Cells were treated with vehicle or oroxylin A for 48 h, and 6 h before harvested, 10 u03bcg ml CHX was added to the medium. p53 protein expression was detected by Western blotting. (d) MDM2 was immunoprecipitated using p53 (Ab6) antibodies. Western blot assays were performed for MDM2, p53. (e) p53 (Ab6) was immunoprecipitated using anti-SIRT3 antibody. Western blot assays were performed for p53 and SIRT3. All the Western blot bands were quantified . ,