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A receptor tyrosine kinase inhibitor, Tyrphostin A9 induces cancer cell death through Drp1 dependent mitochondria fragmentation
Biochemical and Biophysical Research Communications 408 (2011) 465–470
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
A receptor tyrosine kinase inhibitor, Tyrphostin A9 induces cancer cell death through Drp1 dependent mitochondria fragmentation So Jung Park a, Young Jun Park a, Ji Hyun Shin a, Eun Sung Kim a, Jung Jin Hwang b, Dong-Hoon Jin b, Jin Cheon Kim b, Dong-Hyung Cho a,⇑ a b
Graduate School of East-West Medical Science, Kyung Hee University, Gyeoggi-Do 446-701, Republic of Korea Institute for Innovative Cancer Research, Asan Medical Center, Seoul 138-736, Republic of Korea
a r t i c l e
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Article history: Received 8 April 2011 Available online 19 April 2011 Keywords: Mitochondria dynamics Fission Tyrphositn A9 Apoptosis
a b s t r a c t Mitochondria dynamics controls not only their morphology but also functions of mitochondria. Therefore, an imbalance of the dynamics eventually leads to mitochondria disruption and cell death. To identify specific regulators of mitochondria dynamics, we screened a bioactive chemical compound library and selected Tyrphostin A9, a tyrosine kinase inhibitor, as a potent inducer of mitochondrial fission. Tyrphostin A9 treatment resulted in the formation of fragmented mitochondria filament. In addition, cellular ATP level was decreased and the mitochondrial membrane potential was collapsed in Tyr A9-treated cells. Suppression of Drp1 activity by siRNA or over-expression of a dominant negative mutant of Drp1 inhibited both mitochondrial fragmentation and cell death induced by Tyrpohotin A9. Moreover, treatment of Tyrphostin A9 also evoked mitochondrial fragmentation in other cells including the neuroblastomas. Taken together, these results suggest that Tyrphostin A9 induces Drp1-mediated mitochondrial fission and apoptotic cell death. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction As dynamic organelles, mitochondria in healthy cells continuously divide and fuse. Mitochondria are not only necessary for energy generation, but also key components in cell death. Mitochondria dynamics regulate processes associated with mitochondria morphology such as mitochondria biogenesis, localization, and distribution as well as the morphology itself [1]. Several key components in mitochondrial dynamics have been identified. Dynamine-related protein 1 (Drp1) mediates mitochondria and peroxisome membrane fragmentation. Down-regulaton of Drp1 activity not only inhibits mitochondrial fission but also suppresses cytochrome c release, caspase activation, and cell death [2,3]. On the other hand, mitochondria fusion is mediated by mitofusin1 and 2 (Mfn1/2) and optic atrophy protein 1 (Opa1) [4]. Specifically, outer mitochondria membrane (OMM) fusion is mediated by Mfn1/2, while Opa1 activity is associated with inner mitochondria membrane (IMM) fusion. In contrast to fission, mitochondria fusion may function as a cell-protective mechanism. Down-regulation of Mfn1/2 or Opa1 results in mitochondria fragmentation and increases susceptibility to apoptotic stimuli [5,6]. Disruption of this balance contributes to the pathophysiology of neurodegenerative ⇑ Corresponding author. Address: Graduate School of East-West Medical Science, Kyung Hee University 1, Seocheon-Dong, Giheung-Gu, Yongin-Si, Gyeoggi-Do 446701, Republic of Korea. E-mail address: [email protected] (D.-H. Cho). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.04.053
diseases, metabolic disorders, and cancer [1,7,8]. Despite extensive data currently available regarding the machinery of mitochondrial fission and fusion, the precise molecular mechanisms are still not fully understood. Recently, mdivi-1 was identified as a chemical inhibitor of Drp1 [9]. Mdivi-1 inhibits OMM permeabilization, the release of cytochonrome C, and thus prevents apoptotic cell death in HeLa cells [9]. Therefore, identification of regulator of mitochondria dynamics should lead to the development of new therapeutic strategies for treating mitochondria-associated diseases. However, chemical agents modulating mitochondria dynamics are not yet well known. The receptor protein tyrosine kinases such as platelet-derived growth factor receptor (PDGFR) play important roles in regulating cellular functions such as cell proliferation, migration, survival, development as well as cell in many diseases, including cancer [10,11]. Indeed, over-expression of PDGF and their receptor has been reported in several types of cancer including prostate, ovarian, and non-small-cell lung cancer. The inhibition of the tyrosine kinase and their downstream signal pathways has been targeted to cancer therapy [12]. PDGFR activates downstream effectors such as RAS, PI3K, ERK, JNK, Src and Stat proteins that lead to cell proliferation and survival [13]. PDGFR also suppress apoptotic cell death by preventing depolarization of the mitochondrial electropotential gradient and generation of ROS [14]. Tyrphostins (TYRosin PHOSphorylation INhibitors) were originally described as a group of tyrosine kinase inhibitors that compete with substrate binding without affecting ATP [15].
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Currently, several inhibitors for tyrosine kinase have been developed as anti-cancer agents [16–18]. However, the effects of tyrosine kinase inhibitors on mitochondria functions are largely unknown. In the present study, we identified Tyrphostin A9 as a factor of mitochondria fission. Treatment of Tyrphostin A9 highly promoted mitochondria fragmentation and mitochondria membrane depolarization. We also investigated down-regulation of Drp1 suppressed Tyrphostin A9-induced mitochondria fragmentation and cell death. 2. Materials and methods 2.1. Reagents The Lopac 1280 chemical library was from Sigma (St. Louis, MO). The expression plasmid Drp1 dominant negative (K38A) and mito-YFP were a kind gift from van der Bliek AM (University of California at Los Angeles, CA) and from Dr. Gyesoon Yoon (Ajou University, Korea). siRNA targeting Drp1 (50 - GAG GUU AUU GAA CGA CUC A) and negative scrambled siRNA (50 - CCU ACG CCA CCA AUU UCG U) were synthesized from Bioneer (Daejeon, Korea). Tyrphostin A9 and CCCP were purchased from Sigma (St. Louis, MO, USA). 2.2. Cell culture and stable transfection HeLa, SK-N-MC, and SH-SY5Y cells were obtained from the American Type Culture Collection (ATCC). All cells were cultured at 37 °C in a 5% CO2 incubator and maintained in DMEM containing 10% FBS and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA). For stable cell lines, the cells were transfected with pmito-YFP using Lipofectamin according to manufacturer’s protocol (Invitrogen, Carlsbad, CA). Stable transfectants were selected by growth in selection medium containing 1 mg/ml of G418 for 10 days. After single cell cloning, the stable clones were selected under fluorescence microscope. 2.3. Chemical screening for regulator of mitochondria dynamics HeLa cells stably expressing mito-YFP (HeLa/mito-YFP) were seeded in 96-well plates at 1500 cells per well. 24 h after seeding, chemicals were added individually to each well at a final concentration of 10 lM. Then the change of mitochondrial morphology was observed under fluorescence microscopy (Olympus X71). 2.4. Western blotting Cells were extracted with 2 Laemmli sample buffer (Bio-Rad, Hercules, CA), separated by SDS–polyacrylamide gel electrophoresis, and then transferred to PVDF membrane. After blocking with skim milk in TBST, the membranes were incubated with specific primary antibodies. And then the membranes were incubated with HRP-conjugated secondary antibodies (Pierce, Rockford, IL). AntiDrp1 was from BD (San Jose, CA); anti-caspase-3 antibody was from Cell Signaling Technology (Beverly, MA); anti-Actin antibody was from Millipore (Temecula, CA). 2.5. Measurement of mitochondria membrane potential and ATP level HeLa cells (1 105) were treated with Tyrphosin A9 for 8 h. The Mitochondrial membrane potential was examined with a unique fluorescent cationic dye, JC-1 (5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolylcarbocyanine iodide, BD, San Jose, CA) that detects loss of signal of mitochondrial membrane potential, then the depolarized mitochondria was measured with NucleoCounter
NC-3000 (Chemometec, Denmark). HeLa cells were treated with Tyrphosin A9 and then total ATP level was measured with ATP Fluorometric Assay kit (Biovision, Sandiego, CA) according to manufacturer’s protocol. 2.6. Cell viability assay and apoptosis analysis Cell viability was measured by MTT (Sigma, St. Louis, MO) assay. Cells seeded in 96-well plates were incubated at 37 °C in 5% CO2 for 24 h, the medium was replaced with 100 ll of fresh medium with drugs. At the end of the incubation, 10 ll of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide),5 mg/ml] was added. After mixing with DMSO, absorbance of the solution was read with spectrophotometer (VictorX3, Perkinelmer). Apoptotic cell death was determined tryphan blue exclution assay and morphological change of nucleus with DAPI staining assay. The results were expressed as the means ± SD. The probability of statistical differences between experimental groups was determined by the Student’s t test. 3. Results and discussion 3.1. Tyrphostin A9 induces mitochondria fragmentation in HeLa cells Mitochondria are key components in cell death by mediating extrinsic as well as intrinsic signaling pathways. Mitochondria dynamics not only control their morphology but also regulate their functions [1,7]. To identify chemical modulator of mitochondria dynamics, we developed a cell-based screening system using HeLa cells that stably expressing fluorescent protein fused with mitochondria tracker ((mito-YFP), HeLa/Mito-YFP). Used this assay, we screened a LOPAC 1280 chemical library (a collection of pharmacologically active compounds). Based on the screening results, we selected Tyrphostin A9 [(3,5-bis(1,1-dimethylethyl)-4hydroxyphenyl-methylene)-propanedinitrile, C18H22N2O] for further analysis as a potent inducer of mitochondria fragmentation (Fig. 1A). In other to confirm the screening result, HeLa/mito-YFP cells were treated with Tyrphostin A9 for different dosage (0.1– 20 lM) and time point (0.5–6 h). The results showed that mitochondria became excessively fragmented after treatment with Tyrphostin A9 in a dose- and time dependent manner (Fig. 1B and C). 3.2. Tyrphostin A9-induced mitochondria fragmentation results in mitochondrial dysfunction Mitochondria undergo excessive fragmentation during apoptosis and down-regulation of Drp1 suppresses cell death, implying mitochondria fission is directly involved in apoptosis [19]. Moreover, inhibition of some tyrosine kinases causes mitochondria dysfunction and sensitize cancer cells to cell death [15,20] Therefore, we next examined the effect of Tyrphostin A9 on mitochondria functions. Disruption of mitochondrial permeability transition is an important step in the induction of mitochondria-mediated cell death. We used a unique fluorescent cation dye, JC-1, to examine mitochondrial membrane collapse. Treatment with Tyrphostin A9 resulted in an increased red-colored JC-1, indicating that Tyrphostin A9 strongly induces mitochondria membrane depolarization in HeLa cells (Fig. 2A). The electrochemical gradient of mitochondria is also important in ATP generation. Thus, we next examined cellular ATP levels following treatment of Tyrphostin A9. We observed that the cellular ATP levels were gradually decreased by Tyrphostin A9 (Fig. 2B). These results suggest that Tyrphostin A9 disrupts the mitochondrial membrane potential. Tyrphostins are known to suppress cell proliferation by inhibiting receptor tyrosine kinase signaling [15]. We investigated the effect of Tyrphostin A9
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Fig. 1. Tyrphostin A9 induces mitochondrial fragmentation in HeLa cells (A) Representative fluorescence micrographs of mitochondrial morphology before and after Tyrphostin A9 treatment. HeLa cells stably expressing mito-YFP (HeLa/mito-YFP) were exposed to Tyrphostin A9 for 3 h and then were imaged using fluorescence microscope. (B) HeLa/mito-YFP cells were exposed to increasing concentrations of Tyrphostin A9 (0.5, 1, 5, 10, and 20 uM) and then mitochondrial fragmentation was monitored after 6 h. (C) HeLa/mito-YFP cells were treated with Tyrphostin A9 (10 lM) and fragmented mitochondria was determined at indicated time. Data are represented by the mean ± SEM (n > 3).
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Fig. 2. Tyrphostin A9 promote mitochondria dysfunction and cell death in HeLa cells. (A and B) HeLa cells were incubated with Tyrphostin A9 (0.5, 5 lM) for 8 h. The mitochondria permeability transition was assessed with a mitochondria membrane potential detection kit, JC-1 and total ATP levels were determined after 8 h. (C and D) HeLa cells were exposed to Tyrphostin A9. After 24 h, cell viability was determined by MTT assay and caspase-3 activation was examined by detecting cleaved caspase-3. (E) HeLa cells were treated with Tyrphostin A9 (0.5 lM, 5 lM) and the cell proliferation rate was determined using a CCK8 cell proliferation assay. The results are presented as the daily proliferation rate compared to that of the control. Data are represented by the mean ± SEM (n > 3).
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3.4. Tyrphostin A9 also induces mitochondria fragmentation in neuroblastoma cells
on proliferation and cell viability in HeLa cells. The Western blot analysis showed that caspase-3 was significantly activated following Tyrphostin A9 treatment and this was prevented by pan caspase inhibitor, zVAD (Fig. 2D). In addition, Tyrphostin A9 also suppressed cell proliferation in HeLa cells (Fig. 2E). Taken together, our results suggest that Tyrphostin A9 actively contributes to cytotoxicity in the HeLa cells.
Next, we examined whether Tyrphostin A9 induced mitochondria fission in other cell types. To answer this question, we generated the stable cell line with SK-N-MC and SH-SY5Y (a neuroblastoma cell line) using mito-YFP (SK-N-MC/mito-YFP, SY5Y/mito-YFP) and investigated the ability of Tyrphostin A9 to induce mitochondria fragmentation in these cells. In non-treated cells, mitochondria with an intermediated filaments structure were observed, whereas treatment with Tyrphostin A9 resulted in a strong induction of fragmented structures in SK-N-MC and SH-SY5Y cells (Fig. 4A, and data not shown). Similar with the results in HeLa cells, mitochondria fragmentation highly increased in a dose-dependent manner by Tyrphostin A9 in SK-N-MC cells. These results indicate that Tyrphostin A9 also induces mitochondria fission in different types of cells including neuroblastoma. Disruption of balance on mitochondria fission and fusion contributes to the pathophysiology of many diseases [1,7]. Thus, modulation of mitochondria dynamics should lead to the development of new therapeutic strategies for treating mitochondria associated diseases such as neurodegenerative diseases, metabolic disorders, and cancer. In this study, we identified Tyrphostin A9 as a potent regulator of mitochondria dynamics. Tyrphostin A9 was originally synthesized as an inhibitor of receptor tyrosine kinase [21]. Tyrosine kinases are involved in regulation of multiple cellular processes including cell proliferation and cell death. Over-expression or over-activation of tyrosine kinases is strongly associated with development of many types of cancer. Thus, specific inhibitors to tyrosine kinases have
3.3. Tyrphostin A9 induces Drp1-dependent apoptotic cell death in HeLa cells Mitochondria fission is controlled by Drp1, therefore, we addressed the effects of Drp1 on Tyrphostin A9-induced mitochondria fragmentation. Mitochondria suppressed Drp1 activity by siRNA resulted in formation of highly interconnected filament structures (Fig. 3A). Western blot analysis confirmed the reduced expression of Drp1 by specific siRNA. The mitochondria fragmentation induced by Tyrphostin A9 was inhibited by over-expression of a dominant negative Drp1 mutant (K38A) as well as down-regulation of Drp1 with siRNA in HeLa cells (Fig. 3B and C). These results suggest mitochondria fragmentation by Tyrphostin A9 is dependent on Drp1 activity. Since excessive mitochondria fragmentation impacts on cell death, we next investigated the role of Drp1 on Tyrphostin A9-mediated cell death. HeLa cells were transfected with either non-targeting scrambled siRNA or Drp1 specific siRNA and then, exposed to Tyrphostin A9 to examine the effect on cell death. As shown in Fig. 3D, suppressed expression of Drp1 was decreased apoptotic cell death compared with that of control cells, suggesting that Tyrphostin A9 induces apoptotic cell death through a Drp1mediated pathway.
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Fig. 3. Down-regulation of Drp1 prevents Tyrphosin A9-induced mitochondria fragmentation. (A) HeLa/mito-YFP cells were transfected with control scrambled siRNA (Scram) or specific siRNA against Drp1 (siDrp1), and mitochondria morphology was monitored. Reduced expression of Drp1 by siRNA was confirmed by Western blotting (bottom). (B) After 3 days with siRNA transfection, the cells were treated with Tyrphostin A9 (10 lM) for 1.5 h and mitochondrial fragmentation was counted. (C) HeLa/mitoYFP cells transfected with a control empty vector (pcDNA) or a dominant negative mutant of Drp1 (K38A) were treated with CCCP (1 lM) or Tyrphostin A9 (10 uM) for 1.5 h and then mitochondrial fragmentation was counted. (D) HeLa cells were transfected with a control scrambled siRNA or Drp1 siRNA. After 72 h, the cells were incubated with Tyrpohostin A9 for 24 h and then, apoptotic cells were determined by counting of apoptotic nuclei. Data are represented by the mean ± SEM (n > 3).
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Tyr A9 (uM) Fig. 4. Tyrphosin A9 also induces mitochondria fragmentatioin in neuroblastoma cells (A) SK-N-MC cells stably expressing mito-YFP (SK-N-MC/mito-YFP) were treated with Tyrphostin A9 (5 lM) for 3 h and then were imaged using fluorescence microscope. (B) SK-N-MC/mito-YFP cells were exposed to increasing dosages of Tyrphostin A9 (0.1– 20 lM) and then mitochondrial fragmentation was measured after 6 h. The data means ± SEM.
been considered as attractive targets to cancer treatment in the past several years [12,17,22]. Recent studies have reported that inhibition of tyrosine kinase induces apoptosis through activation of the mitochondria-mediated (intrinsic) pathway. In fact, the tyrosine kinase inhibitor, Erlotinib (trade name Tarceva, an EGFR inhibitor) up-regulates pro-apoptotic Bcl-2 family protein, BIM and PUMA [23,24]. Another receptor tyrosine kinase inhibitor Sorafenib (trade name Nexavar) also induces apoptotic cell death by up-regulation of proapoptotic protein and mitochondria-dependent oxidative stress [25,26]. Although mitochondria can be considered a cellular target for tyrosine kinase inhibitors, the precise mechanism of their effect on mitochondria has not been fully elucidated. Here, we first investigated the effect of a tyrosine kinase inhibitor on mitochondria dynamics. Tyrphostin A9 treatment dramatically triggers mitochondria fragmentation, decrease of cellular ATP levels, and depolarization of mitochondria membrane potential, thus leading to reduced cell proliferation and survival. Moreover inhibition of Drp1 by both siRNA and a dominant negative mutant efficiently suppressed mitochondria fragmentation (Fig. 3), demonstrating that Drp1 is also a key player in Tryrphostin A9-mediated mitochondria fission. Tyrosine kinase inhibitors block the activation of downstream kinases and suppress the signaling. Drp1 activity is regulated by various posttranslational modifications during mitochondria fission [1]. For instance, phosphorylation of Drp1 by CDK1/Cyclin B, CaMK, or CDK5 facilitates mitochondria fragmentation whereas PKA-induced phosphorylation of Drp1 results in the suppression of Drp1 activity [27–31]. We also examine the phosphorylation of Ser 161 on Drp1 (target site for CDK1 kinase) after treatment of Tyrphostin A9. However, the phosphor-Drp1 protein level was not changed by Tryphostin A9 (data not shown). Thus, Additional studies will be required to clarify the effect of tyrosine kinase on Drp1 activity in mitochondria fragmentation. In summary, from a cell-based screen, we identified Tyrphostin A9 as a potent inducer of mitochondria fission in several different types of cells. Our studies suggest that Tyrphostin A9 promotes mitochondria fragmentation and cell death via a Drp1 dependent process.
4. Conflict of interest The authors have declared that no conflict of interest exists.
Acknowledgments We thank Dr. van der Bliek AM (UCAL, CA) and G Yoon (Ajou University, Korea) for providing Drp1 and mito-YFP plasmid. This study was supported by a grant of The Korean Health 21 RD Project, Ministry of Health Welfare Republic of Korea (A062254 and A090013). References [1] D.H. Cho, T. Nakamura, S.A. Lipton, Mitochondrial dynamics in cell death and neurodegeneration, Cell. Mol. Life Sci. 67 (2010) 3435–3447. [2] S. Frank, B. Gaume, E.S. Bergmann-Leitner, W.W. Leitner, E.G. Robert, F. Catez, C.L. Smith, R.J. Youle, The role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis, Dev. Cell 1 (2001) 515–525. [3] Y. Yoon, K.R. Pitts, M.A. McNiven, Mammalian dynamin-like protein DLP1 tubulates membranes, Mol. Biol. Cell 12 (2001) 2894–2905. [4] S. Hoppins, J. Nunnari, The molecular mechanism of mitochondrial fusion, Biochim. Biophys. Acta. 1793 (2009) 20–26. [5] A. Olichon, L. Baricault, N. Gas, E. Guillou, A. Valette, P. Belenguer, G. Lenaers, Loss of OPA1 perturbates the mitochondrial inner membrane structure and integrity, leading to cytochrome c release and apoptosis, J. Biol. Chem. 278 (2003) 7743–7746. [6] D.F. Suen, K.L. Norris, R.J. Youle, Mitochondrial dynamics and apoptosis, Genes Dev. 22 (2008) 1577–1590. [7] D.C. Chan, Mitochondria: dynamic organelles in disease, aging, and development, Cell 125 (2006) 1241–1252. [8] S. Grandemange, S. Herzig, J.C. Martinou, Mitochondrial dynamics and cancer, Semin. Cancer Biol. 19 (2009) 50–56. [9] A. Cassidy-Stone, J.E. Chipuk, E. Ingerman, C. Song, C. Yoo, T. Kuwana, M.J. Kurth, J.T. Shaw, J.E. Hinshaw, D.R. Green, J. Nunnari, Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization, Dev. Cell 14 (2008) 193–204. [10] N.J. Dibb, S.M. Dilworth, C.D. Mol, Switching on kinases: oncogenic activation of BRAF and the PDGFR family, Nat. Rev. Cancer 4 (2004) 718–727. [11] R.Jr. Roskoski, Sunitinib: a VEGF and PDGF receptor protein kinase and angiogenesis inhibitor, Biochem. Biophys. Res. Commun. 356 (2007) 323–328. [12] J.T. Hartmann, M. Haap, H.G. Kopp, H.P. Lipp, Tyrosine kinase inhibitors – a review on pharmacology, metabolism and side effects, Curr. Drug Metab. 10 (2009) 470–481. [13] A.V. Jones, N.C. Cross, Oncogenic derivatives of platelet-derived growth factor receptors, Cell. Mol. Life Sci. 61 (2004) 2912–2923. [14] J. M Lee, Inhibition of p53-dependent apoptosis by the KIT tyrosine kinase: regulation of mitochondrial permeability transition and reactive oxygen species generation, Oncogene 17 (1998) 1653–1662. [15] A. Levitzki, E. Mishani, Tyrphostins and other tyrosine kinase inhibitors, Annu. Rev. Biochem. 75 (2006) 93–109. [16] A. Jimeno, M. Hidalgo, Pharmacogenomics of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, Biochim. Biophys. Acta. 1766 (2006) 217–229. [17] J. Homsi, A.I. Daud, Spectrum of activity and mechanism of action of VEGF/ PDGF inhibitors, Cancer Control 14 (2007) 285–294. [18] F. Ciardiello, G. Tortora G, EGFR antagonists in cancer treatment, N. Engl. J. Med. 358 (2008) 1160–1174. [19] N. Ishihara, M. Nomura, A. Jofuku, H. Kato, S.O. Suzuki, K. Masuda, H. Otera, Y. Nakanishi, I. Nonaka, Y. Goto, N. Taguchi, H. Morinaga, M. Maeda, R. Takayanagi,
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
A receptor tyrosine kinase inhibitor, Tyrphostin A9 induces cancer cell death through Drp1 dependent mitochondria fragmentation So Jung Park a, Young Jun Park a, Ji Hyun Shin a, Eun Sung Kim a, Jung Jin Hwang b, Dong-Hoon Jin b, Jin Cheon Kim b, Dong-Hyung Cho a,⇑ a b
Graduate School of East-West Medical Science, Kyung Hee University, Gyeoggi-Do 446-701, Republic of Korea Institute for Innovative Cancer Research, Asan Medical Center, Seoul 138-736, Republic of Korea
a r t i c l e
i n f o
Article history: Received 8 April 2011 Available online 19 April 2011 Keywords: Mitochondria dynamics Fission Tyrphositn A9 Apoptosis
a b s t r a c t Mitochondria dynamics controls not only their morphology but also functions of mitochondria. Therefore, an imbalance of the dynamics eventually leads to mitochondria disruption and cell death. To identify specific regulators of mitochondria dynamics, we screened a bioactive chemical compound library and selected Tyrphostin A9, a tyrosine kinase inhibitor, as a potent inducer of mitochondrial fission. Tyrphostin A9 treatment resulted in the formation of fragmented mitochondria filament. In addition, cellular ATP level was decreased and the mitochondrial membrane potential was collapsed in Tyr A9-treated cells. Suppression of Drp1 activity by siRNA or over-expression of a dominant negative mutant of Drp1 inhibited both mitochondrial fragmentation and cell death induced by Tyrpohotin A9. Moreover, treatment of Tyrphostin A9 also evoked mitochondrial fragmentation in other cells including the neuroblastomas. Taken together, these results suggest that Tyrphostin A9 induces Drp1-mediated mitochondrial fission and apoptotic cell death. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction As dynamic organelles, mitochondria in healthy cells continuously divide and fuse. Mitochondria are not only necessary for energy generation, but also key components in cell death. Mitochondria dynamics regulate processes associated with mitochondria morphology such as mitochondria biogenesis, localization, and distribution as well as the morphology itself [1]. Several key components in mitochondrial dynamics have been identified. Dynamine-related protein 1 (Drp1) mediates mitochondria and peroxisome membrane fragmentation. Down-regulaton of Drp1 activity not only inhibits mitochondrial fission but also suppresses cytochrome c release, caspase activation, and cell death [2,3]. On the other hand, mitochondria fusion is mediated by mitofusin1 and 2 (Mfn1/2) and optic atrophy protein 1 (Opa1) [4]. Specifically, outer mitochondria membrane (OMM) fusion is mediated by Mfn1/2, while Opa1 activity is associated with inner mitochondria membrane (IMM) fusion. In contrast to fission, mitochondria fusion may function as a cell-protective mechanism. Down-regulation of Mfn1/2 or Opa1 results in mitochondria fragmentation and increases susceptibility to apoptotic stimuli [5,6]. Disruption of this balance contributes to the pathophysiology of neurodegenerative ⇑ Corresponding author. Address: Graduate School of East-West Medical Science, Kyung Hee University 1, Seocheon-Dong, Giheung-Gu, Yongin-Si, Gyeoggi-Do 446701, Republic of Korea. E-mail address: [email protected] (D.-H. Cho). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.04.053
diseases, metabolic disorders, and cancer [1,7,8]. Despite extensive data currently available regarding the machinery of mitochondrial fission and fusion, the precise molecular mechanisms are still not fully understood. Recently, mdivi-1 was identified as a chemical inhibitor of Drp1 [9]. Mdivi-1 inhibits OMM permeabilization, the release of cytochonrome C, and thus prevents apoptotic cell death in HeLa cells [9]. Therefore, identification of regulator of mitochondria dynamics should lead to the development of new therapeutic strategies for treating mitochondria-associated diseases. However, chemical agents modulating mitochondria dynamics are not yet well known. The receptor protein tyrosine kinases such as platelet-derived growth factor receptor (PDGFR) play important roles in regulating cellular functions such as cell proliferation, migration, survival, development as well as cell in many diseases, including cancer [10,11]. Indeed, over-expression of PDGF and their receptor has been reported in several types of cancer including prostate, ovarian, and non-small-cell lung cancer. The inhibition of the tyrosine kinase and their downstream signal pathways has been targeted to cancer therapy [12]. PDGFR activates downstream effectors such as RAS, PI3K, ERK, JNK, Src and Stat proteins that lead to cell proliferation and survival [13]. PDGFR also suppress apoptotic cell death by preventing depolarization of the mitochondrial electropotential gradient and generation of ROS [14]. Tyrphostins (TYRosin PHOSphorylation INhibitors) were originally described as a group of tyrosine kinase inhibitors that compete with substrate binding without affecting ATP [15].
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Currently, several inhibitors for tyrosine kinase have been developed as anti-cancer agents [16–18]. However, the effects of tyrosine kinase inhibitors on mitochondria functions are largely unknown. In the present study, we identified Tyrphostin A9 as a factor of mitochondria fission. Treatment of Tyrphostin A9 highly promoted mitochondria fragmentation and mitochondria membrane depolarization. We also investigated down-regulation of Drp1 suppressed Tyrphostin A9-induced mitochondria fragmentation and cell death. 2. Materials and methods 2.1. Reagents The Lopac 1280 chemical library was from Sigma (St. Louis, MO). The expression plasmid Drp1 dominant negative (K38A) and mito-YFP were a kind gift from van der Bliek AM (University of California at Los Angeles, CA) and from Dr. Gyesoon Yoon (Ajou University, Korea). siRNA targeting Drp1 (50 - GAG GUU AUU GAA CGA CUC A) and negative scrambled siRNA (50 - CCU ACG CCA CCA AUU UCG U) were synthesized from Bioneer (Daejeon, Korea). Tyrphostin A9 and CCCP were purchased from Sigma (St. Louis, MO, USA). 2.2. Cell culture and stable transfection HeLa, SK-N-MC, and SH-SY5Y cells were obtained from the American Type Culture Collection (ATCC). All cells were cultured at 37 °C in a 5% CO2 incubator and maintained in DMEM containing 10% FBS and 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA). For stable cell lines, the cells were transfected with pmito-YFP using Lipofectamin according to manufacturer’s protocol (Invitrogen, Carlsbad, CA). Stable transfectants were selected by growth in selection medium containing 1 mg/ml of G418 for 10 days. After single cell cloning, the stable clones were selected under fluorescence microscope. 2.3. Chemical screening for regulator of mitochondria dynamics HeLa cells stably expressing mito-YFP (HeLa/mito-YFP) were seeded in 96-well plates at 1500 cells per well. 24 h after seeding, chemicals were added individually to each well at a final concentration of 10 lM. Then the change of mitochondrial morphology was observed under fluorescence microscopy (Olympus X71). 2.4. Western blotting Cells were extracted with 2 Laemmli sample buffer (Bio-Rad, Hercules, CA), separated by SDS–polyacrylamide gel electrophoresis, and then transferred to PVDF membrane. After blocking with skim milk in TBST, the membranes were incubated with specific primary antibodies. And then the membranes were incubated with HRP-conjugated secondary antibodies (Pierce, Rockford, IL). AntiDrp1 was from BD (San Jose, CA); anti-caspase-3 antibody was from Cell Signaling Technology (Beverly, MA); anti-Actin antibody was from Millipore (Temecula, CA). 2.5. Measurement of mitochondria membrane potential and ATP level HeLa cells (1 105) were treated with Tyrphosin A9 for 8 h. The Mitochondrial membrane potential was examined with a unique fluorescent cationic dye, JC-1 (5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolylcarbocyanine iodide, BD, San Jose, CA) that detects loss of signal of mitochondrial membrane potential, then the depolarized mitochondria was measured with NucleoCounter
NC-3000 (Chemometec, Denmark). HeLa cells were treated with Tyrphosin A9 and then total ATP level was measured with ATP Fluorometric Assay kit (Biovision, Sandiego, CA) according to manufacturer’s protocol. 2.6. Cell viability assay and apoptosis analysis Cell viability was measured by MTT (Sigma, St. Louis, MO) assay. Cells seeded in 96-well plates were incubated at 37 °C in 5% CO2 for 24 h, the medium was replaced with 100 ll of fresh medium with drugs. At the end of the incubation, 10 ll of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide),5 mg/ml] was added. After mixing with DMSO, absorbance of the solution was read with spectrophotometer (VictorX3, Perkinelmer). Apoptotic cell death was determined tryphan blue exclution assay and morphological change of nucleus with DAPI staining assay. The results were expressed as the means ± SD. The probability of statistical differences between experimental groups was determined by the Student’s t test. 3. Results and discussion 3.1. Tyrphostin A9 induces mitochondria fragmentation in HeLa cells Mitochondria are key components in cell death by mediating extrinsic as well as intrinsic signaling pathways. Mitochondria dynamics not only control their morphology but also regulate their functions [1,7]. To identify chemical modulator of mitochondria dynamics, we developed a cell-based screening system using HeLa cells that stably expressing fluorescent protein fused with mitochondria tracker ((mito-YFP), HeLa/Mito-YFP). Used this assay, we screened a LOPAC 1280 chemical library (a collection of pharmacologically active compounds). Based on the screening results, we selected Tyrphostin A9 [(3,5-bis(1,1-dimethylethyl)-4hydroxyphenyl-methylene)-propanedinitrile, C18H22N2O] for further analysis as a potent inducer of mitochondria fragmentation (Fig. 1A). In other to confirm the screening result, HeLa/mito-YFP cells were treated with Tyrphostin A9 for different dosage (0.1– 20 lM) and time point (0.5–6 h). The results showed that mitochondria became excessively fragmented after treatment with Tyrphostin A9 in a dose- and time dependent manner (Fig. 1B and C). 3.2. Tyrphostin A9-induced mitochondria fragmentation results in mitochondrial dysfunction Mitochondria undergo excessive fragmentation during apoptosis and down-regulation of Drp1 suppresses cell death, implying mitochondria fission is directly involved in apoptosis [19]. Moreover, inhibition of some tyrosine kinases causes mitochondria dysfunction and sensitize cancer cells to cell death [15,20] Therefore, we next examined the effect of Tyrphostin A9 on mitochondria functions. Disruption of mitochondrial permeability transition is an important step in the induction of mitochondria-mediated cell death. We used a unique fluorescent cation dye, JC-1, to examine mitochondrial membrane collapse. Treatment with Tyrphostin A9 resulted in an increased red-colored JC-1, indicating that Tyrphostin A9 strongly induces mitochondria membrane depolarization in HeLa cells (Fig. 2A). The electrochemical gradient of mitochondria is also important in ATP generation. Thus, we next examined cellular ATP levels following treatment of Tyrphostin A9. We observed that the cellular ATP levels were gradually decreased by Tyrphostin A9 (Fig. 2B). These results suggest that Tyrphostin A9 disrupts the mitochondrial membrane potential. Tyrphostins are known to suppress cell proliferation by inhibiting receptor tyrosine kinase signaling [15]. We investigated the effect of Tyrphostin A9
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Fig. 1. Tyrphostin A9 induces mitochondrial fragmentation in HeLa cells (A) Representative fluorescence micrographs of mitochondrial morphology before and after Tyrphostin A9 treatment. HeLa cells stably expressing mito-YFP (HeLa/mito-YFP) were exposed to Tyrphostin A9 for 3 h and then were imaged using fluorescence microscope. (B) HeLa/mito-YFP cells were exposed to increasing concentrations of Tyrphostin A9 (0.5, 1, 5, 10, and 20 uM) and then mitochondrial fragmentation was monitored after 6 h. (C) HeLa/mito-YFP cells were treated with Tyrphostin A9 (10 lM) and fragmented mitochondria was determined at indicated time. Data are represented by the mean ± SEM (n > 3).
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Fig. 2. Tyrphostin A9 promote mitochondria dysfunction and cell death in HeLa cells. (A and B) HeLa cells were incubated with Tyrphostin A9 (0.5, 5 lM) for 8 h. The mitochondria permeability transition was assessed with a mitochondria membrane potential detection kit, JC-1 and total ATP levels were determined after 8 h. (C and D) HeLa cells were exposed to Tyrphostin A9. After 24 h, cell viability was determined by MTT assay and caspase-3 activation was examined by detecting cleaved caspase-3. (E) HeLa cells were treated with Tyrphostin A9 (0.5 lM, 5 lM) and the cell proliferation rate was determined using a CCK8 cell proliferation assay. The results are presented as the daily proliferation rate compared to that of the control. Data are represented by the mean ± SEM (n > 3).
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3.4. Tyrphostin A9 also induces mitochondria fragmentation in neuroblastoma cells
on proliferation and cell viability in HeLa cells. The Western blot analysis showed that caspase-3 was significantly activated following Tyrphostin A9 treatment and this was prevented by pan caspase inhibitor, zVAD (Fig. 2D). In addition, Tyrphostin A9 also suppressed cell proliferation in HeLa cells (Fig. 2E). Taken together, our results suggest that Tyrphostin A9 actively contributes to cytotoxicity in the HeLa cells.
Next, we examined whether Tyrphostin A9 induced mitochondria fission in other cell types. To answer this question, we generated the stable cell line with SK-N-MC and SH-SY5Y (a neuroblastoma cell line) using mito-YFP (SK-N-MC/mito-YFP, SY5Y/mito-YFP) and investigated the ability of Tyrphostin A9 to induce mitochondria fragmentation in these cells. In non-treated cells, mitochondria with an intermediated filaments structure were observed, whereas treatment with Tyrphostin A9 resulted in a strong induction of fragmented structures in SK-N-MC and SH-SY5Y cells (Fig. 4A, and data not shown). Similar with the results in HeLa cells, mitochondria fragmentation highly increased in a dose-dependent manner by Tyrphostin A9 in SK-N-MC cells. These results indicate that Tyrphostin A9 also induces mitochondria fission in different types of cells including neuroblastoma. Disruption of balance on mitochondria fission and fusion contributes to the pathophysiology of many diseases [1,7]. Thus, modulation of mitochondria dynamics should lead to the development of new therapeutic strategies for treating mitochondria associated diseases such as neurodegenerative diseases, metabolic disorders, and cancer. In this study, we identified Tyrphostin A9 as a potent regulator of mitochondria dynamics. Tyrphostin A9 was originally synthesized as an inhibitor of receptor tyrosine kinase [21]. Tyrosine kinases are involved in regulation of multiple cellular processes including cell proliferation and cell death. Over-expression or over-activation of tyrosine kinases is strongly associated with development of many types of cancer. Thus, specific inhibitors to tyrosine kinases have
3.3. Tyrphostin A9 induces Drp1-dependent apoptotic cell death in HeLa cells Mitochondria fission is controlled by Drp1, therefore, we addressed the effects of Drp1 on Tyrphostin A9-induced mitochondria fragmentation. Mitochondria suppressed Drp1 activity by siRNA resulted in formation of highly interconnected filament structures (Fig. 3A). Western blot analysis confirmed the reduced expression of Drp1 by specific siRNA. The mitochondria fragmentation induced by Tyrphostin A9 was inhibited by over-expression of a dominant negative Drp1 mutant (K38A) as well as down-regulation of Drp1 with siRNA in HeLa cells (Fig. 3B and C). These results suggest mitochondria fragmentation by Tyrphostin A9 is dependent on Drp1 activity. Since excessive mitochondria fragmentation impacts on cell death, we next investigated the role of Drp1 on Tyrphostin A9-mediated cell death. HeLa cells were transfected with either non-targeting scrambled siRNA or Drp1 specific siRNA and then, exposed to Tyrphostin A9 to examine the effect on cell death. As shown in Fig. 3D, suppressed expression of Drp1 was decreased apoptotic cell death compared with that of control cells, suggesting that Tyrphostin A9 induces apoptotic cell death through a Drp1mediated pathway.
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Fig. 3. Down-regulation of Drp1 prevents Tyrphosin A9-induced mitochondria fragmentation. (A) HeLa/mito-YFP cells were transfected with control scrambled siRNA (Scram) or specific siRNA against Drp1 (siDrp1), and mitochondria morphology was monitored. Reduced expression of Drp1 by siRNA was confirmed by Western blotting (bottom). (B) After 3 days with siRNA transfection, the cells were treated with Tyrphostin A9 (10 lM) for 1.5 h and mitochondrial fragmentation was counted. (C) HeLa/mitoYFP cells transfected with a control empty vector (pcDNA) or a dominant negative mutant of Drp1 (K38A) were treated with CCCP (1 lM) or Tyrphostin A9 (10 uM) for 1.5 h and then mitochondrial fragmentation was counted. (D) HeLa cells were transfected with a control scrambled siRNA or Drp1 siRNA. After 72 h, the cells were incubated with Tyrpohostin A9 for 24 h and then, apoptotic cells were determined by counting of apoptotic nuclei. Data are represented by the mean ± SEM (n > 3).
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Tyr A9 (uM) Fig. 4. Tyrphosin A9 also induces mitochondria fragmentatioin in neuroblastoma cells (A) SK-N-MC cells stably expressing mito-YFP (SK-N-MC/mito-YFP) were treated with Tyrphostin A9 (5 lM) for 3 h and then were imaged using fluorescence microscope. (B) SK-N-MC/mito-YFP cells were exposed to increasing dosages of Tyrphostin A9 (0.1– 20 lM) and then mitochondrial fragmentation was measured after 6 h. The data means ± SEM.
been considered as attractive targets to cancer treatment in the past several years [12,17,22]. Recent studies have reported that inhibition of tyrosine kinase induces apoptosis through activation of the mitochondria-mediated (intrinsic) pathway. In fact, the tyrosine kinase inhibitor, Erlotinib (trade name Tarceva, an EGFR inhibitor) up-regulates pro-apoptotic Bcl-2 family protein, BIM and PUMA [23,24]. Another receptor tyrosine kinase inhibitor Sorafenib (trade name Nexavar) also induces apoptotic cell death by up-regulation of proapoptotic protein and mitochondria-dependent oxidative stress [25,26]. Although mitochondria can be considered a cellular target for tyrosine kinase inhibitors, the precise mechanism of their effect on mitochondria has not been fully elucidated. Here, we first investigated the effect of a tyrosine kinase inhibitor on mitochondria dynamics. Tyrphostin A9 treatment dramatically triggers mitochondria fragmentation, decrease of cellular ATP levels, and depolarization of mitochondria membrane potential, thus leading to reduced cell proliferation and survival. Moreover inhibition of Drp1 by both siRNA and a dominant negative mutant efficiently suppressed mitochondria fragmentation (Fig. 3), demonstrating that Drp1 is also a key player in Tryrphostin A9-mediated mitochondria fission. Tyrosine kinase inhibitors block the activation of downstream kinases and suppress the signaling. Drp1 activity is regulated by various posttranslational modifications during mitochondria fission [1]. For instance, phosphorylation of Drp1 by CDK1/Cyclin B, CaMK, or CDK5 facilitates mitochondria fragmentation whereas PKA-induced phosphorylation of Drp1 results in the suppression of Drp1 activity [27–31]. We also examine the phosphorylation of Ser 161 on Drp1 (target site for CDK1 kinase) after treatment of Tyrphostin A9. However, the phosphor-Drp1 protein level was not changed by Tryphostin A9 (data not shown). Thus, Additional studies will be required to clarify the effect of tyrosine kinase on Drp1 activity in mitochondria fragmentation. In summary, from a cell-based screen, we identified Tyrphostin A9 as a potent inducer of mitochondria fission in several different types of cells. Our studies suggest that Tyrphostin A9 promotes mitochondria fragmentation and cell death via a Drp1 dependent process.
4. Conflict of interest The authors have declared that no conflict of interest exists.
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