Vol. 42, No. 1 Biol. Pharm. Bull. 42, 123–129 (2019) 123 Regular Article

Targeting Pyruvate M2 and II, Pachymic Acid Impairs Metabolism and Induces Mitochondrial Apoptosis Guopeng Miao,* Juan Han, Jifeng Zhang, Yihai Wu, and Guanhe Tong* Department of Bioengineering, Huainan Normal University; Huainan 232038, China. Received September 20, 2018; accepted October 18, 2018; advance publication released online October 30, 2018

Pachymic acid (PA), a triterpenoid from Poria cocos, has various pharmacological effects, including anti-inflammatory, anti-cancer, anti-aging, and insulin-like properties. PA has gained considerable research attention, but the mechanism of its anti-cancer effects remains unclear. In this study, M2 (PKM2) was discovered as a PA target via the drug affinity responsive target stability. Molecular docking and assay revealed that PA is a competing activator of PKM2, and mimics the natural activator, fructose-1,6-bisphosphate. PKM2 activation should augment the flux of . However, decreased glu- cose uptake and lactate production after PA treatment was observed in SK-BR-3 breast carcinoma cells, indicating a blockage or downregulation of glycolysis. The potential of previously reported triterpenoids in blocking hexokinase II (HK2) activity inspired us to investigate the inhibition effect of PA on HK2 activity.

Molecular docking and enzyme assay confirmed that PA was an inhibitor of HK2, with an IC50 of 5.01 µM. The possible consequences of glycometabolic regulation by PA, such as dissociation of HK2 from the mito- chondria, release of mitochondrial cytochrome (Cyt) c, depletion of ATP, and generation of reactive oxygen species, were further validated. Furthermore, the details of the possible linkage of targeting PKM2 and HK2 with previously reported actions of PA were discussed. The results of our study provided valuable informa- tion on the anti-cancer mechanisms of PA. Key words pachymic acid; target; pyruvate kinase; hexokinase; reactive oxygen species; apoptosis

INTRODUCTION protection against proteolysis conferred on the target protein by interacting with small molecules.12) In this study, pyruvate Pachymic acid (PA) is a lanostane-type triterpenoid and kinase isoform M2 (PKM2) and hexokinase II (HK2) were major component of the alcoholic extract of Poria cocos. This found potential targets of PA, through DARTS and molecu- triterpenoid possesses various pharmacological effects, includ- lar docking, respectively. In vitro experiments showed that ing anti-inflammatory, anti-cancer, anti-aging, and insulin-like PA was an inhibitor of HK2 and an activator of PKM2. The properties.1–5) decreased glucose uptake and lactate production, HK2 dis-

PA inhibits phospholipase A2, which is responsible for the sociation from mitochondria, cytochrome c (Cyt c) release, of the sn-2 position of membrane glycerophospho- and ROS generation supported the functions of PA in glucose lipids, to release arachidonic acid, a precursor of eicosanoids, metabolism and illuminated the mechanism of the anti-cancer including prostaglandins (PGs) and leukotrienes.2,6) Several effect of PA. studies have revealed that PA can inhibit lipopolysaccharide- and oxidative-induced inflammation by suppressing extracel- MATERIALS AND METHODS lular signal-regulated kinase 1/2 (ERK 1/2) and p38 signaling pathways7) and nuclear factor (NF)-κB.8) In lung cancer cells, Materials The PA used in this study was purchased from PA induces apoptosis by activating reactive oxygen species Dasf-Bio, Nanjing, China, and it has a purity higher than 98%. (ROS)-dependent c-Jun N-terminal kinase (JNK) and endo- Unless otherwise indicated, chemicals and reagents used in plasmic reticulum (ER) stress pathways.4) In the experiment this study were of analytical grade. that investigated the effects of PA on the invasion of breast DARTS with Cancer Lysates DARTS was per- carcinoma cells, the NF-κB signal pathway was suppressed by formed according to the method of Lomenick et al., with some PA, resulting in the downregulation of matrix metalloprotein- modifications.13) SK-BR-3 breast carcinoma cells were lysed ase-9, which is an extremely relevant enzyme for tumor inva- in buffered saline (PBS) containing 0.5% Triton sion. However, PA did not alter the phosphorylation states of X-100, protease inhibitor (cOmplete™, Sigma-Aldrich, St. mitogen-activated protein (MAPKs), including ERK, Louis, MO, U.S.A.) and phosphatase inhibitor cocktail (P8340, JNK, and p38 kinase.3) Moreover, PA suppresses the growth Sigma-Aldrich). Protein concentrations were determined using and invasiveness of EJ bladder cancer, osteosarcoma, and gas- a bicinchoninic acid assay kit (Beyotime, China), and bovine tric cancer cells.9–11) PA has gained research attention, but the albumin was used as the standard. All the above steps were mechanisms of its anti-inflammation and anti-cancer effects performed on ice. PA (final concentration of 10 µM) or di- remain unclear. methyl sulfoxide (DMSO) was added into the cell lysates and Drug affinity responsive target stability (DARTS) is a incubated at 25°C for 30 min. Samples were digested enzymat- quick and straightforward approach to identify potential pro- ically with pronase (Sigma-Aldrich) at an enzyme:lysate (w/w) tein targets for small molecules. This approach relies on the ratio of 1 : 600 for 30 min at 25°C. The resulting mixtures

* To whom correspondence should be addressed. e-mail: [email protected]; [email protected] © 2019 The Pharmaceutical Society of Japan 124 Biol. Pharm. Bull. Vol. 42, No. 1 (2019) were separated via sodium dodecyl sulfate-polyacrylamide gel and 6-PGA were measured using an ATP colorimetric/fluoro- electrophoresis (SDS-PAGE) and were stained with Coomassie metric assay kit and 6-PGA colorimetric assay kit (BioVision, blue. Gel lanes showing remarkable differences in intensity U.S.A.), respectively, in accordance with the manufacturer’s were excised manually and subjected to an in-gel digestion instructions. All contents measured were expressed as per- procedure.14) Peptides were analyzed via high-resolution centages of the vector control. LC-MS/MS, and raw data were analyzed through MaxQuant Statistics All data were reported as the mean ± standard (version 1.2.2.5) using standard settings. Proteins with more deviation (S.D.) of three replicates. Significant differences than three peptides were considered as reliable proteins. were determined through ANOVA, homogeneity of vari- Molecular Docking The crystal structures of human ance test, and Duncan’s multiple range test for at least three PKM2 (PDB code: 4IP7) and HK2 (PDB code: 2NZT) were treatment groups and Student’s t-test for comparison of two obtained from the Protein Data Bank. Docking was conducted groups. Statistical analysis was performed using SPSS (SPSS, through SystemsDock (http://systemsdock.unit.oist.jp/iddp/ IBM, U.S.A.) at a statistical significance level of 0.05. All ex- home/index).15) periments were repeated twice. Spectrophotometric Assay for Enzyme Activity HK activity was measured through a coupled reaction with RESULTS glucose-6-phosphate dehydrogenase. The reaction mixture contained 100 mM Tris HCl (pH 8.0), 5 mM MgCl2, 100 mM PK Was Discovered as a Candidate PA Target by DARTS glucose, 0.8 mM ATP, 1 mM nicotinamide adenine dinucleo- To understand the anti-cancer mechanism of PA, DARTS was tide phosphate (NADP), and 3 units of glucose-6-phosphate applied to find possible targets of PA besides phospholipase dehydrogenase. The NADP reduction in UV absorbance at A2. Enzyme hydrolysis broke the complete protein (bands 340 nm was monitored at the start of the reaction and after in the negative control, NC) into peptide fragments (Fig. 1). 5 min.16) Pyruvate kinase (PK) activity was determined using Addition of PA protected some peptides from digestion, and a lactate dehydrogenase-linked assay. The reaction mixture resulted in blue bands (31–45 kDa) darker than those of the contained 50 mM Tris HCl (pH 7.5), 100 mM KCl, 10 mM untreated control. The top three possible bands were cut and

MgCl2, 0.6 mM (PEP), 0.9 mM analyzed via LC-MS/MS. MS data revealed that the three ADP, 0.12 mM β-reduced nicotinamide adenine dinucleotide bands, from low to high positions, may contain PK, antitryp- (NADH), and 4.8 U/mL lactate dehydrogenase. PK activ- sin, and , respectively (Table 1). An- ity was calculated at 25 °C by monitoring the change in absorbance at 340 nm from 0 to 20 min.17) PA, bromopyru- vic acid (BA), or TEPP-46 [the thieno(3,2-b) pyrrole(3,2-d)- pyridazinone, PubChem CID: 44246499] was dissolved in DMSO and diluted by 10-fold with cell lysis buffer, and dif- ferent concentrations (5–100 µM) were used. Approximately 30 µL SK-BR-3 cell lysate (20 ng/µL) was incubated with 3 µL PA, BA, or a dissolving vector at 37°C for 10 min. Afterward, 300 µL reaction mixture was added. Glucose Uptake Assay After PA treatment for 24 h, the media of SK-BR-3 cell cultures were collected via centrifu- gation and diluted with water to 4000-fold. Glucose uptake assay was conducted using an Amplex Red glucose assay kit (Invitrogen, U.S.A.) following the manufacturer’s instructions. Results were expressed as percentages of the vector control. Mitochondria Isolation and Protein Fractionation After treatment with 100 µM PA for 12 h at room temperature, the mitochondria and cytosol of SK-BR-3 cells were separated through conventional differential centrifugation.18) The pellet and resulting supernatant were eluted with the SDS sample Fig. 1. DARTS Results for Pronase-Digested SK-BR-3 Breast Carci- loading buffer, separated in 10% SDS polyacrylamide gel, and noma Cell Lysates with or without PA Protection immunoblotted for HK2, Cyt c, and HSP60 using appropriate The NC had no proteinase addition. (Color figure can be accessed in the online version.) antibodies. Measurement of Lactate, ATP, ROS, and 6-Phosphoglu- Table 1. Possible Target Proteins Identified by LCMS* conic Acid (6-PGA) After 24 h incubation with PA or vector solvent, the accumulation of cellular lactate was determined Sequence Unique MW Protein names Matches coverage Score using a lactic acid production detection kit (KeyGen, Nanjing, peptides [kDa] China) in accordance with the manufacturer’s instructions. [%] ROS concentrations were determined using di(acetoxymethyl Pyruvate kinase 19 6 19.1 37.558 87.033 ester) (6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate) Phosphoglycerate kinase 9 7 26.4 44.614 270.54 (Thermo Fisher Scientific, U.S.A.) according to the method Alpha-1-antitrypsin 8 3 13.6 40.234 323.31 of Wang and Joseph.19) The contents of ROS were calculated *: “Score” measures how well an acquired spectrum matches the theoretical frag- from a standard curve of H2O2 (50–1000 µM) and were ex- ment masses, while “Matches” indicates the intensity of the protein in the unique pressed as percentages of the control. The contents of ATP band. Vol. 42, No. 1 (2019) Biol. Pharm. Bull. 125

Fig. 2. Molecular Docking and Enzyme Assay for PA and PKM2 (a) Detailed protein–ligand interactions between PKM2 and FBP (left) or PA (right); (b) Docking scores of PKM2 with FBP and PA. (c) Enzyme activity of PKM2 in cell lysate after treatment with different concentrations of TEPP-46 and PA (with or without FBP). (Color figure can be accessed in the online version.) titrypsin is involved in cancer development and is a potential target of triterpenoids.20,21) However, as mentioned above, evi- dence directed the dysfunction of glycolysis as a PA mecha- nism. Thus, we selected PK or phosphoglycerate kinase as the study subject. PK has important roles in tumor cell growth, and it contains more peptide matches than phosphoglycerate kinase (Table 1). Thus, among the potential targets discovered through DARTS, PK was selected for following investigations. PA Served as a Competing Activator of PKM2 Sys - temsDock is a web server for network pharmacology-based prediction and analysis. This server has an elaborately de- signed scoring function for molecular docking to evaluate the protein–ligand binding potential.15) The PKM2 isoform is the major PK in cancer cells and is overexpressed in tumor cells induced by some oncogenes and tumor suppressors.22) Molecular docking revealed that PA could bind PKM2 in the pocket of its natural activator, fructose-1,6-bisphosphate Fig. 3. (a) Glucose Uptake and (b) Lactate Production in SK-BR-3 Cells (FBP), and share six residues (Fig. 2a). The dock- after 24 h Treatment with 10 or 100 µM PA *, p < 0.05 vs. vector control (0 µM PA). ing score (pKd/pKi) of PA (8.39) was considerably higher that of FBP (5.29) (Fig. 2b), indicating the advantage of PA than FBP in PKM2 binding. Both of PA and the positive control PKM2 activity remained the same, which indicated that FBP (TEPP-46) stimulated the activity of PKM2 in the cell lysate, could compete with PA in binding PKM2. and adding FBP attenuated the increasing trend caused by PA Decreased Glucose Uptake and Lactate Production PA treatment (Fig. 2c). This result corroborated the results of When PKM2 is activated under glucose-rich conditions, the molecular docking. Regardless of FBP addition, the maximum level of glucose uptake increases, and most pyruvate is rapidly 126 Biol. Pharm. Bull. Vol. 42, No. 1 (2019)

Fig. 4. Molecular Docking and Enzyme Assay for PA and HK2 (a) Detailed protein–ligand interactions between HK and (left) G6P or (right) PA. (b) Docking scores of HK2 with G6P and PA. (c) Enzyme activity of HK2 in cell lysate after treatment with different concentrations of PA or BA. (Color figure can be accessed in the online version.) converted to lactate.23) Thus, we tested if PA could increase is a direct inhibitor of HK2.25) We speculated that this phe- glucose uptake and lactate production. However, after PA ap- nomenon was caused by the endogenous chemicals from the plication, glucose uptake and lactate production decreased in cell lysate. a dose-dependent manner (Fig. 3), indicating a blockage or PA Induced Mitochondrial Dysfunction, ATP Depletion, downregulation of glycolysis. and ROS Generation After 12 h treatment with 100 µM PA, PA Directly Inhibited HK2 Activity A recent study has the dissociation of HK2 from the mitochondria and the release discovered that several triterpenoids in fungi Ganoderma si- of mitochondrial Cyt c were observed through immunoblot nense, especially (22E,24R)-6β-methoxyergosta-7,9(11),22-trie analysis in a dose-dependent manner (Fig. 5a). Mitochondrial ne-3β,5α-diol, directly inhibit HK2 activity,16) Similar to PA, dysfunction may affect ATP production and ROS generation. this triterpenoid contains four cyclic rings and a side chain on ATP depletion of up to 0.32 ± 0.08-fold relative to that of the five-membered D ring.16) As a key enzyme in the glycolyt- the control was observed after 100 µM PA treatment for 12 h ic pathway, HK2 catalyzes the first step of glycolysis by phos- (Fig. 5b). Meanwhile, the PA treatment induced ROS produc- phorylating glucose to glucose-6-phosphate (G6P).24) Thus, tion to reached up to 5.99 ± 0.50-fold relative to that of the we examined the inhibition effect of PA on HK2. Molecular control (Fig. 5b). HK2 inhibition and PKM2 activation will docking results showed that HK2 could bind with PA in the decrease the pooling of glycolytic intermediates, which should pocket for G6P (Fig. 4a). Furthermore, the affinity of PA to have been shunted to the pentose phosphate pathway (PPP) HK2 was considerably higher than that of G6P; the molecular for NADPH generation and ROS scavenging.26) After the PA docking scores were 8.18 and 5.31, respectively (Fig. 4b). PA treatment, the level of PPP intermediate, namely 6-PGA, de- substantially inhibited the HK2 activity in the cell lysate, and creased in a dose-dependent manner (Fig. 5b). showed an IC50 of 5.01 µM, which was higher than the 3.07 µM of BA (Fig. 4c). HK2 activity increased after treatment with a DISCUSSION low dose of PA (5 µM). A similar phenomenon was observed in the MDA-MB-231 cell lysate treated with metformin, which PA is an active component of P. cocos, and it possesses var- Vol. 42, No. 1 (2019) Biol. Pharm. Bull. 127

high rate of aerobic glycolysis is frequently observed; this high glycolysis rate is known as the Warburg effect, which is believed to benefit tumor cell growth and survival.32) As a key enzyme in the glycolytic pathway, HK2 is overexpressed in cancer cells, and it is an important target for cancer therapy.33) The potential of triterpenoids in blocking HK2 activity in- spired us to investigate the possible inhibitory effect of PA on HK2.16) In silico and in vitro experiments proved this hypoth- esis (Fig. 4). In tumor cells, HK2 strategically binds to the transmem- brane channels formed by voltage-dependent anion channel (VDAC). This interaction facilitates the growth and prolif- eration of tumors by reducing the sensitivity to the feedback inhibition of G6P, preferred access to ATP, and protection from proteolytic degradation.34) In contrast, the HK2 disso- ciation from the mitochondria will impair tumor growth and induce apoptosis, which is commonly observed in tumor cells treated with HK2 inhibitors, such as metformin, oroxylin A, and methyl jasmonate.25,35,36) Moreover, we hypothesized that PA can induce the detachment of HK2 from the mitochondrial membrane. After the PA treatment, mitochondrial HK2 dis- sociation in a dose-dependent manner was observed (Fig. 5a). The dissociation of HK2 and VDAC will induce the release of mitochondrial Cyt c through either the Cyt c-conducting con- duit formed by Bax and Bak on the outer mitochondrial mem- brane (OMM) or the direct rupture of OMM resulting from sustained VDAC closure.37) Thus, we parallelly examined the release of Cyt c from the mitochondria after the PA treatment, and positive results were obtained (Fig. 5a). The closure of VDAC results in a diminution of mitochon- drial ADP, thereby decreasing the respiratory rate and stimu- Fig. 5. (a) PA-Induced HK2 Dissociation from the Mitochondria, Cyt c Release, (b) ATP Depletion, Generation of ROS, and Declination of 6-PGA lation of ROS generation due to the highly reduced state of the 38) *, p < 0.05 vs. vector control (0 µM PA). components of the electron transport chain. Thus, we exam- ined the influence of PA on the intracellular ROS contents and ATP level. PA stimulated the production of ROS and depletion ious pharmacological effects. However, its action mechanisms of ATP (Fig. 5b); this result was consistent with the work of are unclear. In this study, PKM2 and HK2 were discovered as Jeong et al.9) Furthermore, through the inhibition of HK2 and the new targets of PA. activation of PKM2, PA decreased the intermediate PPP level PKM2 catalyzes the conversion of phosphoenolpyruvate for NADPH generation and ROS scavenging, which may have (PEP) and ADP into ATP and pyruvate, which is the final contributed to the stimulation of ROS.26) reaction in glycolysis.24) When PKM2 is mainly in its inactive ROS can activate intrinsic and extrinsic apoptosis pathways dimeric form, which is the case in tumor cells, all glycolytic and modulate the pro-apoptotic molecules, including MAPK, intermediates above the PK accumulate and are channeled into Bax, and Bcl2; moreover, it can regulate the expression lev- synthetic processes, such as nucleic acid, phospholipid, and els of various transcription factors, including Sp1, AP1, and amino acid syntheses, to support tumor cell proliferation.27) NF-κB, and other pro-oncogenic genes that are engaged in Furthermore, the dimeric form of PKM2 has transcriptional cancer cell proliferation, survival, and metastasis.39) In the 28) regulation functions that benefit cancer cells. Previous stud- study of Jeong et al., blocking of ROS by N-acetyl-L-cysteine, ies have shown that the active tetrameric form of PKM2 can which is a ROS scavenger, effectively reduced the PA-induced be allosterically activated by activators, such as FBP.29,30) As apoptosis.9) Moreover, ROS-mediated ER stress is associated an FBP competitor, PA may increase the ratio of tetrameric/ with apoptosis initiation, which has been observed in PA- dimeric state of PKM2, which needs in-depth investigation. treated lung cancer cells.4,40) In summary, ROS generation Emerging evidence has implicated PKM2 as a critical by PA may be a fundamental mechanism for the anti-cancer regulator of metabolism in immune cells. Moreover, PKM2 activity of PA. expression is substantially increased in lipopolysaccharide- In conclusion, two new PA targets, PKM2 and HK2, were activated macrophages, mainly in a monomeric/dimeric con- discovered in this study. PA is an activator of PKM2 and an formation and phosphorylated state with decreased activity.31) inhibitor of HK2. In the study of Tee et al., increased glucose

Hence, other than inhibiting phospholipase A2, an in-depth consumption as a result of PKM2 activation by TEPP-46 investigation on the role of PA in inflammation is interesting. resulted in sensitized antiproliferative effects of the toxic The activation of PKM2 should have increased the uptake glucose analog, 2-deoxy-D-glucose. Combination treatment of glucose and production of lactate. However, opposite results resulted in reduced viability of a range of cell lines in stan- 41) were obtained in this study (Fig. 3). In malignant tumors, a dard cell culture conditions. Moreover, 2-deoxy-D-glucose 128 Biol. Pharm. Bull. Vol. 42, No. 1 (2019) competitively inhibited the production of G6P from glucose, small-molecule target identification, in Chemical Biology. (2015), which resembles that of PA. This study supported the benefi- Springer. p. 287–298. cial effects of dual targeting of PA to PKM2 and HK2. Pos- 13) Lomenick B, Jung G, Wohlschlegel JA, Huang J. Target identifica- sible consequences of this dual efficacy, such as dissociation tion using drug affinity responsive target stability (DARTS). Cur- rent Protocols in Chemical Biology, 3, 163–180 (2011). of HK2 from the mitochondria, release of the mitochondrial 14) Shevchenko A, Tomas H, Havli J, Olsen JV, Mann M. In-gel diges- Cyt c, depletion of ATP, generation of ROS, and declination tion for mass spectrometric characterization of proteins and pro- of PPP intermediate, were also validated. The results of our teomes. Nat. Protoc., 1, 2856–2860 (2006). study provide valuable information on the anti-cancer mecha- 15) Hsin K-Y, Matsuoka Y, Asai Y, Kamiyoshi K, Watanabe T, Kawa- nisms of PA. oka Y, Kitano H. systemsDock: a web server for network pharma- cology-based prediction and analysis. Nucleic Acids Res., 44 (W1), Acknowledgments This study was supported by the Edu- W507–W513 (2016). cational Commission (KJ2016A668) and the National Natural 16) Bao F, Yang K, Wu C, Gao S, Wang P, Chen L, Li H. New natural Science Foundation (1708085QC52) of the Anhui Province of inhibitors of hexokinase 2 (HK2): Steroids from Ganoderma si- China. nense. Fitoterapia, 125, 123–129 (2018). 17) Chen J, Xie J, Jiang Z, Wang B, Wang Y, Hu X. Shikonin and its analogs inhibit cancer cell glycolysis by targeting tumor pyruvate Conflict of Interest The authors declare no conflict of kinase-M2. Oncogene, 30, 4297–4306 (2011). interest. 18) Achanta G, Sasaki R, Feng L, Carew JS, Lu W, Pelicano H, Keat- ing MJ, Huang P. Novel role of p53 in maintaining mitochondrial REFERENCES genetic stability through interaction with DNA Pol γ. EMBO J., 24, 3482–3492 (2005). 1) Huang YC, Chang WL, Huang SF, Lin CY, Lin HC, Chang TC. 19) Wang H, Joseph JA. 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