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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.

Zhao K, Zhou Y, Qiao C, Ni T, Li Z, Wang X, Guo Q, Lu N, Wei L - J Hematol Oncol (2015)

Bottom Line: In vivo, effects of oroxylin A was investigated in nude mice xenograft tumor-inoculated MCF-7 or HCT116 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.

View Article: PubMed Central - PubMed

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.

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- 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.

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Oroxylin A inhibits the transcription of MDM2 via PTEN. (A) MCF-7 and HCT116 cells were transfected with an MDM2 promoter luciferase reporter plasmid (pGL3Basic-Mdm-P1-luc) and then treated with oroxylin A for 48 h. Luciferase activity was normalized to Renilla activity and expressed as luciferase/Renilla relative units. (B) H1299 cells were transfected with an MDM2 promoter luciferase reporter plasmid (pGL3Basic-Mdm-P1-luc) and then treated with oroxylin A for 48 h. Luciferase activity was measured. (C) Wt-PTEN plasmids were respectively co-transfected with MDM2 promoter luciferase reporter plasmids (pGL3Basic-Mdm-P1-luc, pGL3Basic-Mdm-T1-luc, or pGL3Basic-Mdm-P1-P2-luc) into PC3M cells. Luciferase activity was measured. (D) PTEN siRNA were respectively co-transfected with MDM2 promoter luciferase reporter plasmids (pGL3Basic-Mdm-P1-luc, pGL3Basic-Mdm-T1-luc, or pGL3Basic-Mdm-P1-P2-luc) into DU145 cells. Luciferase activity was measured. (E) Cells were treated with oroxylin A for 48 h. Western blot assays were performed for PTEN. (F) Nucleus and cytosolic fractions were isolated after treatment and subjected to Western blot analysis for PTEN. (G) Immunofluorescence experiment performed in MCF-7 and HCT116 cells upon oroxylin A treatment using antibodies specific to PTEN and DAPI. (H) PTEN siRNA were respectively co-transfected with MDM2 promoter luciferase reporter plasmids (pGL3Basic-Mdm-P1-luc, pGL3Basic-Mdm-T1-luc, or pGL3Basic-Mdm-P1-P2-luc) into DU145 cells. Cells were then treated with 200 μΜ oroxylin A for 48 h. Luciferase activity was measured. (I) MCF-7 and HCT116 cells were transfected with siRNA targeting PTEN or with a non-targeting control siRNA, then incubated with 200 μM oroxylin A for 48 h. The mRNA expression of MDM2 was detected by Quantitative RT-PCR. (J) Cells were transfected with siRNA targeting PTEN or with a non-targeting control siRNA and incubated with 200 μM oroxylin A for 48 h. Western blot assays were performed for p53, MDM2, and PTEN. All the Western Blot bands were quantified. Bars, SD; *p < 0.05 or **p < 0.01 versus non-treated control.
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Fig5: Oroxylin A inhibits the transcription of MDM2 via PTEN. (A) MCF-7 and HCT116 cells were transfected with an MDM2 promoter luciferase reporter plasmid (pGL3Basic-Mdm-P1-luc) and then treated with oroxylin A for 48 h. Luciferase activity was normalized to Renilla activity and expressed as luciferase/Renilla relative units. (B) H1299 cells were transfected with an MDM2 promoter luciferase reporter plasmid (pGL3Basic-Mdm-P1-luc) and then treated with oroxylin A for 48 h. Luciferase activity was measured. (C) Wt-PTEN plasmids were respectively co-transfected with MDM2 promoter luciferase reporter plasmids (pGL3Basic-Mdm-P1-luc, pGL3Basic-Mdm-T1-luc, or pGL3Basic-Mdm-P1-P2-luc) into PC3M cells. Luciferase activity was measured. (D) PTEN siRNA were respectively co-transfected with MDM2 promoter luciferase reporter plasmids (pGL3Basic-Mdm-P1-luc, pGL3Basic-Mdm-T1-luc, or pGL3Basic-Mdm-P1-P2-luc) into DU145 cells. Luciferase activity was measured. (E) Cells were treated with oroxylin A for 48 h. Western blot assays were performed for PTEN. (F) Nucleus and cytosolic fractions were isolated after treatment and subjected to Western blot analysis for PTEN. (G) Immunofluorescence experiment performed in MCF-7 and HCT116 cells upon oroxylin A treatment using antibodies specific to PTEN and DAPI. (H) PTEN siRNA were respectively co-transfected with MDM2 promoter luciferase reporter plasmids (pGL3Basic-Mdm-P1-luc, pGL3Basic-Mdm-T1-luc, or pGL3Basic-Mdm-P1-P2-luc) into DU145 cells. Cells were then treated with 200 μΜ oroxylin A for 48 h. Luciferase activity was measured. (I) MCF-7 and HCT116 cells were transfected with siRNA targeting PTEN or with a non-targeting control siRNA, then incubated with 200 μM oroxylin A for 48 h. The mRNA expression of MDM2 was detected by Quantitative RT-PCR. (J) Cells were transfected with siRNA targeting PTEN or with a non-targeting control siRNA and incubated with 200 μM oroxylin A for 48 h. Western blot assays were performed for p53, MDM2, and PTEN. All the Western Blot bands were quantified. Bars, SD; *p < 0.05 or **p < 0.01 versus non-treated control.

Mentions: Our previous results suggested that oroxylin A could inhibit p53 degradation by downregulating MDM2 expression instead of influencing p53 mRNA level, which was mediated by SIRT3. The result that oroxylin A increased p53 levels suggested that oroxylin A must act through negative feedback on MDM2 transcription through the P2 promoter in wt-p53 cancer cells. However, the expression of SIRT3 still influenced the level of MDM2 in p53- cells (Figure 4B). This inferred the possibility that oroxylin A may function via a p53-independent mechanism to regulate MDM2 transcription. For this purpose, a genomic DNA fragment containing the P1 promoter regions of the MDM2 gene only was ligated to a luciferase reporter gene. A luciferase assay showed that oroxylin A inhibited the transcription of MDM2 in wt-p53 MCF-7 and HCT116 cells (Figure 5A) as well as in p53- H1299 cells (Figure 5B).Figure 5


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.

Zhao K, Zhou Y, Qiao C, Ni T, Li Z, Wang X, Guo Q, Lu N, Wei L - J Hematol Oncol (2015)

Oroxylin A inhibits the transcription of MDM2 via PTEN. (A) MCF-7 and HCT116 cells were transfected with an MDM2 promoter luciferase reporter plasmid (pGL3Basic-Mdm-P1-luc) and then treated with oroxylin A for 48 h. Luciferase activity was normalized to Renilla activity and expressed as luciferase/Renilla relative units. (B) H1299 cells were transfected with an MDM2 promoter luciferase reporter plasmid (pGL3Basic-Mdm-P1-luc) and then treated with oroxylin A for 48 h. Luciferase activity was measured. (C) Wt-PTEN plasmids were respectively co-transfected with MDM2 promoter luciferase reporter plasmids (pGL3Basic-Mdm-P1-luc, pGL3Basic-Mdm-T1-luc, or pGL3Basic-Mdm-P1-P2-luc) into PC3M cells. Luciferase activity was measured. (D) PTEN siRNA were respectively co-transfected with MDM2 promoter luciferase reporter plasmids (pGL3Basic-Mdm-P1-luc, pGL3Basic-Mdm-T1-luc, or pGL3Basic-Mdm-P1-P2-luc) into DU145 cells. Luciferase activity was measured. (E) Cells were treated with oroxylin A for 48 h. Western blot assays were performed for PTEN. (F) Nucleus and cytosolic fractions were isolated after treatment and subjected to Western blot analysis for PTEN. (G) Immunofluorescence experiment performed in MCF-7 and HCT116 cells upon oroxylin A treatment using antibodies specific to PTEN and DAPI. (H) PTEN siRNA were respectively co-transfected with MDM2 promoter luciferase reporter plasmids (pGL3Basic-Mdm-P1-luc, pGL3Basic-Mdm-T1-luc, or pGL3Basic-Mdm-P1-P2-luc) into DU145 cells. Cells were then treated with 200 μΜ oroxylin A for 48 h. Luciferase activity was measured. (I) MCF-7 and HCT116 cells were transfected with siRNA targeting PTEN or with a non-targeting control siRNA, then incubated with 200 μM oroxylin A for 48 h. The mRNA expression of MDM2 was detected by Quantitative RT-PCR. (J) Cells were transfected with siRNA targeting PTEN or with a non-targeting control siRNA and incubated with 200 μM oroxylin A for 48 h. Western blot assays were performed for p53, MDM2, and PTEN. All the Western Blot bands were quantified. Bars, SD; *p < 0.05 or **p < 0.01 versus non-treated control.
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Related In: Results  -  Collection

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Fig5: Oroxylin A inhibits the transcription of MDM2 via PTEN. (A) MCF-7 and HCT116 cells were transfected with an MDM2 promoter luciferase reporter plasmid (pGL3Basic-Mdm-P1-luc) and then treated with oroxylin A for 48 h. Luciferase activity was normalized to Renilla activity and expressed as luciferase/Renilla relative units. (B) H1299 cells were transfected with an MDM2 promoter luciferase reporter plasmid (pGL3Basic-Mdm-P1-luc) and then treated with oroxylin A for 48 h. Luciferase activity was measured. (C) Wt-PTEN plasmids were respectively co-transfected with MDM2 promoter luciferase reporter plasmids (pGL3Basic-Mdm-P1-luc, pGL3Basic-Mdm-T1-luc, or pGL3Basic-Mdm-P1-P2-luc) into PC3M cells. Luciferase activity was measured. (D) PTEN siRNA were respectively co-transfected with MDM2 promoter luciferase reporter plasmids (pGL3Basic-Mdm-P1-luc, pGL3Basic-Mdm-T1-luc, or pGL3Basic-Mdm-P1-P2-luc) into DU145 cells. Luciferase activity was measured. (E) Cells were treated with oroxylin A for 48 h. Western blot assays were performed for PTEN. (F) Nucleus and cytosolic fractions were isolated after treatment and subjected to Western blot analysis for PTEN. (G) Immunofluorescence experiment performed in MCF-7 and HCT116 cells upon oroxylin A treatment using antibodies specific to PTEN and DAPI. (H) PTEN siRNA were respectively co-transfected with MDM2 promoter luciferase reporter plasmids (pGL3Basic-Mdm-P1-luc, pGL3Basic-Mdm-T1-luc, or pGL3Basic-Mdm-P1-P2-luc) into DU145 cells. Cells were then treated with 200 μΜ oroxylin A for 48 h. Luciferase activity was measured. (I) MCF-7 and HCT116 cells were transfected with siRNA targeting PTEN or with a non-targeting control siRNA, then incubated with 200 μM oroxylin A for 48 h. The mRNA expression of MDM2 was detected by Quantitative RT-PCR. (J) Cells were transfected with siRNA targeting PTEN or with a non-targeting control siRNA and incubated with 200 μM oroxylin A for 48 h. Western blot assays were performed for p53, MDM2, and PTEN. All the Western Blot bands were quantified. Bars, SD; *p < 0.05 or **p < 0.01 versus non-treated control.
Mentions: Our previous results suggested that oroxylin A could inhibit p53 degradation by downregulating MDM2 expression instead of influencing p53 mRNA level, which was mediated by SIRT3. The result that oroxylin A increased p53 levels suggested that oroxylin A must act through negative feedback on MDM2 transcription through the P2 promoter in wt-p53 cancer cells. However, the expression of SIRT3 still influenced the level of MDM2 in p53- cells (Figure 4B). This inferred the possibility that oroxylin A may function via a p53-independent mechanism to regulate MDM2 transcription. For this purpose, a genomic DNA fragment containing the P1 promoter regions of the MDM2 gene only was ligated to a luciferase reporter gene. A luciferase assay showed that oroxylin A inhibited the transcription of MDM2 in wt-p53 MCF-7 and HCT116 cells (Figure 5A) as well as in p53- H1299 cells (Figure 5B).Figure 5

Bottom Line: In vivo, effects of oroxylin A was investigated in nude mice xenograft tumor-inoculated MCF-7 or HCT116 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.

View Article: PubMed Central - PubMed

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.

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- 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.

Show MeSH
Related in: MedlinePlus