Mitochondria-targeted Re(I) complexes bearing guanidinium as ligands and their anticancer activity.

JBIC Journal of Biological Inorganic Chemistry https://doi.org/10.1007/s00775-020-01827-7 ORIGINAL PAPER Mitochondria‑targeted Re(I) complexes bearing guanidinium as ligands and their anticancer activity Shu‑Fen He1,2 · Nan‑Lian Pan1 · Bing‑Bing Chen1 · Jia‑Xin Liao1 · Min‑ying Huang1 · Hai‑Jun Qiu1 · Dong‑Chun Jiang1 · Jun‑Jie Wang1 · Jia‑Xi Chen1 · Jing Sun1 Received: 18 August 2020 / Accepted: 12 October 2020 © Society for Biological Inorganic Chemistry (SBIC) 2020 Abstract As the “powerhouse” of a cell, mitochondria maintain energy homeostasis, synthesize ATP via oxidative phosphoryla- tion, generate ROS signaling molecules, and modulate cell apoptosis. Herein, three Re(I) complexes bearing guanidinium derivatives have been synthesized and characterized. All of these complexes exhibit moderate anticancer activity in HepG2, HeLa, MCF-7, and A549 cancer cells. Mechanism studies indicate that complex 3, [Re(CO)3(L)(Im)](PF ) , can selectively 6 2 localize in the mitochondria and induce cancer cell death through mitochondria-associated pathways. In addition, complex 3 can effectively depress the ability of cell migration, cell invasion, and colony formation. Graphic abstract Keywords Re(I) complex · Cytotoxicity · Mitochondria · Apoptosis Introduction Shu-Fen He and Nan-Lian Pan have contributed equally to this work. Mitochondria, as essential organelles in most eukaryotic Electronic supplementary material The online version of this cells, are related to many biological activities, such as article (https ://doi.org/10.1007/s0077 5-020-01827 -7) contains supplementary material, which is available to authorized users. energy metabolism and regulation of programmed cell death [1–3]. Mitochondria have the ability to control the activation Extended author information available on the last page of the article Vol.:(0112 33456789) JBIC Journal of Biological Inorganic Chemistry of programmed cell death by regulating the translocation lines was performed. The mechanism of action of complex of proapoptotic proteins from the mitochondrial intermedi- 3 was investigated in detail, which was found to selectively ate space to cytosol [4]. Numerous studies have developed localize in the mitochondria, and cause mitochondria dys- mitochondria-targeting anticancer drugs and reported their function, leading to cellular apoptosis. Meanwhile, complex promising application in chemotherapy and diagnosis of sev- 3 exhibited marked inhibition in wound closure and led to eral disorders [5–8]. potent inhibition of cell migration. Over the past decades, researches have placed increas- ing focus on investigating metal complexes and their anti- Results and discussion cancer activity. Among these compounds, Re(I) complexes have emerged as promising alternatives [9–17]. Compared Synthesis, characterization, photophysical to other metal complexes that have entered clinical trials, properties, and lipophilicity such as platinum [18], ruthenium [19], and titanium [20], studies on Re(I) complexes have only been recently initi- ated. As a new class of anticancer drugs, a key advantage Ligand L was obtained based on the previous work [26]. of Re(I) complexes is their rich spectroscopic properties. Complex 1 was synthesized by reacting L and Re(CO) Cl in 5 For instance, these complexes exhibit high photo stability, methanol/methanol and heated at 80 °C under argon for 4 h long-lived phosphorescence, which simplifies time-resolved in the dark. Then, the crude product was purified via recrys- detection, and large Stokes shifts, which minimize the pos- tallization from CH CN/CH CH OCH CH . Complexes 3 3 2 2 3 sible self-quenching effect [21]. The emission properties of 2 and 3 were achieved by refluxing different monodentate these complexes can be altered by the addition of different ligands (Py for 2, Im for 3) with complex 1 in methanol or ligands. Furthermore, combining various recognition groups tetrahydrofuran under argon for 24 h. The synthetic routes and biological molecules into these complexes is anticipated of these complexes are shown in Scheme S1 (ESM). The to result in the development of new probes and anticancer three complexes were fully characterized by mass spectrom- agents [22]. etry, 1H NMR spectroscopy (Figs. S1–S6), and elemental In this work, three Re(I) tricarbonyl complexes were analysis. The UV–Vis spectra of complexes 1–3 in phos- synthesized by combining biologically active guanidinium phate buffered saline (PBS), CH CN, and CH Cl were also 3 2 2 groups [23] as ligands (Fig. 1). It was reported that the pres- investigated. The relatively intense absorption bands in the ence of the guanidine group in these compounds is often UV region at approximately 250–330 nm can be assigned directly related to their biological activities, as exemplified to intraligand (π → π*) absorption, and the relatively lower by antimalarial [24] and antitumor agents [25]. Such par- energy bands at 380–430 nm are attributed to MLCT (metal- ticular properties reveal that the molecular mode-of-action to-ligand charge-transfer) absorption (Fig. S7) [27]. Mean- of guanidine metabolites is not only related to a possible role while, three complexes showed good stability in different as the active species, but can also be more specific. Yet, the solvents for at least 48 h revealed by the UV–Vis spectra. relationship between guanidine properties and antimalarial/ Upon 405 nm excitation, all complexes exhibited yellow antitumor effects is still unknown. A systematic evaluation emission in PBS, CH CN, and CH Cl at 298 K (Fig. S8). 3 2 2 of the characterization, photophysical properties, and in vitro The lipophilicities of three Re(I) complexes were examined anticancer effects of these complexes on several cancer cell using the flask-shaking method. These Log Po/w values Fig. 1 The chemical structures CO CO OC H OC H of Re(I) complexes 1–3 N N N N Re NH2 Re NH2 N N N N OC Cl N H NH2 OC N N H NH2 PF 6 PF 1 2 6 2 CO OC H N N Re NH2 N N OC N NH2 N H PF HN 6 2 3 1 3 JBIC Journal of Biological Inorganic Chemistry for 1–3 were determined to be − 0.21, − 0.14, and 0.38, It is generally known that small molecules penetrate into respectively. the plasma membrane main through energy-dependent or energy-independent pathways [28, 29]. The reduced effi- In vitro cytotoxicity ciency of cellular uptake in HepG2 cells incubated with 3 at lower temperature (4 °C) or CCCP metabolic inhibitor The in vitro antiproliferative activities of all Re(I) complexes was determined by the confocal microscopic data (Fig. 2b). against several human cancer lines (HepG2, HeLa, MCF- Meanwhile, the endocytic inhibitor chloroquine had no influ- 7, and A549) were examined by MTT assay (Table 1 and ence on the penetration function of 3 into the cell membrane. Fig. S9). Three complexes exhibited moderate anticancer The results show that complex 3 enters HepG2 cells via an activity to the cancer cell lines, with complex 3 displaying energy-dependent pathway. highest anticancer efficacy. This may be attributed to the different lipophilicities of these complexes. Generally, high ROS detection Log Po/w can shorten the time of complex to enter cells, which means that the cytotoxicity will increase. Although As the major sites of cellular ROS production, mitochon- this is not the sole factor affecting the cytotoxicity of the dria are one of the most important mediators of cell death. complex. Notably, complexes 1 and 3 showed high selectiv- Many studies have proven that mitochondrial dysfunction ity to HepG2 cells. Especially, the cytotoxicity of complex can obviously improve intracellular ROS levels [30]. There- 3 ( IC = 13.2 ± 0.8 μM) to HepG2 cells was higher than 50 fore, intracellular ROS levels effected by complex 3 were cisplatin ( IC = 18.2 ± 1.2 μM), which indicates that 3 may 50 checked though a DCFH-DA fluorescence assay. Confocal be a suitable candidate for cancer therapy. microscopic analysis of DCFH-DA labeled Re(I)-treated Cellular uptake mechanism HepG2 cells showed that complex 3 caused an obvious con- centration-dependent increase in the ROS levels after 12 h of incubation (Fig. 3a). The same results can be achieved by Complex 3 was selected as the targeting compound to fur- flow cytometry (Fig. 3b). At the concentration of 80 μM, the ther explore its superior anticancer mechanism. The intra- mean fluorescence intensity (MFI) of DFC in 3-treated cells cellular distribution of Re(I) complexes can be indicated by increased 3.3-fold compared to the control cells. These find- fluorescence microscopy, according to the rich photophysi- ings indicate that 3 could induce intracellular ROS elevation, cal properties. Figure S10 reveals that complex 3 completely which plays a critical role in cell death. penetrated into the cytoplasm after 12 h of co-incubated with HepG2 cells, revealing green fluorescence. Meanwhile, com- plex 3 displayed high coincidence of colocalization with the ATP detection and cell cycle arrest organelle-specific stain M itoTracker® Red CMXRos (MTR), as indicated by a Pearson’s colocalization coefficient of 0.91 As the ATP generators, mitochondria mediate essential with MTR. On the other hand, the colocalization between 3 cell functions, such as control of the ATP content and cell and Lyso-tracker Red (LTR) can be ignored under the same cycle arrest [31]. Moreover, cell cycle arrest may occur in conditions. All results suggest that complex 3 can specifi- response to the blockage of macromolecule biosynthesis cally target mitochondria (Fig. 2a). caused by the reduction of ATP and mitochondria dysfunc- tion [32]. To gain further insight into the mechanism of the inhibi- tory effects of the complex, we measured their impact on Table 1 Cytotoxic effects of three Re(I) complexes 1–3 on different intracellular ATP levels and cell cycle inhibition. As shown cancer cell linesa (The in vitro antiproliferative activities of Re(I) in Fig. 4a, the ATP levels of 3-treated cells (80 μM 3: 40.1%) complexes were examined by MTT assay) were obviously lower compared with the control cells Compounds IC (μM) (100%) and decreased in a concentration-dependent manner. 50 Cell cycle arrest in HepG2 cells induced by complex 3 was HepG2 HeLa MCF-7 A549 analyzed by flow cytometry. It can be seen from Fig. 4b and 1 22.5 ± 1.9 188.3 ± 3.2 30.6 ± 0.7 47.2 ± 1.5 Fig. S11, complex 3 caused an obvious accumulation of cells 2 40.8 ± 0.3 87.9 ± 0.9 53.2 ± 1.5 50.1 ± 1.0 in the S phase. After a 24 h Re(I) treatment, the percentage 3 13.2 ± 0.8 45.2 ± 1.1 39.6 ± 2.3 29.3 ± 0.4 of cells in S phase increased from 21.7 ± 1.2% (control) to Cisplatin 18.2 ± 1.2 15.3 ± 2.2 14.5 ± 0.5 15.2 ± 1.5 35.4 ± 1.7% (3: 80 μM), indicating that the effect of 3 on cell cycle progression is concentration-dependent (Table S1). a Cell lines were treated with complexes 1–3 for 48 h in the dark. Data Meanwhile, the proportions of cells were reduced in G2/M are presented as means ± standard deviations obtained at least three independent experiments phase (control: 61.2 ± 2.3%, 80 μM 3: 46.3 ± 0.9%). All 1 3 JBIC Journal of Biological Inorganic Chemistry Fig. 2 a, b Confocal micro- scopic images of HepG2 cells incubated with complex 3 (50 μM, 12 h), MTR (100 nM, 0.5 h), and LTR (100 nM, 0.5 h). Complex 3 excitation at 405 nm, and MTR and LTR excitation at 552 nm. c The confocal images of HepG2 cells after incubation with 3 (50 μM) at indicated condi- tions: (1) HepG2 cells were incubated with 3 at 37 °C for 12 h; (2) HepG2 cells were incubated with 3 at 4 °C for 12 h; (3) HepG2 cells were incubated with 3 at 37 °C for 12 h after pre-incubation with 20 μM CCCP at 37 °C for 1 h; (4) HepG2 cells were incubated with 3 at 37 °C for 12 h after pre-incubation with 50 μM chloroquine at 37 °C for 1 h these results suggest that complex 3 can affect mitochon- microscopy, which are described as follows. Necrotic cells drial integrity. were stained with Hoechst 33342 to observe the typical apoptotic changes, such as cell shrinkage, plasma mem- Induction of apoptosis brane blebbing, and chromatin condensation, and compare with normal morphology (Fig. 5a). Following a treatment The ability of complex 3 inducing apoptosis of HepG2 with complex 3 for 12 h, the percentage of cells in early and cells was investigated by Hoechst 33342 and Annexin V/ late apoptotic stages increased in a concentration-dependent PI labeling. In general, an apoptotic cell was following manner from 11.6% (control) to 84.2% (3: 80 μM). More- with morphological changes [33]. Therefore, the morpho- over, the percentage of abnormal nuclei in late apoptotic logical changes of HepG2 cells were observed by confocal stages was determined 81.5% (Fig. 5b). 1 3 JBIC Journal of Biological Inorganic Chemistry Fig. 3 Effects of complex 3 on ROS production. HepG2 cells were incubated with 3 and labeled with DCFH-DA, and then analyzed using confo- cal microscopy (a) and flow cytometry (b) Since the activation of caspase is an important hallmark Transwell chamber assay is widely used to evaluate cell of apoptosis [34], the caspase-Glo assay was carried out to migration and invasion in vitro [38, 39]. In this experiment, study the effect of Re(I) complex on caspase-3/7 activity. It the invasive HepG2 cells were seeded on a Matrigel-coated can be seen from Fig. S12, complex 3 displayed a moderate membrane and treated with complex 3 for 24 h (Fig. 6c, d). effect on the activation of caspase-3/7 after a 24 h treatment Compared with the control, complex 3 inhibited the can- in HepG2 cells. The activation of caspase-3/7 increased cer cell invasion in a dose-inhibition manner, which further approximately 1.37-fold compared to the control cells. confirm the anti-metastasis activity of the Re(I) complex. For malignant cancer cells, especially cancer stem cells, Inhibition of cell migration, invasion, and colony the formation of colony is a vital feature [37]. To investi- formation gate the influence of complex 3 on the colony formation, the colony-forming efficiency of HepG2 cells in the absence Metastasis is a complex biological process that contains the and presence of 3 was assessed [40]. As shown in Fig. 6e, digestion of extracellular matrix, cell migration and invasion f, the colony formation of HepG2 cells was inhibited after to lymph nodes in circulation system, and growth of tumors incubation with different concentrations of complex 3. and angiogenesis at new sites [35]. Therefore, we detected The colony-forming efficiency decreased by 69.2 ± 1.36% the effects of 3-treatment on HepG2 cell motility by a wound (3: 20 μM), 62.8 ± 3.07% (3: 60 μM), and 5.8 ± 0.08% (3: healing assay, as described in previous reports [36, 37]. In 80 μM) compared with the control (100%). Based on these the vehicle control cells, after 24-h incubation, the distance findings, complex 3 can notably repress the invasion ability of the border become smaller, and almost disappeared after of HepG2 cells. 48 h of incubation (Fig. 6a, b). However, the migration of Western blot analysis the Re(I)-treated cells was obviously suppressed, and the borders exhibited a significant concentration- and time- dependent manner. The results indicate that complex 3 has The above results suggest that complex 3 could locate in an inhibition capability in wound closure. the mitochondria, and induce apoptosis, migration, and 1 3 JBIC Journal of Biological Inorganic Chemistry frequently lower in comparison to non-neoplastic cells which allow the small molecule agents to enter the tumor cell mitochondria. To further prove the anticancer action mechanism, we studied the expression level of related pro- teins in HepG2 cells treated with complex 3. As shown in Fig. 7, complex 3 can up-regulate the expression of Bax pro- tein and down-regulate the expression of Bcl-2 protein. As a pair of homologous-related proteins, Bax and Bcl-2 promote apoptosis and anti-apoptosis, respectively; thus, their expres- sion levels are directly related to the regulation of apopto- sis [42, 43]. Currently, it is believed that the regulation of apoptosis is mainly mediated by the inhibition or activation of mitochondrial-related signals. We found that complex 3 up-regulates the expression levels of Cytochrome C and PARP. Cytochrome C is the key protein for mitochondria to initiate apoptosis, and plays an important role in redox energy metabolism [44], and PARP has been identified as an important marker of apoptosis [45]. In addition, Caspase-3 protein expression was down-regulated, which further con- firms that complex 3 could affect the cell cycle and induce apoptosis by the caspase pathway. Furthermore, complex 3 could down-regulate the expressions of MMP-2 and VEGF, indicating its inhibitory ability of migration, invasion, and angiogenesis of HepG2 cells. Conclusions In this work, three new Re(I) complexes bearing guanidin- ium ligands were synthesized and characterized. All com- Fig. 4 a Intracellular ATP levels in HepG2 cells after incubated with plexes show moderate antitumor activity to the tested cancer complex 3 at different concentrations. b Effects of 3 on the distribu- cells. The intracellular distribution studies suggest that com- tion of HepG2 cells in cell cycle population at indicated conditions plex 3 is selectively localized in the mitochondria. Antican- for 24-h treatment (*p < 0.05) cer mechanism studies indicate that 3 can induce cancer cell apoptosis by ROS elevation, ATP reduction, caspase activa- tion, and cell cycle arrest in the S phase. Meanwhile, com- invasion of HepG2 cells. It is believed that the mitochon- plex 3 joins in some main cancerous events, including the dria-targeting compounds could reduce oxygen consump- inhibition of cell migration, invasion, and colony formation. tion, release Cytochrome C and activate a caspase pathway In summary, our work provides useful insight for researching to apoptosis which is specific in cancer cells [41]. Because and designing new metal anticancer agents. the mitochondrial membrane potentials in cancer cells are 1 3 JBIC Journal of Biological Inorganic Chemistry Fig. 5 a Hoechst 33,342 labeled HepG2 cells after incubated with complex 3. b Flow cytometric quantification of Annexin V/PI stained HepG2 cells after incubation with 3 for 24 h 1 3 JBIC Journal of Biological Inorganic Chemistry Fig. 6 a Wound-healing assay performed on 3-treated HepG2 cells. b cells invasiveness in Matrigel-transwell assay. E, f Inhibition of col- The corresponding migration distance of 3-treated HepG2 cells (20, ony formation of HepG2 cells induced by 3 at indicated concentra- 50, and 100 μM) at 0, 24, and 48 h. c, d The effect of 3 on HepG2 tions for 24 h 1 3 JBIC Journal of Biological Inorganic Chemistry 9. Knopf KM, Murphy BL, MacMillan SN, Baskin JM, Barr MP, Boros E, Wilson JJ (2017) J Am Chem Soc 139:14302–14314 10. Kitanovic I, Can S, Alborzinia H, Kitanovic A, Pierroz V, Leoni- dova A, Pinto A, Spingler B, Ferrari S, Molteni R, Steffen A, Met- zler-Nolte N, Wölfl S, Gasser G (2014) Chem Eur J 20:2496–2507 11. Yang J, Cao Q, Zhang H, Hao L, Zhou D, Gan Z, Li Z, Tong YX, Ji LN, Mao ZW (2018) Biomaterials 176:94–105 12. He L, Pan ZY, Qin WW, Li Y, Tan CP, Mao ZW (2019) Dalton Trans 48:4398–4404 13. Yip MH, Lo KW (2018) Coord Chem Rev 361:138–163 14. Capper MS, Enriquez Garcia A, Macia N, Lai B, Lin JB, Nomura M, Alihosseinzadeh A, Ponnurangam S, Heyne B, Shemanko CS, Jalilehvand F (2020) J Biol Inorg Chem 25:759–776 15. Leonidova A, Gasser G (2014) ACS Chem Biol 9–10:2180–2193 16. Capper MS, Packman H, Rehkämper M (2020) ChemBioChem 21:2111–2115 17. Konkankit CC, Vaughn BA, Huang Z, Boros E, Wilson JJ (2020). Dalton Trans. https: //doi.org/10.1039/d0dt01 097a 18. Wheate NJ, Walker S, Craig GE, Oun R (2010) Dalton Trans 39:8113–8127 19. Bergamo A, Sava G (2011) Dalton Trans 40:7817–7823 20. Kostova I (2009) Anti-Cancer Agents Med Chem 9:827–842 21. Lo KK (2015) Acc Chem Res 48:2985–2995 Fig. 7 Western blot analysis of complex 3 on the expression of Bax, 22. Lo KK, Zhang KY, Li SP (2011) Eur J Inorg Chem Bcl-2, Cytochrome C, PARP, Caspase-3, MMP-2, and VEGF. HepG2 24:3551–3568 cells were incubated with indicated concentrations of 3 for 24 h 23. Mishra A, Batra S (2013) Curr Top Med Chem 13:2011–2025 24. Liu Z, Sadler PJ (2014) Accounts Chem Res 47:1174–1185 25. Leung CH, Zhong HJ, Chan DSH, Ma DL (2013) Coord Chem Rev 257:1764–1776 Acknowledgments This work was supported by the National Natural 26. Chen WX, Song XD, Chen JX, Zhao XH, Xing JH, Ren JR, Wu Science Foundation of China (21101034), the Science and Technol- T, Sun J (2016) Polyhedron 110:274–281 ogy Plan of Guangdong Province (2016A020217020), the Scientific 27. Louie MW, Fong TTH, Lo KKW (2011) Inorg Chem Research Project of Guangdong Provincial Bureau of Traditional Chi- 50:9465–9471 nese Medicine (20182070), and “Group-type” special support project 28. Li CY, Yu MX, Sun Y, Wu YQ, Huang CH, Li FY (2011) J Am for Education Talents in University (4SG19045G, 4SG19053G and Chem Soc 133:11231–11239 4SG19057G). 29. Puckett CA, Barton JK (2008) Biochemistry 47:11711–11716 30. Trachootham D, Alexandre J, Huang P (2009) Nat Rev Drug Dis- Compliance with ethical standards cov 8:579–591 31. Cao JJ, Tan CP, Chen MH, Wu N, Yao DY, Liu XG, Ji LN, Mao ZW (2017) Chem Sci 8:631–640 Conflict of interest The authors declare no competing financial inter- 32. DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB (2008) ests. Cell Metab 7:11–20 33. Yan YY, Su XD, Liang YJ, Zhang JY, Shi CJ, Lu Y, Gu LQ, Fu LW (2008) Mol Cancer Ther 7:1688–1697 References 34. Jänicke RU, Sprengart ML, Wati MR, Porter AG (1998) J Biol Chem 273:9357–9360 35. Kwong WL, Lam KY, Lok CN, Lai YT, Lee PY, Che CM (2016) 1. Guerra F, Arbini AA, Moro L (2017) Biochim Biophys Acta Angew Chem Int Ed Engl 55:13524–13528 1858:686–699 36. Liang XJ, Choi Y, Sackett DL, Park JK (2008) Cancer Res 2. Galluzzi L, Morselli E, Kepp O, Vitale I, Rigoni A, Vacchelli E, 68:5267–5272 Michaud M, Zischka H, Castedo M, Kroemer G (2009) Mol Asp 37. Wang FX, Chen MH, Lin YN, Zhang H, Tan CP, Ji LN, Mao Med 31:1–20 ZW (2017) Appl Mater Interfaces 9:42471–42481 3. Kumar R, Han J, Lim HJ, Ren WX, Lim JY, Kim JH, Kim JS 38. Xu XL, Li SQ, Lin YW, Chen JH, Xie LP (2013) Transl Med (2014) J Am Chem Soc 136:17836–17843 11:276–288 4. Fulda S, Galluzzi L, Kroemer G (2010) Nat Rev Drug Discov 39. Zhang J, Liu H, Hou LD, Wang G, Zhang R, Huang YX, Chen 9:447–464 XY, Zhu JS (2017) Mol Cancer 16:151–159 5. Battogtokh G, Choi YS, Kang DS, Park SJ, Shim MS, Huh KM, 40. Xu XL, Li SQ, Lin YW, Chen H, Hu ZH, Mao YQ, Xu X, Wu Cho YY, Lee JY, Lee HS, Kang HC (2018) Acta Pharm Sin B J, Zhu Y, Zheng XY, Luo JD, Xie LD (2013) J Transl Med 8:862–880 11:276–284 6. Sakuma M, Fuchi Y, Usui K, Karasawa S (2019) Chem Asian J 41. Pilkington GJ, Parker K, Murray SA (2008) Semin Cancer Biol 14:3938–3945 18:226–235 7. Ye RR, Cao JJ, Tan CP, Ji LN, Mao ZW (2017) Chem Eur J 42. Korsmeyer SJ, Shutter JR, Veis DJ, Merry DE, Oltvai ZN (1993) 23:15166–15176 Semin Cancer Biol 4:327–332 8. Liu JP, Chen Y, Li GY, Zhang PY, Jin CZ, Zeng LL, Ji LN, Chao 43. Su YT, Chang HL, Shyue SK, Lan SH (2005) Biochem Pharmacol H (2015) Biomaterials 56:140–153 70:229–241 1 3 JBIC Journal of Biological Inorganic Chemistry 44. Ow YL, Green DR, Hao Z, Mak TW (2008) Nat Rev Mol Cell Publisher’s Note Springer Nature remains neutral with regard to Biol 9:532–542 jurisdictional claims in published maps and institutional affiliations. 45. Lewis JS, Meeke K, Osipo C, Ross EA, Kidawi N, Li T, Bell E, Chandel NS, Jordan VC (2005) J Natl Cancer Inst 97:1746–1759 Affiliations Shu‑Fen He1,2 · Nan‑Lian Pan1 · Bing‑Bing Chen1 · Jia‑Xin Liao1 · Min‑ying Huang1 · Hai‑Jun Qiu1 · Dong‑Chun Jiang1 · Jun‑Jie Wang1 · Jia‑Xi Chen1 · Jing Sun1 * Jia-Xi Chen 1 School of Pharmacy, Guangdong Medical University, cppcc@qq.com Dongguan 523808, China * Jing Sun 2 Department of Pharmacy, Dongguan People’s Hospital, sunjing@gdmu.edu.cn Dongguan 523059, China 1 3