Sophoridine Inhibits Human Colorectal Cancer Progression Via

Published OnlineFirst October 1, 2019; DOI: 10.1158/1541-7786.MCR-19-0553

Cell Fate Decisions Molecular Cancer Research Sophoridine Inhibits Human Colorectal Cancer Progression via Targeting MAPKAPK2 Rui Wang1, Hongwei Liu1, Yingying Shao1, Kailong Wang1, Shuangshuang Yin1, Yuling Qiu2, Honghua Wu1, Erwei Liu1, Tao Wang1, Xiumei Gao1, and Haiyang Yu1

Abstract

Radian Sophorae flavescentis is a traditional Chinese medicine The inhibition effects are further confirmed by Western blot: commonly used to treat cancer in China. However, its active Sophoridine significantly decreases phospho-MAPKAPK2 components and underlying mechanism remain ambiguous. (Thr222) in a time-dependent manner, but there is no obvious In this study, we have screened the pharmacokinetic para- change in its total expression in colorectal cancer cells. Clinical meters of the main chemical constituents of Radian Sophorae studies have shown that a higher level of MAPKAPK2 is asso- flavescentis by Traditional Chinese Medicine Systems Pharma- ciated with a poorer percent survival rate (prognosis). Further- cology (TCMSP) Database and Analysis Platform and have more, a higher level of MAPKAPK2 is positively associated with found that Sophoridine is one of the best antitumor active the enrichment of downregulation of apoptosis and autophagy ingredients. We have found that MAPKAPK2 is a potential by gene set enrichment analysis, as well as upregulation of target for Sophoridine by the PharmMapper and KEGG datab- proliferation and cell-cycle arrest. Taken together, our results Xase analysis. Moreover, we have found that Sophoridine suggest that the MAPKAPK2 plays a key role in Sophoridine- selectively inactivates phospho-MAPKAPK2 (Thr222) and inhibited growth and invasion in colorectal cancers. directly binds into the ATP site of MAPKAPK2 by molecular docking. Furthermore, we have found out a direct binding Implications: These studies show that Sophoridine may be a between MAPKAPK2 and Sophoridine by cellular thermal promising therapeutic strategy that blocks tumorigenesis in shift assay and drug affinity responsive targets stability assay. colorectal cancers.

Introduction Traditional Chinese medicine (TCM) has a long history of application and a significant contribution to modern medi- Colorectal cancer is one of the most frequent causes of cancer- cine (9, 10). As important sources of active natural products, related morbidity and mortality globally (1–3).Despite the benefits TCM shows unique advantages (11–13). Radix Sophorae Flaves- of high-quality early screening and detection, surgery, and new centis (the dried roots of Sophora Flavescens Ait) is widely used in chemotherapeutic agents for improving the treatment of advanced China, Japan, and some European countries for its various phys- and metastatic colorectal cancers, the 5-year survival rate for iologic functions (14, 15). It contains several major effective advanced colorectal cancers is less than 10% (4). There is still a components such as Matrine, Sophoridine, Oxymatrine, etc. lack of optimal treatment strategies for colorectal cancers. Current- Sophoridine, an active quinolizidine alkaloid compound, dis- ly, anti-EGFR agents (bevacizumab, ramucirumab, regorafenib, ziv- plays various biological properties such as anticancer activity, aflibercept) are used in combination with chemotherapy as a antiviral activity, antifibrotic activity, antimicrobial activity, anti- standard of care for the first-line therapy of metastatic colorectal inflammatory activity, etc. (16–19). In particular, recent studies cancer (5–7). Although these agents are designed to restrain tumor- have revealed that it exhibits potent anticancer effects in different selective proliferation, they can cause serious toxic effects, affect tumor cell lines and animal models (20); however, the exact normal tissues, and sometimes severely interfere with therapeutic underlying mechanism of the anticancer effect of Sophoridine is success and the quality of life of patients (8). Therefore, the remaining unclear. development of novel, effective treatment approaches is urgently In this study, we screened the pharmacokinetic parameters of needed to improve clinical outcomes of colorectal cancer patients. the main chemical constituents of Radian Sophorae flavescentis by Traditional Chinese Medicine Systems Pharmacology (TCMSP) Database and Analysis Platform database and found 1Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin, China. 2School of Pharmacy, Tianjin that Sophoridine is one of the best antitumor active ingredi- Medical University, Tianjin, China. ents.Wehaveidentified that Sophoridine is an effective inhibitor of MAPKAPK2 by directly interacting with the ATP site of Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). MAPKAPK2, leading to the repression of multiple oncogenic processes in colorectal cancers. Clinical studies have shown Corresponding Author: Haiyang Yu, Tianjin University of Traditional Chinese that a higher level of MAPKAPK2 is associated with a poorer Medicine, 312 Anshan Road, Nankai District, Tianjin 300193, China. Phone: 86-22- 5959-6166; Fax: 86-22-5959-6166; E-mail: [email protected] percent survival rate (prognosis). Furthermore, a higher level of MAPKAPK2 was positively associated with the enrichment Mol Cancer Res 2019;17:2469–79 of downregulation of apoptosis and autophagy by gene set doi: 10.1158/1541-7786.MCR-19-0553 enrichment analysis (GSEA), as well as upregulation of prolif- 2019 American Association for Cancer Research. eration and cell-cycle arrest.

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Taken all together, these results show that Sophoridine may be 24 hours. Subsequently, medium was discarded and added solu- recognized as an attractive drug candidate by targeting MAP- tion of cell counting to each well, followed by 1 hour of incubation. KAPK2 for colorectal cancers therapy. The absorbance was detected at 450 nm using a microplate reader.

Colony formation assay Materials and Methods Human colorectal cancer cells (1 103) were seeded into Cell culture 6-well plates. After 24 hours, cells were treated with Sophoridine Human colorectal cancer cell lines HCT116, RKO, and SW480 at the indicated concentrations for 48 hours. Cells were then were obtained from Cell Resource Center of the Chinese Academy cultured in fresh medium for another week. Colonies were fixed of Sciences in 2017 (CAMS, PUMC, Beijing, China). The cells with 4% paraformaldehyde and stained with 0.1% crystal violet. being used were used within 1 month after resuscitation (passage Photographs were acquired in indicated time periods, and the cell number between 9 and 30). The cell lines were identified using a numbers were counted. short tandem repeat analysis and tested for mycoplasma using MycoAlert (Lonza) in these cell lines. All cell lines were cultured in Immunofluorescence assay DMEM supplemented with 10% FBS and 1% penicillin/strepto- Cells were seeded in 24-well plates and treated with Sophor- mycin in a humidified atmosphere under 5% CO2 at 37 C. idine at indicated concentrations for 48 hours. The cells were Sophoridine (purity 98%) was purchased from Shanghai Yua- washed in cold PBS and then fixed with 4% paraformaldehyde nye Bio-Technology and dissolved in dimethyl sulfoxide (DMSO) followed by 5 minutes of permeabilization with 0.1% Triton X- to prepare a 10 mmol/L stock solution for storage at 20C. 100. Then we blocked with 1% BSA containing 1% goat serum for 30 minutes. After incubation with anti-LC3 overnight at 4C, cells Cell viability assay were exposed to corresponding secondary antibodies for 1 hour at Cell viability was measured using CCK-8 assay (Dojindo). room temperature, and then stained with DAPI (40,6-diamidino- Human colorectal cancer cells were seeded into flat-bottom 96- 2-phenylindole). Cells were observed by confocal laser scanning well plates (5 103 cells/well) and treated with Sophoridine at microscopy and quantified manually the acquired images with indicated concentrations for another 48 hours after plating for Image J software.

Figure 1. Sophoridine suppresses growth and induces apoptosis in CRC cells. A, The photo of Radian Sophorae ﬂavescentis. B, The Chemical structure of Sophoridine. C, The clonogenicity of RKO, SW480, and HCT116 cells was determined after treatment with Sophoridine at indicated concentrations for 14 days. D–H, CRC cells were treated with indicated concentrations of Sophoridine for 48 hours. D–F, Cell viability was determined using CCK8 assay. G, The protein levels of Bcl-2 and Bax were detected by Western blot assays. H, The percentage of apoptotic cells was determined by ﬂow cytometer. For C–G, data are shown as mean SD (n ¼ 3); , P < 0.05; , P < 0.01; and , P < 0.001 compared with control (Student t test). All the Western data shown are representative of at least three independent experiments.

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Western blot assays FITC and propidium iodide (BD Biosciences) according to the Standard Western blot analysis was performed as previously manufacturer's instructions. In a limited time, the percentages of described (21, 22). Antibodies against SQSTM1/p62 (D5E2), Bax apoptotic cells were analyzed using flow cytometry (Attune NxT; (D2E11), Bcl-2, and Cyclin D1 were purchased from Cell Signaling Invitrogen). For cell-cycle analysis, harvested cells were fixed in Technology. Antibodies against p27, LC-3, and MAPKAPK2 were 75% ethanol overnight at 20C. On the following day, the cells purchased from Proteintech. b-Actin and Phospho-MAPKAPK2 were recovered by centrifugation and washed in cold PBS. There- (Thr222) antibodies were purchased from ABClonal. Full scans of after, they were incubated in 0.5 mL PI/RNase staining Solution Western blot assays are shown in Supplementary Figs. S4 to S7. (Invitrogen) in the dark at room temperature for 30 minutes. Cell cycle was determined and analyzed by flow cytometry. Plasmids transfection Expression vector of human MAPKAPK2 was designed and Real-time PCR purchased from Genechem. We transfected with 0.8 mg of DNA Total RNA was extracted as previously described (23). RNA construct using Lipofectamine 2000 (Invitrogen) according to the quantity and purity were determined by using a NanoDrop 2000 manufacturer's instructions. (Thermo Scientific). Total RNA was reverse transcribed with HiFi- Script cDNA Synthesis Kit (Cowin Biotech). Then, real-time PCR Flow cytometry analysis was performed in triplicate with UltraSYBR mixture (Cowin Colorectal cancer cells were treated with sop for 48 hours. The Biotech) using 7500 RT-PCR System (Applied Biosystems, Life cells were collected with EDTA-free trypsin and washed with ice- Technologies). The expression of genes was normalized to the cold PBS for 2 times. For cell apoptosis analysis, all cells were Actin gene. The primers used are listed in the Supplementary resuspended with binding buffer and incubated with Annexin V– Table S1.

Figure 2. Sophoridine induces cell-cycle arrest and promotes autophagy in CRC cells. A and B, Western blotting analysis shows that the protein expression of Cyclin D1 and p27 was determined in SW480 and RKO cells treated with indicated doses of Sophoridine for 48 hours. C, Cells were treated with indicated concentrations of Sophoridine for 48 hours. Cell-cycle distribution was detected by ﬂow cytometer. D and E, The protein levels of p62 and LC-3 were determined by Western blot assays. F, Cells were treated with DMSO or Sophoridine. The expression of LC-3 was determined by immunoﬂuorescence staining. Right, relative level of LC-3.

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Table 1. Top ten pharmacophore candidates identiﬁed by PharmMapper Pharma model Norm ﬁt Sample Name Uniplot 2p3g_v 0.9707 MAPKAPK2 MAP kinase-activated protein kinase 2 P49137 3gam_v 0.8849 NQO2 Ribosyldihydronicotinamide dehydrogenase (quinone) P16083 1shj_v 0.8818 CASP7 Caspase-7 CASP7_HUMAN 1e7a_v 0.8008 ALB Serum albumin ALBU_HUMAN 2zas_v 0.7294 ESRRG Estrogen-related receptor gamma P62508 2o65_v 0.702 PIM1 Proto-oncogene serine/threonine-protein kinase Pim-1 PIM1_HUMAN 2ipw_v 0.6914 AKR1B1 Aldose reductase ALDR_HUMAN 3fzk_v 0.6896 HSPA8 Heat shock cognate 71 kDa protein P11142 1fdu_v 0.6888 HSD17B1 Estradiol 17-beta-dehydrogenase 1 P14061

Cellular thermal shift assays ture. Subsequently, cells were lysed and proteins were sepa- Cellular thermal shift assays (CETSA) were performed rated, and the corresponding index was determined by West- to determine the direct binding between Sophoridine and ern blot assays. MAPKAPK2 in cellular. Colorectal cancer cells were pretreated with DMSO or Sophoridine for 48 hours, chilled on ice, Drug afﬁnity responsive targets stability assay washed with PBS plus protease inhibitor cocktail, and then The drug afﬁnity responsive targets stability (DARTS) assay was collected and heated for 3 minutes at appropriate tempera- conducted as described above (24, 25). To prepare DARTS

Figure 3. Network pharmacology analysis of the candidate targets. A, D-T network is the intersection of drug targets and CRC-associated genes. B, The mRNA levels of the candidate targets were detected by real-time PCR. C, PPI network of potential target proteins (color distribution according to topological parameter degree). D, Relative drug-mediated genes was analyzed by ClueGO.

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samples, 1 107 colorectal cancer cells were lysed in 2.4 mL Molecular docking M-PER buffer with protease inhibitors, centrifuged, collected Docking simulations were operated using the Discovery- proteins, and then added 10 TNC buffer. Lysates were equally Studio 2017 R2 molecular modeling software. The three- divided into two parts for 1 hour at room temperature with DMSO dimensional (3D) structures of the Sophoridine molecule were or Sophoridine, and incubated with 1 mg/mL pronase at room generated with ChemDraw and were energy minimized with temperature for 5 minutes. The reaction was stopped by adding CHARMm force ﬁeld. The initial 3D geometric coordinates of protease inhibitors, and samples were stored at 20C standby. MAPKAP kinase 2 (PDBcode: 2JBO) were obtained from the

Figure 4. Sophoridine targeting inhibits phosphorylation of MAPKAPK2. A–C, Cells were treated with or without 160 mmol/L of Sophoridine for 48 hours and subsequently heated at different temperature for 3 minutes. After freeze-thaw cycles for cell lysis, the soluble MAPKAPK2 protein levels bound to a drug were visualized by Western blot assays. Right, relative band intensity of MAPKAPK2. D, Cells were incubated with 160 mmol/L of Sophoridine for or PBS for 1 hour at room temperature and digested with Pronase for 5 minutes at room temperature. MAPKAPK2 protein levels were tested by western blot assays. E–G, Docking model of Sophoridine with MAPKAPK2. E, The interaction pattern of Sophoridine with the residues. F, 2D diagram between the receptor and ligand. G, Sophoridine binding with the pocket is composed of hydrogen bonds. H, SW480 and RKO cells were treated with indicated concentration of Sophoridine for 48 hours. The protein levels of p-MAPKAPK2 were detected by Western blot assays. Total MAPKAPK2 expressions were detected as the internal control.

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Figure 5. Sophoridine promotes the apoptotic and autophagic capacities and induces cell-cycle arrest via MAPKAPK2 inactivation. A–I, Cells transfected with MAPKAPK2 (MAPKAPK2 Vec) or empty vector (Control Vec) followed by Sophoridine treatment. A, D, F, and H, The protein levels of MAPKAPK2, Bcl-2, Bax, Cyclin D1, p27, and LC-3 were detected by Western blot assays. B, The cell viability was measured by CCK8 assay. C, The colony formation capability was detected by clonogenic assay. E, The percentage of apoptotic cells was measured by ﬂow cytometer. (Continued on the following page.)

Protein Databank (PDB). Then, the protein structure was pre- Results pared by removing water molecules and adding hydrogen. Sophoridine suppresses growth and induces apoptosis in CDOCKER protocols were employed as docking approaches colorectal cancer cells and calculated the predicted binding energy (kcal mol 1). The The photo of Radian Sophorae flavescentis is shown in Fig. 1A. complex structure with the most favorable binding-free ener- The pharmacokinetic parameters of the main chemical constitu- gies was selected as the optimal docked conformation for later ents of Radian Sophorae flavescentis were screened by TCMSP experimental verification. database in Supplementary Table S2, and Sophoridine is one of the best antitumor active ingredients. As shown in Supplementary Database of colorectal cancer patients Fig. S1A and Supplementary Table S3, the pharmacokinetics Clinical data can be obtained via the publically available The properties were elucidated of Sophoridine in rat by high perfor- Cancer Genome Atlas and Gene Expression Omnibus (GEO) mance liquid chromatography. The result could provide mean- datasets. The expression level of MAPKAPK2 in colorectal cancer ingful reference for further clinical medication of Sophoridine. patients was analyzed by Kaplan–Meier estimate. GSEA was used The chemical structure of Sophoridine is shown in Fig. 1B. As to identify the association of MAPKAPK2 expression with bio- shown in Fig. 1C, clonogenicity of colorectal cancer cell lines logical processes of colorectal cancer cells by GSEA 3.0 software (RKO, SW480, and HCT116) was dramatically reduced by (http://www. broadinstitute.org/gsea/). Sophoridine for 48 hours. The CCK8 assay was used to detect the cytotoxic effects of Sophoridine against colorectal cancer cell Statistical analysis lines and two nontumorigenic cell lines (HKC and LX-2). Con- The data were represented as mean SD. Two-tailed unpaired sistently, as evidenced by decreased cell viability, Sophoridine Student t test was used for comparing two groups of data. One- strongly inhibited cell proliferation in colorectal cancer cells ANOVA was used to compare multiple groups of data. Survival (Fig. 1D–F). However, Sophoridine did not affect the cell viability analysis was determined using the Kaplan–Meier estimates and of HKC and LX-2 cells (Supplementary Fig. S1B and S1C). In vivo, the log-rank test. The variation (P < 0.05) was considered statis- the indexes had no statistical difference of routine blood test and tically significant. serum biochemical measurements, compared with the control

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Figure 5. (Continued.)G, Cell-cycle distribution was detected by flow cytometer. I, The LC-3 was detected by IF analysis. Right, relative level of LC-3. For B, C, G, and I, data are shown as mean SD (n ¼ 3); , P < 0.01; , P < 0.005; and , P < 0.001 compared with Vector control; #, P < 0.01 and ###, P < 0.001 compared with Vector control–transfected cells treated with Sophoridine (Student t test). All the Western blot data shown are representative of at least three independent experiments. group, in Supplementary Tables S4 and S5, and no obvious expression of Cyclin D1, while markedly increased the expression abnormalities in histopathology after the Sophoridine adminis- of p27 in a dose-dependent manner. The cell-cycle arrest effects tration in mice (Supplementary Fig. S1D). These results suggest were further confirmed by employing flow cytometry detection. that Sophoridine showed no obvious drug toxicity under condi- As shown in Fig. 2C, Sophoridine significantly raised cell number tions of potent antitumor efficacy. To investigate cell apoptosis at G0–G1 phase after 48-hour exposure, accompanied by regulation effect of Sophoridine on colorectal cancer cells, we decreased cell number at G2–M phase. Moreover, we also performed Western blot assays. As shown in Fig. 1G, after expo- explored the protein levels of classical autophagy markers by sure to Sophoridine, colorectal cancer cells showed a downregu- Western blot assays and immunofluorescence staining. As shown lation of Bcl-2 and an upregulation of Bax, which result in a dose- in Fig. 2D–F, Sophoridine markedly reduced the expression of dependent increase of the ratio of Bax/Bcl-2. The apoptotic effects p62, whereas dramatically increased the expression of LC-3B were further confirmed by employing Annexin V staining by puncta dose-dependently. Collectively, these results indicate that treating colorectal cancer cells with Sophoridine (Fig. 1H). Taken Sophoridine induces cell-cycle arrest and promotes autophagy of together, these results suggest that Sophoridine suppresses growth colorectal cancer cells. and induces apoptosis of colorectal cancer cells. Potential target prediction and screening by network Sophoridine induces cell-cycle arrest and promotes autophagy pharmacology in colorectal cancer cells To investigate potential targets of Sophoridine in colorectal To investigate cell-cycle arrest promotion effect of Sophoridine cancer cells, 116 pharmacophore candidates were predicted via on colorectal cancer cells, we performed Western blot assays. As pharmMapper (http://www.lilab-ecust.cn/pharmmapper/). The indicated in Fig. 2A and B, Sophoridine significantly reduced the ranked list of hit target pharmacophore models is sorted by

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normalized fit score in descending order (Supplementary together, these results indicate that Sophoridine promotes Table S6), and the top ten were displayed in Table 1. To improve apoptosis and autophagy and induces cell-cycle arrest through the specificity, 3,298 colorectal cancer–associated genes were targeting MAPKAPK2. retrieved from the disGeNET (http://www.disgenet.org) database (Supplementary Table S7). A total of 67 potential targets of Clinical significance of the MAPKAPK2 in colorectal cancer Sophoridine identified in colorectal cancer–associated genes were To further investigate the clinical outcome of MAPKAPK2 in selected for constructing the drug-target (D-T) network (Fig. 3A). colorectal cancer patient, we subjected them to Kaplan–Meier Thus, we examined changes in the transcriptional levels of these analysis in GEO data set. Data revealed that higher MAPKAPK2 genes in colon cancer cells after treatment with Sophoridine. As expression was associated with poorer percent disease-specific shown in Fig. 3B, Sophoridine can significantly inhibit the survival (DSS) (GSE17536, P ¼ 0.020, Fig. 6A), disease-free expression of MAPK14, BRAF, FGFR1, and other genes. To make survival (DFS) (GSE17536, P ¼ 0.039, Fig. 6B), and overall a deep exploring of the action mechanism of Sophoridine in survival (OS) (GSE17536, P ¼ 0.025, Fig. 6C). Moreover, GEO colorectal cancer cells, we used String (https://string-db.org/) to database revealed that MAPKAPK2 level is high in colorectal obtain protein interactions and then constructed protein–protein cancer tissue compared with that in normal colon tissue interaction (PPI) network of genes associated with drug mediated (GSE110225, P ¼ 0.043, Fig. 6D). Furthermore, according to the by Cytoscape 3.2.1 (Fig. 3C), and the key topological parameter level of MAPKAPK2 from GSE17536, we estimated that higher degree was analyzed. The biological functions of these potential level of MAPKAPK2 was positively correlated with enrichment of targets were performed by Cytoscape plugin, ClueGO. These downregulation of apoptosis and autophagy by GSEA, as well as results suggest that these genes are involved in the development upregulation of proliferation and cell-cycle arrest (Fig. 6E–H). of various cancers and are closely related to MAPK VENTS, Taken together, these results indicate that MAPKAPK2 may be a signaling through FGFR, VEGF pathways, etc. (Fig. 3D). Based prognosis marker in colorectal cancer patients. on the above analysis, we predict that Sophoridine can inhibit the Collectively, our results show that the MAPKAPK2 plays an development of colorectal cancer cells by targeting MAPKAPK2. important role in Sophoridine-regulated apoptosis, autophagy, and cell-cycle arrest in colorectal cancer. Sophoridine promotes the apoptotic and autophagic capacities and induces cell-cycle arrest via MAPKAPK2 inactivation To detect whether MAPKAPK2 is a direct target of Sophoridine, Discussion we employed the CETSAs. As shown in Fig. 4A–C, Sophoridine Sophoridine, an active quinolizidine alkaloid compound, has treatment significantly shifted the MAPKAPK2 melting curve com- been proven to possess extensive physiologic activities (16, 17). pared with control. Moreover, our DARTS data suggested that Previous studies have revealed that Sophoridine displays prom- Sophoridine binds to MAPKAPK2, protecting it from proteolytic inent anticancer biological effects (20). However, the underlying cleavage (Fig. 4D). To search the promising target for binding mode molecular mechanisms are still elucidated. In this study, we have of Sophoridine in MAPKAPK2, molecular docking simulation illustrated that Sophoridine promotes apoptosis and autophagy experiments were performed between Sophoridine and MAP- and induces cell-cycle arrest via targeting MAPKAPK2, leading to KAPK2 by employing Discovery Studio 2017 R2 software. The the blocking of the growth and development of colorectal cancers. CDOCKER docking result revealed that Sophoridine can bound Network pharmacology is one of the strategies for discovering into the ATP site of MAPKAP kinase 2 (PDBcode: 2JBO), and extend new drugs (26–28). In recent years, it has played an increasingly into the sub pockets for the adenine moiety and the a-phosphate, important role in the research and development of new drugs, surrounded by key residues (LEU193, LEU70, ALA91, VAL78, such as target identification, mechanism of action, discovery and LYS93), thus blocking the ATP-binding site fully (Fig. 4E–G). optimization of drug lead, and preclinical efficacy and safety Furthermore, to further evaluate the MAPKAPK2 inhibitory effect, evaluation (29–31). Network pharmacology is often studied by we detected the constitutive activation of MAPKAPK2 in colorectal integrating multidisciplinary molecular networks, such as chem- cancer cells by the specific antibodies against phospho-MAPKAPK2 ical informatics, bioinformatics, and systems biology (32, 33). Thr222. As shown in Fig. 4H and Supplementary Fig. S2A, Sophor- Radix Sophorae Flavescentis is very commonly used in China, Japan, idine significantly reduced the phosphorylation level of MAP- and some European countries for its various physiologic func- KAPK2 (Thr222) and MAPKAPK2 activity, but there was no big tions (17). Sophoridine is one of major bioactive components difference in its total expression in colorectal cancer cells. Interest- from Radix Sophorae Flavescentis. Previous studies have identified ingly, Sophoridine did not affect the p38 (upstream activators of Sophoridine contains several biological properties (16, 20). In MAPKAPK2) activity and significantly reduced the docking inter- this study, we predicted 116 pharmacophore candidates via action of p38-MAPKAPK2 in Supplementary Fig. S2B and S2C. PharmMapper (http://www.lilab-ecust.cn/pharmmapper/) and Next, we found that MAPKAPK2 overexpression in colorectal found 67 potentially targets of Sophoridine from 3,298 colorectal cancer cells strongly attenuated the inhibitory effect of Sophor- cancer–associated genes, and then we constructed the D-T net- idine on colony formation and cell viability (Fig. 5A–C). In work through these targets. Moreover, we have found that addition, ectopic MAPKAPK2 expression dramatically recov- Sophoridine can significantly inhibit the expression of MAPK14, ered MAPKAPK2-regulated cell-cycle arrest and MAPKAPK2- BRAF, FGFR1, and other genes by real-time PCR. Furthermore, we induced apoptosis and autophagy (Fig. 5D–I). Moreover, we used String (https://string-db.org/) to obtain protein interactions found that knockdown of endogenous MAPKAPK2 by siRNA and then constructed PPI network of genes associated with drug has a similar effect with Sophoridine-regulated cell-cycle arrest, mediated by Cytoscape 3.2.1. And we analyzed the key topolog- apoptosis, and autophagy. Furthermore, knockdown of endog- ical parameter degree and performed the biological functions of enous MAPKAPK2 further enhanced the anticolorectal cancer these potential targets by Cytoscape plugin, ClueGO. Taken effect of Sophoridine (Supplementary Fig. S3A–S3D). Taken together, the results suggest that these genes are involved in the

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Figure 6. Clinical significance of the MAPKAPK2 in CRC. A–C, Kaplan–Meier plots of the PFS, DFS, and OS of CRC patients, stratified by expression of MAPKAPK2. Data obtained from the dataset of GEO (GSE17536). D, MAPKAPK2 expression level in CRC tissue compared with in normal colon tissue (GSE110225). E–H, Identification of gene sets enriched in phenotypes associated with MAPKAPK2 by GSEA using GSE17536 data. development of various cancers and are closely related to MAPK angiogenesis, migration, survival, and proliferation (35). Litera- EVENTS, signaling through FGFR, VEGF pathways, etc., and we ture reports have shown that deletion of MAPKAPK2 conduces to predicted that MAPKAPK2 may target colorectal cancer cells DNA damage and apoptosis through impaired phosphorylation involved in the treatment with Sophoridine. of MDM2 and subsequently enhances the primary regulator of MAPKAPK2, also called MK2, is known to be acted as a down- p53 stability in skin cancer (45). Past studies have reported that stream signaling protein of p38MAPK and regulates a cascade of MAPKAPK2 promotes the invasion and metastasis via regulation critical biological processes including inflammatory responses, of MMP-2/9 mRNA half-life in bladder cancer (46). In the present nuclear export, stress, and DNA damage (34). Depending on these study, we have found that ectopic MAPKAPK2 expression signif- processes, MAPKAPK2 regulates transcript stability, expression of icantly blocks Sophoridine-regulated apoptosis, autophagy, and diverse proteins, and the phosphorylation involved in numerous cell-cycle arrest of colorectal cancer. Clinical studies have shown important cellular phenomenon, such as cell cycle, senescence, cell that MAPKAPK2 expression is associated with poor prognosis. migration, cell proliferation, and apoptosis (35–37). Systemic side Furthermore, we estimated that a higher level of MAPKAPK2 was effects are a major obstacle to the conversion of developed positively associated with the enrichment of downregulation of p38MAPK inhibitors into successful new drugs. This is the fore- apoptosis and autophagy by GSEA, as well as upregulation of most cause of failure in the clinical trials. To solve this problem and proliferation and cell-cycle arrest. Collectively, our results dem- effectively inhibit p38MAPK, researchers turned their focus to onstrate that the MAPKAPK2 plays a major role in Sophoridine- many of its downstream targets, such as MAPKAPK2. Previous regulated tumorigenesis in colorectal cancers. studies have shown that targeting MAPKAPK2 to interdict its In summary, we have screened the pharmacokinetic parameters downstream events is as good as direct upstream inhibition of of the main chemical constituents of Radian Sophorae flavescentis the p38MAPK pathway without obvious side effects of p38MAPK by TCMSP database and have found that Sophoridine is one of the inhibitors (38–40). In the present study, we have demonstrated a best antitumor active ingredients. Our study elucidates that direct binding between MAPKAPK2 and Sophoridine by DARTS Sophoridine induces apoptosis, autophagy, and cell-cycle arrest assay and CETSA. Furthermore, we have found that Sophoridine through targeting MAPKAPK2, which leads to inhibiting the selectively inactivates phospho-MAPKAPK2 (Thr222) and directly tumor development and progression of colorectal cancers. These binds into the ATP site of MAPKAPK2 by molecular docking. The studies show that Sophoridine may be a promising therapeutic blockage effects are further determined by Western blot: Sophor- strategy that blocks tumorigenesis in colorectal cancers. idine markedly reduces phospho-MAPKAPK2 (Thr222) in a dose- dependent manner, but there is no obvious deference in its total Disclosure of Potential Conflicts of Interest expression in colorectal cancer cells. No potential conflicts of interest were disclosed. Past reports have demonstrated the expression of MAPKAPK2 in a multitude of cell types like cancers, smooth muscle cells, and Authors' Contributions – endothelial cells (41 44). A recent study has elucidated that Conception and design: H. Yu MAPKAPK2 plays a key role in colon cancer processes via axis Acquisition of data (provided animals, acquired and managed patients, inhibition of Hsp27, which finally results in promoting cell provided facilities, etc.): R. Wang, H. Liu, Y. Shao, K. Wang

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Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Ministry of Science and Technology of China (2018ZX09735-002 to T. Wang), computational analysis): S. Yin, Y. Qiu, H. Wu, E. Liu, T. Wang, X. Gao, H. Yu and Natural Science Foundation of Tianjin City (No. 15PTCYSY00030 to Z. Li). Writing, review, and/or revision of the manuscript: H. Yu Study supervision: H. Yu The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in Acknowledgments accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This work was supported by grants from National Natural Science Founda- tion of China (81603253, 21711540293, and 81873089 to H. Yu, and Received May 24, 2019; revised August 23, 2019; accepted September 27, 81602614 and 81973570 to Y. Qiu), Important Drug Development Fund, 2019; published ﬁrst October 1, 2019.

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www.aacrjournals.org Mol Cancer Res; 17(12) December 2019 2479

Sophoridine Inhibits Human Colorectal Cancer Progression via Targeting MAPKAPK2

Rui Wang, Hongwei Liu, Yingying Shao, et al.

Mol Cancer Res 2019;17:2469-2479. Published OnlineFirst October 1, 2019.

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