This journal is©The Royal Society of Chemistry 2017 Chem. Commun.

Cite this:DOI: 10.1039/c7cc02789c

Identification of actin as a direct proteomic targetof berberine using an affinity-based chemicalprobe and elucidation of its modulatory role inactin assembly†

Chae-Min Yi,‡a Jihyun Yu,‡a Hyunbin Kim,‡b Na-Rae Lee,a Sang Won Kim,a

Noh-Jin Lee,a Jun Lee,a Jihye Seong,*bc Nam-Jung Kim*a and Kyung-Soo Inn *ab

Despite the diverse pharmacological activities of berberine, including

anti-cancer and anti-inflammatory effects, the direct proteomic

targets of berberine have remained largely unknown. Here, we have

identified actin as a direct proteomic target of berberine using an

affinity-based chemical probe. In addition, we found that actin

assembly was significantly modulated by berberine in vitro at the

biochemical level and cellular level.

Berberine, one of the active ingredients found in many medicinalplants including Berberis vulgaris, is a natural benzylisoquinolinealkaloid.1–3 It shows various biological activities, especially anti-cancer effects.4–17 Driven by various reports about the pharmaco-logical activities of berberine, tremendous efforts have been madetoward its clinical use, but further development into novel drugcandidates has been relatively limited, partly due to insufficientinformation about berberine’s direct targets and mode of action.Recently, several studies have reported that berberine could bindand stabilize human telomeric G–quadruplex structures, formedat the single-stranded overhang of telomeric DNA, leadingto potent anticancer activity in cancerous cells.18–20 Giventhat berberine has various biological activities and its complexformation with telomeric G–quadruplex DNA can explain only asmall amount of its anti-cancer activity, it is assumed that anothertarget might be associated with its various pharmacologicalactivities including anti-cancer effects and further efforts tosystemically investigate the cellular targets of berberine aresignificantly needed. To date, a direct proteomic target of

berberine has scarcely been explored, although its complexationwith DNA has been studied as a mode of its action.21 In recentdecades, affinity-based chemical probes have been widely usedand considered as powerful tools for identifying proteomictargets of biologically active small molecules.22–25 In an effortto find the proteomic target of berberine, we considered itworthwhile to synthesize and use an affinity-based chemicalprobe. In this study, we synthesized an affinity-based chemicalprobe of berberine and thus directly pulled down the cellularproteins, leading to identification of the molecular target bymass analysis. We also showed that the identified target can bemodulated by berberine in vitro at the biochemical level andcellular level.

Based on a previous study on its structure–activity relationship,26

focusing on the anti-cancer activity of berberine, we designedand synthesized the chemical probe berberine biotinylatedprobe (BBP, 4), composed of berberine, the hydrophilic linkerfrom the C-9 position of berberine, and biotin (Scheme 1).BBP (4) was concisely and efficiently synthesized via 2 stepsequences from known compound 1,26 which was easily con-verted into compound 2 by simple propargylation of the phenolmoiety without a base. BBP (4) was finally obtained through

Scheme 1 Synthesis of probe 4: (a) propargyl bromide, DMF, 80 1C,100%; and (b) 3, CuSO4, Na ascorbate, t-BuOH, H2O, rt, 28%.

a Department of Fundamental Pharmaceutical Sciences, Graduate School,

Kyung Hee University, Seoul 02447, Republic of Korea. E-mail: innks@khu.ac.kr,

kimnj@khu.ac.krb KHU-KIST Department of Converging Science and Technology, Graduate School,

Kyung Hee University, Seoul 02447, Republic of Koreac Convergence Research Center for Diagnosis, Treatment and Care System of

Dementia, Korea Institute of Science and Technology (KIST), Seoul 02792,

Republic of Korea. E-mail: jseong@kist.re.kr

† Electronic supplementary information (ESI) available: Experimental proceduresand characterization data. See DOI: 10.1039/c7cc02789c‡ These authors contributed equally to this work.

Received 11th April 2017,Accepted 7th June 2017

DOI: 10.1039/c7cc02789c

rsc.li/chemcomm

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click chemistry of compound 2 with commercially availableazido biotin (3) in the presence of CuSO4 and sodium ascorbate.

Using synthesized BBP (4), we performed the target identifi-cation process on berberine. The overall process of targetidentification is briefly described in Fig. 1A. The proteome ina whole cell lysate of Jurkat T-cells was treated with BBP (20 mM)and incubated for 2 hours with or without free berberine(40 mM). Via enrichment with streptavidin-resin and a sequen-tial wash-out process, the proteome labeled with BBP (4) wasseparated out by an insoluble solid support and isolated fromthe obtained proteome mixture. Then, proteome labeled withBBP or biotin-azide was released from the streptavidin beadand run on an SDS-PAGE gel, followed by silver staining. Asshown in Fig. 1B, a protein band around 40 kDa in size wasdetected from the sample co-precipitated with BBP, suggestingits specific interaction with BBP. Moreover, the band intensitywas drastically decreased in the presence of free berberine,indicating a specific interaction between the correspondingprotein and berberine. Mass-spectrometry (MS/MS) analysis ofthe excised band identified the peptides covering 25.07% ofactin, indicating that BBP could physically bind with actin(Fig. 1C and Fig. S1, ESI†). Taken together, this means that

actin might be a direct proteomic target of berberine. Tofurther confirm the interaction between berberine and actin,an in vitro binding assay and surface plasmon resonance assay(SPR) using purified rabbit actin were performed. In line withthe initial pull-down assay result, BBP successfully co-precipitatedwith purified actin protein, and co-incubation with free berberinecompletely abrogated the interaction (Fig. 1D). In addition, freeberberine showed dose-dependent binding with the immobilizedactin in SPR analysis, which confirms the direct interaction ofberberine with actin (Fig. 1E).

Considering our results and previous reports showing thatberberine inhibits cancer cell migration and metastasis, weassumed that these anti-cancer properties might be associatedwith an inhibitory role of berberine in actin assembly.27 Therefore,we examined whether berberine could affect the in vitro actinpolymerization process. Assembly of monomeric pyrene G-actininto filamentous F-actin was monitored by measuring pyrenefluorescence.28 As depicted in Fig. 2A, the addition of berberineresulted in dose-dependent inhibition of actin polymerization,suggesting that berberine is capable of directly suppressing theactin assembly process through direct interaction. Furthermore,disassembly of pre-assembled filamentous actin was also drama-tically facilitated by the addition of berberine in a dose-dependentmanner (Fig. 2B). This might be due to a shift from the dynamicequilibration state to the disassembly process by suppression ofactin polymerization by berberine.

Next, we used a macrophage podosome reformation assay todetermine whether berberine can inhibit actin polymerizationat the cellular level. Starved RAW macrophage cells were incubatedwith or without berberine for 3 hours and treated with PP2 (CAS172889-27-9) to disrupt existing podosomes, followed by washingand incubation in serum-containing media for 30 minutes toallow podosome reformation. As shown in Fig. 3A, berberinetreatment resulted in a significant decrease in podosomeformation, further confirming the inhibitory role of berberinein actin assembly.

To examine the effect of berberine on actin architecture incells, NIH3T3 cells were briefly treated with a low concentrationof Triton X-100 (0.0025%, 10 min, 4 1C) to increase the cellpermeability and then treated with a vehicle or berberine.Three hours after treatment, fragmented actin filaments were

Fig. 1 Identification of actin as a direct interacting protein of berberine(BBR). Panel A shows the scheme of affinity purification of berberineinteracting proteins and the proteomic identification process by LC-MS/MS. Panel B shows silver stained proteins co-precipitated with BBP. Panel Cshows the peptides of actin (red) detected by LC-MS/MS analysis. Panel Dshows the result of the direct pull-down assay using purified actin and BBP.Panel E shows SPR sensorgrams obtained on an actin coated chip at differentconcentrations of BBR.

Fig. 2 Modulation of in vitro actin assembly by berberine. Panel A showsthe inhibition of pyrene-actin polymerization by berberine (BBR). Panel Bshows the induction of pyrene-actin depolymerization by berberine (BBR).Polymerization and depolymerization of pyrene actin were analyzed bymonitoring fluorescence signals.

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increased in berberine (100 mM) treated cells as analyzed byAlexa Fluor 594 phalloidin staining. The cells treated withberberine for 48 hours showed significantly disrupted actinfilament networks compared to vehicle treated cells (Fig. 3B).

In summary, we identified actin as a potential direct targetof berberine using an affinity-based probe and elucidated thatberberine is capable of modulating actin polymerization. Sup-pression of actin polymerization by berberine may provide areasonable explanation for its biological activities such asinhibition of cell migration and cancer cell invasion. This resultprovided that berberine could directly bind to actin, which hasbeen a crucial target for metastatic cancers, implying thatberberine would serve as a useful tool for biological sciences

related in its pharmacology and novel scaffolds for develop-ment of anti-cancer drugs.

This study was supported by the Basic Science ResearchProgram through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning(NRF-2014R1A1A2058039 and NRF-2016R1A2B4015169).

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Fig. 3 Effect of berberine on the cellular actin architecture. Panel A showsthe effect of berberine on the podosome reformation of RAW cells. PanelB shows the effect of berberine on cell actin cytoskeleton structure ofNIH3T3 cells. The actin network of the cells treated with a vehicle (DMSO)or berberine was visualized by staining using Alexa Fluor 594 phalloidin andanalyzed by fluorescence microscopy.

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