Antimalarial Drug Pyrimethamine Plays a Dual Role in Antitumor

Published OnlineFirst January 14, 2019; DOI: 10.1158/1535-7163.MCT-18-0936

Small Molecule Therapeutics Molecular Cancer Therapeutics Antimalarial Drug Pyrimethamine Plays a Dual Role in Antitumor Proliferation and Metastasis through Targeting DHFR and TP Huijuan Liu1,2, Yuan Qin1,3, Denghui Zhai1,3, Qiang Zhang1,3,JuGu1,3, Yuanhao Tang1,3, Jiahuan Yang1,3, Kun Li1,3, Lan Yang3, Shuang Chen3, Weilong Zhong1,3, Jing Meng1,3, Yanrong Liu3, Tao Sun1,3, and Cheng Yang1,3

Abstract

Pyrimethamine (Pyr), an antimalarial drug that targeting can inhibit the epithelial–mesenchymal transition (EMT), plasmodium dihydrofolate reductase (pDHFR), has been metastasis and invasion of lung cancer cells. These results proved to have antitumor activity. However, its direct target indicated that hDHFR is not the only target of Pyr. We further on cancer cells remains unclear. Methotrexate (MTX) is a found that thymidine phosphorylase (TP), an enzyme that is widely used anticancer drug that blocks human dihydrofolate closely associated with the EMT of cancer cells, is also a target reductase (hDHFR). In this work, we examined the anticancer protein of Pyr. The data retrieved from the Cancer Genome effects of Pyr in vitro and in vivo. Our results showed that Atlas (TCGA) database revealed that TP overexpression is hDHFR and pDHFR have similar secondary and three-dimen- associated with poor prognosis of patients with lung cancer. sional structures and that Pyr can inhibit the activity of hDHFR In conclusion, Pyr plays a dual role in antitumor proliferation in lung cancer cells. Although Pyr and MTX can inhibit the and metastasis by targeting DHFR and TP. Pyr may have proliferation of lung cancer cells by targeting DHFR, only Pyr potential clinical applications for the treatment of lung cancer.

Introduction target in anticancer drug development. In fact, DHFR inhibitors, Pyrimethamine (2,4-diamino-5-p-chlorophenyl-6-ethyl-pyri- such as methotrexate (MTX), have been applied in cancer treat- midine, Pyr) has been clinically used as antimalarial drugs (1). ment (10). The effect of Pyr on hDHFR has not been previously Pyr exerts its antimalarial effect by targeting plasmodium dihy- reported. drofolate reductase (pDHFR; ref. 2). DHFR is an essential enzyme MTX is extensively used in chemotherapy for several cancer in the synthesis of folic acid, which is a cofactor required for DNA types, including lung cancer, leukemia, lymphoma, breast cancer, synthesis. In addition to its antimalarial effects, Pyr exhibits the and head and neck cancers (11–13). Previous studies showed that activity of inducing apoptosis of tumor cells through cathepsin MTX treatment may also result in undesirable side-effects. For B–dependent and caspase–dependent apoptotic pathways (3, 4). example, MTX might induce lethal interstitial lung diseases, Pyr can also inhibit the STAT3 pathway in breast cancer cells (5). including pulmonary fibrosis in some cases (14). High doses of Pyr also has a broad range of effects in non–small cell lung MTX can also inflict structural and functional injury to the cancers (6). However, the target of Pyr has not been elucidated gastrointestinal tract (15), cause inflammatory response, and alter before. absorptive capacity (16–18). Some in vitro studies have shown Human DHFR (hDHFR) is a core enzyme in folate metabolism. that MTX may induce EMT of epithelial cells. MTX can promote It plays a key role in the biosynthesis of nucleic acids and is closely the migration and invasion of RLE/Abca3 cells and increase the associated with thymidylate synthase in purine and pyrimidine expression of TGF-b (19). MTX can also inflict damage on alveolar production (7–9). Given these characteristics, hDHFR is a crucial epithelial cells and promote the epithelial–mesenchymal transition (EMT) of epithelial cells (20, 21). During EMT, cells lose their typical epithelial characteristics and acquire mesenchymal 1 State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, traits (22). Cancer cells undergoing EMT lose their cell–cell Nankai University, Tianjin, China. 2College of Life Sciences, Nankai University, – 3 connection, cell matrix contact, and normal epithelial polarity Tianjin, China. Tianjin Key Laboratory of Molecular Drug Research, Tianjin fi International Joint Academy of Biomedicine, Tianjin, China. while gaining mesenchymal characteristics. These modi cations may enhance the migratory and invasive ability of cancer cells. Note: Supplementary data for this article are available at Molecular Cancer Given that hDHFR is a target of antitumor drug development Therapeutics Online (http://mct.aacrjournals.org/). and pDHFR is a target of Pyr in plasmodium, we first investigated H. Liu, Y. Qin, D. Zhai, and Q. Zhang contributed equally to the article. whether Pyr demonstrates antitumor activity by inhibiting Corresponding Authors: Huijuan Liu, No. 38 Tongyan Road, Haihe River Edu- hDHFR in tumor cells. We found that Pyr not only inhibits the cation Park, Jinnan District, Tianjin 300353, China. Phone: 22-6537-8882; E-mail: proliferation of cancer cells but also suppresses the migration of [email protected]; Tao Sun, [email protected]; Cheng Yang, lung cancer cells, whereas MTX could only inhibit the prolifera- [email protected] tion of cancer cells. These results suggested that DHFR is not the doi: 10.1158/1535-7163.MCT-18-0936 only target of Pyr. We found that Pyr might play a dual role in 2019 American Association for Cancer Research. antitumor proliferation and migration by synergistic targeting

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DHFR and thymidine phosphorylase (TP). TP is a nucleoside- Scientiﬁc). Data were analyzed using GraphPad and a log plot of metabolizing enzyme that has a crucial association with tumor cell viability (%) against the concentrations of drugs was con- migration and invasion (23). structed. The IC50 was also calculated from the plot.

Real-time cell proliferation monitoring Materials and Methods Cell proliferation assays were performed with the real time cell Protein sequence alignment and structural analysis analyzer (RTCA). Background impedance was measured with 50 ClustalX was used to blast the primary structure of Human mL of culture medium. NCI-H460 and A549 cells were seeded into dihydrofolate reductase (hDHFR) and Plasmodium falciparum plates (E-plate 16, ACEA Biosciences) with 100 mL of medium per Dihydrofolate Reductase (pDHFR). The secondary structure ele- well. Subsequently, the plates were monitored on the xCELLi- ments alignment of pDHFR and hDHFR was generated by the gence RTCA Dual Plate instrument (ACEA Biosciences) at 37Cin Esprint 3.0 server. Three-dimensional structures were aligned by a humidified atmosphere with 5% CO2. Pyr (15 mmol/L) and MTX using PYMOL. The crystal structures of the hDHFR–MTX complex (30 mmol/L) were added to the plate after the cells entered the (PDB code 1u72) and pDHFR–Pyr complex (PDB code 1j3j) were logarithmic growth period. The experiments were repeated three downloaded from the Protein Data Bank. Molecular docking was times. performed using Schrodinger software. MTX in hDHFR–MTX complex was extracted from crystal structures, and the pocket Live/dead and apoptosis analyses was used as the central docking location. Live/dead fixable dead cell stain kits (Invitrogen) were used to evaluate the effect of Pyr on cells in accordance with the manu- MD simulation facturer's instructions. Cell viability was analyzed through flow Energy minimizations and MD simulations were performed cytometry (Millipore guava easyCyte). with the Pmemd module of the Amber 14 package. To simulate An Annexin V-FITC/PI apoptosis detection kit (Nanjing Kaiji the normal physiological reaction temperature, the entire MD Biotechnology Development Co., Ltd.) was also used to evaluate system was gradually heated to 310 K. Periodic boundary condi- the effect of Pyr on cell apoptosis in accordance with the man- tions were used in the NPT ensemble and the SHAKE algorithm ufacturer's protocol. The cells (1 106) were evenly spread in a was applied to constrain all covalent bonds that involved hydro- 6-well plate and the drug was added after adherence. After 24 gen atoms. The cutoff values for nonbonded interactions were set hours, the cells were collected, Annexin V-FITC and PI were at 10 Å. Finally, the RMSD of the initial structure from the sequentially added according to the instructions. Experiments simulated positions was used to evaluate the stability of the entire were repeated three times. simulation. Wound-healing assay Binding-free energy calculations For the wound-healing assay, NCI-H460 and A549 cells were Thebinding-freeenergies(DGbind) of the ligands with grown on 24-well plates to 100% confluence. A 100 mm wound proteins were calculated through the MM–PBSA procedure in was scratched using sterile pipette tips, and the exfoliated cells AMBER14. The binding-free energy for each molecular species were washed off three times with PBS, and then Pyr (7.5 or (complex, protein, and ligand) was computed by using the 15 mmol/L) or MTX (15 mmol/L) was added to cells cultured in D ¼ þ equation Gbind Gcomplex (Gprotein Gligand). serum-free medium. Cell migration ability was assessed by mea- suring the movement of cells in the scratches in the wells. The Cell culture wound closure rate after 24 and 48 hours was measured and The cancer cell lines NCI-H460, NCI-H446, A549, HepG2, normalized to length at 0 hours. After 48 hours, images of the MHCC97L, LLC, MCF-7, ASPC-1, PCNA-1, SGC-7901, HT-29, wounds were acquired under light microscopy (Nikon, Japan). SW480 and PC-3 were obtained from KeyGen Biotech (Nanjing, The relative length values of the individual wounds were counted China) in 2013 and authenticated by STR genotyping. Mycoplasma according to the normalized length of 0 hours. was analyzed using the Mycoplasma qPCR Detection Kit (Sigma) before experiment. Cells were grown in medium supplemented Invasion assays with 10% FBS and maintained at 37 C in a humidified atmo- In this assay, 24-transwell plates were used. A total of 5 104 sphere containing 5% CO2. cells were placed on the top chamber inserts, which were coated with Matrigel (BD Biosciences). After incubation with Pyr (7.5 or Cell viability assay 15 mmol/L) or MTX (15 mmol/L) for 24 hours, the cells were The effects of Pyr and MTX on cell viability were determined stained with 0.1% crystal violet. Invading cells were visualized and through the MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- counted in six randomly selected fields under an inverted micro- phenytetrazoliumromide) assay. Pyr and MTX were purchased scope (100). from Meilun Biotechnology Co., LTD., and the chemical structures of them were showed in Supplementary Materials (Supplemen- Western blot analysis tary Fig. S1). A total of 5 103 cells were seeded in 96-well culture After treatment with different drugs, proteins were extracted plates. Then, the cells were treated with various concentrations from NCI-H460 cells and analyzed through western blot analysis. MTX and Pyr (0–200 mmol/L). After 24, 48, and 72 hours of After the culture medium was aspirated, each dish was washed incubation, the cells were stained with MTT. Then, the culture with PBS, and protein lysis buffer was added (containing protease medium was removed, and cells were lysed using DMSO. Finally, and phosphatase inhibitors) to extract the proteins. The proteins the optical density (OD) values of the solution were determined at were separated by 10% polyacrylamide gel electrophoresis, trans- 570 nm by using a microplate reader (Multiskan FC, Thermo ferred to a polyvinylidene fluoride (PVDF) membrane (the PVDF

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membrane was activated by methanol), and blocked with 5% MMP9. All antibodies were obtained from Affinity and diluted skim milk. Proteins were incubated with primary antibodies at the rate of 1:50. Brown-stained cytoplasm, nuclei, or mem- against b-actin (Affinity, 1:5,000), E-cadherin (Affinity, branes in cells were considered positive. Staining intensity was 1:1,000), Vimentin (Affinity, 1:1,000 dilution), Ki-67 (Affinity, scored as follows: none (0), weak brown (1þ), moderate brown 1:1,000), MEK2 (Affinity, 1:1,000), ERK2 (Affinity, 1:1,000), and (2þ), and strong brown (3þ). The percentage of stained cells GAPDH (Affinity, 1:5,000). The samples were incubated with was divided into five classes: 0 for negative cells, 1 for 1%–25%, primary antibody overnight in a rotator at 4C. Blots were further 2 for 25%–50%, 3 for 50%–75%, and 4 for >75%. incubated with horseradish peroxidase–labeled secondary antibodies (Affinity, 1:5,000). Finally, target proteins were visualized Biacore assay and protein thermal shift assay using ECL substrate reagents (Millipore). Biacore 3000 instrument (GE Healthcare) was used in the experiment. TP was immobilized on CM5 sensor chips in accor- Immunofluorescent staining dance with the instructions provided with the Biacore Amini NCI-H460 cells seeded on a cell-climbing slice were incubated Coupling Kit. Pyr was diluted in running buffer at different for 24 hours with Pyr (7.5 or 15 mmol/L) or MTX (15 mmol/L) in concentrations and injected into TP-immobilized CM5 sensor 24-well culture plates. The cells were fixed in 3.7% paraformal- chips. The concentrations of Pyr were 0, 0.25, 0.5, 1, 2, 4, and dehyde for 15 minutes and then treated with 0.1% Triton X-100 8 mmol/L. The surface of the control chip was prepared in the for 10 minutes, after which the cells were incubated with 3% BSA same manner for data correction. BIA evaluation software was for 30 minutes. The cells were then incubated overnight with adopted for data analysis. primary antibodies at 4C. Cells were washed four times with PBS Thermal shift assay (TSA) was performed using SYPRO Orange and incubated for 30 minutes with secondary antibodies. Finally, (Life Sciences) as the shift reporter dye. Briefly, 11.4 mg of protein the cells were covered with DAPI for 15 minutes. Proteins were was incubated with Pyr at a ratio of 1:10 or 1:20 for 20 minutes, visualized through confocal microscopy (Nikon, Japan). dye was added, and the reactions were monitored in real time (Bio-Rad MiniOpticon; excitation, 490; emission, 575 nm) from Animal studies 29Cto95C with a rate of change of 1C/min. The melt curve is C57BL/6J mice (male, 5–6-weeks-old) were maintained in a represented as normalized data and calculated as d(fluorescence)/ specific pathogen-free animal care facility. The mice were allowed d (temperature). to acclimate for 7 days before the experiment. All animal studies were carried out in accordance with National Institutes of Health TP activity assay Animal Use Guidelines and the current Chinese Regulations and TP activity was reflected by intercellular thymine concentration, Standards for the Use of Laboratory Animals. All animal proce- which was detected through LC–MS–MS (24). NCI-H460 cells dures were approved on the basis of guidelines of the Animal were incubated for 24 hours with Pyr (30 mmol/L), 5UIR Ethics Committee of the Tianjin International Joint Academy of (30 mmol/L), and MTX (30 mmol/L). Next, 1 107 cells were Biotechnology and Medicine. Lung cancer xenografts were estab- lysed with ice-cold 80% methanol. After centrifugation, 0.05 mgof 7 13 15 lished by subcutaneously injecting 1 10 cells (suspended in U- C10 and U- N2 thymidine (Sigma) were added to the super- saline) into the flanks of the mice. After the tumors volume natant as the internal control. The polar metabolites in the reached approximate 100 mm3, the mice were randomly divided supernatant were separated, dried, and reconstituted with the LC into four groups (n ¼ 5). Pyr (7.5 or 15 mg/kg), MTX (7.5 mg/kg) mobile phase. Intercellular thymidine level was measured or saline were orally administered to the mice once a day. Tumor through LC–MS–MS in the positive-ion mode and expressed as volume and body weight were measured daily after tumor inoc- ng/1 107 cells. All experiments were repeated independently at ulation. Tumor volumes were calculated in accordance with the least twice. formula V ¼ ab2/2 (a ¼ length and b ¼ width). After 2 weeks of treatment, all mice were euthanized. The xenografts and lungs Effect of Pyr on TP induced EMT were resected and measured. Metastases in lung tissues were The methods of wound-healing assay, invasion assays and observed by using a stereoscopic microscope and detected western blot were same as mentioned above. A549 cells were through hematoxylin/eosin staining. used in the experiments. Cells were divided into four groups: control (treated with solvent), TP (treated with TP 10 ng/mL), Pyr Hematoxylin/eosin staining (treated with Pyr 15 mmol/L) and PyrþTP (Pyr 15 mmol/L com- Tumor and lung tissues were fixed in 10% formaldehyde, bined with TP 10 ng/mL). dehydrated, and embedded in paraffin wax. Then, 4-mm sections of the tissues were stained with hematoxylin and eosin. Digital Proteomics analysis images were acquired under microscopy (Nikon, Japan). Proteomics analysis was used to identify the differentially expressed proteins of A549 cells treated versus non-treated Immunohistochemical analysis with Pyr (7.5 mmol/L), which were significantly regulated Tissues were deparaffinized and rehydrated through incuba- (|logFC|>1.5) in the samples treated with Pyr. To initially explore tion with xylene and decreasing concentrations of ethanol. which functions and pathways have changed in the Pyr groups, we Endogenous peroxidase activity was blocked with 3% hydrogen used metascape website (http://metascape.org/) to perform GO peroxide. The microwave antigen retrieval technique was used and KEGG enrichment analysis. Protein-protein interaction (PPI) for antigen retrieval. Samples were incubated overnight network was analyzed using STRING website (www.string-db. with primary antibodies at 4C after blocking with rabbit org/) and Cytoscape software. To get more reliable data, we only polyclonal anti–E-cadherin, rabbit polyclonal anti-vimentin, chose the interactions of the combination score >0.9. To further rabbit polyclonal anti-MMP2, and rabbit polyclonal anti- study which proteins play a greater role in the PPI network,

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CentiScape 2.2 plug-in module of Cytoscape was performed to (RMSD) obtained by aligning the three-dimensional structures calculate the degree of connectivity in the PPI network. To better of the proteins was 0.679 (Fig. 2B). On the basis of the alignment understand the biological significance of the PPI network, results, we docked Pyr into the active sites of hDHFR and pDHFR. MCODE plug-in module was used to select most significant The conformations and orientations exhibited by Pyr in the active (MCODE score >10) sub-modules. centers of hDHFR and pDHFR are almost identical. The docking score of Pyr and hDHFR is 7.483, and the docking score of Pyr DHFR activity assay and pDHFR is 7.140 (Fig. 2B). Therefore, Pyr may have similar Commercially available Human DHFR ELISA Kit (Wuhan binding capacities for hDHFR and pDHFR. We ran 50 ns molec- Elabscience Biotechnology Co., Ltd.) was used for assaying of ular dynamics (MD) simulations for the complexation of hDHFR the activity of the DHFR in lung cancer cells. NCI-H460 cells were with Pyr, pDHFR with Pyr, and hDHFR with MTX. We analyzed used to test the activity of DHFR. The cells (1 106) were evenly the RMSD values provided by the simulations to illustrate the spread in a 6-well plate and the drug was added after adherence. dynamic stability of the three complexes and to ensure the After 24 hours, the cell supernatant was collected, centrifuged at rationality of the following analysis (Fig. 2C). The RMSD of each 1,000 g for 20 minutes to remove impurities and cell debris, and system tended to converge. This tendency indicated that the the supernatant was collected. Then the activity of DHFR was systems are stable and in equilibrium. To further compare the tested according to the product manual. binding of Pyr to hDHFR and pDHFR, we calculated the binding- free energies of all three systems by using the MM-PBSA program TCGA data analysis in AMBER. As shown in Fig. 2D, the binding capacities of Pyr for The data of DHFR expression levels were obtained from hDHFR and pDHFR are almost the same. The binding capacity of "Human Protein Atlas" database (https://www.proteinatlas.org/ Pyr–hDHFR complex was weaker than MTX–hDHFR complex. ENSG00000228716-DHFR/cell). Survival data of patients with The hydrogen bond interactions that occur between the drug and lung cancer were downloaded from TCGA. There are 982 patients protein play an important role in the binding of the inhibitor to with lung cancer and 12 normal ones. Overall survival and kinase in the three protein-inhibitor systems. Our results showed disease-free survival was analyzed according to the expression of that Pyr and MTX formed stable hydrogen bonds with Ile 7, Tyr TP or DHFR in 982 cases of lung cancer patient (using the Kaplan– 121, and Val 115 in hDHFR. The above results suggested that Meier method and evaluated using the log-rank test). According to similar to MTX, Pyr can act as a hDHFR inhibitor. the data, the FPKM (fragments per kilobase of exon per million fragments mapped) value of more than 2.8 is classified as "high," Pyr inhibits the proliferation of lung cancer cells in vitro and the FPKM value of less than 2.8 is classified as "low." We used the MTT assay to detect the effect of Pyr on cell viability GraphPad was used for mapping. at 24, 48, and 72 hours. As shown in Fig. 3A, the IC50 values of Pyr for NCI-H460 cells at 24, 48, and 72 hours treatment are 98.17, 64.31, and 37.60 mmol/L, respectively. As shown in Fig. 3B, the Results IC50 values of Pyr for A549 cells at 24, 48, and 72 hours treatment Pyr exerts an inhibitory effect on lung cancer cells are 83.37, 40.57, and 28.07 mmol/L, respectively. Next, we used a Pyr is a known inhibitor of pDHFR. However, its effect on real-time cell analyzer (RTCA) to demonstrate the effects of Pyr on hDHFR has not been verified. We used DHFR assay kits to test the the proliferation of NCI-H460 and A549 cells. We found that Pyr effect of Pyr on hDHFR in lung cancer cells. Our results showed inhibited the proliferation of NCI-H460 and A549 cells in a dose- that both MTX and Pyr could inhibit the activity of DHFR in lung dependent manner (Fig. 3C and E). We also used the live/dead cancer cells (Fig. 1A). MTX, a clinically used chemotherapy drug assay to explore the effect of Pyr (15 and 30 mmol/L) on NCI-H460 targeting DHFR, exerts an inhibitory effect on different tumor and A549 cells. The percentage of cell death increased in a dose- cells. So we detected the inhibitory effect of Pyr on different dependent manner (Fig. 3D and F). We used an Annexin V-FITC/ tumors cells. Fig 1B and C showed the inhibitory effects of MTX propidium iodide (PI) kit to examine the effect of Pyr on cell and Pyr on various types of tumor cells in vitro. Our findings apoptosis and found that Pyr can effectively induce apoptosis in showed that Pyr showed inhibitory activity to a variety of cell the NCI-H460 and A549 cells in a dose-dependent manner lines, such as MCF-7, NCI-H460, NCI-H446 and so on. The (Fig. 3G and H). The western blot analysis results also showed expression levels of DHFR in several cell lines from the Human that Pyr treatment decreased the expression level of Ki67, which is Protein Atlas Database are displayed in Fig. 1D. The results of a marker of cell proliferation (Fig. 3I). correlation analysis showed that the IC50 values of MTX and Pyr for cancer cells were positively correlated with the expression level Pyr can inhibit the migration, invasion, and EMT of lung cancer of DHFR (Fig. 1E and F). cell lines We performed the wound-healing assay to investigate the pDHFR and hDHFR possess similar three-dimensional ability of Pyr to inhibit the migration of NCI-H460 (Fig. 4A) and structures A549 (Fig. 4B) cells. The migration ability of cells increased after We aligned orthologous pDHFR and hDHFR sequences (down- 48 hours of treatment with MTX. By contrast, wounds were loaded from UniProt) to identify the residues and secondary widened under Pyr treatment. This behavior indicated that Pyr structures that were shared by these enzymes. pDHFR and hDHFR treatment inhibited the migration of cancer cells. We also detected shared low amino acid sequence identity (Fig. 2A). Aligning the the effect of Pyr on cell invasiveness (Fig. 4C). The number of cells secondary and three-dimensional structures of hDHFR and that invaded through the Matrigel-coated filter decreased under pDHFR revealed that the structures of the two enzymes are mainly Pyr treatment relative to the control. We used western blot analysis differentiated by an a-helix (circled in red), which is consistent and immunofluorescent staining to detect the effect of Pyr and with previous report (25). The root–mean–square deviation MTX on the expression of EMT markers E-cadherin and vimentin.

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Figure 1. Pyr exerts an inhibitory effect on various types of tumor cells, which is positively correlated with the expression level of DHFR. A, DHFR activity of NCI-H460 cells under Pyr or MTX treatment. The experiment was performed in triplicate. Results are shown as means SD (, P < 0.01). B, Inhibition rate of MTX (50 mmol/L) for different cancer cell lines. C, Inhibition rate of Pyr (50 mmol/L) for different cancer cell lines. D, Expression level of DHFR in different cancer cell lines obtained from the Human Protein Atlas Database. E, Correlation between the inhibition rate of MTX and the expression level of DHFR in different cancer cell lines. F, Correlation between the inhibition rate of Pyr and the expression level of DHFR in different cancer cell lines.

We found that Pyr decreased the expression of vimentin and Pyr inhibits tumor growth and metastasis in vivo increased the expression of E-cadherin in NCI-H460 cells (Fig. 4D We examined the effect of Pyr on Lewis lung cancer (LLC) and E), whereas MTX exerted the opposite effect. The same results xenografts in C57BL/6J mice. The body weights of mice in the MTX were observed in A549 cell lines (Supplementary Figs. S2 and S3). treatment group decreased relative to those of mice in the model

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Figure 2. Pyr can bind to human DHFR (hDHFR). A, Alignment of the hDHFR and pDHFR sequence through the Esprint 3.0 server. B, Three-dimensional structure alignment of hDHFR and pDHFR (RMSD) and the major differences between the two proteins are marked with red circles. Binding mode of Pyr during docking in the active site of hDHFR and pDHFR (hDHFR-Pyr are shown in blue, and pDHFR-Pyr are shown in yellow.). C, Analysis of RMSD values of Pyr–hDHFR, Pyr– pDHFR, and MTX–DHFR complexes. D, Binding-free energies of Pyr–hDHFR, Pyr–pDHFR, and MTX–DHFR complexes calculated using the MM-PBSA program in AMBER.

group. No signiﬁcant change was noted between the model group increased after MTX treatment. Immunohistochemical results of and Pyr treatment group (Fig. 5A). Tumor growth was suppressed tumor tissues revealed that E-cadherin expression levels were in the MTX and Pyr treatment groups relative to that in the model higher in the Pyr treatment group than those in the control groups group (Fig. 5B and C). As shown in Fig. 5D–F, the number of and the expression levels of vimentin, MMP2, and MMP9 metastatic tumor nodes on lung surfaces drastically decreased decreased in the Pyr treatment groups (Fig. 5G). Western-blot after Pyr treatment. Meanwhile, the extent of lung metastasis analysis showed the same results (Supplementary Fig. S4).

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Figure 3. Pyr can inhibit the viability and proliferation of lung cancer cells in vitro. A, Survival rates of NCI-H460 cells treated for 24, 48, and 72 hours with Pyr. B, Survival rates of A549 cells treated for 24, 48, and 72 hours with Pyr. C, Real-time proliferation curve of NCI-H460 cells treated with 15 and 30 mmol/L Pyr assayed with a real-time cell analyzer (RTCA). D, Proportion of dead NCI-H460 cells after treatment with 15 and 30 mmol/L Pyr detected with the live/dead assay kit. E, Real- time proliferation curve of A549 cell under treatment with 15 and 30 mmol/L Pyr assayed by the RTCA. F, Proportion of dead A549 cells after treatment with 15 and 30 mmol/L Pyr detected with the live/dead assay kit. G and H, Effect of Pyr on cell apoptosis detected by Annexin V-FITC/PI kit. I, Effect of Pyr on the marker of cell proliferation Ki67 as detected through western blot analysis. Each experiment was performed in triplicate. Results are shown as means SD (, P < 0.05 and , P < 0.01).

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Figure 4. Pyr inhibits the migration, invasion, and EMT of lung cancer cells. A, Effect of Pyr or MTX on the migration of NCI-H460 at 24 and 48 hours. B, Effects of Pyr and MTX on the migration of A549 cells at 24 and 48 hours. C, Transwell chambers were used for the invasion assay, and images were obtained under 200 magniﬁcation. NCI-H460 and A549 cells were treated with Pyr or MTX. D, Changes of E-cadherin and vimentin expression in NCI-H460 cells treated with Pyr or MTX (Western blot assay). b-Actin was used as the loading control. E, Changes of E-cadherin and vimentin expression in NCI-H460 cells treated with Pyr or MTX (immunoﬂuorescence assay). Each experiment was performed in triplicate. Results are shown as means SD (, P < 0.05 and , P < 0.01).

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Figure 5. Pyr can inhibit tumor growth and metastasis in Lewis lung cancer (LLC) xenografts, whereas MTX can only inhibit the tumor growth. A, Body weight (g) changes in the animals with LLC xenografts after treatment with Pyr or MTX. B, Changes in the tumor volume of LLC xenografts after treatment with Pyr or MTX. C, Representative images of LLC xenograft tumor tissues treated with Pyr or MTX. D and E, Number of metastatic tumor nodes in lung tissues. The metastasis of LLC xenografts is inhibited by Pyr but is promoted by MTX. F, Pathological sections of metastatic tumor nodes in lung tissues observed through hematoxylin/ eosin staining (40). G, Effect of Pyr and MTX on the expression of EMT markers in LLC xenograft tumor tissues, as observed through immunohistochemistry analysis (40). Brown or yellow staining was considered as positive expression. Each experiment was performed in triplicate. The results are shown as means SD (, P < 0.05 and , P < 0.01).

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Pyr targets TP and inhibits its activity tissues (Fig. 8A and B). The results from the ULCAN database Pyr could not only inhibit the proliferation of lung cancer cells revealed that the mRNA levels of the two proteins in lung cancer like MTX, but also inhibit EMT, migration, and invasion of lung tissues were elevated relative to those in normal lung tissues cancer cells. Thus, we speculated that Pyr may have another EMT- (Fig. 8C). We also analyzed the samples in the TCGA database associated target in tumor cells. Pyr is a pyrimidine analog. TP on the basis of pathology grade and clinical stage. The mRNA plays an important role in tumor migration, and invasion. The expression of DHFR was positively correlated with clinical phase I, chemical structures of Pyr and thymidine were similar. So we clinical phase II, and clinical phase III. And the mRNA expression hypothesized that TP may be another target of Pyr. We performed of TP was positively correlated with clinical phase II and clinical molecular docking simulations to compare the binding scores of phase III (Fig. 8D). We analyzed the effect of DHFR and TP the TP inhibitors (TPI, 5-Iodouracil, and 5-fluorouracil), Pyr, and expression on survival status. DHFR and TP overexpression were MTX. We found that Pyr and TP inhibitors have similar docking associated with poor prognosis (Fig. 8E–G). To further investigate scores and that MTX cannot enter the active center of TP (Fig. 6A; the relationship between DHFR/TP expression and EMT, we Supplementary Fig. S5). We used LC–MS–MS to detect thymine analyzed the correlation between the mRNA expression levels of concentration in cells treated with 5UIR and Pyr, which can reflect DHFR/TP and EMT markers E-cadherin (gene name: CDH1) and TP activity. The results showed that Pyr inhibited the activity of TP vimentin (gene name: VIM). We found that TP was positively (Fig. 6B). Moreover, we verified the interaction between Pyr and correlated with vimentin expression and negatively correlated TP through the Biacore assay (Kd ¼ 6.19 0.78 mmol/L; Fig. 6C). with E-cadherin expression. However, the expression levels of The TSA assay also showed that Pyr could bind with TP (Supple- DHFR and EMT markers were not correlated (Fig. 8H). mentary Fig. S6). To further explore the inhibitory effect of Pyr on TP, we conducted a wound-healing assay. Our results showed that TP Discussion induces the migration and invasion of lung cancer cells. Pyr Pyrimethamine (Pyr) is a pyrimidine derivative, which inter- inhibited the TP-induced migration and invasion of cancer cells feres with the regeneration of tetrahydrofolic acid from dihydro- (Fig. 6D and E). Compared with the control treatment, TP folate by targeting DHFR of the plasmodium. Because pDHFR and promoted the expression of ERK2 and MEK2, whereas Pyr sup- hDHFR possess similar three-dimensional structures, so Pyr was pressed the expression of ERK2 and MEK2 (Fig. 6F). TP increased used for anticancer drug research. Our results showed that Pyr the expression of vimentin and decreased the expression of could bind to hDHFR, but the binding capacities of Pyr and E-cadherin, and Pyr reversed the changes of EMT markers, which hDHFR were weaker than MTX and hDHFR. We found that Pyr showed that Pyr inhibited the EMT induced by TP (Fig. 6F). could effectively inhibit the proliferation of many cancer cell lines, and the effect is equivalent to MTX in vitro, which suggested that Effects of Pyr on proteomics profiles of lung cancer cells DHFR was a driving force for tumor cell proliferation, and even Proteomics analysis was used to identify the differentially mild inhibition could significantly affect the proliferation of cells. expressed proteins of A549 cells treated or non-treated with Pyr. Besides, we found that Pyr inhibited the EMT, migration, and Gene Ontology (GO) analysis results showed that the differential invasion of lung cancer cells. We further demonstrated that TP proteins were enriched in the functions of pyridine nucleotide might be another target protein of Pyr, which plays an important metabolic process, apoptotic signaling pathway, regulation of role in EMT. cell-cycle G2–M phase transition, cell cycle phase transition, and DHFR, a folate-dependent enzyme that is related to DNA extracellular matrix organization. The KEGG pathway analysis synthesis in cancer cells, has become a crucial target enzyme of revealed that the differential proteins were mainly involved in antitumor drugs (26). DHFR positively regulates the proliferation several pathways, including programmed cell death, focal adhe- of tumor cells, and its expression is markedly elevated in tumor sion, extracellular matrix organization, collagen formation, col- cells. MTX is an antitumor drug that targets DHFR and exhibits lagen degradation, and cell-cycle pathway. Protein–protein inter- inhibitory activity against lung cancer, breast cancer, acute leu- action (PPI) network was shown in Fig. 7B. The hub proteins were kemia, and other malignancies (27–30). Pyr is an antimalarial marked in red, such as HSP90AA1, CKAP5, NEDD4L, and CPSF1. drug targeting plasmodium dihydrofolate reductase (pDHFR). It The sub-networks (MCODE score > 10) from PPI network were can induce tumor cell apoptosis through the bilateral mechan- shown in Fig. 7C–E. The functions of the sub-networks mainly isms of caspases and cathepsins (3). Pyr can also inhibit tumor associated with cell-cycle phase transition, protein ubiquitina- growth by regulating the activity of matrix metalloproteinases tion, collagen degradation, and nucleic acid binding. These results (MMPs) and telomerase (4, 31). Pyr can influence the activity of indicated that Pyr affected not only the proliferation-related STAT3 in TUBO and TM40D-MB metastatic breast cancer cells (5). pathways (e.g., pyridine nucleotide metabolic process, apoptotic The target protein of Pyr in human cancer cells has not been signaling pathway, and cell-cycle pathway) of tumor cells, but also reported. Our results showed that the tertiary structure of pDHFR migration and invasion-related pathways (eg. Focal adhesion, is highly similar to that of hDHFR. Pyr can block the proliferation Extracellular matrix organization, Collagen formation, and Col- of tumor cells by binding to hDHFR. lagen degradation) of tumor cells, which is consistent with the Although MTX can inhibit the proliferation of tumor cells, it functions of Pyr observed in vivo and in vitro. induces some adverse side effects. Methotrexate (MTX) can lead to alveolar epithelial cell injury followed by pulmonary fibrosis as a DHFR and TP are associated with lung cancer malignancy result of the recruitment and proliferation of myofibroblasts. MTX We analyzed the expression of DHFR and TP in tissues from 982 induces the EMT-like phenotype of A549 cells accompanied by patients with lung cancer included in the TCGA database. We increased interleukin-6 (IL-6) and transforming growth factor found that the expression levels of DHFR and TP in lung cancer (TGF)-b1, and enhance the migration of A549 cells (20, 21). tissues were significantly higher than that in normal human lung MTX can also induce the EMT of type II alveolar epithelial

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Figure 6. Pyr can bind to TP and inhibit TP-induced migration, invasion, and EMT of lung cancer cells. A, Molecular docking results for TP and Pyr. B, Thymine concentration of NCI-H460 cells treated with 5-UIR, Pyr, and MTX. Intercellular thymine concentration was used as an index of TP activity. C, Interaction between Pyr and TP veriﬁed through Biacore assay (Kd ¼ 6.190.78 mmol/L). D, Pyr can inhibit the TP-induced migration of cancer cells. Wound-healing assay was used in this experiment. E, Pyr can inhibit the TP-induced invasion of cancer cells. Transwell chambers were used for the invasion assay, and images were obtained under 200 magniﬁcation. F, E-Cadherin, vimentin, MEK2, and ERK2 expression levels of NCI-H460 cells in different treatment groups. The GAPDH blot served as the loading control. Data are presented as the means of three experiments, and error bars represent the standard deviation (, P < 0.05 and , P < 0.01).

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Figure 7. The effect of Pyr on proteomics proﬁles of A549 cells. A, Gene Ontology (GO) analysis and KEGG analysis results of the differentially expressed proteins in Pyr- treated cells. B, Protein–protein interaction (PPI) network of the differentially expressed proteins (the interactions of the combination score >0.9). The hub proteins were marked in red. C–E, The three sub-networks (MCODE score >10) obtained from PPI network.

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Figure 8. TP and DHFR are associated with lung cancer prognosis. A, Expression levels of DHFR protein in normal human lung tissues (n ¼ 6) and lung cancer tissues (n ¼ 36). B, Expression levels of TP protein in normal human lung tissues (n ¼ 6) and lung cancer tissues (n ¼ 46). C, mRNA expression levels of DHFR and TP in normal human lung tissues (n ¼ 12) and lung cancer tissues (n ¼ 982). D, Relationship between the mRNA expression level of DHFR/TP and the clinical stage of patients with lung cancer. E–G, Effect of the mRNA expression levels of TP and DHFR on the median survival time of patients with lung cancer. TP and DHFR are associated with lung cancer prognosis. H, Relationship of TP and DHFR expression level with EMT markers expression. There is a correlation between the mRNA expression of TP and EMT markers. I, The simpliﬁed schematic diagram of Pyr and MTX antitumor mechanism. The data of DHFR expression levels were obtained from "Human Protein Atlas" database. Survival data of patients with lung cancer were downloaded from TCGA. Results are shown as means SD (, P < 0.05; , P < 0.01; , P < 0.001).

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RLE/Abca3 cells like TGF-b1 in vitro (19). Our results showed high TP expression have poor prognoses and suggested that TP can that low doses of MTX can cause the migration and invasion of be a target in the development of drugs against lung cancer. lung cancer cells and promote EMT of A549 and NCI-H460 cells In conclusion, Pyr not only targets DHFR to repress the pro- in vitro. In LLC xenografts in C57BL/6J mice, MTX increased the liferation of lung cancer cells but also inhibits EMT of lung cancer metastatic tumor nodes on lung tissue. cells through the TP/MEK2/ERK2/MMPs pathway and thereby We found that Pyr not only inhibits the proliferation of lung impeding the migration and invasion of tumor cells (Fig. 8I). Pyr cancer cells but also blocks the EMT, migration, and invasion of may have potential clinical applications as a novel dual effective lung cancer cells. Therefore, we speculated that Pyr inhibited the anti-lung cancer drug. EMT of lung cancer cells through other target. The chemical fl structure of Pyr is similar to that of thymidine, a substrate of TP, Disclosure of Potential Con icts of Interest fl which indicate that TP may be a target of Pyr. The molecular No potential con icts of interest were disclosed. docking simulations and Biacore assay results revealed that Pyr Authors' Contributions has binding ability with TP. Pyr can also inhibit the enzymatic Conception and design: H. Liu, Y. Qin, T. Sun, C. Yang activity of TP. Development of methodology: Y. Qin, Q. Zhang TP is closely related to the migration, invasion, and EMT of Acquisition of data (provided animals, acquired and managed patients, tumor cells and is an important enzyme in the pyrimidine provided facilities, etc.): H. Liu, Y. Qin, D. Zhai, J. Yang, K. Li, L. Yang pathway. TP expression in tumor tissue is elevated relative to that Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Gu, Y. Tang, W. Zhong in normal tissue, and low TP expression is associated with pro- Writing, review, and/or revision of the manuscript: H. Liu, Y. Qin, D. Zhai, longed patient survival (32, 33). TP can stimulate the migration of S. Chen, T. Sun, C. Yang human endothelial cells by specifically activating integrins a5b1 Administrative, technical, or material support (i.e., reporting or organizing and aVb (34). TP exerts a chemotactic effect on endothelial cells data, constructing databases): J. Meng, Y. Liu in vitro, induces tumor angiogenesis, and promotes tumor cell Study supervision: C. Yang – migration in vivo (35 37). TP facilitates cell matrix degradation Acknowledgments and tumor invasion by promoting MMP2/9 expression. TP affects This study was founded by Foundation for National Natural Science Funds of the expression of MMPs through the MAPK/Erk2 pathway (38). China (Grant No. 81572838 and 81703581), National Science and Technology Given that the activation of the Erk pathway plays an important Major Project (Grant No. 2018ZX09736005), Tianjin science and technology role in EMT (39), TP may also influence the EMT of cancer cells. In innovation system and the condition of platform construction plan (Grant this work, we verified that TP could induce the EMT of lung cancer No.14TXSYJC00572), Post-doctoral innovative talent support program (Grant cells. Because the induction of EMT is the primary mechanism by No. BX20180150). which epithelial cancer cells acquire malignant phenotypes that The costs of publication of this article were defrayed in part by the payment of promote metastasis, TP may become a target for the development page charges. This article must therefore be hereby marked advertisement in of anti-tumor metastasis drugs. In this work, we found that Pyr accordance with 18 U.S.C. Section 1734 solely to indicate this fact. inhibits TP-induced ERK2 and MEK2 expression and that Pyr reverses the EMT of cancer cells induced by TP. The results of the Received August 16, 2018; revised November 16, 2018; accepted January 11, TCGA data analysis indicated that patients with lung cancer with 2019; published first January 14, 2019.

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Antimalarial Drug Pyrimethamine Plays a Dual Role in Antitumor Proliferation and Metastasis through Targeting DHFR and TP

Huijuan Liu, Yuan Qin, Denghui Zhai, et al.

Mol Cancer Ther 2019;18:541-555. Published OnlineFirst January 14, 2019.

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