Chem Biol Drug Des 2015 Research Article
Synthesis, Characterization, and Anticancer Activity of Novel Lipophilic Emodin Cationic Derivatives Xiang Yang1, Wenna Zhao1, Xiufang Hu1, Xianxiao Hao1, Fang Hong1, Jianlong Wang1, Liping Xiang1, Yunhui Zhu1, Yaofeng Yuan1,2, Rodney J.Y. Ho1,3, Wenfeng Wang1,2,* and Jingwei Shao1,2,3,* 1
College of Chemistry, Fuzhou University, Fuzhou, Fujian 350108, China 2 Research Institute of Photocatalysis, State Key Laboratory of Photocatalysis on Energy and Environment, Fuzhou, Fujian 350002, China 3 Department of Pharmaceutics, University of Washington, Seattle, WA 98105, USA *Corresponding authors: Jingwei Shao, shaojingwei@ fzu.edu.cn; Wenfeng Wang, [email protected] Seventeen novel emodin derivatives were synthesized, and the structures were confirmed by IR, H NMR, MS, and elemental analysis. The cytotoxic activity of the derivatives was evaluated against A375, BGC-823, HepG2, and HELF cells by MTT assay. Compound 9a with highest potency and low toxicity was selected to further investigate its detailed molecular mechanism. The lead compound 9a induced a loss of the mitochondrial transmembrane potential (MΨm), an increase in reactive oxygen species (ROS), release of cytochrome c and activation of caspase-3 and caspase-9. In addition, the confocal study showed that emodin derivative 9a (containing asymmetric hydrocarbon tails) was mainly localized in mitochondria, demonstrating a key role of the mitochondria-mediated apoptosis pathway in cancer cells. Taken together, the results demonstrate that embodin derivative 9a preferentially regulates the ROS-mediated apoptosis in A375 cells through the induction of cytochrome c expression and activation of caspase-3 and caspase-9 proteins. Key words: apoptosis, emodin derivative, reactive oxygen species, synthesis Received 9 January 2015, revised 15 May 2015 and accepted for publication 4 June 2015
It has been known for many years that mitochondria of cancer cells have significantly higher transmembrane potential than normal cells (1,2). Taking advantage of this biological property has been a popular strategy for anticancer drugs to improve their therapeutic selectivity (3,4). Delocalized lipophilic cations (DLC), which possess highly hydrophobic ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12612
structures and positive charge, are able to accumulate in the mitochondria of cancer cell due to the highly negatively charged microenvironment in the mitochondrial matrix (5,6). Some DLCs, such as Rhodamine-123 (7), MKT-077 (8), and F16 (9) (Figure S1), have been found to exhibit some degree of efficacy in killing cancer cell. One obvious character of DLC is that their positive charge delocalizes to almost whole molecule rather than localizes in one atom. In our recent work (10,11), a series of emodin quaternary ammonium salt derivatives whose positive charges localize in N atom were synthesized. All the emodin quaternary ammonium salts containing two long carbon chains with moderate length in N cation showed good anticancer activities both in vitro and in vivo, which indicates that localized lipophilic cations (LLC) also possess high anticancer activity. But some questions remain unanswered. (i) Is mitochondria also the target of emodin LLC? (ii) Is the position of emodin where lipophilic group attached important to the anticancer activity of emodin LLC? (iii) Can other emodin LLCs, such as emodin quaternary phosphonium salt and emodin sulphonium salt, show high anticancer activities like emodin quaternary ammonium salt? (iv) Emodin tertiary amine, tertiary amine oxide carry less positive charge compared with emodin quaternary ammonium salts, can they exhibit the similar anticancer activities as those of emodin quaternary ammonium salts? In this study, we have synthesized various novel emodin LLCs as well as emodin tertiary amine, emodin tertiary amine oxide, in an attempt to improve the anticancer activity and to explore the structure–activity relationships. The cytotoxicity of these compounds was all tested by MTT assay. The cell apoptosis and reactive oxygen species (ROS) generation induced by these compounds were also tested by acridine orange (AO)/ethidium bromide (EB) staining, DAPI staining, and flow cytometry (FCM) analysis. The intracellular localization of drugs was studied using confocal microscopy. Based on these work, the four questions mentioned above are answered in this study.
Experiment General Emodin was purchased from China Xi’an Sino-Herb Biotechnology Co. Ltd. (Xian, Shanxi Province, China), in 62% 1
Yang et al.
purity. After two repeats of wash (by 5% NaHCO3) and extraction (2% Na2CO3), the purity of the emodin can reach about 90%. Silica gel (100–200 mesh) used in column chromatography was provided by Tsingtao Marine Chemistry Co. Ltd (Tsingtao, Shandong Province, China). All solvents had been distilled before they were used, and other reagents were obtained from commercial suppliers in analytically chemically pure forms. Melting points were measured on WRS-1B micromelting apparatus and were uncorrected. 1H NMR and 13C NMR spectra were recorded on BRUKER AV (400M) spectrometer with tetramethylsilane as an internal standard in CDCl3. The chemical shift values are on d scale and the coupling constants (J) are in Hz. Electrospray ionization (ESI) mass spectra were measured on an Agilent 1100 IC/MSD TrapXCT and were reported as m/z. Elemental analyses, conducted by the Service Center of Elemental Analysis of Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences.
Synthesis The methods to give other derivatives and characterization data of compounds were showed in Supporting information.
Cell lines and culture A375, BGC-823, HepG2, and HELF cell lines were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cells were maintained in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 mg/mL) in a humidified atmosphere of 5.0% CO2 at 37 °C.
Cell viability assay The cytotoxicity of the derivatives was determined by MTT assay as previously described with minor modification (12– 16). Briefly, emodin, paclitaxel, and compounds 5a–18 were dissolved in dimethyl sulfoxide (DMSO) and diluted with culture medium (final DMSO concentration of 0.1%), respectively. Paclitaxel was used as a positive control. Various cells including A375, BGC823, HepG2, and HELF were treated with indicated concentrations of different emodin derivatives for 24 h. Cell viability was determined by detecting the absorbance at 570 nm in a microquant plate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and triplicate wells were analyzed at each dose. The concentration of the compound which gives the 50% growth inhibition value was defined as the IC50.
Detection of cell apoptosis by AO/EB staining A375 cells were incubated with compound 9a (4 lM) for 24 h. The cells were then trypsinized and suspended in 2
the medium at cell density of 1 9 106 mL. Cell suspension (25 lL) was stained with 1 lL AO/EB, and the stained cells were visualized with a fluorescent microscope (Leica Co. Ltd, Oberkochen, Germany) preset at excitation 510 nm to assess apoptotic and necrosis morphological changes of the cells caused by compound 9a.
Detection of cell apoptosis by FCM Cell apoptosis was quantified by FCM analysis using Annexin V-FITC/PI apoptosis detection kit according to the manufacture’s protocol as previously described (13,14). Apoptotic cells were those stained with Annexin V-FITC+/ PI (early apoptotic cells) and Annexin V-FITC+/PI+ (late apoptotic cells).
Detection of cell apoptosis by DAPI staining The cells were treated with compound 9a (2 lM) for 24 h and then washed once in PBS followed by fixation in cold methanol: acetic acid (3:1) for 10 min. After washing thrice in PBS for 5 min, these cells were then stained with 1 lg/ mL DAPI solution for 5 min at room temperature. The cells were washed twice with PBS and stained nuclei were observed using a fluorescence microscope (Leica).
Detection of intracellular ROS 2,7-Dichlorodihydrofluorescein diacetate (Invitrogen, Waltham, MA, USA) was used to detect intracellular ROS level. DCF-DA is cleaved intracellularly by non-specific esterases and oxidized by ROS to the fluorescent 2,7dichlorofluorescein (DCF). After washing once with PBS, treated cells were incubated with 20 lM DCF-DA in serum-free DMEM at 37 °C for 30 min before analysis by FCM.
Measurement of mitochondrial membrane potential Mitochondrial membrane potential (MΨm) was measured using 3,30 -dihexyloxacarbocyanine iodide (DiOC6) (Invitrogen) by FCM as described previously (13,14). Briefly, the treated cells were incubated with 50 nM DiOC6 in serumfree DMEM for 30 min at 37 °C in a 5% CO2 incubator, then washed and resuspended in PBS at 1 9 106 cells/ mL, and then analyzed by FCM.
Measurement of caspase activity Activities of caspase-3 and caspase-9 were measured using Caspase Activity Assay Kit (Biyotime Co. Ltd, Sichou, Jiangsu Province, China) according to the manufacturer’s instructions as previously described (13). Briefly, A375 cells were treated with compound 9a for 24 h. Lysates were incubated at 37 °C for 4 h. Samples were measured with a microplate reader (Tecan Infinite 200 €nnedorf, Switzerland) at an absorbance of PRO, Ma Chem Biol Drug Des 2015
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405 nm. Caspase activity is reported as the ratio of the fluorescence output in treated sample relative to control which is normalized to 1.0.
Western blotting analysis Western blots were performed as previously described (15,16). Briefly, cells were collected and resuspended in whole cell lysis buffer. Total protein content was quantified using the Bradford assay. After separated by SDS–PAGE under reducing conditions, proteins were transferred onto polyvinylidene difluoride (PVDF) membrane, followed by immunoblotting. Total proteins expression were normalized to b-actin levels.
Measurement of subcellular localization The fluorescent mitochondrial probe, Mito-Tracker Green, was used to measure the subcellular localization of compound 9a in A375 cells, as described previously (17). A375 cells at final density 105 cells/mL were cultured in Petri dish for study of intracellular localization of compound 9a. At 70% confluence, the cells were exposed to compound 9a (2 lM) at 37 °C in a 5% CO2 incubator for 24 h. The old medium was then aspirated and the cells were washed twice with prewarmed fresh PBS and incubated in the medium containing 100 nM of the fluorescent mitochondrial probe (Mito-Tracker Green, emitted at 488 nm) for 30 min. After being washed twice with PBS, the intracellular localization of compound 9a (emitted at 568 nm) was examined on a laser confocal microscope (Olympus FluoView FV1000, Tokyo, Japan).
Statistical analysis The data were presented as mean standard deviations of three determinations. Statistical analysis was performed using Student’s t-test and one-way analysis of variance. Multiple comparisons of the means were performed by the least significant difference (LSD) test. A probability value of 50 lM was given. The results are listed in Table S1. Our recent reports showed that there was an obvious correlation between the length of carbon chains attached to the quarter N cation of emodin and anticancer activity, only those quaternary ammonium salts that contained two long carbon chains with moderate length had high anticancer activity. In this study, we introduce a decyloxyl and not a methoxyl in 3-position of emodin and synthesize the compounds 5a–5c. As a result, the anticancer activities of 5a are lower than those of compounds 5b and 5c that both contain two long carbon chains with moderate length in quarter N cation, which indicates that the replacement of methoxyl by decyloxyl does not change structure–activity relationship drastically. That is to say, the anticancer activity of emodin is not 3
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sensitive to the change of length of carbon chain in 3position. Among the compound 9a–9e, compound 9e contains two dodecyl groups in quarter N cation and shows the lowest cytotoxicities against cancer cells. The other emodin quaternary ammonium salts (9a–9d) all contain two long chains with 8–10 carbon atoms in quarter N cation and all show higher anticancer activity than paclitaxel. Among which, the quaternary ammonium salt 9a containing two long chains with 8 and 9 carbon atoms, respectively, exhibits the highest anticancer activity. Such a structure–activity relationship indicates that the length of carbon chain attached to the N cation of emodin quaternary ammonium salts has a large effect on the anticancer activity of emodin derivatives. It is very likely that this region is an activity sensitive region that interacts with target point strongly. Four triphenyl quaternary phosphonium salts 11a–11d and one trioctyl quaternary phosphonium salt 11e all exhibit higher anticancer activities than paclitaxel. The length of link arm between emodin moiety and triphenyl moiety of compound 11d is much longer than that of 11a, but the two emodin quaternary phosphonium salts possess almost equal anticancer activity. The reason of which is likely because their lipophilic property both come from triphenyl phosphine group rather than link arm. As phenyl and octyl both are good lipophilic group, compounds 11a–11e all exhibit high anticancer activities, but their toxicities to normal cell are some higher than those of emodin quaternary ammonium salts. Emodin sulphonium salt 14 exhibits a moderate anticancer activity to HepG2 cell, but emodin thioether 16 shows no anticancer activity due to lack of positive charge and sufficient lipophilic property. Emodin tertiary amine 17 exhibits a high anticancer activity because tertiary amine can carry positive charge by protonation, but tertiary amine oxide 18 exhibits a relative low anticancer activity due to its relative weak proton affinity. As human melanoma cell line A375 shows good sensitivity to emodin quaternary ammonium salts, the most active compound 9a is chosen to further investigate its growthinhibitory mechanism in vitro on human melanoma A375 cell.
Compound 9a induces cell death in apoptotic fashion To examine whether 9a induced apoptosis in A375 cells, we conducted AO and EB staining, DAPI staining and FCM analysis with Annexin V-FITC/PI staining to access the cell membrane integrity and the typical characteristics of apoptosis (Figure 1). Morphological analysis using AO/ EB staining showed that compound 9a treated cell displayed morphological characteristics of apoptosis, such as 4
plasma membrane blebbing, congregated chromatin, and nucleolus pyknosis, meanwhile many cells edge damage occurred, healthy cells were absent, shown as intense red particles under the microscope, while the control cells showed uniform green-yellow fluorescence, indicating that the morphology of the cells was intact as evidenced by the evenly distributed chromatin in the nucleolus of healthy cells (Figure 1A). FCM analysis further showed that compound 9a induced a significant increase in the percentage of annexin-V/PI and annexin-V+/PI cells, suggesting an increase in the number of apoptosis cells (Figure 1B). For further assessment of apoptosis, morphological changes of nuclei were visualized following DNA staining by the fluorescent dye, 40 ,6-diamidine-20 -phenylindole dihydrochloride-DAPI (Figure 1C). As shown in Figure 1C, the cells treated with compound 9a showed morphological changes of apoptosis, including a condensed and fragmented nuclear structure and decreased cell size by DAPI staining.
Changes of cellular ROS level in the effects of compound 9a We next assessed intracellular ROS production in HepG2, BGC823, and A375 cell lines using 2,7dichlorodihydrofluorescein diacetate (DCF-DA) fluorescence dye. As shown in Figure S2, exposure of cells to compound 9a (1 lM) elicited significant (p < 0.05, p < 0.01) elevation of cellular ROS level compared to those of untreated cells, and the levels of intracellular ROS were significantly high (p < 0.01), while incubation of cells with relative high concentrations of compound 9a (2 lM), which led to a 3.6-, 6.1-, and 7.2-fold increase in the ROS production level compared to those of untreated cells, respectively.
The abilities to generate ROS for various emodin derivatives In order to ascertain whether the difference between emodin and emodin quaternary ammonium salts is relative to their abilities to generate ROS, the abilities of emodin and several emodin quaternary ammonium salts to generate ROS are tested and the result is listed in Figure S3. Thereinto, the synthesis of compound w3 has been reported in our early work, and its structure is listed in Figure S3. Compound w3 is water soluble and without any anticancer activity. Figure S3 shows that its ability to generate ROS is even lower than that of control group, which implies that the lipophilic property plays an important role for the ability to generate ROS. But excessive lipophilic property (9e) is unfavorable of both anticancer activity and the ability to generate ROS, so the anticancer activity and the ability to generate ROS of compound 9e are both lower than those of compound 9a. That is to say, there is a positive correlation between the anticancer activity and the ability to generate ROS. Chem Biol Drug Des 2015
Lipophilic Emodin Cationic Derivatives A
B
C
Figure 1: Compound 9a induces cell death in apoptotic fashion in A375 cells. (A) Fluorescence images of AO and ethidium bromide-stained A375 cells. (B) Representative dot-plots results by flow cytometry from three independent experiments illustrating 9a-induced apoptotic status. (C) Apoptosis of A375 cells detected by DAPI staining.
Compound 9a causes accelerated loss of mitochondrial membrane potential The DΨm of BGC823, HepG2, and A375 cells was measured following a 24 h of incubation with different concentrations of compound 9a (2, 4, 8 lM). As shown in Figure 2, compound 9a diminished the DΨm in a concentration-dependent manner compared to control in all three cells. The DΨm levels were significantly (p < 0.05, p < 0.01) diminished, while incubation of cells with relative high concentrations of compound 9a (4, 8 lM), which led to a 49% and 78% decrease in DΨm, respectively, compared to those of untreated A375 cells.
Caspases and cytochrome c are involved in compound 9a-induced apoptosis To further determine the roles of caspases in compound 9a-induced apoptosis, we first assessed caspase-3 and caspase-9 activities after 9a treatment for 24 h in A375 cell line. As shown in Figure 3A, compound 9a induced an increase in the activities of caspase-3 and caspase-9, indiChem Biol Drug Des 2015
Figure 2: Effects of compound 9a on mitochondrial membrane potential of HepG2, BGC823, and A375 cells. Cells were treated with compound 9a (2, 4, and 8 lM) resulted in a concentrationdependent decrease in mitochondrial DΨ m. Results are expressed as the percent change in DΨ m of the treated cells compared to the untreated control. Statistical difference from controls: *p < 0.05, **p < 0.01.
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B
Figure 3: Caspases and cytochrome c are involved in compound 9a-induced apoptosis. A. Compound 9a induced activations of caspase-3 and caspase-9 in A375 cells. B. Effect of compound 9a on the expression of cytochrome c in A375 cells by Western blot analysis. *p < 0.05, **p < 0.01.
cating that ROS elicited from 9a was involved in the 9ainduced caspase activation. Release of cytochrome c from mitochondria into cytoplasm is thought to be an absolute requirement for the activation of caspase-9 in the intrinsic apoptosis pathway. We finally used Western blot analysis to assess the effect of compound 9a on the expression of cytochrome c in A375 cells. The efficiencies of cytochrome c were shown in Figure 3B. Compound 9a induced a concentrationdependent increase in cytochrome c expression.
in mitochondria of A375 (Figure S4B,C), and to a lesser degree, perhaps in cytoplasm and other subcellular organelles (Figure S4C), but not in the nuclear (Figure S4A–D). According to our data, we summarize the signaling pathways related to compound 9a-induced apoptosis of A375 cells in Figure 4. ROS and DΨm elicited from 9a trigger the intrinsic apoptotic pathways. Cytochrome c preferentially trigger caspase-3 and caspase-9 activation to dominate 9a-induced classical apoptosis in A375 cells.
Conclusion Subcellular localization of compound 9a in A375 cells After 24 h of incubation of A375 with compound 9a (2 lM), the distribution of compound 9a within the cells was observed using confocal laser microscopy with the aid of subcellular probes. Different emission wavelengths were applied to corresponding probes of subcellular organelles. Mitochondria was stained green with emission wavelength at 488 nm (Figure S4A). Compound 9a exhibited red at 568 nm (Figure S4B). Under the confocal laser scanning microscope, compound 9a was primarily located
The above study result indicate that (i) the length of carbon chains in 3-position of emodin has no obvious effect on the anticancer activities of emodin quaternary ammonium salt, but the length of carbon chains in N cation has a close relationship with anticancer activity; (ii) both lipophilicity and positive charge play an important role on the anticancer activity of emodin derivatives; (iii) when cancer cell is treated with emodin quaternary ammonium salt, its mitochondrial membrane potential decreases and ability to generate ROS increases. (iv) Molecular mechanistic studies demonstrated that compound 9a may induce cell apoptosis by inducing loss of MΨm, increasing ROS level, release of cytochrome c, and activation of caspase-3 and caspase-9 in A375 cells. These findings can serve as the basis for further research on the chemical modifications, structure–activity relationships, and bioactivity of emodin and other anthraquinone compound.
Acknowledgments This study is supported by the Natural Science Foundation of Fujian Province (No. 2013J01361 and 2014J01364) and the National Natural Science Foundation of China (No. J1103303, 81201709, and 81472767) and National Health and Family Planning Commission (WKJ-FJ-15).
Conflicts of interests Figure 4: Schematic diagram showing apoptotic pathways induced by compound 9a in A375 cells.
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signaling
No potential conflicts of interests were disclosed. Chem Biol Drug Des 2015
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References 1. Chen L.B. (1988) Mitochondrial membrane potential in living cells. Annu Rev Cell Biol;4:155–181. 2. Davis S., Weiss M.J., Wong J.R., Lampidis T.J., Chen L.B. (1985) Mitochondrial and plasma membrane potentials cause unusual accumulation and retention of rhodamine 123 by human breast adenocarcinomaderived MCF-7 cells. J Biol Chem;260:13844–13850. 3. Wang F., Ogasawara M.A., Huang P. (2010) Small mitochondria-targeting molecules as anti-cancer agents. Mol Aspects Med;31:75–92. 4. Biasutto L., Dong L.F., Zoratti M., Neuzil J. (2010) Mitochondrially targeted anti-cancer agents. Mitochondrion;10:670–681. 5. Modica-Napolitano J.S., Aprille J.R. (2001) Delocalized lipophilic cations selectively target the mitochondria of carcinoma cells. Adv Drug Deliv Rev;49:63–70. 6. Rajaputra P., Nkepang G., Watley R., You Y. (2013) Synthesis and in vitro biological evaluation of lipophilic cation conjugated photosensitizers for targeting mitochondria. Bioorg Med Chem;21:379–387. 7. Bernal S.D., Lampidis T.J., Summerhayes I.C., Chen L.B. (1982) Rhodamine-123 selectively reduces clonal growth of carcinoma cells in vitro. Science;218:1117–1119. 8. Koya K., Li Y., Wang H., Ukai T., Tatsuta N., Kawakami M., Shishido T., Chen L.B. (1996) MKT-077, a novel rhodacyanine dye in clinical trials, exhibits anticarcinoma activity in preclinical studies based on selective mitochondrial accumulation. Cancer Res;56:538–543. 9. Fantin V.R., Leder P. (2004) F16, a mitochondriotoxic compound, triggers apoptosis or necrosis depending on the genetic background of the target carcinoma cell. Cancer Res;64:329–336. 10. Shao J., Zhang F., Bai Z., Wang C., Yuan Y., Wang W. (2012) Synthesis and antitumor activity of emodin quaternary ammonium salt derivatives. Eur J Med Chem;56:308–319. 11. Wang W., Bai Z., Zhang F., Wang C., Yuan Y., Shao J. (2012) Synthesis and biological activity evaluation of emodin quaternary ammonium salt derivatives as potential anticancer agents. Eur J Med Chem;56:320–331. 12. Shao J.W., Dai Y.C., Xue J.P., Wang J.C. (2011) In vitro and in vivo anticancer activity evaluation of ursolic acid derivatives. Eur J Med Chem;46:2652–2661. 13. Wang J.C., Jiang Z., Xiang L.P., Li Y.F., Ou M.R., Yang X., Shao J.W., Lu Y.S., Lin L.F., Chen J.Z., Dai Y., Jia L. (2014) Synergism of ursolic acid derivative US597 with 2-deoxy-D-glucose to preferentially induce tumor cell death by dual-targeting of apoptosis and glycolysis. Sci Rep;4:5006. 14. Shao J.W., Dai Y.C., Zhao W.N., Xie J.J., Xue J.P., Ye J.H., Jia L. (2013) Intracellular distribution and mechanisms of actions of photosensitizer Zinc (II)-phthalocyanine solubilized in Cremophor EL against human hepatocellular carcinoma HepG2 cells. Cancer Lett;1:49–56.
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15. Dong H.Y., Yang X., Xie J.J., Xiang L.P., Li Y.F., Ou M.R., Chi T., Liu Z.H., Yu S.H., Gao Y., Chen J.Z., Shao J.W., Jia L. (2015) UP12, a novel ursolic acid derivative with potential for targeting multiple signaling pathways in hepatocellular carcinoma. Biochem Pharmacol;2:151–162. 16. Xiang L.P., Chi T., Tang Q., Yang X., Ou M.R., Chen X.F., Yu X.B., Chen J., Ho R.J., Shao J.W., Jia L. (2015) A pentacyclic triterpene natural product, ursolic acid and its prodrug US597 inhibit targets within cell adhesion pathway and prevent cancer metastasis. Oncotarget;6:9295–9312. 17. Shao J.W., Xue J.P., Dai Y.C., Liu H., Chen N.S., Jia L., Huang J.L. (2012) Inhibition of human hepatocellular carcinoma HepG2 by phthalocyanine photosensitiser PHOTOCYANINE: ROS production, apoptosis, cell cycle arrest. Eur J Cancer;13:2086–2096.
Supporting Information Additional Supporting Information may be found in the online version of this article: Appendix S1. Synthesis. Table S1. In vitro activity of synthesized compounds against various cancer cells and normal cell. Scheme S1. The synthetic route of emodin derivatives with a long carbon chain in 3-position. Scheme S2. The synthetic route of emodin derivatives 9a–9e. Scheme S3. The synthetic route of emodin quaternary phosphonium salts. Scheme S4. The synthetic route of emodin sulphonium and emodin thioether. Scheme S5. The synthetic route of emodin tertiary amine and emodin tertiary amine oxide. Figure S1. The structures of some delocalized lipophilic cations. Figure S2. Increase in intracellular reactive oxygen species (ROS) level in HepG2, BGC823, and A375 cells. Figure S3. The abilities to generate reactive oxygen species (ROS) for emodin and its quaternary ammonium salt derivatives (control, A; Emodin, B; 3a, C; 11e, D; 14a, E) and the mean fluorescence of 2,7- dichlorofluorescein (DCF) is shown (F). Figure S4. Intracellular distribution of compound 9a in A375 localized by fluorescence phase-contrast image.
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Synthesis, Characterization, and Anticancer Activity of Novel Lipophilic Emodin Cationic Derivatives Xiang Yang1, Wenna Zhao1, Xiufang Hu1, Xianxiao Hao1, Fang Hong1, Jianlong Wang1, Liping Xiang1, Yunhui Zhu1, Yaofeng Yuan1,2, Rodney J.Y. Ho1,3, Wenfeng Wang1,2,* and Jingwei Shao1,2,3,* 1
College of Chemistry, Fuzhou University, Fuzhou, Fujian 350108, China 2 Research Institute of Photocatalysis, State Key Laboratory of Photocatalysis on Energy and Environment, Fuzhou, Fujian 350002, China 3 Department of Pharmaceutics, University of Washington, Seattle, WA 98105, USA *Corresponding authors: Jingwei Shao, shaojingwei@ fzu.edu.cn; Wenfeng Wang, [email protected] Seventeen novel emodin derivatives were synthesized, and the structures were confirmed by IR, H NMR, MS, and elemental analysis. The cytotoxic activity of the derivatives was evaluated against A375, BGC-823, HepG2, and HELF cells by MTT assay. Compound 9a with highest potency and low toxicity was selected to further investigate its detailed molecular mechanism. The lead compound 9a induced a loss of the mitochondrial transmembrane potential (MΨm), an increase in reactive oxygen species (ROS), release of cytochrome c and activation of caspase-3 and caspase-9. In addition, the confocal study showed that emodin derivative 9a (containing asymmetric hydrocarbon tails) was mainly localized in mitochondria, demonstrating a key role of the mitochondria-mediated apoptosis pathway in cancer cells. Taken together, the results demonstrate that embodin derivative 9a preferentially regulates the ROS-mediated apoptosis in A375 cells through the induction of cytochrome c expression and activation of caspase-3 and caspase-9 proteins. Key words: apoptosis, emodin derivative, reactive oxygen species, synthesis Received 9 January 2015, revised 15 May 2015 and accepted for publication 4 June 2015
It has been known for many years that mitochondria of cancer cells have significantly higher transmembrane potential than normal cells (1,2). Taking advantage of this biological property has been a popular strategy for anticancer drugs to improve their therapeutic selectivity (3,4). Delocalized lipophilic cations (DLC), which possess highly hydrophobic ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12612
structures and positive charge, are able to accumulate in the mitochondria of cancer cell due to the highly negatively charged microenvironment in the mitochondrial matrix (5,6). Some DLCs, such as Rhodamine-123 (7), MKT-077 (8), and F16 (9) (Figure S1), have been found to exhibit some degree of efficacy in killing cancer cell. One obvious character of DLC is that their positive charge delocalizes to almost whole molecule rather than localizes in one atom. In our recent work (10,11), a series of emodin quaternary ammonium salt derivatives whose positive charges localize in N atom were synthesized. All the emodin quaternary ammonium salts containing two long carbon chains with moderate length in N cation showed good anticancer activities both in vitro and in vivo, which indicates that localized lipophilic cations (LLC) also possess high anticancer activity. But some questions remain unanswered. (i) Is mitochondria also the target of emodin LLC? (ii) Is the position of emodin where lipophilic group attached important to the anticancer activity of emodin LLC? (iii) Can other emodin LLCs, such as emodin quaternary phosphonium salt and emodin sulphonium salt, show high anticancer activities like emodin quaternary ammonium salt? (iv) Emodin tertiary amine, tertiary amine oxide carry less positive charge compared with emodin quaternary ammonium salts, can they exhibit the similar anticancer activities as those of emodin quaternary ammonium salts? In this study, we have synthesized various novel emodin LLCs as well as emodin tertiary amine, emodin tertiary amine oxide, in an attempt to improve the anticancer activity and to explore the structure–activity relationships. The cytotoxicity of these compounds was all tested by MTT assay. The cell apoptosis and reactive oxygen species (ROS) generation induced by these compounds were also tested by acridine orange (AO)/ethidium bromide (EB) staining, DAPI staining, and flow cytometry (FCM) analysis. The intracellular localization of drugs was studied using confocal microscopy. Based on these work, the four questions mentioned above are answered in this study.
Experiment General Emodin was purchased from China Xi’an Sino-Herb Biotechnology Co. Ltd. (Xian, Shanxi Province, China), in 62% 1
Yang et al.
purity. After two repeats of wash (by 5% NaHCO3) and extraction (2% Na2CO3), the purity of the emodin can reach about 90%. Silica gel (100–200 mesh) used in column chromatography was provided by Tsingtao Marine Chemistry Co. Ltd (Tsingtao, Shandong Province, China). All solvents had been distilled before they were used, and other reagents were obtained from commercial suppliers in analytically chemically pure forms. Melting points were measured on WRS-1B micromelting apparatus and were uncorrected. 1H NMR and 13C NMR spectra were recorded on BRUKER AV (400M) spectrometer with tetramethylsilane as an internal standard in CDCl3. The chemical shift values are on d scale and the coupling constants (J) are in Hz. Electrospray ionization (ESI) mass spectra were measured on an Agilent 1100 IC/MSD TrapXCT and were reported as m/z. Elemental analyses, conducted by the Service Center of Elemental Analysis of Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences.
Synthesis The methods to give other derivatives and characterization data of compounds were showed in Supporting information.
Cell lines and culture A375, BGC-823, HepG2, and HELF cell lines were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cells were maintained in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 mg/mL) in a humidified atmosphere of 5.0% CO2 at 37 °C.
Cell viability assay The cytotoxicity of the derivatives was determined by MTT assay as previously described with minor modification (12– 16). Briefly, emodin, paclitaxel, and compounds 5a–18 were dissolved in dimethyl sulfoxide (DMSO) and diluted with culture medium (final DMSO concentration of 0.1%), respectively. Paclitaxel was used as a positive control. Various cells including A375, BGC823, HepG2, and HELF were treated with indicated concentrations of different emodin derivatives for 24 h. Cell viability was determined by detecting the absorbance at 570 nm in a microquant plate reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and triplicate wells were analyzed at each dose. The concentration of the compound which gives the 50% growth inhibition value was defined as the IC50.
Detection of cell apoptosis by AO/EB staining A375 cells were incubated with compound 9a (4 lM) for 24 h. The cells were then trypsinized and suspended in 2
the medium at cell density of 1 9 106 mL. Cell suspension (25 lL) was stained with 1 lL AO/EB, and the stained cells were visualized with a fluorescent microscope (Leica Co. Ltd, Oberkochen, Germany) preset at excitation 510 nm to assess apoptotic and necrosis morphological changes of the cells caused by compound 9a.
Detection of cell apoptosis by FCM Cell apoptosis was quantified by FCM analysis using Annexin V-FITC/PI apoptosis detection kit according to the manufacture’s protocol as previously described (13,14). Apoptotic cells were those stained with Annexin V-FITC+/ PI (early apoptotic cells) and Annexin V-FITC+/PI+ (late apoptotic cells).
Detection of cell apoptosis by DAPI staining The cells were treated with compound 9a (2 lM) for 24 h and then washed once in PBS followed by fixation in cold methanol: acetic acid (3:1) for 10 min. After washing thrice in PBS for 5 min, these cells were then stained with 1 lg/ mL DAPI solution for 5 min at room temperature. The cells were washed twice with PBS and stained nuclei were observed using a fluorescence microscope (Leica).
Detection of intracellular ROS 2,7-Dichlorodihydrofluorescein diacetate (Invitrogen, Waltham, MA, USA) was used to detect intracellular ROS level. DCF-DA is cleaved intracellularly by non-specific esterases and oxidized by ROS to the fluorescent 2,7dichlorofluorescein (DCF). After washing once with PBS, treated cells were incubated with 20 lM DCF-DA in serum-free DMEM at 37 °C for 30 min before analysis by FCM.
Measurement of mitochondrial membrane potential Mitochondrial membrane potential (MΨm) was measured using 3,30 -dihexyloxacarbocyanine iodide (DiOC6) (Invitrogen) by FCM as described previously (13,14). Briefly, the treated cells were incubated with 50 nM DiOC6 in serumfree DMEM for 30 min at 37 °C in a 5% CO2 incubator, then washed and resuspended in PBS at 1 9 106 cells/ mL, and then analyzed by FCM.
Measurement of caspase activity Activities of caspase-3 and caspase-9 were measured using Caspase Activity Assay Kit (Biyotime Co. Ltd, Sichou, Jiangsu Province, China) according to the manufacturer’s instructions as previously described (13). Briefly, A375 cells were treated with compound 9a for 24 h. Lysates were incubated at 37 °C for 4 h. Samples were measured with a microplate reader (Tecan Infinite 200 €nnedorf, Switzerland) at an absorbance of PRO, Ma Chem Biol Drug Des 2015
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405 nm. Caspase activity is reported as the ratio of the fluorescence output in treated sample relative to control which is normalized to 1.0.
Western blotting analysis Western blots were performed as previously described (15,16). Briefly, cells were collected and resuspended in whole cell lysis buffer. Total protein content was quantified using the Bradford assay. After separated by SDS–PAGE under reducing conditions, proteins were transferred onto polyvinylidene difluoride (PVDF) membrane, followed by immunoblotting. Total proteins expression were normalized to b-actin levels.
Measurement of subcellular localization The fluorescent mitochondrial probe, Mito-Tracker Green, was used to measure the subcellular localization of compound 9a in A375 cells, as described previously (17). A375 cells at final density 105 cells/mL were cultured in Petri dish for study of intracellular localization of compound 9a. At 70% confluence, the cells were exposed to compound 9a (2 lM) at 37 °C in a 5% CO2 incubator for 24 h. The old medium was then aspirated and the cells were washed twice with prewarmed fresh PBS and incubated in the medium containing 100 nM of the fluorescent mitochondrial probe (Mito-Tracker Green, emitted at 488 nm) for 30 min. After being washed twice with PBS, the intracellular localization of compound 9a (emitted at 568 nm) was examined on a laser confocal microscope (Olympus FluoView FV1000, Tokyo, Japan).
Statistical analysis The data were presented as mean standard deviations of three determinations. Statistical analysis was performed using Student’s t-test and one-way analysis of variance. Multiple comparisons of the means were performed by the least significant difference (LSD) test. A probability value of 50 lM was given. The results are listed in Table S1. Our recent reports showed that there was an obvious correlation between the length of carbon chains attached to the quarter N cation of emodin and anticancer activity, only those quaternary ammonium salts that contained two long carbon chains with moderate length had high anticancer activity. In this study, we introduce a decyloxyl and not a methoxyl in 3-position of emodin and synthesize the compounds 5a–5c. As a result, the anticancer activities of 5a are lower than those of compounds 5b and 5c that both contain two long carbon chains with moderate length in quarter N cation, which indicates that the replacement of methoxyl by decyloxyl does not change structure–activity relationship drastically. That is to say, the anticancer activity of emodin is not 3
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sensitive to the change of length of carbon chain in 3position. Among the compound 9a–9e, compound 9e contains two dodecyl groups in quarter N cation and shows the lowest cytotoxicities against cancer cells. The other emodin quaternary ammonium salts (9a–9d) all contain two long chains with 8–10 carbon atoms in quarter N cation and all show higher anticancer activity than paclitaxel. Among which, the quaternary ammonium salt 9a containing two long chains with 8 and 9 carbon atoms, respectively, exhibits the highest anticancer activity. Such a structure–activity relationship indicates that the length of carbon chain attached to the N cation of emodin quaternary ammonium salts has a large effect on the anticancer activity of emodin derivatives. It is very likely that this region is an activity sensitive region that interacts with target point strongly. Four triphenyl quaternary phosphonium salts 11a–11d and one trioctyl quaternary phosphonium salt 11e all exhibit higher anticancer activities than paclitaxel. The length of link arm between emodin moiety and triphenyl moiety of compound 11d is much longer than that of 11a, but the two emodin quaternary phosphonium salts possess almost equal anticancer activity. The reason of which is likely because their lipophilic property both come from triphenyl phosphine group rather than link arm. As phenyl and octyl both are good lipophilic group, compounds 11a–11e all exhibit high anticancer activities, but their toxicities to normal cell are some higher than those of emodin quaternary ammonium salts. Emodin sulphonium salt 14 exhibits a moderate anticancer activity to HepG2 cell, but emodin thioether 16 shows no anticancer activity due to lack of positive charge and sufficient lipophilic property. Emodin tertiary amine 17 exhibits a high anticancer activity because tertiary amine can carry positive charge by protonation, but tertiary amine oxide 18 exhibits a relative low anticancer activity due to its relative weak proton affinity. As human melanoma cell line A375 shows good sensitivity to emodin quaternary ammonium salts, the most active compound 9a is chosen to further investigate its growthinhibitory mechanism in vitro on human melanoma A375 cell.
Compound 9a induces cell death in apoptotic fashion To examine whether 9a induced apoptosis in A375 cells, we conducted AO and EB staining, DAPI staining and FCM analysis with Annexin V-FITC/PI staining to access the cell membrane integrity and the typical characteristics of apoptosis (Figure 1). Morphological analysis using AO/ EB staining showed that compound 9a treated cell displayed morphological characteristics of apoptosis, such as 4
plasma membrane blebbing, congregated chromatin, and nucleolus pyknosis, meanwhile many cells edge damage occurred, healthy cells were absent, shown as intense red particles under the microscope, while the control cells showed uniform green-yellow fluorescence, indicating that the morphology of the cells was intact as evidenced by the evenly distributed chromatin in the nucleolus of healthy cells (Figure 1A). FCM analysis further showed that compound 9a induced a significant increase in the percentage of annexin-V/PI and annexin-V+/PI cells, suggesting an increase in the number of apoptosis cells (Figure 1B). For further assessment of apoptosis, morphological changes of nuclei were visualized following DNA staining by the fluorescent dye, 40 ,6-diamidine-20 -phenylindole dihydrochloride-DAPI (Figure 1C). As shown in Figure 1C, the cells treated with compound 9a showed morphological changes of apoptosis, including a condensed and fragmented nuclear structure and decreased cell size by DAPI staining.
Changes of cellular ROS level in the effects of compound 9a We next assessed intracellular ROS production in HepG2, BGC823, and A375 cell lines using 2,7dichlorodihydrofluorescein diacetate (DCF-DA) fluorescence dye. As shown in Figure S2, exposure of cells to compound 9a (1 lM) elicited significant (p < 0.05, p < 0.01) elevation of cellular ROS level compared to those of untreated cells, and the levels of intracellular ROS were significantly high (p < 0.01), while incubation of cells with relative high concentrations of compound 9a (2 lM), which led to a 3.6-, 6.1-, and 7.2-fold increase in the ROS production level compared to those of untreated cells, respectively.
The abilities to generate ROS for various emodin derivatives In order to ascertain whether the difference between emodin and emodin quaternary ammonium salts is relative to their abilities to generate ROS, the abilities of emodin and several emodin quaternary ammonium salts to generate ROS are tested and the result is listed in Figure S3. Thereinto, the synthesis of compound w3 has been reported in our early work, and its structure is listed in Figure S3. Compound w3 is water soluble and without any anticancer activity. Figure S3 shows that its ability to generate ROS is even lower than that of control group, which implies that the lipophilic property plays an important role for the ability to generate ROS. But excessive lipophilic property (9e) is unfavorable of both anticancer activity and the ability to generate ROS, so the anticancer activity and the ability to generate ROS of compound 9e are both lower than those of compound 9a. That is to say, there is a positive correlation between the anticancer activity and the ability to generate ROS. Chem Biol Drug Des 2015
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B
C
Figure 1: Compound 9a induces cell death in apoptotic fashion in A375 cells. (A) Fluorescence images of AO and ethidium bromide-stained A375 cells. (B) Representative dot-plots results by flow cytometry from three independent experiments illustrating 9a-induced apoptotic status. (C) Apoptosis of A375 cells detected by DAPI staining.
Compound 9a causes accelerated loss of mitochondrial membrane potential The DΨm of BGC823, HepG2, and A375 cells was measured following a 24 h of incubation with different concentrations of compound 9a (2, 4, 8 lM). As shown in Figure 2, compound 9a diminished the DΨm in a concentration-dependent manner compared to control in all three cells. The DΨm levels were significantly (p < 0.05, p < 0.01) diminished, while incubation of cells with relative high concentrations of compound 9a (4, 8 lM), which led to a 49% and 78% decrease in DΨm, respectively, compared to those of untreated A375 cells.
Caspases and cytochrome c are involved in compound 9a-induced apoptosis To further determine the roles of caspases in compound 9a-induced apoptosis, we first assessed caspase-3 and caspase-9 activities after 9a treatment for 24 h in A375 cell line. As shown in Figure 3A, compound 9a induced an increase in the activities of caspase-3 and caspase-9, indiChem Biol Drug Des 2015
Figure 2: Effects of compound 9a on mitochondrial membrane potential of HepG2, BGC823, and A375 cells. Cells were treated with compound 9a (2, 4, and 8 lM) resulted in a concentrationdependent decrease in mitochondrial DΨ m. Results are expressed as the percent change in DΨ m of the treated cells compared to the untreated control. Statistical difference from controls: *p < 0.05, **p < 0.01.
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B
Figure 3: Caspases and cytochrome c are involved in compound 9a-induced apoptosis. A. Compound 9a induced activations of caspase-3 and caspase-9 in A375 cells. B. Effect of compound 9a on the expression of cytochrome c in A375 cells by Western blot analysis. *p < 0.05, **p < 0.01.
cating that ROS elicited from 9a was involved in the 9ainduced caspase activation. Release of cytochrome c from mitochondria into cytoplasm is thought to be an absolute requirement for the activation of caspase-9 in the intrinsic apoptosis pathway. We finally used Western blot analysis to assess the effect of compound 9a on the expression of cytochrome c in A375 cells. The efficiencies of cytochrome c were shown in Figure 3B. Compound 9a induced a concentrationdependent increase in cytochrome c expression.
in mitochondria of A375 (Figure S4B,C), and to a lesser degree, perhaps in cytoplasm and other subcellular organelles (Figure S4C), but not in the nuclear (Figure S4A–D). According to our data, we summarize the signaling pathways related to compound 9a-induced apoptosis of A375 cells in Figure 4. ROS and DΨm elicited from 9a trigger the intrinsic apoptotic pathways. Cytochrome c preferentially trigger caspase-3 and caspase-9 activation to dominate 9a-induced classical apoptosis in A375 cells.
Conclusion Subcellular localization of compound 9a in A375 cells After 24 h of incubation of A375 with compound 9a (2 lM), the distribution of compound 9a within the cells was observed using confocal laser microscopy with the aid of subcellular probes. Different emission wavelengths were applied to corresponding probes of subcellular organelles. Mitochondria was stained green with emission wavelength at 488 nm (Figure S4A). Compound 9a exhibited red at 568 nm (Figure S4B). Under the confocal laser scanning microscope, compound 9a was primarily located
The above study result indicate that (i) the length of carbon chains in 3-position of emodin has no obvious effect on the anticancer activities of emodin quaternary ammonium salt, but the length of carbon chains in N cation has a close relationship with anticancer activity; (ii) both lipophilicity and positive charge play an important role on the anticancer activity of emodin derivatives; (iii) when cancer cell is treated with emodin quaternary ammonium salt, its mitochondrial membrane potential decreases and ability to generate ROS increases. (iv) Molecular mechanistic studies demonstrated that compound 9a may induce cell apoptosis by inducing loss of MΨm, increasing ROS level, release of cytochrome c, and activation of caspase-3 and caspase-9 in A375 cells. These findings can serve as the basis for further research on the chemical modifications, structure–activity relationships, and bioactivity of emodin and other anthraquinone compound.
Acknowledgments This study is supported by the Natural Science Foundation of Fujian Province (No. 2013J01361 and 2014J01364) and the National Natural Science Foundation of China (No. J1103303, 81201709, and 81472767) and National Health and Family Planning Commission (WKJ-FJ-15).
Conflicts of interests Figure 4: Schematic diagram showing apoptotic pathways induced by compound 9a in A375 cells.
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No potential conflicts of interests were disclosed. Chem Biol Drug Des 2015
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Supporting Information Additional Supporting Information may be found in the online version of this article: Appendix S1. Synthesis. Table S1. In vitro activity of synthesized compounds against various cancer cells and normal cell. Scheme S1. The synthetic route of emodin derivatives with a long carbon chain in 3-position. Scheme S2. The synthetic route of emodin derivatives 9a–9e. Scheme S3. The synthetic route of emodin quaternary phosphonium salts. Scheme S4. The synthetic route of emodin sulphonium and emodin thioether. Scheme S5. The synthetic route of emodin tertiary amine and emodin tertiary amine oxide. Figure S1. The structures of some delocalized lipophilic cations. Figure S2. Increase in intracellular reactive oxygen species (ROS) level in HepG2, BGC823, and A375 cells. Figure S3. The abilities to generate reactive oxygen species (ROS) for emodin and its quaternary ammonium salt derivatives (control, A; Emodin, B; 3a, C; 11e, D; 14a, E) and the mean fluorescence of 2,7- dichlorofluorescein (DCF) is shown (F). Figure S4. Intracellular distribution of compound 9a in A375 localized by fluorescence phase-contrast image.
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