Profiling of Multiple Targets of Artemisinin Activated by Hemin in

Letters pubs.acs.org/acschemicalbiology

Profiling of Multiple Targets of Artemisinin Activated by Hemin in Cancer Cell Proteome Yiqing Zhou,†,§ Weichao Li,†,‡,§ and Youli Xiao*,† †

CAS Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China ‡ University of Chinese Academy of Sciences, Beijing 100039, China S Supporting Information *

ABSTRACT: The antimalarial drug artemisinin is found to have diverse biological activities ranging from anti-inflammatory to anticancer properties; however, as of today, the cellular targets and mechanism of action of this important compound have remained elusive. Here, we report the global protein target profiling of artemisinin in the HeLa cancer cell proteome using a chemical proteomics approach. In the presence of hemin, multiple proteins were targeted by artemisinin probe through covalent modification. Further studies revealed that reducing of hemin to heme by protein thiols was essential for endoperoxide activation and subsequent protein alkylation. Artemisinin may exert its synergistic therapeutic anticancer effects via modulation of a variety of cellular pathways through acting on multiple targets.

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human cancer cells from numerous studies, and hundreds of cytotoxic artemisinin derivatives have been reported in the past two decades,15,16 the direct protein target(s) of this structural and functional unique natural product are poorly studied. Dihydroartemisinin (DHA) binds human translationally controlled tumor protein (hTCTP, or fortilin) and reduces the intracellular levels of hTCTP via ubiquitination and subsequent proteasome-mediated degradation in cancer cell lines.17 Recently, activity- and affinity-based chemical probes have been widely utilized in target profiling of many biologically active small molecules.18 A fluorescent probe was successfully applied in living cell imaging, which revealed mitochondrial localization of artemisinin.19 Efferth and co-workers utilized an affinity tube bounded with artemisinin for interacting protein profiling with the assistance of a bioinformatics study. Nearly 20 proteins were specifically captured by the affinity matrix and identified as artemisinin binding proteins in the cancer cell proteome.20 Most recently, kelch-like ECH-associated protein 1 (Keap1) was identified as a direct binding partner by biotinylated artemisinin, which accounts for its immunosuppressive activity.21 Multiple proteins related to different pathways were targeted by the probe. However, no competition experiment was performed to validate the specificity of these binding proteins. It should be noted that the interaction between artemisinin and most of the reported target proteins are noncovalent and

rtemisinin (Qinghaosu), a sesquiterpene lactone containing a 1,2,4-trioxane motif extracted from the medicinal plant species wormwood (Artemisia annua L.),1,2 has been widely used for the treatment of multidrug-resistant strains of Plasmodium falciparum.3 Besides the potent antimalarial activity, numerous studies have unearthed the anticancer, antiangiogenesis, antiviral, and antifungal properties of artemisinin and its various derivatives.4−6 Although molecular targets and the mode of action of artemisinin are still under debate, it is widely accepted that inside the parasite, the endoperoxide bridge is activated and cleaved through an iron- or heme-dependent mechanism, which releases carbon-centered radicals that can alkylate parasitic proteins.7 Radiolabeled artemisinin reacts covalently with several parasitic proteins, while the inactive analogue deoxyarteether cannot label any proteins.8 In early years, translationally controlled tumor protein (Pf TCTP)9 and sarco/endoplasmic reticulum Ca2+-ATPase (PfATP6, or SERCA)10 were two identified physiological-related targets of artemisinin in Plasmodium falciparum. The inhibitory effect of artemisinins on falcipain-2 (Pf FP2)11 and histidine-rich protein (Pf HRP-2)12 was discovered later. Recently, with supergenomic network compression and molecular dynamics simulation studies, two functionally significant proteins, membrane glutathione S-transferase (Pf MGST or Pf EXP1)13 and phosphatidylinositol-3-kinase (Pf PI3K),14 were found to be inhibited by artemisinins in Plasmodium falciparum. The anticancer mechanism of artemisinin has been proposed to be similar to that of antimalaria, which could be attributed to the rapid uptake and high concentration of iron in cancer cells.7 Although artemisinin has been shown to be effective against © XXXX American Chemical Society

Received: December 17, 2015 Accepted: February 8, 2016

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DOI: 10.1021/acschembio.5b01043 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology

Figure 1. Chemical proteomics profiling of cellular targets of artemisinin. (A) Structures of artemisinin probe ART-yne and its derivatives. (B) Work flow of target identification in this study.

Figure 2. Model studies with recombinant GST proteins. (A) Inhibitory effect of artemisinin on enzyme activity of five members of the GST protein family. (B) Inhibitory effect of artemisinin (ART), probe (ART-yne), and deoxyartemisinin (D-ART) on GSTK1 enzyme activity. (C) Heminmediated probe labeling of GSTK1 can be competed by artemisinin (ART) but not by deoxyartemisinin (D-ART). (D) Labeling of GSTK1 by increasing concentrations of ART-yne with 10 μM of hemin. (E) Labeling of GSTK1 by increasing concentrations of hemin with 10 μM of ARTyne.

reversible, while multiple studies revealed that the inhibitory effect of artemisinin on cancer cells is iron-dependent. Since an iron-triggered artemisinin radical is more likely to covalently alkylate cellular proteins, in this study we attempted to profile covalent-tagged artemisinin binding proteins in a cancer cell proteome through a chemical proteomics approach (Figure 1A). By utilizing a chemical probe of artemisinin activated by hemin, about 80 proteins were enriched and identified as artemisinin-alkylated targets. Furthermore, we proposed that the actual activator of artemisinin could be heme, which reduces from hemin by cellular electron donors such as protein

thiols. As a structurally distinct natural product, artemisinin may exert its unique anticancer activity through alkylation of multiple key proteins related to cell viability under an iron-rich environment within cancer cells.

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RESULTS AND DISCUSSION First, to identify the cellular targets and further elucidate the mode of actions of artemisinin, an alkyne-containing chemical probe ART-yne was designed and synthesized (Figure 1B). Each synthetic step was readily performed using literature procedures with slight modifications (Supporting InformaB


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Figure 3. Gel-based profiling of artemisinin binding proteins in HeLa cell lysates and mode of action study. (A) Hemin-mediated ART-yne profiling of HeLa cell lysates. (B) Competition of ART-yne (10 μM) labeling profiles with four competitors at 100 μM (10×): artemisinin (ART), dihydroartemisinin (DHA), artesunate (ASU), and deoxyartemisinin (D-ART). (C) Effect of inorganic FeSO4 and EDTA on hemin-mediated ARTyne labeling. (D) Chemical structure of hemin-probe adducts. (E) Quantification of the formation of hemin-probe adducts in different redox conditions by LC-MS. (F) The effect of reducing agent NaVc, oxidative reagent H2O2, and thiol-blocking reagents (IAA and NEM) on heminmediated ART-yne labeling of HeLa cell lysates. (G) Same experiment in F on recombinant human GSTK1 protein.

GSTK1 was also inhibited by artemisinin (IC50 = 2.8 μM, Figure 2A). The inhibitory effect of the probe ART-yne is retained, while that of the inactive analogue deoxyartemisinin (D-ART) at 10 μM is much weaker, indicating the significant role of the endoperoxide moiety in GSTK1 inhibition (Figure 2B). To further demonstrate the interaction between artemisinin and GSTK1, recombinant human GSTK1 protein was incubated with the “clickable” probe ART-yne, followed by Cu(I)-catalyzed click reaction with tetramethylrhodamine (TAMRA)-azide and SDS-PAGE separation. Protein labeling was evaluated by in-gel fluorescence scanning. Unfortunately,

tion).19 To demonstrate the feasibility of the probe, we explored the inhibitory and labeling effect of ART-yne on one known class of artemisinin target proteins, glutathione-Stransferases (GSTs). Artemisinin inhibits the enzyme activity of human GSTP1 with an IC50 value of 2 μM;22 another watersoluble analogue, artesunate (ASU), potentially inhibits EXP1, a malarial membrane GST family protein with an IC50 value of 184 nM.13 In this study, the inhibition of GSTs was investigated using the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH) by monitoring the UV absorbance at 340 nm as previously described.23 Among five GSTs screened in our study, the results suggested that besides GSTP1, human C


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Figure 4. Ingenuity Pathway Analysis (IPA) bioinformatics analysis of artemisinin binding proteins and proposed binding mode. (A) Cellular localization of artemisinin-binding proteins. (B) Functional classification of artemisinin-binding proteins. (C) Top 5 molecular and biological functions to which artemisinin-binding proteins are classified. (D) Binding mode of heme-activated artemisinin with proteins. (E) Binding mode of artemisinin with heme-containing or -bounded proteins.

10 μM of ART-yne did not label the recombinant protein (Figure 2C, lane 2). It seems that the endoperoxide moiety was not activated, and no detectable covalent linkage is formed under these conditions. As hemin was found to play a significant role in artemisinin-Pf TCTP interaction,9 we then performed the labeling experiment in the presence of hemin (10 μM). The results showed that GSTK1 was nicely labeled by ART-yne (Figure 2C, lane 4). The band intensities obviously decreased by pretreatment of GSTK1 with excess artemisinin, but not with deoxyartemisinin, suggesting the key role of the endoperoxide moiety in protein alkylation (Figure 2C, lanes 5 and 6). Finally, the fluorescent intensities of protein bands increased as more hemin and probes were used, indicating a hemin-dependent mechanism in protein alkylation by the probe (Figure 2D,E). However, further increasing the hemin concentration (>20 μM) reduced labeling efficiency (Figure 2E, lanes 6 and 7), which could be due to the competition effect caused by probe alkylation of an excess amount of hemin.24

Next, to profile binding proteins of artemisinin in the proteome, we attempted to label human cervical cancer HeLa cell lysates with ART-yne. To mimic the intracellular heme content in HeLa cells (∼0.5 pmol μg−1 protein),25 10 μM of hemin was added to probe-treated cell lysates with a protein concentration of 1 mg mL−1. Multiple bands were labeled by 10 μM of ART-yne in the presence of hemin, and all the labeling experiments performed on GSTK1 protein were repeatable on cell lysates (Figure 3A,B). Additionally, we tried to investigate whether inorganic iron could activate the probe. No labeling was detected when 1.0 mM of FeSO4 was used instead of hemin. Meanwhile, the addition of EDTA to either hemin or FeSO4 treated samples failed to induce any labeling (Figure 3C, lanes 2−4), indicating the core function of hemin-iron in probe activation. Then, to investigate the modes of action of hemin, especially the valence state of iron within the porphyrin complex during probe activation, we incubated ART-yne with hemin (both 100 D


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ACS Chemical Biology μM) in the presence of H2O (vehicle), H2O2 (1.0 mM), and sodium ascorbate (NaVc, 1.0 mM) which served as a reductant of the trivalent iron of hemin. It is proposed that heme serves as both initiator and receptor of carbon-centered radicals of artemisinin,24 and later on artemisinin-hemin adducts were separated and characterized.26 Thus, in our case, the yield of probe-hemin adducts would reflect the level of endoperoxide activation. According to LC-MS results, hemin-probe adducts (m/z = 1104.5 and 1044.5) were detected in all three of the reaction groups. Compared with the vehicle group, the addition of H2O2 reduced the yield to less than 30%, while the yield increased by more than 2 fold by NaVc treatment (Figure 3D,E). Actually, the formation of such adducts in the vehicle group has been suggested as a background signal caused by hemin reduction under mass spectrometry conditions.26 Similarly, we examined the role of heme/hemin during probe activation and labeling in cell lysates. Predictably, the addition of NaVc increased the labeling efficiency while H2O2 nearly blocked the labeling (Figure 3F, lanes 1−3). Since the click reaction was not affected by H 2 O2 or NaVc at this concentration (Supporting Information Figure S2), these results suggested that heme activated artemisinin more efficiently than hemin. However, it is still uncertain whether hemin was able to trigger endoperoxide activation under physiological conditions because protein thiols might serve as electron donors that reduce hemin.9 Pretreatment of cell lysates with iodoacetamide (IAA) and N-ethylmaleimide (NEM), two irreversible thiol-blocking reagents, nearly diminished all the labeling (Figure 3F, lanes 4 and 6), indicating that free thiols were involved in protein alkylation. To exclude the side effect of oxidative/reductive species in cell lysate, we further performed these experiments on GSTK1, and similar results were obtained (Figure 3G, lanes 4 and 6). More importantly, the addition of NaVc to IAA/NEM treated cell lysates recovered the labeling (Figure 3F, lanes 5 and 7), while similar treatment on GSTK1 showed even higher labeling efficiency than that before IAA/NEM blocking (Figure 3G, lanes 5 and 7). Since the alkylation sites of the probe can only be amino acid residues other than cysteine, it can be postulated that free thiols do serve as electron donors during artemisinin activation. Alkylation of cysteine residue by artemisinin-derived radicals is also possible but may not be site-specific. Hemin-activated ART-yne labeling of proteins under nonreducing conditions is undetectable, and the actual activator is very possibly heme with bivalent iron. Encouraged by the cell lysate labeling results, we proceeded to enrich and identify specific protein targets of artemisinin through a chemical proteomics approach. In the presence of hemin, HeLa cell lysates were incubated with ART-yne, followed by a “click” reaction with biotin-azide. After enrichment by pull-down with streptavidin beads, bounded proteins were subjected to reductive-alkylation by dithiothreitol (DTT) and iodoacetamide (IAA), followed by on-beads trypsin digestion. The digested peptides were analyzed by Thermo QExactive LC-MS/MS mass spectrometry. To reduce falsepositive target proteins, we filtered the mass spectrometry results of the positive group (probe + hemin) for proteins with at least five spectra counts and with at least 2-fold spectra counts enrichment over the competition group (probe + hemin in the presence of artemisinin). A total of 79 proteins were identified as artemisinin targets according to the criteria above. These proteins include the two known artemisinin targets, TCTP (fortilin) and SERCA-2. The full list was provided in the

Supporting Information (Table S1). It is not surprising that artemisinin targets a large number of proteins because free radicals are highly reactive species with less selectivity. Previous studies have shown multiple signaling pathways were modulated by artemisinin in human cancer cells, including NF-κB, survivin, NOXA, HIF-1α, and so on,27 which could be the result of less-selective alkylation of proteins by an artemisinin-derived radical. Finally, to reveal the key biological pathways through proteomics data, we analyzed the artemisinin-competed proteome using the Ingenuity Pathway Analysis (IPA) bioinformatics resource. Graphical views of subcellular localization and molecular functional types of the 79 identified artemisinin binding proteins are shown in Figure 4A and B. Further gene ontology (GO) analysis revealed that artemisinin target proteins were distributed within different cellular compartments (Supporting Information Figure S4). The potential molecular and cellular functions of artemisininenriched proteins were also analyzed. The top categories included molecule transport, protein trafficking, cell death and survival, nucleic acid metabolism, and small molecular biochemistry (Figure 4C). The main biological network associated with cancer, organismal injury and abnormalities, and reproductive system disease was represented by the software, where 28 proteins are involved in this network with a score of 57 (Supporting Information Figure S5). It is of interest that transferrin receptor protein 1 (TfR1), which makes up the major iron transport system in cancer cells,28 plays important roles in this network. Cancer cells require high rates of iron intake due to their rapid growth and proliferation, and TfR1 is overexpressed on the surface of various human cancer cell lines.29−31 Besides the well described mechanism regarding iron-induced radical formation, another report showed a nonclassical pathway in which dihydroartemisinin (DHA) may exert its anticancer activity through regulating TfR1 and depleting cellular iron.32 Our results suggested that the alkylation property of artemisinins might explain their regulatory effect on TfR1. Although the alkylation of proteins by heme-activated artemisinin seems less selective, both the iron-activated radical mechanism and TfR1 mediated irondepletion pathway could be more specific to cancer cells or tumor tissues in vivo. In conclusion, we have synthesized a clickable probe capable of proteome-wide profiling of potential targets of artemisinin. To be admitted, we cannot rule out the possibility that some of the identified targets could be nonspecific modifications by the artemisinin radical due to random alkylation on the protein surface. Combined with the results reported by other research groups,33,34 we proposed that at least three modes were involved in artemisinin alkylation: (1) Artemisinin specifically and noncovalently binds protein, such as SERCA-2, then a covalent bond formed by heme activation (Figure 4D, upper panel). (2) Artemisinin nonspecifically attached to the surface of proteins, and a covalent bond formed by heme activation, which mainly including the high abundance proteins such as tubulin, actin, etc. (Figure 4D, lower panel). (3) Artemisinin alkylates heme-containing proteins through heme or amino acid residues nearby (Figure 4E), such as cytochrome b-c1 complex subunit 1. Also, it is possible that artemisinin targets certain proteins through more than one mode mentioned above. Among the targeted proteins, glutathione S-transferases are ubiquitous and abundant enzymes involved in cellular detoxification and transport of endogenous and xenobiotic E



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compounds.35 The inhibition of enzymatic activity and mRNA expression of multiple human GSTs by artemisinin compounds has been reported.36 EXP1, a membrane GST from Plasmodium falciparum, catalyzes the conjugation of glutathione to artesunate, which could account for the detoxification and drug resistance in malaria.13 In the present study, four human GST members, GSTP1, GSTK1, GSTO1, and microsomal GST3 (MGST3), were targeted by our probe. The interaction between ART-yne and recombinant human GSTP1 and GSTO1 proteins was validated (Supporting Information Figure S3). However, artemisinin did not inhibit the enzyme activity of GSTO1 (Figure 2A); therefore it is possible that the interaction is a nonspecific surface attachment (Figure 4D, lower panel). The inhibition of both GSTP1 and GSTK1 were in the micromole range; thus artemisinin may interact with the catalytic domains of these two GSTs (Figure 4D, upper panel). However, we have not detected any high quality artemisininmodified peptide fragments after trypsin digestion by mass spectrometry (data not shown), which could be due to the low abundance of multiple kinds of artemisinin-modified peptides caused by nonselective radical alkylation. MGST3, functionally similar to Plasmodium falciparum EXP1, could be one of the human GST candidates for artemisinin detoxification in cancer cells. The following recombinant expression and enzyme activity study is ongoing at this moment. Taken together, the data presented here illustrate the use of a chemical probe to uncover both protein targets and mechanisms of action of a structurally and functional unique nature product, artemisinin. Taking advantage of the endoperoxide moiety and increased requirement of iron by cancer cells, targeted treatment of cancer by artemisinin could be achieved. Since the probe ART-yne is not cell-permeable, the physiological relevance of artemisinin binding proteins activated by hemin in cell lysate requires further confirmation; future work will focus on the design of cell-permeable activitybased probes for in vivo studies. When this paper was under review, an important chemical proteomics report on artemisinin targets in Plasmodium falciparum was published.37 More than 120 proteins were covalently targeted by an artemisinin probe. Twenty-four common proteins were targeted by heme-activated artemisinin probes in both systems, namely, living parasites of the literature and the HeLa cell lysate of our study (Supporting Information Table S1). Our study showed the mechanism of hemin reduction, endoperoxide activation and protein alkylation in vitro. Both papers provided evidence for the heme/heam as the true iron source responsible for endoperoxide activation and multitarget mechanism of artemisinin.

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METHODS

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AUTHOR INFORMATION

Corresponding Author

*Phone: 86-21-54924226. E-mail: [email protected]. Author Contributions §

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grants 21342013, 21572243, and 21502206), the State Key Basic Research Program of China (Grant 2013CB734000), the “Thousand Talents Program” young investigator award, the Shanghai Pujiang Program (Grant 13PJ1409100), the Key Projects of Shanghai C o m m it t e e o f S c i e n c e a n d T e c h n o l o g y ( G r a n t 15JC1400402), the National Key Laboratory of Plant Molecular Genetics, and the National Key Laboratory of Bioorganic and Natural Product Chemistry. We thank Dr. Yuanhong Shan and Dr. Yining Liu in the Core Facility Centre of the Institute of Plant Physiology and Ecology for mass spectrometry assistance.

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REFERENCES

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Synthesis of probe, procedures of biological experiments, and full list of artemisinin binding proteins are provided in the Supporting Information.

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.5b01043. The synthetic method of ART-yne, biological experimental procedures, and bioinformatics analysis (PDF) F


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Profiling of Multiple Targets of Artemisinin Activated by Hemin in

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