Oncogene (2011) 30, 4297–4306 & 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11 www.nature.com/onc ORIGINAL ARTICLE Shikonin and its analogs inhibit cancer cell glycolysis by targeting tumor pyruvate kinase-M2 J Chen1, J Xie1, Z Jiang2, B Wang1, Y Wang2 and X Hu1 1Cancer Institute (a Key Laboratory For Cancer Prevention & Intervention, National Ministry of Education, China; a Key Laboratory of Molecular Biology in Medical Sciences, Zhejiang, China), The Second Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang, China and 2Department of Chemistry, Zhejiang University, Hangzhou, China We recently reported that shikonin and its analogs were et al., 2008; Hsu and Sabatini, 2008; Vander Heiden a class of necroptotic inducers that could bypass cancer et al., 2009). Thus, glycolysis, the major pathway for drug resistance. However, the molecular targets of energy generation, is vital for the proliferation and shikonin are not known. Here, we showed that shikonin survival of cancer cells (DeBerardinis et al.,2008;Vander and its analogs are inhibitors of tumor-specific pyruvate Heiden et al., 2009). Why then cancer cells shift energy kinase-M2 (PKM2), among which shikonin and its production from Krebs cycle to glycolysis? Although it is enantiomeric isomer alkannin were the most potent and not completely understood, it is believed that pyruvate showed promising selectivity, that is, shikonin and kinase (PK) has important roles in this shift (Mazurek alkannin at concentrations that resulted in over 50% et al., 2002, 2005; Mazurek and Eigenbrodt, 2003; inhibition of PKM2 activity did not inhibit PKM1 and Mazurek, 2007; Christofk et al.,2008). pyruvate kinase-L (PKL). Shikonin and alkannin signifi- In mammalian cells, there are two PK genes: one cantly inhibited the glycolytic rate, as manifested by encodes pyruvate kinase-M1 (PKM1) and PKM2, and cellular lactate production and glucose consumption in the other encodes PKL and PKR. Thus, PKM1 and drug-sensitive and resistant cancer cell lines (MCF-7, PKM2, or PKL and PKR, are the isoforms resulting MCF-7/Adr, MCF-7/Bcl-2, MCF-7/Bcl-xL and A549) from alternative splicing (Noguchi et al., 1986, 1987). that primarily express PKM2. HeLa cells transfected PKM1 and PKM2 are the primary isoforms in adult with PKM1 showed reduced sensitivity to shikonin- or tissue and embryonic tissues, respectively, and PKL and alkannin-induced cell death. To the best of our knowledge, PKR are exclusively expressed in liver and red blood shikonin and alkannin are the most potent and specific cells, respectively (Imamura et al., 1973; Ibsen, 1977). inhibitors to PKM2 reported so far. As PKM2 universally Tumor cells almost ubiquitously express PKM2, which is expresses in cancer cells and dictates the last rate-limiting also called tumor M2-PK (Altenberg and Greulich, step of glycolysis vital for cancer cell proliferation and 2004). PKM2 has been shown to have an important role survival, enantiomeric shikonin and alkannin may have in cancer cell metabolism and growth, because inhibition potential in future clinical application. of PKM2 by peptide aptamer inhibited cell growth Oncogene (2011) 30, 4297–4306; doi:10.1038/onc.2011.137; (Spoden et al., 2008, 2009) and PKM2 knockdown by published online 25 April 2011 RNA interference or displacement of PKM2 with PKM1 (Christofk et al., 2008) significantly reduced the ability Keywords: shikonin; alkannin; pyruvate kinase-M2; of human cancer cell lines to form tumor in nude mice. glycolysis; inhibitor As PKM2 is necessary for cancer cells’ aerobic glycolysis, which is a hallmark of cancer metabolism and the major energy source essential for cancer cell growth and survival (Warburg, 1956; Mazurek et al., 2002, 2005; Mazurek Introduction and Eigenbrodt, 2003; Gatenby and Gillies, 2004; Mazurek, 2007; Christofk et al., 2008; DeBerardinis One of the metabolic characteristics of cancer cells is et al., 2008; Hsu and Sabatini, 2008; Vander Heiden et al., Warburg effect, that is, even in the presence of ample 2009), PKM2 is a potential molecular target for oxygen, cancer cells use glucose through glycolysis and disrupting glucose metabolism in cancer cells (Christofk ferment the end product pyruvate to lactic acid rather et al., 2008; Spoden et al., 2008, 2009). than completely oxidizing it through Krebs cycle In the last four decades, shikonin, alkannin and their (Warburg, 1956; Gatenby and Gillies, 2004; DeBerardinis derivatives have been investigated as potential anticancer drugs for various aspects of cancer treatment (Ahn et al., 1995; Papageorgiou VP et al., 1999; Bailly, 2000; Kim Correspondence: Professor X Hu, Cancer Institute, The Second et al., 2001; Masuda et al., 2003, 2004; Nakaya and Affiliated Hospital, Zhejiang University School of Medicine, 88 Miyasaka, 2003; Han et al., 2007; Hu and Xuan, 2008; Jiefang Road, Hangzhou, Zhejiang 310009, China. E-mail: [email protected] Xuan and Hu, 2009; Yang et al.,2009),including Received 3 October 2010; revised 3 March 2011; accepted 23 March aclinicalstudy(Guoet al., 1991), which indicated 2011; published online 25 April 2011 that a shikonin mixture was effective in treating Shikonin–potent inhibitor to pyruvate kinase M2 J Chen et al 4298 19 patients of later-stage lung cancer who were not methods) to extract cellular proteins (MCF-7) of suitable for operation, radiotherapy and chemotherapy. interest. The proteins bound to crystal shikonin were We previously reported that shikonin and its analogs subjected to sodium dodecyl sulfate (SDS)–PAGE, and induced cell death with characteristics of necroptosis, a the protein bands were sliced for subsequent mass basic cell death defined by Degterev et al. and further spectroscopy. One of the bands was identified to be studied in depth by the same group of scientists (Degterev PKM2 (Figure 2); the other proteins potentially bound et al., 2005, 2008; Hitomi et al., 2008). We found that to crystal shikonin have been shown in Supplementary shikonin and its analogs showed similar cytotoxicity Figures 1–13. Alternatively, we used shikonin-conju- toward cancer cells regardless of P-gp, MRP1, BCRP1, gated Sepharose-6B to extract cellular proteins from Bcl-1 or Bcl-xL expression levels, indicating that these MCF-7 cell lysate, among which PKM2 was detected by compounds were capable to bypass multiple defense lines mass spectroscopy. Purified recombinant PKM2, PKM1 of cancer cells against anticancer drugs (Han et al.,2007; and PKL could also bind to shikonin-conjugated Xuan and Hu, 2009). In this study, we tried to investigate Sepharose-6B (Supplementary Figure 14). These results the molecular targets of shikonin. Using solid-phase showed that PKM2 was a potential target of shikonin. shikonin to extract cellular proteins that were subse- quently analyzed by mass spectroscopy, we found that Shikonin and its enantiomeric alkannin are potent PK isozyme-M2 (PKM2) was one of the proteins inhibitors of PKM2 but not PKM1 and PKL potentially bound by shikonin. We then determined the An enzyme assay was performed according to the reported inhibitory activity and specificity of shikonin and its method for identification of compound-3 (N-(3-carboxy-4- analogs (Figure 1) toward PKM2, PKM1 and PKL. Our hydroxy)phenyl-2,5,-dimethylpyrrole) as PKM2 inhibitor, study shows that shikonin and alkannin are potent and the most potent inhibitor discovered from a screening of specific inhibitors to PKM2, an enzyme that dictates the over 100 000 compounds (Vander Heiden et al., 2010). last rate-limiting step of glycolysis, which is essential for Shikonin and alkannin (the optical isomer of shikonin) cancer cells’ proliferation and survival. showed comparable inhibitory activities, more potent than other shikonin analogs (Figures3bandc).Intheabsence Results of D-fructose-1,6-bisphosphate (FBP), the IC50 of both shikonin and alkannin was 0.3 mM, about 100-fold lower PKM2 is a potential target of shikonin than that of compound-3 (37.4 mM) (Figure 3c). In the In order to find out the molecular targets of shikonin, presence of FBP (125 mM), the IC50s of shikonin and we used solid-phase shikonin (see Materials and alkannin were 0.8 and 0.9 mM, respectively, about 50-fold lower than that of compound-3 (50 mM) (Figure 3d). Although allosteric activation of enzyme activities by FBP is concentration dependent (Figure 3a), we used FBP (125 mM) for the enzyme assay. First, FBP (125 mM) significantly activated PKM2 (Figure 3a), so that the results of this study could be compared with the reported data (Vander Heiden et al., 2010). In fact, the IC50 of compound-3 determined in this study is similar to the reported data. Second, according to available data (Hirayama et al., 2009), intracellular FBP levels were lower than 125 mM. As we used a lactate dehydrogenase (LDH)-coupled enzyme assay for measurement of PKM2 activity, it is necessary to rule out that LDH activity is not affected by shikonin and alkannin. The results showed that LDH was not inhibited by shikonin, alkannin and the analogs (Figure 3e). Despite that the three isoenzymes could all bind to solid-phase shikonin, shikonin and alkannin showed promising selectivity toward PKM2, PKM1 and PKL (Figure 4). The IC50s of shikonin toward PKM1 and PKL were about 20- and 10-fold higher than that toward PKM2. The IC50s of alkannin toward PKM1 and PKL were also about 20- and 10-fold higher than that toward PKM2. Shikonin and alkannin inhibit the rates of cellular lactate production and glucose consumption Figure 1 (a) The chemical structures of shikonin and its analogs. The direct consequence of cellular PKM2 inhibition is (b) The enantiomeric shikonin and alkannin. slowdown of the rate from phosphoenolpyruvate (PEP) Oncogene Shikonin–potent inhibitor to pyruvate kinase M2 J Chen et al 4299 Figure 2 Identification of PKM2 as a potential target of shikonin by solid-phase shikonin extraction and mass spectroscopy. (a) SDS–PAGE of cellular proteins extracted by solid-phase shikonin.