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. 2015 Oct 13;6(31):31216-32.
doi: 10.18632/onco_target.5157.

Apigenin blocks IKKα activation and suppresses prostate cancer progression

Sanjeev Shukla  1   2 , Rajnee Kanwal  1   2 , Eswar Shankar  1   2 , Manish Datt  3 , Mark R Chance  3 , Pingfu Fu  4 , Gregory T MacLennan  5 , Sanjay Gupta  1   2   6   7   8
Affiliations

Apigenin blocks IKKα activation and suppresses prostate cancer progression

Sanjeev Shukla et al. Onco_target. .

Abstract

IKKα has been implicated as a key regulator of oncogenesis and driver of the metastatic process; therefore is regarded as a promising therapeutic _target in anticancer drug development. In spite of the progress made in the development of IKK inhibitors, no potent IKKα inhibitor(s) have been identified. Our multistep approach of molecular modeling and direct binding has led to the identification of plant flavone apigenin as a specific IKKα inhibitor. Here we report apigenin, in micro molar range, inhibits IKKα kinase activity, demonstrates anti-proliferative and anti-invasive activities in functional cell based assays and exhibits anticancer efficacy in experimental tumor model. We found that apigenin directly binds with IKKα, attenuates IKKα kinase activity and suppresses NF-ĸB/p65 activation in human prostate cancer PC-3 and 22Rv1 cells much more effectively than IKK inhibitor, PS1145. We also showed that apigenin caused cell cycle arrest similar to knockdown of IKKα in prostate cancer cells. Studies in xenograft mouse model indicate that apigenin feeding suppresses tumor growth, lowers proliferation and enhances apoptosis. These effects correlated with inhibition of p-IKKα, NF-ĸB/p65, proliferating cell nuclear antigen and increase in cleaved caspase 3 expression in a dose-dependent manner. Overall, our results suggest that inhibition of cell proliferation, invasiveness and decrease in tumor growth by apigenin are mediated by its ability to suppress IKKα and downstream _targets affecting NF-ĸB signaling pathways.

Keywords: NF-ĸB signaling; apigenin; cell cycle; prostate cancer; therapeutic _target.

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Conflict of interest statement

CONFLICTS OF INTEREST

None of the authors have any relationships that they anticipate can be construed as resulting in an actual, potential, or perceived conflict of interest with regard to this manuscript submitted for review.

Figures

Figure 1
Figure 1. Expression of IKKα/β and their phosphorylation in various representative benign and prostate cancer tissues
A. Protein expression of IKKα, IKKβ, p-IKKα (Ser176) and p-IKKβ (Ser177) in paired benign and cancer specimens was analyzed by Western blotting; cytokeratin18 expression served as loading control. A modest increase in IKKα and IKKβ expression was observed in cancer specimens compared to benign tissue; whereas a significant increase in p-IKKα and p-IKKβ was observed in cancer specimens. B. Relative density of bands showing protein expression in benign and cancer specimens. Mean ± SD; **P < 0.05, compared to benign tissue. C. Paraffin-embedded (4.0 μm) sections from benign and prostate cancer were used for p-IKKα/β (Ser177/176) expression by immunohistochemistry. Phosphorylated levels of IKKα/β was detected both in the nucleus and in the cytoplasm of malignant cells and was more intense in the cytoplasm. Magnified at x20 and x40. Details are described in ‘materials and methods’ section.
Figure 2
Figure 2. Silencing effect of IKKα and IKKβ on cell cycle in human prostate cancer cells
A. PC-3 and B. 22Rv1 cells were transfected with IKKα and IKKβ shRNA retroviral particles, a pool of viral particle containing 3 _target specific constructs and one scrambled and one with negative shRNA that encode 19–25 nt (plus hairpin) designed to knockdown gene expression, selected under polybrene and used after 15–20 passage, stained with propidium iodide (50 mg/ml) and subjected to cell cycle analysis by flow cytometry. Percentage of cells in G0-G1, S and G2-M phase were calculated using Mod-fit computer software and are represented in the right side of the histograms. Knockdown of IKKα in both cell lines resulted in significant accumulation of cells in the G1 phase whereas IKKβ knockdown resulted in arrest in S phase of the cell cycle. Details are described in ‘materials and methods’ section.
Figure 3
Figure 3. Molecular modeling of the interaction between apigenin and IKKα/β
A. Apigenin a. and PS1145 b. docked in to the pocket of IKKα. Apigenin is represented as sticks with carbon atoms in cyan and oxygen atoms in red; PS1145 is represented in sticks with carbon atoms in magenta, nitrogen atoms in blue, and chlorine atom in green. Structure of IKKα is depicted as surface model. Schematic illustration of interaction between apigenin c. and PS1145 d. with different amino acid residues in the pocket of IKKα is demonstrated. B. Apigenin a. and PS1145 b. docked in to the pocket of IKKβ. Apigenin is represented as sticks with carbon atoms in cyan and oxygen atoms in red; PS1145 is represented in sticks with carbon atoms in magenta, nitrogen atoms in blue, and chlorine atom in green. Structure of IKKβ is depicted as surface model. Schematic illustration of interaction between apigenin c. and PS1145 d. with different amino acid residues in the pocket of IKKβ is shown. Details are described in ‘materials and methods’ section.
Figure 4
Figure 4. Effect of apigenin and PS1145 on IKKα and IKKβ phosphorylation in human prostate cancer cells
A. PC-3 and B. 22Rv1 cells were treated with indicated doses with apigenin and PS1145 for 16 h and IKKα and IKKβ kinase activity was determined using PathScan® Phospho-IKKα (Ser176/180) and PathScan® Phospho-IKKβ (Ser177/181) Sandwich ELISA Kit following vendor's protocol. Kinase activity is depicted as fold change. A significant decrease in IKKα/β phosphorylation in dose-dependent fashion, which was more pronounced for IKKα than IKKβ. Mean ± SD; **P < 0.05, compared to vehicle treated control. Details are described in ‘materials and methods’ section.
Figure 5
Figure 5. Apigenin binding to IKKα and IKKβ by ex vivo pull down assay
A. PC-3 and B. 22Rv1 cells were used for whole cell lysate precipitated with sepharose 4B beads, sepharose 4B-apigenin and sepharose 4B-PS1145 coupled beads. Whole cell lysate (input control, lane 1), precipitate with sepharose 4B beads (negative control, lane 2), sepharose 4B-apigenin coupled beads (lane 3) and sepharose 4B-PS1145 coupled beads were applied to SDS-PAGE, and detected with antibodies against IKKα and IKKβ after transferring the membrane. An increased binding of IKKα was observed in sepharose B-apigenin coupled beads in both cell lines whereas modest binding was observed for IKKβ with apigenin. Details are described in ‘materials and methods’ section.
Figure 6
Figure 6. Effect of apigenin on IKKα/β its phosphorylation and NF-ĸB/p65 protein expression in human prostate cancer cells
A. PC-3 and B. 22Rv1 cells were treated with indicated doses and times with 20 μM apigenin for 16 h and protein expression of NF-ĸB/p65, IKKα, IKKβ and their phosphorylation was determined by Western blot analysis. A significant decrease in IKKα/β phosphorylation, IKKα and NF-ĸB/p65 in dose- and time- dependent fashion was observed. Relative density of bands showing time course change in the protein expression of p-IKKα/β and NF-ĸB/p65 is shown in the right panel. Details are described in ‘materials and methods’ section.
Figure 7
Figure 7. Effect of apigenin on sub-cellular distribution of IKKα, IKKβ and its phosphorylated forms in human prostate cancer cells
A. PC-3 and B. 22Rv1 cells were treated with indicated doses of apigenin for 16 h; subjected to preparation of cytosolic and nuclear fractions and protein expression of IKKα, IKKβ and their phosphorylation was determined by Western blot analysis. A significant decrease in p-IKKα in the nuclear fraction and simultaneous increase in p-IKKα expression in the cytosol after apigenin treatment, compared to untreated group in both cell lines. Details are described in ‘materials and methods’ section.
Figure 8
Figure 8. Effect of apigenin on DNA cell cycle and wound healing in prostate cancer cells
A. DNA cell cycle analysis. PC-3 and 22Rv1 cells were synchronized in G0 phase by depleting the nutrients for 36 h (referred as control) and replating at sub confluent densities into complete medium containing vehicle or apigenin at indicated doses for 16 h, stained with PI (50 mg/ml) and analyzed by flow cytometry. Percentage of cells in G0-G1, S and G2-M phase were calculated using Mod-fit computer software and are represented in the right side of the histograms. A marked increase in G0-G1 phase accumulation of cells was observed after apigenin treatment. B. Wound healing assay. PC-3 and 22Rv1 cells were seeded into six-well plates and grown overnight. Then the cells were serum starved for 24 h. A sterile 200 μl pipette tip was used to scratch the cells to form a wound. The cells were washed with PBS and treated with vehicle or apigenin at indicated doses for 6 h and 16 h. Migration of the cells to the wound was visualized with an inverted Olympus phase-contrast microscope. A decrease in wound healing was observed after apigenin treatment in both cell lines. Details are described in ‘materials and methods’ section.
Figure 9
Figure 9. Effect of apigenin intake on the protein expression of IKKα/β and its phosphorylation, NF-ĸB/p65, and markers of proliferation and apoptosis in prostate tumor xenograft specimens obtained from athymic nude mice
A. PC-3 and B. 22Rv1 tumors obtained after tumor implantation and feeding mice with 20- and 50- μg apigenin in 0.2 ml vehicle daily for 8 weeks. Details are described in Supplemental figure 4. Vehicle treated group served as control. Protein expression of IKKα, IKKβ, p-IKKα/β, NF-ĸB/p65, proliferating cell nuclear antigen (PCNA) and cleaved caspase 3 were determined by Western blot analysis. A marked reduction in the protein expression of IKKα and its phosphorylation, NF-ĸB/p65 whereas a modest decrease in p-IKKβ in PC-3 and 22Rv1 tumor xenografts was observed after apigenin intake. A dose-dependent decrease in proliferating nuclear cell antigen (PCNA), and increase in the expression of cleaved caspase 3 was observed in both tumor xenografts. Relative density of bands showing fold change in the protein expression of these protein is shown below. Mean ± SD; **P < 0.05, compared to vehicle treated control. Details are described in ‘materials and methods’ section.
Figure 10
Figure 10. Effect of apigenin intake on the IKKα/β phosphorylation and extent of proliferation and apoptosis in prostate tumor xenograft specimens obtained from athymic nude mice
A. PC-3 tumors and B. 22Rv1 tumors. Immunohistochemical analyses of p-IKKα/β, PCNA and cleaved caspase 3 was performed in mice fed with 20- and 50- μg apigenin in 0.2 ml vehicle daily for 8 weeks. Details are described in Supplemental figure 4. Vehicle treated group served as control. A significant decrease in IKKα/β phosphorylation, marked decrease in proliferation index and enhancement of apoptosis was observed after apigenin intake in PC-3 and 22Rv1 tumor xenografts, compared with control group. Labeling index for p-IKKα/β, proliferation and apoptotic index is shown in the panel on the right. Mean ± SD; **P < 0.05, compared to vehicle treated control. Details are described in ‘materials and methods’ section.
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