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Drug Design, Development and Therapy Volume 9, 2015 - Issue
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Original Research

Plumbagin elicits differential proteomic responses mainly involving cell cycle, apoptosis, autophagy, and epithelial-to-mesenchymal transition pathways in human prostate cancer PC-3 and DU145 cells

Jia-Xuan Qiu1 School of Public Health, Wuhan University, Wuhan, Hubei, People’s Republic of China;2 Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China
,
Zhi-Wei Zhou3 Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA;4 Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, Guizhou, People’s Republic of China
,
Zhi-Xu He4 Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, Guizhou, People’s Republic of China
,
Ruan Jin Zhao5 Center for Traditional Chinese Medicine, Sarasota, FL, USA
,
Xueji Zhang6 Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of China
,
Lun Yang7 Bio-X Institutes, Key Laboratory for the Genetics of Development and Neuropsychiatric Disorders (Ministry of Education), Shanghai Jiao Tong University, Shanghai, People’s Republic of China
,
Shu-Feng Zhou3 Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA;4 Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, Guizhou, People’s Republic of ChinaCorrespondence[email protected]
&
Zong-Fu Mao1 School of Public Health, Wuhan University, Wuhan, Hubei, People’s Republic of ChinaCorrespondence[email protected]
 show all
Pages 349-417 | Published online: 07 Jan 2015

Figures & data

Figure 1 Chemical structure of PLB (5-hydroxy-2-methyl-1,4-naphthoquinone) and effect of PLB on cell viability in PC-3 and DU145 cells.

Notes: PC-3 and DU145 cells were treated with PLB at 0.1 to 20 μM for 24 or 48 hours. (A) Chemical structure of PLB, and (B) cell viability of PC-3 and DU145 cells. Data are the mean ± SD of three independent experiments.
Abbreviation: IC50, half maximal inhibitory concentration; PLB, plumbagin; SD, standard deviation.
Figure 1 Chemical structure of PLB (5-hydroxy-2-methyl-1,4-naphthoquinone) and effect of PLB on cell viability in PC-3 and DU145 cells.

Table 1 Predicted protein targets of PLB

Table 2 Molecular interactions of PLB with selected potential target proteins

Figure 2 Molecular interactions between PLB and selected predicted targets.

Notes: Protein structure identifications from PDB. ABL1 (ID: 1OPL); ACPP (ID: 1CVI); ADH7 (ID: 1D1T); and AKR1C1 (ID: 1IHI).
Abbreviations: ABL1, c-Abl oncogene 1; ACPP, prostate acid phosphatase; ADH7, alcohol dehydrogenase 5; AKR1C1, aldo–keto reductase family 1, member C1; PDB, Protein Data Bank; PLB, plumbagin.
Figure 2 Molecular interactions between PLB and selected predicted targets.

Figure 3 Molecular interactions between PLB and selected predicted targets.

Notes: Protein structure identifications from PDB. AKR1C3 (ID: 1YRO); ALDH1L1 (ID: 1S3I); ASS1 (ID: 2NZ2); and AURKA (ID: 1MUO).
Abbreviations: AKR1C3, aldo–keto reductase family 1, member C3; ALDH1L1, aldehyde dehydrogenase 1 family, member L1; ASS1, argininosuccinate synthase 1; AURKA, aurora kinase A; PDB, Protein Data Bank; PLB, plumbagin.
Figure 3 Molecular interactions between PLB and selected predicted targets.

Figure 4 Molecular interactions between PLB and selected predicted targets.

Notes: Protein structure identifications from PDB. BCAT2 (ID: 1KTA); CA4 (ID: 1G54); and CDKN2A (ID: 1OIQ).
Abbreviations: BCAT2, mitochondrial branched-chain amino-acid transaminase 2; CA4, carbonic anhydrase IV; CDKN2A, cyclin-dependent kinase inhibitor 2A; PDB, Protein Data Bank; PLB, plumbagin.
Figure 4 Molecular interactions between PLB and selected predicted targets.

Figure 5 Molecular interactions between PLB and selected predicted targets.

Notes: Protein structure identifications from PDB. CLK1 (ID: 1Z57); CRABP2 (ID: 1CBS); and ESR1/NR3A1 (ID:1GWQ).
Abbreviations: CLK1, CDC-like kinase 1; CRABP2, cellular retinoic acid binding protein 2; ESR1/NR3A1, estrogen receptor-α; PDB, Protein Data Bank; PLB, plumbagin.
Figure 5 Molecular interactions between PLB and selected predicted targets.

Table 3 The top enriched clusters (Enrich score >3) by the DAVID database for the target list of PLB derived from molecular docking calculations

Table 4 The top enriched KEGG pathways (FDR <0.1) by the DAVID database for the target list of PLB derived from molecular docking calculations

Table 5 Potential molecular targets, signaling pathways, and cellular functions regulated by PLB in PC-3 cells

Table 6 Potential molecular targets, signaling pathways, and cellular functions regulated by PLB in DU145 cells

Figure 6 Proteomic analysis revealed molecular interactome regulated by PLB in PC-3 cells.

Notes: PC-3 cells were treated with 5 μM PLB for 24 hours and the protein samples were subject to quantitative proteomic analysis. There were 1,225 molecules and 341 related pathways regulated by PLB in PC-3 cells. Red indicates an upregulation; green indicates a downregulation; brown indicates a predicted activation; and blue indicates a predicted inhibition. The intensity of green and red molecule colors indicates the degree of down- or upregulation, respectively. Solid arrows indicate direct interaction and dashed arrows indicate indirect interaction.
Abbreviation: PLB, plumbagin.
Figure 6 Proteomic analysis revealed molecular interactome regulated by PLB in PC-3 cells.

Figure 7 Proteomic analysis revealed molecular interactome regulated by PLB in DU145 cells.

Notes: DU145 cells were treated with 5 μM PLB for 24 hours and the protein samples were subject to quantitative proteomic analysis. There were 267 molecules and 107 related pathways regulated by PLB in DU145 cells. Red indicates an upregulation; green indicates a downregulation; brown indicates a predicted activation; and blue indicates a predicted inhibition. The intensity of green and red molecule colors indicates the degree of down- or upregulation, respectively. Solid arrows indicate direct interaction and dashed arrows indicate indirect interaction.
Abbreviation: PLB, plumbagin.
Figure 7 Proteomic analysis revealed molecular interactome regulated by PLB in DU145 cells.

Figure 8 Proteomic analysis revealed a network of signaling pathways regulated by PLB in PC-3 cells.

Notes: A network of signaling pathways was analyzed by IPA according to the 1,225 molecules and 341 related pathways which were regulated by PLB in PC-3 cells.
Abbreviations: IPA, Ingenuity Pathway Analysis; PLB, plumbagin; TCA, tricarboxylic acid cycle.
Figure 8 Proteomic analysis revealed a network of signaling pathways regulated by PLB in PC-3 cells.

Figure 9 Proteomic analysis revealed networks of signaling pathways regulated by PLB in DU145 cells.

Notes: Networks of signaling pathways were analyzed by IPA according to 267 molecules and 107 related pathways which were regulated by PLB in DU145 cells.
Abbreviations: cAMP, cyclic adenosine monophosphate; IPA, Ingenuity Pathway Analysis; PLB, plumbagin.
Figure 9 Proteomic analysis revealed networks of signaling pathways regulated by PLB in DU145 cells.

Figure 10 PLB regulates cell cycle at G2/M checkpoint in PC-3 cells.

Notes: PC-3 cells were treated with 5 μM PLB for 24 hours and the protein samples were subject to quantitative proteomic analysis. Red indicates an upregulation; green indicates a downregulation; brown indicates a predicted activation. The intensity of green and red molecule colors indicates the degree of down- or upregulation, respectively. Solid arrows indicates direct interaction.
Abbreviations: PLB, plumbagin; UV, ultraviolet.
Figure 10 PLB regulates cell cycle at G2/M checkpoint in PC-3 cells.

Figure 11 PLB regulates apoptosis signaling pathway in PC-3 cells.

Notes: PC-3 cells were treated with 5 μM PLB for 24 hours and the protein samples were subject to quantitative proteomic analysis. Red indicates an upregulation; green indicates a downregulation. The intensity of green and red molecule colors indicates the degree of down- or upregulation, respectively. Solid arrows indicate direct interaction and dashed arrows indicate indirect interaction.
Abbreviation: PLB, plumbagin.
Figure 11 PLB regulates apoptosis signaling pathway in PC-3 cells.

Figure 12 mTOR signaling pathway regulated by PLB in PC-3 cells.

Notes: PC-3 cells were treated with 5 μM PLB for 24 hours and the protein samples were subject to quantitative proteomic analysis. Red indicates an upregulation; green indicates a downregulation. The intensity of green and red molecule colors indicates the degree of down- or upregulation, respectively. Solid arrows indicate direct interaction and dashed arrows indicate indirect interaction.
Abbreviation: PLB, plumbagin.
Figure 12 mTOR signaling pathway regulated by PLB in PC-3 cells.

Figure 13 mTOR signaling pathway regulated by PLB in DU145 cells.

Notes: DU145 cells were treated with 5 μM PLB for 24 hours and the protein samples were subject to quantitative proteomic analysis. Red indicates an upregulation; green indicates a downregulation. The intensity of green and red molecule colors indicates the degree of down- or upregulation, respectively. Solid arrows indicate direct interaction and dashed arrows indicate indirect interaction.
Abbreviation: PLB, plumbagin.
Figure 13 mTOR signaling pathway regulated by PLB in DU145 cells.

Figure 14 PLB regulates epithelial adherent junction signaling pathway in PC-3 cells.

Notes: PC-3 cells were treated with 5 μM PLB for 24 hours and the protein samples were subject to quantitative proteomic analysis. Red indicates an upregulation; green indicates a downregulation; brown indicates a predicted activation. The intensity of green and red molecule colors indicates the degree of down- or upregulation, respectively. Solid arrows indicate direct interaction and dashed arrows indicate indirect interaction.
Abbreviation: PLB, plumbagin.
Figure 14 PLB regulates epithelial adherent junction signaling pathway in PC-3 cells.

Figure 15 PLB-regulated Nrf2-mediated oxidative stress response in PC-3 cells.

Notes: PC-3 cells were treated with 5 μM PLB for 24 hours and the protein samples were subject to quantitative proteomic analysis. Red indicates an upregulation; green indicates a downregulation; brown indicates a predicted activation. The intensity of green and red molecule colors indicates the degree of down- or upregulation, respectively. Solid arrows indicate direct interaction and dashed arrows indicate indirect interaction.
Abbreviations: PLB, plumbagin; UV, ultraviolet.
Figure 15 PLB-regulated Nrf2-mediated oxidative stress response in PC-3 cells.

Figure 16 PLB-regulated Nrf2-mediated oxidative stress response in DU145 cells.

Notes: DU145 cells were treated with 5 μM PLB for 24 hours and the protein samples were subject to quantitative proteomic analysis. Red indicates an upregulation; green indicates a downregulation. The intensity of green and red molecule colors indicates the degree of down- or upregulation, respectively. Solid arrows indicate direct interaction and dashed arrows indicate indirect interaction.
Abbreviations: PLB, plumbagin; UV, ultraviolet.
Figure 16 PLB-regulated Nrf2-mediated oxidative stress response in DU145 cells.

Table 7 Top five canonical pathways regulated by PLB in PC-3 cells

Table 8 Top five canonical pathways regulated by PLB in DU145 cells

Figure 17 PLB inhibits the proliferation of PC-3 and DU145 cells, and induces G2/M arrest in PC-3 cells and G1 arrest in DU145 cells.

Notes: Cell cycle distribution of PC-3 and DU145 cells with the treatment of PLB at 0.1 to 10 μM for 24 hours. (A) Representative flow cytometric plots of cell cycle distribution of PC-3 and DU145, and (B) bar graphs showing the percentage of PC-3 and DU145 cells in G1, S, and G2 phases. Data are the mean ± standard deviation of three independent experiments. *P<0.05; **P<0.01; and ***P<0.001 by one-way analysis of variance.
Abbreviations: PI, propidium iodide; PLB, plumbagin.
Figure 17 PLB inhibits the proliferation of PC-3 and DU145 cells, and induces G2/M arrest in PC-3 cells and G1 arrest in DU145 cells.

Figure 18 Inhibitory effect of PLB on the proliferation of PC-3 and DU145 cells over 72 hours.

Notes: The time course of PLB-induced cell cycle change over 72 hours in PC-3 and DU145 cells. (A) Representative flow cytometric plots of cell cycle distribution of PC-3 and DU145, and (B) bar graphs showing the percentage of PC-3 and DU145 cells in G1, S, and G2 phases. Data are the mean ± standard deviation of three independent experiments. *P<0.05; **P<0.01; and ***P<0.001 by one-way analysis of variance.
Abbreviations: PI, propidium iodide; PLB, plumbagin.
Figure 18 Inhibitory effect of PLB on the proliferation of PC-3 and DU145 cells over 72 hours.

Figure 19 PLB regulates the expression of CDK1/CDC2, cyclin B1, CDK2, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53 in PC-3 cells.

Notes: PC-3 cells were treated with PLB at 0.1, 1, and 5 μM for 24 hours and protein samples were subject to Western blot assay. (A) Representative blots of CDK1/CDC2, cyclin B1, CDK2, cyclin D1, p21 Waf1/Cip1, p27 Kip1, p53, and β-actin in PC-3 cells, and (B) bar graphs showing the relative levels of CDK1/CDC2, cyclin B1, CDK2, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53 in PC-3 cells. Data are the mean ± standard deviation of three independent experiments. *P<0.05; **P<0.01; and ***P<0.001 by one-way analysis of variance.
Abbreviation: PLB, plumbagin.
Figure 19 PLB regulates the expression of CDK1/CDC2, cyclin B1, CDK2, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53 in PC-3 cells.

Figure 20 PLB regulates the expression of CDK1/CDC2, cyclin B1, CDK2, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53 in DU145 cells.

Notes: DU145 cells were treated with PLB at 0.1, 1, and 5 μM for 24 hours and protein samples were subject to Western blot assay. (A) Representative blots of CDK1/CDC2, cyclin B1, CDK2, cyclin D1, p21 Waf1/Cip1, p27 Kip1, p53, and β-actin in DU145 cells, and (B) bar graphs showing the relative levels of CDK1/CDC2, cyclin B1, CDK2, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53 in DU145 cells. Data are the mean ± standard deviation of three independent experiments. *P<0.05; **P<0.01; and ***P<0.001 by one-way analysis of variance.
Abbreviation: PLB, plumbagin.
Figure 20 PLB regulates the expression of CDK1/CDC2, cyclin B1, CDK2, cyclin D1, p21 Waf1/Cip1, p27 Kip1, and p53 in DU145 cells.

Figure 21 Effects of PLB treatment on the expression and phosphorylation levels of PI3K, Akt, mTOR, p38MAPK, and cytochrome c in PC-3 cells.

Notes: PC-3 cells were treated with PLB at 0.1, 1, and 5 μM for 24 hours and protein samples were subject to Western blot assay. (A) Representative blots of p- and t-PI3K, p- and t-Akt, p- and t-mTOR, p- and t-p38MAPK, and cytochrome c in PC-3 cells, and (B) bar graphs showing the relative levels of p/t-PI3K, p/t-Akt, p/t-mTOR, p/tp38MAPK, and cytochrome c in PC-3 cells. Data are the mean ± standard deviation of three independent experiments. *P<0.05; **P<0.01; and ***P<0.001 by one-way analysis of variance.
Abbreviation: PLB, plumbagin.
Figure 21 Effects of PLB treatment on the expression and phosphorylation levels of PI3K, Akt, mTOR, p38MAPK, and cytochrome c in PC-3 cells.
Figure 21 Effects of PLB treatment on the expression and phosphorylation levels of PI3K, Akt, mTOR, p38MAPK, and cytochrome c in PC-3 cells.

Figure 22 Effects of PLB treatment on the expression and phosphorylation levels of PI3K, Akt, mTOR, p38MAPK, and cytochrome c in DU145 cells.

Notes: DU145 cells were treated with PLB at 0.1, 1, and 5 μM for 24 hours and protein samples were subject to Western blot assay. (A) Representative blots of p- and t-PI3K, p- and t-Akt, p- and t-mTOR, p- and t-p38MAPK, and cytochrome c in DU145 cells, and (B) bar graphs showing the relative levels of p/t-PI3K, p/t-Akt, p/t-mTOR, p/tp38MAPK, and cytochrome c in DU145 cells. Data are the mean ± standard deviation of three independent experiments. *P<0.05; **P<0.01 by one-way analysis of variance.
Abbreviation: PLB, plumbagin.
Figure 22 Effects of PLB treatment on the expression and phosphorylation levels of PI3K, Akt, mTOR, p38MAPK, and cytochrome c in DU145 cells.
Figure 22 Effects of PLB treatment on the expression and phosphorylation levels of PI3K, Akt, mTOR, p38MAPK, and cytochrome c in DU145 cells.

Figure 23 Dose effect of PLB on the expression level of selected EMT markers in PC-3 cells.

Notes: PC-3 cells were treated with PLB at 0.1, 1, and 5 μM for 24 hours and protein samples were subject to Western blot assay. (A) Representative blots of E-cadherin, N-cadherin, snail, slug, TCF-8/ZEB1, vimentin, β-catenin, ZO-1, and β-actin in PC-3 cells treated with PLB at 0.1, 1, and 5 μM for 24 hours, and (B) bar graphs showing the levels of E-cadherin, N-cadherin, snail, slug, TCF-8/ZEB1, vimentin, β-catenin, and ZO-1 in PC-3 cells. Data represent the mean ± standard deviation of three independent experiments.*P<0.05; **P<0.01; ***P<0.001 by one-way analysis of variance.
Abbreviations: EMT, epithelial–mesenchymal transition; PLB, plumbagin.
Figure 23 Dose effect of PLB on the expression level of selected EMT markers in PC-3 cells.
Figure 23 Dose effect of PLB on the expression level of selected EMT markers in PC-3 cells.

Figure 24 Dose-effect of PLB on the expression level of selected EMT markers in DU145 cells.

Notes: DU145 cells were treated with PLB at 0.1, 1, and 5 μM for 24 hours and protein samples were subject to Western blot assay. (A) Representative blots of E-cadherin, N-cadherin, snail, slug, TCF-8/ZEB1, vimentin, β-catenin, ZO-1, and β-actin in DU145 cells treated with PLB at 0.1, 1, and 5 μM for 24 hours, and (B) bar graphs showing the levels of E-cadherin, N-cadherin, snail, slug, TCF-8/ZEB1, vimentin, β-catenin, and ZO-1 in DU145 cells. Data represent the mean ± standard deviation of three independent experiments. *P<0.05; **P<0.01; ***P<0.001 by one-way analysis of variance.
Abbreviations: EMT, epithelial–mesenchymal transition; PLB, plumbagin.
Figure 24 Dose-effect of PLB on the expression level of selected EMT markers in DU145 cells.
Figure 24 Dose-effect of PLB on the expression level of selected EMT markers in DU145 cells.

Figure 25 Effects of PLB on the expression level of selected EMT markers in PC-3 cells over 48 hours.

Notes: PC-3 cells were treated with 5 μM PLB over 48 hours and protein samples were subject to Western blot assay. (A) Representative blots of E-cadherin, N-cadherin, vimentin, β-catenin, and β-actin in PC-3 cells, and (B) bar graphs showing the levels of E-cadherin, N-cadherin, vimentin, and β-catenin in PC-3 cells. Data represent the mean ± standard deviation of three independent experiments. *P<0.05; **P<0.01; ***P<0.001 by one-way analysis of variance.
Abbreviations: EMT, epithelial–mesenchymal transition; PLB, plumbagin.
Figure 25 Effects of PLB on the expression level of selected EMT markers in PC-3 cells over 48 hours.

Figure 26 Effects of PLB on the expression level of selected EMT markers in DU145 cells over 48 hours.

Notes: DU145 cells were treated with 5 μM PLB over 48 hours and protein samples were subject to Western blot assay. (A) Representative blots of E-cadherin, N-cadherin, vimentin, β-catenin, and β-actin in DU145 cells, and (B) bar graphs showing the levels of E-cadherin, N-cadherin, vimentin, and β-catenin in DU145 cells. Data represent the mean ± standard deviation of three independent experiments. *P<0.05; **P<0.01 by one-way analysis of variance.
Abbreviations: EMT, epithelial–mesenchymal transition; PLB, plumbagin.
Figure 26 Effects of PLB on the expression level of selected EMT markers in DU145 cells over 48 hours.

Figure 27 The role of Sirt-1 in PLB-induced EMT inhibition in PC-3 and DU145 cells.

Notes: Cells were treated with PLB at 0.1, 1, and 5 μM for 24 hours and protein samples were subject to Western blot assay. (A) Representative blots of Sirt 1 and β-actin in PC-3 and DU145 cells; (B) bar graphs showing the relative expression level of Sirt-1 in PC-3 and DU145 cells; (C) representative blots of E-cadherin, N-cadherin, and β-actin in PC-3 and DU145 cells; and (D) bar graphs showing the relative expression level of E-cadherin and N-cadherin in PC-3 and DU145 cells. Data are the mean ± standard deviation of three independent experiments. *P<0.05; **P<0.01 by one-way analysis of variance.
Abbreviations: EMT, epithelial–mesenchymal transition; PLB, plumbagin; STL, sirtinol.
Figure 27 The role of Sirt-1 in PLB-induced EMT inhibition in PC-3 and DU145 cells.
Figure 27 The role of Sirt-1 in PLB-induced EMT inhibition in PC-3 and DU145 cells.

Figure 28 Effect of PLB on the intracellular ROS generation in PC-3 and DU145 cells.

Notes: Intracellular ROS level in PC-3 (A) and DU145 (B) cells treated with PLB at 0.1, 1, and 5 μM for 24 hours; and intracellular ROS level in PC-3 (C) and DU145 (D) cells treated with 5 μM PLB over 72 hours. Data are the mean ± standard deviation of three independent experiments. *P<0.05; **P<0.01, and ***P<0.001 by one-way analysis of variance.
Abbreviations: Apo, apocynin; PLB, plumbagin; ROS, reactive oxygen species.
Figure 28 Effect of PLB on the intracellular ROS generation in PC-3 and DU145 cells.

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