- Email: [email protected]
3′-Geranyl-mono-substituted chalcone Xanthoangelovl induces apoptosis in human leukemia K562 cells via activation of mitochondrial pathway
Chemico-Biological Interactions 261 (2017) 103e107
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
30 -Geranyl-mono-substituted chalcone Xanthoangelovl induces apoptosis in human leukemia K562 cells via activation of mitochondrial pathway Yuou Teng, Lixin Wang, Huan Liu, Yuan Yuan, Qian Zhang, Meng Wu, Luyao Wang, Haomeng Wang, Zhen Liu*, Peng Yu** Key Lab of Industrial Fermentation Microbiology, Tianjin Key Lab of Industrial Microbiology, Sino-French Joint Lab of Food Nutrition/Safety and Medicinal Chemistry, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 September 2016 Received in revised form 7 November 2016 Accepted 22 November 2016 Available online 29 November 2016
30 -Geranyl-mono-substituted chalcone Xanthoangelol (1b), a chalcone derivative, was previously reported to show selective cytotoxicity against human chronic myelogenous leukemia K562 cells with a half-maximal inhibitory concentration (IC50) of 3.98 mM. In the present study, we investigated the molecular mechanism underlying the cytotoxicity of 1b in K562 cells. Treatment with compound 1b caused K562 cells to adopt a typical apoptotic morphology. Flow cytometric analysis also confirmed the presence of an apoptotic cell population following treatment of Annexin-V-FITC and propidium iodide (PI) double-labeled K562 cells with 1b. Furthermore, we observed dissipation of the mitochondrial membrane potential, caspase-3 activation, and a reduction of the Bcl-2/ Bax ratio in these cells, which suggest that the mitochondrial apoptotic pathway is induced by 1b in K562 cells. Collectively, our findings demonstrate that compound 1b notably induces mitochondrialmediated apoptosis in K562 cells, which might have a potential anticancer activity. © 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Chalcones Apoptosis Caspase-3 Bcl-2
1. Introduction Chalcones are natural or synthetic compounds belonging to the flavonoid family with wide distribution in the flowers, leaves, bark, and roots of liquorice, saffron, and other natural plants. They possess many pharmacological and biological activities, such as antitumor [1e3], antidiabetic [4], antimalarial [5], antioxidant [6], antifungal [7], anti-angiogenic [8], and anti-inflammatory activities [9]. In recent years, many natural chalcone products and their analogs were found to exhibit high cytotoxic activity in tumors. Zi and Simoneau [10] found that Flawkawain A extracted from the roots of plants, activated caspase-9 and caspase-3, thereby inducing apoptosis via the mitochondrial apoptotic pathway in human bladder cancer cells. Furthermore, in vivo studies on tumor-bearing mice confirmed Flawkawain A's antitumor efficacy. Hsu et al. [11] found that isoliquiritigenin could induce apoptosis in HepG2 cells
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Z. Liu), [email protected] (P. Yu). http://dx.doi.org/10.1016/j.cbi.2016.11.025 0009-2797/© 2016 Elsevier Ireland Ltd. All rights reserved.
by regulating certain cellular factors, such as p53, p21, and Bax. Saxena et al. [12] showed that chalcone derivatives induced A549 cell apoptosis by promoting the expression of p35. Boumendjel et al. [13] reported that several chalcone analogs induced G2/M cell cycle arrest in K562 cells. It is well known that apoptosis is the result of a highly complicated cascade of cellular events causing cell rounding and shrinkage, chromatin condensation, DNA fragmentation, shedding of small cellular fragments, and loss of adhesion [14]. In particular, it has been established that many chemotherapeutic agents induce cancer cell apoptosis via a mitochondria-dependent pathway [15e17]. The mitochondrial pathway is highly regulated by Bcl-2 family members [18,19]. The Bcl-2 family of proteins is thought to be the key players regulating mitochondrial membrane permeabilization (MMP) in caspase-dependent apoptosis. The Bcl-2 family consists of both pro-apoptotic and anti-apoptotic members, such as Bax and Bcl-2 [20]. We previously reported the design, synthesis, and in vitro cytotoxicity of a series of novel chalcone derivatives, which could be used as anticancer agents [21]. The 3’-geranyl-mono-substituted chalcone Xanthoangelol (1b) showed good and selective cytotoxic activity (IC50 ¼ 3.98 mM). In the present study, we investigated the
104
Y. Teng et al. / Chemico-Biological Interactions 261 (2017) 103e107
molecular mechanism underlying the cytotoxic activity of 1b against human chronic myelogenous leukemia K562 cells. 2. Materials and methods 2.1. Cell lines and culture conditions The K562 cell line was obtained from the Shanghai Institutes of Biological Sciences (Shanghai, China). Cells were grown at 37 C in RPMI-1640 supplemented with 10% fetal bovine serum, 2.05 mM glutamine, and 1% penicillin/streptomycin in a humidified atmosphere containing 5% CO2. The medium was replaced once every third day. 2.2. Cytotoxic activity assay The cytotoxic activity was measured using an MTT assay [22]. Briefly, 100 mL of K562 cell suspension was cultured in 96-well plates at a density of 5 104 cells/mL. After 2 h, different concentrations of 1b (1e10,000 nM) were added to each well and the cells were incubated for another 48 h. The MTT assay was performed using a thermo microplate reader. The DMSO-treated controls were assigned a cell viability value of 100%. The inhibitory concentrations (IC50) were obtained by nonlinear regression using GraphPad Prism 5.0. For each treatment, the IC50 value was calculated from three independent experiments.
2.6. Flow cytometric analysis of the mitochondrial membrane potential To measure the effects of 1b on the mitochondrial membrane potential (DJm), K562 cells were treated with DMSO or 1b (30 mM) for 12, 24, or 48 h. Then, cells were stained with 100 nM tetramethylrhodamine methyl ester (TMRM; Invitrogen, USA) for 30 min in the dark at room temperature, and analyzed by flow cytometry. TMRM is the most specific agent for measuring changes in DJm. 2.7. Western blot analysis Anti-caspase-3, anti-PARP, anti-Bcl-2, anti-Bcl-xl, and anti-Bax were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Other anti-antibodies were purchased from Cell Signaling Technology (CST, Boston, MA). Cells were lysed in a lysis buffer containing 10 mM Hepes-Na, 150 mM Na2SO4, 1 mM EDTA, 3% CHAPS, 1 mM phenylmethylsulfonyl fluoride, and 10 mg/mL of both aprotinin and leupeptin. For the western blot analysis of total cell lysates, samples were prepared by mixing an aliquot of cell lysate with an equal volume of 2 Laemmli's sample buffer and heating at 100 C. The samples were separated by SDS-PAGE and electrotransferred to PVDF membranes (Millipore, Bedford, MA). The membranes were probed with the aforementioned antibodies and incubated with Alexa Fluor® 680 Goat Anti-Mouse IgG (HþL) and Alexa Fluor® 680 Goat Anti-Rabbit IgG (HþL) followed by detection using the Odyssey western blotting detection system (Amersham Pharmacia Biotech).
2.3. Flow cytometric analysis of apoptosis Apoptotic cells were assayed by the Annexin-V-FITC Apoptosis Detection Kit (BD Biosciences, USA) according to the manufacturer's instructions. Briefly, K562 cells were treated with DMSO or 1b (30 mM) for 12 h, 24 h, 36 h, or 48 h. Cells were harvested, washed twice with ice-cold PBS, and resuspended in 1 binding buffer at a concentration of 1 106 cells/mL, after which they were stained with 5 mL of Annexin-V-FITC and 5 mL of PI (50 mg/mL) for 15 min in the dark at 25 C, and analyzed by flow cytometry.
2.8. Statistical analysis
2.4. Analysis of combination effect
3. Results
Briefly, 100 mL of K562 cell suspension was cultured in 96-well plates at a density of 5 104 cells/mL for 2 h. Then, cells were incubated with 4-PBA or TNF-a for 0.5 h. Different concentrations of 1b (1e10,000 nM) were added to each well and cells were cultured for another 48 h. Then, an MTT assay was performed using a thermo microplate reader. The DMSO treated controls were assigned a cell viability value of 100%. The IC50 values were obtained by nonlinear regression using GraphPad Prism 5.0. For each treatment, the IC50 value was calculated from three independent experiments.
3.1. Morphological changes in 1b-treated K562 cells
Data were expressed as means ± S.D. or percentage and analyzed for statistical significance using one-way analysis of variance (ANOVA) followed by Dunnett's test. P-value less than or equal to 0.05 was considered to be statistically significant. Statistical analyses were carried out in GraphPad Prism 5 (GraphPad Software, San Diego, CA, U.S.A.).
Treatment with the highly potent cytotoxic compound 1b (IC50 ¼ 3.98 mM) (Fig. 1A) caused K562 cells to adopt a typical apoptotic morphology (cell shrinkage and/or blebbing; Fig. 1B). The ball-like bodies appeared as early as 12 h after treatment with 30 mM 1b. The number of apoptotic cells increased in a timedependent manner, whereas the total number of cells decreased. These results indicated that compound 1b might inhibit K562 cell proliferation by inducing apoptosis.
2.5. Caspase-3 activity assay Caspase-3 activity was evaluated by the Apo-ONE ® Homogeneous Caspase-3/7 Assay Kit (Promega) according to the manufacturer's instructions. Briefly, K562 cells were cultured in 6-well plates with either DMSO or 1b (30 mM) for 48 h. To each well, 100 mL of Apo-ONE ® Homogeneous Caspase-3/7 buffer with 1 mL Caspase Substrate Z-DEVD-R110 (100 ) was added and cells were cultured for 0.5 h. The release of the fluorochrome following peptide cleavage was kinetically monitored at room temperature (excitation at 485 nm and emission at 521 nm). Cells treated with staurosporine (0.3 mM) for 6 h were used as a positive control.
3.2. Compound 1b induced apoptosis in K562 cells Flow cytometric analysis of cells double-labeled with AnnexinV-FITC and PI revealed the presence of apoptotic K562 cells following 1b treatment (Fig. 2A). The apoptotic rates (Annexin Vþ/ PI and Annexin Vþ/PIþ) of 1b-treated K562 cells increased to 62.8% of total cells compared to 9.8% in the control. As shown in Fig. 2A, treatment with 1b for different time periods showed that the apoptosis rates increased in a time-dependent manner. These results suggested that 1b inhibits the proliferation of K562 cells by inducing apoptosis in a time-dependent manner.
Y. Teng et al. / Chemico-Biological Interactions 261 (2017) 103e107
105
Fig. 1. Morphological changes in 1b-treated K562 cells. (A) Structure and in vitro cell proliferation inhibitory activity of 1b. (B) Morphological changes induced by 1b treatment (30 mM) for 12, 24, 36 or 48 h.
3.3. Cooperative effects of 1b and 4-PBA or TNF-a
3.4. Caspase-3 activity assay
There are three types of apoptotic pathways: the death receptor pathway, mitochondrial pathway, and endoplasmic reticulum pathway. Combination treatment with 1b and the widely used apoptosis inhibitors was used to determine the type of apoptotic pathway through which 1b induces apoptosis in K562 cells. As shown in Fig. 2B, treatment of 1b-treated K562 cells with the endoplasmic reticulum apoptosis inhibitor (4-PBA) or the death receptor inhibitor (TNF-a) did not affect the cell viability significantly. The result showed that the endoplasmic reticulum apoptosis inhibitor and the death receptor inhibitor did not affect 1b-treated K562 cells, thus confirming that the apoptotic pathway triggered by 1b in K562 cells was not the death receptor pathway or the endoplasmic reticulum pathway.
The caspase cascade is one of the most important events in the mitochondrial apoptosis pathway [23]. To determine whether caspase-3 was involved in 1b-induced apoptosis, K562 cells were treated with DMSO, 0.3 mM staurosporine (positive control), or 30 mM 1b, and caspase-3 activation was then determined. As shown in Fig. 3A, compared to the positive control, 1b-treated K562 cells exhibited increased caspase-3 activation. This result indicates that the caspase-3-dependent pathway was involved in 1b-induced apoptosis. From the experimental results, compound 1b was confirmed to induce apoptosis of K562 cells. To study the molecular mechanism by which compound 1b induces apoptosis, we investigated the effect of 1b on the expression of caspase-3 and PARP by using
Fig. 2. 1b induced apoptosis in K562 cells. (A) K562 cells were treated with 30 mM 1b for different periods of time. Flow cytometry analyses of K562 cells under various treatments using double staining with Annexin V (horizontal line) and propidium iodide (PI, vertical line). (B) The cooperative effect of 1b on K562 cells with inhibitor protection.
106
Y. Teng et al. / Chemico-Biological Interactions 261 (2017) 103e107
Fig. 3. Effects of 1b treatment on caspase-3 activity and the expression levels of caspase-3 and PARP proteins. (A) Caspase-3 activation was quantified by means of a fluorescencebased method. (B) Expression of caspase-3 and PARP proteins during 1b-induced apoptosis.
western blotting. As shown in Fig. 3B, compared with DMSOtreated K562 cells, cells treated with 1b showed a dosedependent increase in caspase-3 activation. Furthermore, western blot analysis revealed increased levels of cleaved PARP following treatment with 1b, providing biochemical confirmation for the induction of apoptosis in the K562 cell line. 3.5. 1b induced apoptosis in K562 cells through the mitochondrial pathway Mitochondria play a significant role in apoptosis of certain cancer cell lines. To clarify whether the mitochondrial pathway is involved in 1b-induced apoptosis, K562 cells were treated with DMSO (0.5%) or 1b (30 mM) for 12, 24, or 48 h, and then with a membrane potential sensing dye, TMRM. Compared to DMSOtreated K562 cells, the 1b-treated cells showed a significant decrease in mitochondrial membrane potential from 90.9% (DMSO) to 61.9% (1b) at 12 h, from 93.0% (DMSO) to 70.9% (1b) at 24 h, and from 94.6% (DMSO) to 61.9% (1b) at 48 h (Fig. 4A). These results show that 1b can cause a reduction in the mitochondrial membrane potential, which may induce apoptosis in K562 cells via the mitochondrial pathway.
Western blot analysis was performed to investigate the protein expression of Bcl-2, Bax, and Bcl-xl in K562 cells treated with different concentrations of compound 1b. Alpha-tubulin was used as an internal loading control. As shown in Fig. 4B, western blot results suggest that Bcl-2 and Bcl-xl protein levels dramatically decreased with 1b treatment, whereas the expression level of the Bax protein increased. Moreover, the ratio of Bcl-2/Bax in the treated groups clearly decreased with increasing 1b concentrations (Fig. 4C). These results indicated that in K562 cells, compound 1b initiates programmed cell death via the mitochondrial apoptotic pathway. 4. Discussion The results of this study confirm that compound 1b has excellent cytotoxic activity against human chronic myelogenous leukemia cells K562 (IC50 ¼ 3.98 mM). As shown in Fig. 1B, an apoptotic cell population was clearly observed after K562 cells were treated with 1b for 48 h. Treatment with 1b caused K562 cells to take on a typical apoptotic morphology, characterized by cell shrinkage and/ or blebbing. Furthermore, as shown in Fig. 2A, 1b inhibits the proliferation of
Fig. 4. Effects of 1b treatment on the mitochondrial membrane potential and the expression level of Bcl-2 family proteins. (A) The effect of 1b on K562 cells' mitochondrial membrane potential (DJm). (B) Expression of Bcl-2, Bax, and Bcl-xl during 1b-induced apoptosis. (C) Effect of 1b on the Bcl-2/Bax ratio in K562 cells.
Y. Teng et al. / Chemico-Biological Interactions 261 (2017) 103e107
K562 cells by inducing apoptosis in a time-dependent manner. To determine the apoptotic pathway triggered by 1b in K562 cells, we examined the effects of 1b in combination with TNF-a (the death receptor pathway inhibitor) or 4-PBA (the endoplasmic reticulum pathway inhibitor). The apoptotic pathway induced by 1b was not the death receptor pathway or the endoplasmic reticulum pathway (Fig. 2B). The results suggest that 1b induces apoptosis via the mitochondrial pathway. Caspases, a family of cysteine proteases, play a crucial role in apoptosis regulation with caspase-3 being the main player in the execution of apoptotic cell death [24]. In this study, through the use of both a Caspase-3/7 Assay Kit and western blot analysis, we found that 1b causes caspase-3 activation (Fig. 3). Moreover, as shown in Fig. 4, we found that 1b causes the dissipation of the mitochondrial membrane potential (DJm). Importantly, noticeable changes in the protein levels of Bcl-2, Bax, and Bcl-xl were observed. The Bcl-2/Bax ratio in the treated groups decreased significantly with increasing 1b concentrations. In conclusion, as compared with chalcone, 1b had a low cytotoxicity and improved cell proliferation inhibitory activity. We provide strong evidence that compound 1b inhibits the growth of human chronic myelogenous leukemia K562 cells via the induction of apoptosis. The results indicate that compound 1b inhibits cell proliferation through the initiation of the mitochondrial apoptotic pathway. Studies are underway to clarify the action mechanism and the target molecule of 1b. Although 1b has potent cytotoxicity as compared with chalcone, the IC50 of 1b against K562 is not very high (3.98 mM). Thus, future studies are warranted for the design and synthesis of various types of chalcone derivatives with excellent cytotoxicity and low toxicity. Competing interests The authors declare that there is no conflicts interest. Acknowledgement This work was supported jointly by National Natural Science Foundation of China (31301142 and 31601203) and International Science & Technology Cooperation Program of China (2013DFA31160). We are thankful to the Research Centre of Modern Analytical Technology, Tianjin University of Science and Technology for high resolution mass spectrum analysis. We would like to thank Editage [www.editage.cn] for English language editing. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2016.11.025. References [1] T. Okuyama, M. Takata, J. Takayasu, T. Hasegawa, H. Tokuda, A. Nishino, H. Nishino, A. Iwashima, Anti-tumor-promotion by principles obtained from
107
Angelica keiskei, Planta Med. 57 (1991) 242e246. [2] R. Nishimura, K. Tabata, M. Arakawa, Y. Ito, Y. Kimura, T. Akihisa, H. Nagai, A. Sakuma, H. Kohno, T. Suzuki, Isobavachalcone, a chalcone constituent of Angelica keiskei, induces apoptosis in neuroblastoma, Biol. Pharm. Bull. 30 (2007) 1878e1883. [3] Y.L. Hsu, P.L. Kuo, W.S. Tzeng, C.C. Lin, Chalcone inhibits the proliferation of human breast cancer cell by blocking cell cycle progression and inducing apoptosis, Food Chem. Toxicol. 44 (2006) 704e713. [4] T. Enoki, H. Ohnogi, K. Nagamine, Y. Kudo, K. Sugiyama, M. Tanabe, E. Kobayashi, H. Sagawa, I. Kato, Antidiabetic activities of chalcones isolated from a Japanese Herb, Angelica keiskei, J. Agric. Food Chem. 55 (2007) 6013e6017. [5] S.K. Awasthi, N. Mishra, B. Kumar, M. Sharma, A. Bhattacharya, L.C. Mishra, V.K. Bhasin, Potent antimalarial activity of newly synthesized substituted chalcone analogs in vitro, Med. Chem. Res. 18 (2009) 407e420. [6] P.M. Sivakumar, P.K. Prabhakar, M. Doble, Synthesis, antioxidant evaluation, and quantitative structureeactivity relationship studies of chalcones, Med. Chem. Res. 20 (2010) 482e492. [7] K.L. Lahtchev, D.I. Batovska, S.P. Parushev, V.M. Ubiyvovk, A.A. Sibirny, Antifungal activity of chalcones: a mechanistic study using various yeast strains, Eur. J. Med. Chem. 43 (2008) 2220e2228. [8] Y.S. Lee, S.S. Lim, K.H. Shin, Y.S. Kim, K. Ohuchi, S.H. Jung, Anti-angiogenic and anti-tumor activities of 2'-hydroxy-4'-methoxychalcone, Biol. Pharm. Bull. 29 (2006) 1028e1031. [9] F. Jin, X.Y. Jin, Y.L. Jin, D.W. Sohn, S.A. Kim, D.H. Sohn, Y.C. Kim, H.S. Kim, Structural requirements of 2',4',6'-tris(methoxymethoxy) chalcone derivatives for anti-inflammatory activity: the importance of a 2'-hydroxy moiety, Arch. Pharm. Res. 30 (2007) 1359e1367. [10] X. Zi, A.R. Simoneau, A. Flavokawain, A novel chalcone from kava extract, induces apoptosis in bladder cancer cells by involvement of Bax proteindependent and mitochondria-dependent apoptotic pathway and suppresses tumor growth in mice, Cancer Res. 65 (2005) 3479e3486. [11] Y.L. Hsu, P.L. Kuo, C.C. Lin, Isoliquiritigenin induces apoptosis and cell cycle arrest through p53-dependent pathway in Hep G2 cells, Life Sci. 77 (2005) 279e292. [12] H.O. Saxena, U. Faridi, J.K. Kumar, S. Luqman, M.P. Darokar, K. Shanker, C.S. Chanotiya, M.M. Gupta, A.S. Negi, Synthesis of chalcone derivatives on steroidal framework and their anticancer activities, Steroids 72 (2007) 892e900. [13] A. Boumendjel, G. Sotoing Taiwe, E. Ngo Bum, T. Chabrol, C. Beney, V. Sinniger, R. Haudecoeur, L. Marcourt, S. Challal, E. Ferreira Queiroz, F. Souard, M. Le Borgne, T. Lomberget, A. Depaulis, C. Lavaud, R. Robins, J.L. Wolfender, B. Bonaz, M. De Waard, Occurrence of the synthetic analgesic tramadol in an African medicinal plant, Angew. Chem. Int. Ed. Engl. 52 (2013) 11780e11784. [14] Y.S. Wu, S.N. Chen, Apoptotic cell: linkage of inflammation and wound healing, Front. Pharmacol. 5 (2014) 1. [15] D.R. Green, J.C. Reed, Mitochondria and apoptosis, Science 281 (1998) 1309e1312. [16] K. Sinha, J. Das, P.B. Pal, P.C. Sil, Oxidative stress: the mitochondria-dependent and mitochondria-independent pathways of apoptosis, Arch. Toxicol. 87 (2013) 1157e1180. [17] J.D. Robertson, S. Orrenius, Role of mitochondria in toxic cell death, Toxicology 181e182 (2002) 491e496. [18] J. Kale, Q. Liu, B. Leber, D.W. Andrews, Shedding light on apoptosis at subcellular membranes, Cell 151 (2012) 1179e1184. [19] T. Moldoveanu, A.V. Follis, R.W. Kriwacki, D.R. Green, Many players in BCL-2 family affairs, Trends Biochem. Sci. 39 (2014) 101e111. [20] S.J. Korsmeyer, BCL-2 gene family and the regulation of programmed cell death, Cancer Res. 59 (1999) 1693se1700s. [21] H.M. Wang, L. Zhang, J. Liu, Z.L. Yang, H.Y. Zhao, Y. Yang, D. Shen, K. Lu, Z.C. Fan, Q.W. Yao, Y.M. Zhang, Y.O. Teng, Y. Peng, Synthesis and anti-cancer activity evaluation of novel prenylated and geranylated chalcone natural products and their analogs, Eur. J. Med. Chem. 92 (2015) 439e448. [22] Y. Zhou, H.Y. Zhao, K.L. Han, Y. Yang, B.B. Song, Q.N. Guo, Z.C. Fan, Y.M. Zhang, Y.O. Teng, P. Yu, 5-(2-carboxyethenyl) isatin derivative induces G(2)/M cell cycle arrest and apoptosis in human leukemia K562 cells, Biochem. Biophys. Res. Commun. 450 (2014) 1650e1655. [23] M. Poreba, A. Strozyk, G.S. Salvesen, M. Drag, Caspase substrates and inhibitors, Cold Spring Harb. Perspect. Biol. 5 (2013) a008680. [24] H. Wu, X. Che, Q. Zheng, A. Wu, K. Pan, A. Shao, Q. Wu, J. Zhang, Y. Hong, Caspases: a molecular switch node in the crosstalk between autophagy and apoptosis, Int. J. Biol. Sci. 10 (2014) 1072e1083.
Contents lists available at ScienceDirect
Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
30 -Geranyl-mono-substituted chalcone Xanthoangelovl induces apoptosis in human leukemia K562 cells via activation of mitochondrial pathway Yuou Teng, Lixin Wang, Huan Liu, Yuan Yuan, Qian Zhang, Meng Wu, Luyao Wang, Haomeng Wang, Zhen Liu*, Peng Yu** Key Lab of Industrial Fermentation Microbiology, Tianjin Key Lab of Industrial Microbiology, Sino-French Joint Lab of Food Nutrition/Safety and Medicinal Chemistry, College of Biotechnology, Tianjin University of Science and Technology, Tianjin, 300457, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 September 2016 Received in revised form 7 November 2016 Accepted 22 November 2016 Available online 29 November 2016
30 -Geranyl-mono-substituted chalcone Xanthoangelol (1b), a chalcone derivative, was previously reported to show selective cytotoxicity against human chronic myelogenous leukemia K562 cells with a half-maximal inhibitory concentration (IC50) of 3.98 mM. In the present study, we investigated the molecular mechanism underlying the cytotoxicity of 1b in K562 cells. Treatment with compound 1b caused K562 cells to adopt a typical apoptotic morphology. Flow cytometric analysis also confirmed the presence of an apoptotic cell population following treatment of Annexin-V-FITC and propidium iodide (PI) double-labeled K562 cells with 1b. Furthermore, we observed dissipation of the mitochondrial membrane potential, caspase-3 activation, and a reduction of the Bcl-2/ Bax ratio in these cells, which suggest that the mitochondrial apoptotic pathway is induced by 1b in K562 cells. Collectively, our findings demonstrate that compound 1b notably induces mitochondrialmediated apoptosis in K562 cells, which might have a potential anticancer activity. © 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Chalcones Apoptosis Caspase-3 Bcl-2
1. Introduction Chalcones are natural or synthetic compounds belonging to the flavonoid family with wide distribution in the flowers, leaves, bark, and roots of liquorice, saffron, and other natural plants. They possess many pharmacological and biological activities, such as antitumor [1e3], antidiabetic [4], antimalarial [5], antioxidant [6], antifungal [7], anti-angiogenic [8], and anti-inflammatory activities [9]. In recent years, many natural chalcone products and their analogs were found to exhibit high cytotoxic activity in tumors. Zi and Simoneau [10] found that Flawkawain A extracted from the roots of plants, activated caspase-9 and caspase-3, thereby inducing apoptosis via the mitochondrial apoptotic pathway in human bladder cancer cells. Furthermore, in vivo studies on tumor-bearing mice confirmed Flawkawain A's antitumor efficacy. Hsu et al. [11] found that isoliquiritigenin could induce apoptosis in HepG2 cells
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Z. Liu), [email protected] (P. Yu). http://dx.doi.org/10.1016/j.cbi.2016.11.025 0009-2797/© 2016 Elsevier Ireland Ltd. All rights reserved.
by regulating certain cellular factors, such as p53, p21, and Bax. Saxena et al. [12] showed that chalcone derivatives induced A549 cell apoptosis by promoting the expression of p35. Boumendjel et al. [13] reported that several chalcone analogs induced G2/M cell cycle arrest in K562 cells. It is well known that apoptosis is the result of a highly complicated cascade of cellular events causing cell rounding and shrinkage, chromatin condensation, DNA fragmentation, shedding of small cellular fragments, and loss of adhesion [14]. In particular, it has been established that many chemotherapeutic agents induce cancer cell apoptosis via a mitochondria-dependent pathway [15e17]. The mitochondrial pathway is highly regulated by Bcl-2 family members [18,19]. The Bcl-2 family of proteins is thought to be the key players regulating mitochondrial membrane permeabilization (MMP) in caspase-dependent apoptosis. The Bcl-2 family consists of both pro-apoptotic and anti-apoptotic members, such as Bax and Bcl-2 [20]. We previously reported the design, synthesis, and in vitro cytotoxicity of a series of novel chalcone derivatives, which could be used as anticancer agents [21]. The 3’-geranyl-mono-substituted chalcone Xanthoangelol (1b) showed good and selective cytotoxic activity (IC50 ¼ 3.98 mM). In the present study, we investigated the
104
Y. Teng et al. / Chemico-Biological Interactions 261 (2017) 103e107
molecular mechanism underlying the cytotoxic activity of 1b against human chronic myelogenous leukemia K562 cells. 2. Materials and methods 2.1. Cell lines and culture conditions The K562 cell line was obtained from the Shanghai Institutes of Biological Sciences (Shanghai, China). Cells were grown at 37 C in RPMI-1640 supplemented with 10% fetal bovine serum, 2.05 mM glutamine, and 1% penicillin/streptomycin in a humidified atmosphere containing 5% CO2. The medium was replaced once every third day. 2.2. Cytotoxic activity assay The cytotoxic activity was measured using an MTT assay [22]. Briefly, 100 mL of K562 cell suspension was cultured in 96-well plates at a density of 5 104 cells/mL. After 2 h, different concentrations of 1b (1e10,000 nM) were added to each well and the cells were incubated for another 48 h. The MTT assay was performed using a thermo microplate reader. The DMSO-treated controls were assigned a cell viability value of 100%. The inhibitory concentrations (IC50) were obtained by nonlinear regression using GraphPad Prism 5.0. For each treatment, the IC50 value was calculated from three independent experiments.
2.6. Flow cytometric analysis of the mitochondrial membrane potential To measure the effects of 1b on the mitochondrial membrane potential (DJm), K562 cells were treated with DMSO or 1b (30 mM) for 12, 24, or 48 h. Then, cells were stained with 100 nM tetramethylrhodamine methyl ester (TMRM; Invitrogen, USA) for 30 min in the dark at room temperature, and analyzed by flow cytometry. TMRM is the most specific agent for measuring changes in DJm. 2.7. Western blot analysis Anti-caspase-3, anti-PARP, anti-Bcl-2, anti-Bcl-xl, and anti-Bax were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Other anti-antibodies were purchased from Cell Signaling Technology (CST, Boston, MA). Cells were lysed in a lysis buffer containing 10 mM Hepes-Na, 150 mM Na2SO4, 1 mM EDTA, 3% CHAPS, 1 mM phenylmethylsulfonyl fluoride, and 10 mg/mL of both aprotinin and leupeptin. For the western blot analysis of total cell lysates, samples were prepared by mixing an aliquot of cell lysate with an equal volume of 2 Laemmli's sample buffer and heating at 100 C. The samples were separated by SDS-PAGE and electrotransferred to PVDF membranes (Millipore, Bedford, MA). The membranes were probed with the aforementioned antibodies and incubated with Alexa Fluor® 680 Goat Anti-Mouse IgG (HþL) and Alexa Fluor® 680 Goat Anti-Rabbit IgG (HþL) followed by detection using the Odyssey western blotting detection system (Amersham Pharmacia Biotech).
2.3. Flow cytometric analysis of apoptosis Apoptotic cells were assayed by the Annexin-V-FITC Apoptosis Detection Kit (BD Biosciences, USA) according to the manufacturer's instructions. Briefly, K562 cells were treated with DMSO or 1b (30 mM) for 12 h, 24 h, 36 h, or 48 h. Cells were harvested, washed twice with ice-cold PBS, and resuspended in 1 binding buffer at a concentration of 1 106 cells/mL, after which they were stained with 5 mL of Annexin-V-FITC and 5 mL of PI (50 mg/mL) for 15 min in the dark at 25 C, and analyzed by flow cytometry.
2.8. Statistical analysis
2.4. Analysis of combination effect
3. Results
Briefly, 100 mL of K562 cell suspension was cultured in 96-well plates at a density of 5 104 cells/mL for 2 h. Then, cells were incubated with 4-PBA or TNF-a for 0.5 h. Different concentrations of 1b (1e10,000 nM) were added to each well and cells were cultured for another 48 h. Then, an MTT assay was performed using a thermo microplate reader. The DMSO treated controls were assigned a cell viability value of 100%. The IC50 values were obtained by nonlinear regression using GraphPad Prism 5.0. For each treatment, the IC50 value was calculated from three independent experiments.
3.1. Morphological changes in 1b-treated K562 cells
Data were expressed as means ± S.D. or percentage and analyzed for statistical significance using one-way analysis of variance (ANOVA) followed by Dunnett's test. P-value less than or equal to 0.05 was considered to be statistically significant. Statistical analyses were carried out in GraphPad Prism 5 (GraphPad Software, San Diego, CA, U.S.A.).
Treatment with the highly potent cytotoxic compound 1b (IC50 ¼ 3.98 mM) (Fig. 1A) caused K562 cells to adopt a typical apoptotic morphology (cell shrinkage and/or blebbing; Fig. 1B). The ball-like bodies appeared as early as 12 h after treatment with 30 mM 1b. The number of apoptotic cells increased in a timedependent manner, whereas the total number of cells decreased. These results indicated that compound 1b might inhibit K562 cell proliferation by inducing apoptosis.
2.5. Caspase-3 activity assay Caspase-3 activity was evaluated by the Apo-ONE ® Homogeneous Caspase-3/7 Assay Kit (Promega) according to the manufacturer's instructions. Briefly, K562 cells were cultured in 6-well plates with either DMSO or 1b (30 mM) for 48 h. To each well, 100 mL of Apo-ONE ® Homogeneous Caspase-3/7 buffer with 1 mL Caspase Substrate Z-DEVD-R110 (100 ) was added and cells were cultured for 0.5 h. The release of the fluorochrome following peptide cleavage was kinetically monitored at room temperature (excitation at 485 nm and emission at 521 nm). Cells treated with staurosporine (0.3 mM) for 6 h were used as a positive control.
3.2. Compound 1b induced apoptosis in K562 cells Flow cytometric analysis of cells double-labeled with AnnexinV-FITC and PI revealed the presence of apoptotic K562 cells following 1b treatment (Fig. 2A). The apoptotic rates (Annexin Vþ/ PI and Annexin Vþ/PIþ) of 1b-treated K562 cells increased to 62.8% of total cells compared to 9.8% in the control. As shown in Fig. 2A, treatment with 1b for different time periods showed that the apoptosis rates increased in a time-dependent manner. These results suggested that 1b inhibits the proliferation of K562 cells by inducing apoptosis in a time-dependent manner.
Y. Teng et al. / Chemico-Biological Interactions 261 (2017) 103e107
105
Fig. 1. Morphological changes in 1b-treated K562 cells. (A) Structure and in vitro cell proliferation inhibitory activity of 1b. (B) Morphological changes induced by 1b treatment (30 mM) for 12, 24, 36 or 48 h.
3.3. Cooperative effects of 1b and 4-PBA or TNF-a
3.4. Caspase-3 activity assay
There are three types of apoptotic pathways: the death receptor pathway, mitochondrial pathway, and endoplasmic reticulum pathway. Combination treatment with 1b and the widely used apoptosis inhibitors was used to determine the type of apoptotic pathway through which 1b induces apoptosis in K562 cells. As shown in Fig. 2B, treatment of 1b-treated K562 cells with the endoplasmic reticulum apoptosis inhibitor (4-PBA) or the death receptor inhibitor (TNF-a) did not affect the cell viability significantly. The result showed that the endoplasmic reticulum apoptosis inhibitor and the death receptor inhibitor did not affect 1b-treated K562 cells, thus confirming that the apoptotic pathway triggered by 1b in K562 cells was not the death receptor pathway or the endoplasmic reticulum pathway.
The caspase cascade is one of the most important events in the mitochondrial apoptosis pathway [23]. To determine whether caspase-3 was involved in 1b-induced apoptosis, K562 cells were treated with DMSO, 0.3 mM staurosporine (positive control), or 30 mM 1b, and caspase-3 activation was then determined. As shown in Fig. 3A, compared to the positive control, 1b-treated K562 cells exhibited increased caspase-3 activation. This result indicates that the caspase-3-dependent pathway was involved in 1b-induced apoptosis. From the experimental results, compound 1b was confirmed to induce apoptosis of K562 cells. To study the molecular mechanism by which compound 1b induces apoptosis, we investigated the effect of 1b on the expression of caspase-3 and PARP by using
Fig. 2. 1b induced apoptosis in K562 cells. (A) K562 cells were treated with 30 mM 1b for different periods of time. Flow cytometry analyses of K562 cells under various treatments using double staining with Annexin V (horizontal line) and propidium iodide (PI, vertical line). (B) The cooperative effect of 1b on K562 cells with inhibitor protection.
106
Y. Teng et al. / Chemico-Biological Interactions 261 (2017) 103e107
Fig. 3. Effects of 1b treatment on caspase-3 activity and the expression levels of caspase-3 and PARP proteins. (A) Caspase-3 activation was quantified by means of a fluorescencebased method. (B) Expression of caspase-3 and PARP proteins during 1b-induced apoptosis.
western blotting. As shown in Fig. 3B, compared with DMSOtreated K562 cells, cells treated with 1b showed a dosedependent increase in caspase-3 activation. Furthermore, western blot analysis revealed increased levels of cleaved PARP following treatment with 1b, providing biochemical confirmation for the induction of apoptosis in the K562 cell line. 3.5. 1b induced apoptosis in K562 cells through the mitochondrial pathway Mitochondria play a significant role in apoptosis of certain cancer cell lines. To clarify whether the mitochondrial pathway is involved in 1b-induced apoptosis, K562 cells were treated with DMSO (0.5%) or 1b (30 mM) for 12, 24, or 48 h, and then with a membrane potential sensing dye, TMRM. Compared to DMSOtreated K562 cells, the 1b-treated cells showed a significant decrease in mitochondrial membrane potential from 90.9% (DMSO) to 61.9% (1b) at 12 h, from 93.0% (DMSO) to 70.9% (1b) at 24 h, and from 94.6% (DMSO) to 61.9% (1b) at 48 h (Fig. 4A). These results show that 1b can cause a reduction in the mitochondrial membrane potential, which may induce apoptosis in K562 cells via the mitochondrial pathway.
Western blot analysis was performed to investigate the protein expression of Bcl-2, Bax, and Bcl-xl in K562 cells treated with different concentrations of compound 1b. Alpha-tubulin was used as an internal loading control. As shown in Fig. 4B, western blot results suggest that Bcl-2 and Bcl-xl protein levels dramatically decreased with 1b treatment, whereas the expression level of the Bax protein increased. Moreover, the ratio of Bcl-2/Bax in the treated groups clearly decreased with increasing 1b concentrations (Fig. 4C). These results indicated that in K562 cells, compound 1b initiates programmed cell death via the mitochondrial apoptotic pathway. 4. Discussion The results of this study confirm that compound 1b has excellent cytotoxic activity against human chronic myelogenous leukemia cells K562 (IC50 ¼ 3.98 mM). As shown in Fig. 1B, an apoptotic cell population was clearly observed after K562 cells were treated with 1b for 48 h. Treatment with 1b caused K562 cells to take on a typical apoptotic morphology, characterized by cell shrinkage and/ or blebbing. Furthermore, as shown in Fig. 2A, 1b inhibits the proliferation of
Fig. 4. Effects of 1b treatment on the mitochondrial membrane potential and the expression level of Bcl-2 family proteins. (A) The effect of 1b on K562 cells' mitochondrial membrane potential (DJm). (B) Expression of Bcl-2, Bax, and Bcl-xl during 1b-induced apoptosis. (C) Effect of 1b on the Bcl-2/Bax ratio in K562 cells.
Y. Teng et al. / Chemico-Biological Interactions 261 (2017) 103e107
K562 cells by inducing apoptosis in a time-dependent manner. To determine the apoptotic pathway triggered by 1b in K562 cells, we examined the effects of 1b in combination with TNF-a (the death receptor pathway inhibitor) or 4-PBA (the endoplasmic reticulum pathway inhibitor). The apoptotic pathway induced by 1b was not the death receptor pathway or the endoplasmic reticulum pathway (Fig. 2B). The results suggest that 1b induces apoptosis via the mitochondrial pathway. Caspases, a family of cysteine proteases, play a crucial role in apoptosis regulation with caspase-3 being the main player in the execution of apoptotic cell death [24]. In this study, through the use of both a Caspase-3/7 Assay Kit and western blot analysis, we found that 1b causes caspase-3 activation (Fig. 3). Moreover, as shown in Fig. 4, we found that 1b causes the dissipation of the mitochondrial membrane potential (DJm). Importantly, noticeable changes in the protein levels of Bcl-2, Bax, and Bcl-xl were observed. The Bcl-2/Bax ratio in the treated groups decreased significantly with increasing 1b concentrations. In conclusion, as compared with chalcone, 1b had a low cytotoxicity and improved cell proliferation inhibitory activity. We provide strong evidence that compound 1b inhibits the growth of human chronic myelogenous leukemia K562 cells via the induction of apoptosis. The results indicate that compound 1b inhibits cell proliferation through the initiation of the mitochondrial apoptotic pathway. Studies are underway to clarify the action mechanism and the target molecule of 1b. Although 1b has potent cytotoxicity as compared with chalcone, the IC50 of 1b against K562 is not very high (3.98 mM). Thus, future studies are warranted for the design and synthesis of various types of chalcone derivatives with excellent cytotoxicity and low toxicity. Competing interests The authors declare that there is no conflicts interest. Acknowledgement This work was supported jointly by National Natural Science Foundation of China (31301142 and 31601203) and International Science & Technology Cooperation Program of China (2013DFA31160). We are thankful to the Research Centre of Modern Analytical Technology, Tianjin University of Science and Technology for high resolution mass spectrum analysis. We would like to thank Editage [www.editage.cn] for English language editing. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2016.11.025. References [1] T. Okuyama, M. Takata, J. Takayasu, T. Hasegawa, H. Tokuda, A. Nishino, H. Nishino, A. Iwashima, Anti-tumor-promotion by principles obtained from
107
Angelica keiskei, Planta Med. 57 (1991) 242e246. [2] R. Nishimura, K. Tabata, M. Arakawa, Y. Ito, Y. Kimura, T. Akihisa, H. Nagai, A. Sakuma, H. Kohno, T. Suzuki, Isobavachalcone, a chalcone constituent of Angelica keiskei, induces apoptosis in neuroblastoma, Biol. Pharm. Bull. 30 (2007) 1878e1883. [3] Y.L. Hsu, P.L. Kuo, W.S. Tzeng, C.C. Lin, Chalcone inhibits the proliferation of human breast cancer cell by blocking cell cycle progression and inducing apoptosis, Food Chem. Toxicol. 44 (2006) 704e713. [4] T. Enoki, H. Ohnogi, K. Nagamine, Y. Kudo, K. Sugiyama, M. Tanabe, E. Kobayashi, H. Sagawa, I. Kato, Antidiabetic activities of chalcones isolated from a Japanese Herb, Angelica keiskei, J. Agric. Food Chem. 55 (2007) 6013e6017. [5] S.K. Awasthi, N. Mishra, B. Kumar, M. Sharma, A. Bhattacharya, L.C. Mishra, V.K. Bhasin, Potent antimalarial activity of newly synthesized substituted chalcone analogs in vitro, Med. Chem. Res. 18 (2009) 407e420. [6] P.M. Sivakumar, P.K. Prabhakar, M. Doble, Synthesis, antioxidant evaluation, and quantitative structureeactivity relationship studies of chalcones, Med. Chem. Res. 20 (2010) 482e492. [7] K.L. Lahtchev, D.I. Batovska, S.P. Parushev, V.M. Ubiyvovk, A.A. Sibirny, Antifungal activity of chalcones: a mechanistic study using various yeast strains, Eur. J. Med. Chem. 43 (2008) 2220e2228. [8] Y.S. Lee, S.S. Lim, K.H. Shin, Y.S. Kim, K. Ohuchi, S.H. Jung, Anti-angiogenic and anti-tumor activities of 2'-hydroxy-4'-methoxychalcone, Biol. Pharm. Bull. 29 (2006) 1028e1031. [9] F. Jin, X.Y. Jin, Y.L. Jin, D.W. Sohn, S.A. Kim, D.H. Sohn, Y.C. Kim, H.S. Kim, Structural requirements of 2',4',6'-tris(methoxymethoxy) chalcone derivatives for anti-inflammatory activity: the importance of a 2'-hydroxy moiety, Arch. Pharm. Res. 30 (2007) 1359e1367. [10] X. Zi, A.R. Simoneau, A. Flavokawain, A novel chalcone from kava extract, induces apoptosis in bladder cancer cells by involvement of Bax proteindependent and mitochondria-dependent apoptotic pathway and suppresses tumor growth in mice, Cancer Res. 65 (2005) 3479e3486. [11] Y.L. Hsu, P.L. Kuo, C.C. Lin, Isoliquiritigenin induces apoptosis and cell cycle arrest through p53-dependent pathway in Hep G2 cells, Life Sci. 77 (2005) 279e292. [12] H.O. Saxena, U. Faridi, J.K. Kumar, S. Luqman, M.P. Darokar, K. Shanker, C.S. Chanotiya, M.M. Gupta, A.S. Negi, Synthesis of chalcone derivatives on steroidal framework and their anticancer activities, Steroids 72 (2007) 892e900. [13] A. Boumendjel, G. Sotoing Taiwe, E. Ngo Bum, T. Chabrol, C. Beney, V. Sinniger, R. Haudecoeur, L. Marcourt, S. Challal, E. Ferreira Queiroz, F. Souard, M. Le Borgne, T. Lomberget, A. Depaulis, C. Lavaud, R. Robins, J.L. Wolfender, B. Bonaz, M. De Waard, Occurrence of the synthetic analgesic tramadol in an African medicinal plant, Angew. Chem. Int. Ed. Engl. 52 (2013) 11780e11784. [14] Y.S. Wu, S.N. Chen, Apoptotic cell: linkage of inflammation and wound healing, Front. Pharmacol. 5 (2014) 1. [15] D.R. Green, J.C. Reed, Mitochondria and apoptosis, Science 281 (1998) 1309e1312. [16] K. Sinha, J. Das, P.B. Pal, P.C. Sil, Oxidative stress: the mitochondria-dependent and mitochondria-independent pathways of apoptosis, Arch. Toxicol. 87 (2013) 1157e1180. [17] J.D. Robertson, S. Orrenius, Role of mitochondria in toxic cell death, Toxicology 181e182 (2002) 491e496. [18] J. Kale, Q. Liu, B. Leber, D.W. Andrews, Shedding light on apoptosis at subcellular membranes, Cell 151 (2012) 1179e1184. [19] T. Moldoveanu, A.V. Follis, R.W. Kriwacki, D.R. Green, Many players in BCL-2 family affairs, Trends Biochem. Sci. 39 (2014) 101e111. [20] S.J. Korsmeyer, BCL-2 gene family and the regulation of programmed cell death, Cancer Res. 59 (1999) 1693se1700s. [21] H.M. Wang, L. Zhang, J. Liu, Z.L. Yang, H.Y. Zhao, Y. Yang, D. Shen, K. Lu, Z.C. Fan, Q.W. Yao, Y.M. Zhang, Y.O. Teng, Y. Peng, Synthesis and anti-cancer activity evaluation of novel prenylated and geranylated chalcone natural products and their analogs, Eur. J. Med. Chem. 92 (2015) 439e448. [22] Y. Zhou, H.Y. Zhao, K.L. Han, Y. Yang, B.B. Song, Q.N. Guo, Z.C. Fan, Y.M. Zhang, Y.O. Teng, P. Yu, 5-(2-carboxyethenyl) isatin derivative induces G(2)/M cell cycle arrest and apoptosis in human leukemia K562 cells, Biochem. Biophys. Res. Commun. 450 (2014) 1650e1655. [23] M. Poreba, A. Strozyk, G.S. Salvesen, M. Drag, Caspase substrates and inhibitors, Cold Spring Harb. Perspect. Biol. 5 (2013) a008680. [24] H. Wu, X. Che, Q. Zheng, A. Wu, K. Pan, A. Shao, Q. Wu, J. Zhang, Y. Hong, Caspases: a molecular switch node in the crosstalk between autophagy and apoptosis, Int. J. Biol. Sci. 10 (2014) 1072e1083.