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Role of mitochondrial permeability transition in human hepatocellular carcinoma Hep-G2 cell death induced by rhein
Fitoterapia 91 (2013) 68–73
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Role of mitochondrial permeability transition in human hepatocellular carcinoma Hep-G2 cell death induced by rhein Qiong Du a,b,1, Xiao-Lan Bian c,d,1, Xiao-Le Xu a,b,1, Bin Zhu a,b, Bo Yu a,b,⁎, Qing Zhai a,b,⁎⁎ a b c d
Department of Pharmacy, Fudan University Shanghai Cancer Center, 270 Dong-An Road, Shanghai 200032, China Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China Department of Pharmacy, Luwan Branch of Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Department of Pharmacy, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
a r t i c l e
i n f o
Article history: Received 24 May 2013 Accepted in revised form 9 August 2013 Available online 27 August 2013 Keywords: Cell death Hep-G2 Mitochondrial permeability transition Rhein
a b s t r a c t Rhein, a compound found as a glucoside in the root of rhubarb, is currently a subject of interest for its antitumor properties. The apoptosis of tumor cell lines induced by rhein was observed, and the involvement of mitochondria was established; however, the role of mitochondrial permeability transition (MPT) remains unknown. Here we report that MPT plays an important role in the apoptosis of human hepatocellular carcinoma Hep-G2 cells induced by rhein. After adding rhein to the isolated hepatic mitochondria, swelling effects and the leakage of Ca2+ were observed. These alterations were suppressed by cyclosporin A (CsA), an MPT inhibitor. Furthermore, in Hep-G2 cells, the decrease of ATP production, the loss of mitochondrial transmembrane potential (MTP), the release of cytochrome c (Cyto c), and the activation of caspase 3 were also observed. These toxic effects of rhein can also be attenuated by CsA as well. Moreover, TUNEL assay confirmed that in the presence of CsA, rhein-induced apoptosis was largely inhibited. These results suggest that MPT plays a critical role in the pathogenesis of Hep-G2 cell injury induced by rhein, and imply that MPT may contribute to the anti-cancer activity of rhein. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In China, during 2009, liver cancer ranked second in males and third in females among cancer mortality [1]. The therapies
Abbreviations: CsA, Cyclosporine A; Cyto c, Cytochrome c; DAPI 4′, 6-Diamidino-2-phenylindole; FITC, Fluorescein isothiocyanate; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; HEPES, 2-[4-(2-Hydroxyethyl)1-piperazine] ethanesulfonic acid; HRP, Horseradish peroxidase; MPT, Mitochondrial permeability transition; MTP, Mitochondrial transmembrane potential; PBS, Phosphate-buffered saline; ROS, Reactive oxygen species; SDS–PAGE, Sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TBS, Tris-buffered saline. ⁎ Correspondence to: B. Yu, Department of Pharmacy, Fudan University Shanghai Cancer Center, 270 Dong-An Road, Shanghai 200032, China. Tel.: +86 15821279851. ⁎⁎ Correspondence to: Q. Zhai, Department of Pharmacy, Fudan University Shanghai Cancer Center, 270 Dong-An Road, Shanghai 200032, China. E-mail addresses: [email protected] (B. Yu), [email protected] (Q. Zhai). 1 These authors contributed equally to this work. 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.08.008
for this high-mortality cancer have included surgery, chemical therapy, and targeted therapy, but the 5-year survival rate remains only between 3% and 67% [2]. An alternative therapy for liver cancer is certainly needed. Previous studies have shown that rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid), a compound found in the free state as a glucoside in the root of rhubarb (Rheum palmatum L. or Rheum tanguticum Maxim) [3], inhibited the growth of many cancer cells, including human breast cancer [4], cervical cancer [5], and gastric cancer [6], as well as human hepatocellular carcinomas [7]; however, the mechanism involving mitochondrial pathways in rhein-induced cell death remains unknown. Ca2+ overload, oxidative stress, hypoxia, and cytotoxic drugs may all lead to mitochondrial permeability transition (MPT) [8]. Furthermore, MPT occurs along with the loss of mitochondrial transmembrane potential (MTP), which is significant for mitochondrial ATP production [9], and plays an important role in drug-induced necrosis and apoptosis [10,11]. MPT is defined as an increase of the mitochondrial inner membrane permeability
Q. Du et al. / Fitoterapia 91 (2013) 68–73
to allow ions and solutes with molecular masses up to 1500 Da to leak from the mitochondria leading to matrix swelling. The subsequent breaking of the mitochondrial outer membrane will lead to the release of proteins into the inter-membrane space, such as cytochrome c (Cyto c) and other factors that play a critical role in apoptotic cell death [12]. The present study was undertaken to identify the contribution of mitochondrial components to rhein-induced tumor cell death by using the human hepatocellular carcinoma cell line, Hep-G2, and to investigate the possible mechanisms involved. In this study, we focused on rhein-induced MPT and its role on human hepatocellular carcinoma Hep-G2 cell death. 2. Materials and methods
69
peroxidase (HRP)-labeled secondary antibodies were the products of Cell Signaling Technology (Danvers, MA, USA). 2.2. Preparation of rat liver mitochondria and induction of MPT Hepatic mitochondria were isolated from male Sprague– Dawley rats in a sucrose- 2-[4-(2-Hydroxyethyl)-1-piperazine] ethanesulfonic acid (HEPES) buffer by differential centrifugation [13]. Mitochondria (75 μg mitochondrial protein per ml) were suspended in 1 ml buffer (2 mM HEPES, pH 7.5, 0.25 M sucrose, 10 mM succinate, 1 mM potassium phosphate). MPT was initiated by the addition of rhein with calcium (20 μM final concentration) as indicated in Fig. 1. The progression of the MPT was monitored by the change in absorbance at 540 nm at room temperature.
2.1. Materials 2.3. Measurement of mitochondrial Ca2+ release All chemicals and solvents used were purchased from Sigma (St. Louis, MO, USA) and were of analytical grade unless otherwise stated. RPMI-1640 medium and fetal calf serum were purchased from GIBCO/BRL (Grand Island, NY, USA). Monoclonal antibodies against human glyceraldehyde3-phosphate dehydrogenase (GAPDH), Cyto c, and horseradish
Ca2+ release from the mitochondria was measured as described [14]. Briefly, Arsenazo III (50 μM) was added to the medium as mentioned above. Absorbance of the medium was monitored at the 675/685 nm wavelength pair at room temperature. Rhein was added where indicated.
Fig. 1. Induction of mitochondrial swelling (A) and calcium release (B) by rhein in isolated hepatic mitochondria. (A) Swelling effect was monitored as the decrease of absorbance at 540 nm in energized mitochondria, and rhein was added where arrows indicated. Ca2+ (20 μM final concentration) was used as the control. Note that 20 μM Ca2+ alone could not induce MPT. Mitochondrial swelling was inhibited by adding 1 μM CsA. The data represented a typical experiment conducted at least 3 times with similar results. (B) Induction of calcium release by rhein in isolated mitochondria. Calcium release was monitored as the difference of absorbance at 675 nm and 685 nm in mitochondria energized by succinate. Rhein was added where indicated. Note that 20 μM Ca2+ alone could not induce calcium release. This effect was inhibited by adding 1 μM CsA. The data represent 6 independent tests with similar results.
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2.4. Cell culture Hep-G2 cells were cultured in RPMI-1640 medium containing 10% fetal calf serum, 1% penicillin (100 IU/mL), and 1% streptomycin (100 μg/mL), in a 5% CO2 atmosphere, at 37 °C. 2.5. Cell viability Cells were treated with different concentrations of rhein, and the general viabilities of cultured cells were determined with the Cell Counting Kit-8 (Dojindo Laboratories, Tokyo, Japan). 2.6. Measurement of MTP MTP was evaluated as the accumulation of rhodamine 123 (final concentration of 1.5 μM) according to the methods described by Wu [15]. CsA (1 μM) was used in this procedure as an inhibitor of MPT. Fluorescence readings were taken on a fluorimeter (NOVOstar, BMG LABTECH, Offenburg, Germany) with the excitation wavelength at 485 nm and the emission wavelength at 520 nm.
and removed at 6000 ×g for 25 min. The supernatant was further subjected to centrifugation at 20000 ×g for 60 min, and the final supernatant was used as the cytosolic fraction for the detection of released Cyto c. Between 20 and 40 μg of protein from each sample were loaded on 5% stacking gels and separated by SDS–PAGE, using 12% separating gels, and then analyzed as indicated above. 2.11. Caspase 3 activity assay Caspase 3 activity was measured as described [24]. Briefly, Caspase 3 assay kit (BD Biosciences Pharmingen, San Diego, CA, USA) was used and fluorescence was measured in the fluorimeter (NOVOstar, BMG LABTECH, Offenburg, Germany) with excitation at 380 nm and emission at 460 nm. 2.12. Statistical analyses Results were expressed as means ± SD of six independent experiments, unless otherwise indicated. Statistical analyses were performed using Student's t-test as appropriate, with the level of significance set at p b 0.05.
2.7. Measurement of cellular ATP content
3. Results
Intracellular ATP levels in the presence or absence of CsA (1 μM) were determined using CellTiter-GloTM Luminescent Cell Viability Assay kit according to manufacturer's instructions (Promega, Madison, WI, USA). Bioluminescence was measured with Novostar (BMG LABTECH, Offenburg, Germany).
3.1. Rhein-induced mitochondrial swelling
2.8. TUNEL assay After the induction of apoptosis, Hep-G2 cells were collected and apoptosis was evaluated using the In Situ Cell Death Detection Kit (Roche Applied Science, Penzberg, Germany).
First, we subjected isolated hepatic mitochondria to rhein acute stimuli and monitored the apparent decrease in the absorbance of the mitochondria suspension at 540 nm. As shown in Fig. 1A, rhein-induced mitochondrial swelling in a dose-dependent manner with a quick onset and large magnitude. This swelling effect was fully blocked by CsA, a specific blocker of MPT. This result occurred simultaneously with the Ca2+ release assay (Fig. 1B). These results suggest that rhein targets the MPT complex and might induce the breakdown of MTP.
2.9. Western blotting 3.2. Rhein-induced mitochondrial dysfunction Between 20 and 40 μg of protein from each sample were loaded on 5% stacking gels and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) using 12% separating gels. Proteins were transferred onto nitrocellulose paper and blocked for 1 h with 5% skim milk in Trisbuffered saline (TBS) containing 0.01% NaN3 and 0.1% Tween 20, and then incubated overnight with the primary antibody in TBS/0.1% Tween 20 at 4 °C. After three washes with TBS/0.1% Tween 20, membranes were incubated with HRP-conjugated secondary antibody at 1:5000 dilution for 1 h. Bands were visualized by enhanced chemiluminescence.
To further define MPT induced by rhein in Hep-G2 cells, the membrane potential-sensitive probe, rhodamine 123, was used to detect MTP. Significant reductions in MTP were observed after rhein treatment, indicating mitochondrial dysfunction upon rhein stimuli. Meanwhile, co-treatment of 1 μM CsA blocked the decrease in MTP (Fig. 2A). The loss of MTP may only occur when supplies of ATP are depleted [15]. In this study, the production of ATP was decreased upon rhein treatment (Fig. 2B) and restored by CsA. These data imply the importance of MPT in rhein-induced mitochondrial dysfunction.
2.10. Determination of translocation by Western blotting
3.3. Rhein-induced Cyto c release and caspase 3 activation
The cells were washed twice with ice-cold phosphatebuffered saline (PBS) and then resuspended in isotonic homogenization buffer (250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTANa2, 1 mM EGTA-Na2, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 10 μg/mL pepstatin A, 10 mM Tris-HCl, pH 7.4). After 80 strokes in a Dounce homogenizer, the unbroken cells were spun down at 30 ×g for 10 min. The nuclei and mitochondria fractions were fractionated
Cyto c release and caspase 3 enzyme activation are connected to the breakdown of the mitochondrial membrane. We further investigated the subcellular distribution of Cyto c from mitochondria and the activation of caspase 3. A significant increase of Cyto c release is shown in Fig. 3A. At the same time, activation of caspase 3 (Fig. 3B) was detected in cells treated with rhein. Meanwhile, all of these effects could be blocked by adding CsA to the culture medium.
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Fig. 2. Effects of rhein on (A) MTP decrease and (B) ATP depletion of Hep-G2 cells. Samples were taken after incubation with indicated concentrations of rhein for 48 h. Results are presented as means ± SD, n = 6. Significantly different from the control group at each time point: *p b 0.05, **p b 0.01 or from groups treated with the same concentration of rhein without CsA: # p b 0.05, ##p b 0.01.
3.4. Rhein-induced apoptosis of Hep-G2 cells A significant increase in the percentage of apoptotic cells was detected after rhein treatment, and the addition of CsA in the culture medium alleviated Hep-G2 cell apoptosis induced by rhein (Fig. 4). 4. Discussion The study confirmed that rhein caused damage to Hep-G2 cells through a mitochondria-dependent mechanism and showed, for the first time, that MPT played a key role in this toxic process. Several lines of evidence support this conclusion. First, rhein-induced mitochondrial swelling as well as the release of Ca2+ from isolated mitochondria (Fig. 1), causing
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Fig. 3. (A) Western blot analysis of Cyto c release, and (B) activation of caspase 3 in Rhein-treated Hep-G2 cells after indicated time periods. The insert in (A) is a representative result of 3 independent tests with similar results. Results are presented as the mean ± SD, n = 3. Significantly different from control at each time point: *p b 0.05 and **p b 0.01 or from groups treated with the same concentration of rhein without CsA: #p b 0.05, ## p b 0.01.
membrane depolarization and a decrease in ATP production in Hep-G2 cells (Fig. 2). Second, rhein-induced the release of Cyto c and activated caspase 3 from Hep-G2 cells (Fig. 3). All of these toxic effects could be inhibited by CsA. Finally, CsA protected the cells against rhein-induced apoptosis (Fig. 4). Our results thus suggest that MPT activated by rhein is responsible for rhein-induced mitochondrial and cellular damage. It has been reported that Ca2+ levels are enhanced in cancer cells treated with rhein [16,17]. As a key regulator of mitochondria, Ca2+ combined with other stimuli may cause MPT [18]. Loss of MTP induced by rhein, which represents the damaged mitochondria, has also been reported in other cancer cells [19,20]. However, these phenomena firstly joined each other in the present study. Rhein can act directly on mitochondria, resulting in MPT and the release of Ca2+ and
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Acknowledgements The current study is supported partially by the National Natural Science Foundation of China (No. NSFC81201807 to BY). We thank Sonya Vargulick for excellent comments on manuscript preparation. References
Fig. 4. TUNEL assay for Hep-G2 cell apoptosis. Hep-G2 cells were treated with normal medium (A, D, and G), 100 μM rhein (B, E, and H) and 100 μM rhein + 1 μM CsA (C, F, and I) for 72 h. Cells were stained with 4′,6diamidino-2-phenylindole (DAPI) (A-C), fluorescein isothiocyanate (FITC) (D–F), or merge (G–I). Scale bar is 100 μm. A–I: representative data of 3 similar results. The values shown in J are mean ± SD, n = 3. Significantly different from the control group at each time point: *p b 0.05, **p b 0.01 and ## p b 0.01.
Cyto c into the cytosol; also triggering caspase 3 activation and the subsequent process of cell death. Oxidative injury induced by rhein, is considered a crucial modulator of MPT [21], but was not investigated in the present study. There are reports on the role of reactive oxygen species (ROS) in rhein-induced cancer cell death [20,22,23]; interestingly, however, MPT mediated cell death does not necessarily lead to ROS production [24] and increased ROS can lead to tumorigenesis [25,26]. To disclose the role of ROS in MPT and Hep-G2 cell death induced by rhein, further research should be conducted. Overall, the most important finding of the current study was that MPT plays a critical role in rhein-induced cell toxicity.
Conflict of interest The authors declare that there are no conflicts of interest.
[1] Chen W, Zheng R, Zhang S, Zhao P, Li G, Wu L, et al. Report of incidence and mortality in China cancer registries, 2009. Chin J Cancer Res 2013;25(1):10–21. [2] Schwarz RE, Smith DD. Trends in local therapy for hepatocellular carcinoma and survival outcomes in the US population. Am J Surg 2008;195(6):829–36. [3] Lee JH, Kim JM, Kim C. Pharmacokinetic analysis of rhein in Rheum undulatum L. J Ethnopharmacol 2003;84(1):5–9. [4] Chang CY, Chan HL, Lin HY, Way TD, Kao MC, Song MZ, et al. Rhein induces apoptosis in human breast cancer cells. Evid Based Complement Alternat Med 2012;2012:952504. [5] Ip SW, Weng YS, Lin SY, Mei DY, Tang NY, Su CC, et al. The role of Ca + 2 on rhein-induced apoptosis in human cervical cancer Ca Ski cells. Anticancer Res 2007;27(1A):379–89. [6] Li Y, Xu Y, Lei B, Wang W, Ge X, Li J. Rhein induces apoptosis of human gastric cancer SGC-7901 cells via an intrinsic mitochondrial pathway. Braz J Med Biol Res 2012;45(11):1052–9. [7] Shi P, Huang Z, Chen G. Rhein induces apoptosis and cell cycle arrest in human hepatocellular carcinoma BEL-7402 cells. Am J Chin Med 2008;36(4):805–13. [8] McCommis KS, Baines CP. The role of VDAC in cell death: friend or foe? Biochim Biophys Acta 2012;1818(6):1444–50. [9] Barbu A, Welsh N, Saldeen J. Cytokine-induced apoptosis and necrosis are preceded by disruption of the mitochondrial membrane potential (Deltapsi(m)) in pancreatic RINm5F cells: prevention by Bcl-2 [J]. Mol Cell Endocrinol 2002;190(1–2):75–82. [10] Uyemura SA, Santos AC, Mingatto FE, Jordani MC, Curti C. Diclofenac sodium and mefenamic acid: potent inducers of the membrane permeability transition in renal cortex mitochondria. Arch Biochem Biophys 1997;342(2):231–5. [11] Kim JS, He L, Lemasters JJ. Mitochondrial permeability transition: a common pathway to necrosis and apoptosis. Biochem Biophys Res Commun 2003;304(3):463–70. [12] Bernardi P. The mitochondrial permeability transition pore: a mystery solved? Front Physiol 2013;4:95. [13] Cai Y, Gong LK, Qi XM, Li XH, Ren J. Apoptosis initiated by carbon tetrachloride in mitochondria of rat primary cultured hepatocytes. Acta Pharmacol Sin 2005;26(8):969–75. [14] Li Y, Qi XM, Xue X, Wu XF, Wu YF, Chen M, et al. The relationship between diphenylamine structure and NSAIDs-induced hepatocytes injury [J]. Toxicol Lett 2009;186(2):111–4. [15] Wu EY, Smith MT, Bellomo G, Di Monte D. Relationships between the mitochondrial transmembrane potential, ATP concentration, and cytotoxicity in isolated rat hepatocytes. Arch Biochem Biophys 1990;282(2):358–62. [16] Lin ML, Chen SS, Lu YC, Liang RY, Ho YT, Yang CY, et al. Rhein induces apoptosis through induction of endoplasmic reticulum stress and Ca2+-dependent mitochondrial death pathway in human nasopharyngeal carcinoma cells. Anticancer Res 2007;27(5A):3313–22. [17] Hsia TC, Yang JS, Chen GW, Chiu TH, Lu HF, Yang MD, et al. The roles of endoplasmic reticulum stress and Ca2+ on rhein-induced apoptosis in A-549 human lung cancer cells. Anticancer Res 2009;29(1):309–18. [18] Brookes PS, Yoon Y, Robotham JL, Anders MW, Sheu SS. Calcium, ATP, and ROS: a mitochondrial love-hate triangle. Am J Physiol Cell Physiol 2004;287(4):C817–33. [19] Heo SK, Yun HJ, Park WH, Park SD. Rhein inhibits TNF-alpha-induced human aortic smooth muscle cell proliferation via mitochondrialdependent apoptosis. J Vasc Res 2009;46(4):375–86. [20] Lin S, Fujii M, Hou DX. Rhein induces apoptosis in HL-60 cells via reactive oxygen species-independent mitochondrial death pathway. Arch Biochem Biophys 2003;418(2):99–107. [21] Kowaltowski AJ, Castilho RF, Vercesi AE. Mitochondrial permeability transition and oxidative stress. FEBS Lett 2001;495(1–2):12–5. [22] Lai WW, Yang JS, Lai KC, Kuo CL, Hsu CK, Wang CK, et al. Rhein-induced apoptosis through the endoplasmic reticulum stress, caspase- and mitochondria-dependent pathways in SCC-4 human tongue squamous cancer cells. In Vivo 2009;23(2):309–16. [23] Bironaite D, Ollinger K. The hepatotoxicity of rhein involves impairment of mitochondrial functions [J]. Chem Biol Interact 1997;103(1):35–50.
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using mouse two-stage skin carcinogenesis as a model. PLoS One 2009;4(4):e5284. [26] Ziech D, Franco R, Georgakilas AG, Georgakila S, Malamou-Mitsi V, Schoneveld O, et al. The role of reactive oxygen species and oxidative stress in environmental carcinogenesis and biomarker development. Chem Biol Interact 2010;188(2):334–9.
Contents lists available at ScienceDirect
Fitoterapia journal homepage: www.elsevier.com/locate/fitote
Role of mitochondrial permeability transition in human hepatocellular carcinoma Hep-G2 cell death induced by rhein Qiong Du a,b,1, Xiao-Lan Bian c,d,1, Xiao-Le Xu a,b,1, Bin Zhu a,b, Bo Yu a,b,⁎, Qing Zhai a,b,⁎⁎ a b c d
Department of Pharmacy, Fudan University Shanghai Cancer Center, 270 Dong-An Road, Shanghai 200032, China Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China Department of Pharmacy, Luwan Branch of Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Department of Pharmacy, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
a r t i c l e
i n f o
Article history: Received 24 May 2013 Accepted in revised form 9 August 2013 Available online 27 August 2013 Keywords: Cell death Hep-G2 Mitochondrial permeability transition Rhein
a b s t r a c t Rhein, a compound found as a glucoside in the root of rhubarb, is currently a subject of interest for its antitumor properties. The apoptosis of tumor cell lines induced by rhein was observed, and the involvement of mitochondria was established; however, the role of mitochondrial permeability transition (MPT) remains unknown. Here we report that MPT plays an important role in the apoptosis of human hepatocellular carcinoma Hep-G2 cells induced by rhein. After adding rhein to the isolated hepatic mitochondria, swelling effects and the leakage of Ca2+ were observed. These alterations were suppressed by cyclosporin A (CsA), an MPT inhibitor. Furthermore, in Hep-G2 cells, the decrease of ATP production, the loss of mitochondrial transmembrane potential (MTP), the release of cytochrome c (Cyto c), and the activation of caspase 3 were also observed. These toxic effects of rhein can also be attenuated by CsA as well. Moreover, TUNEL assay confirmed that in the presence of CsA, rhein-induced apoptosis was largely inhibited. These results suggest that MPT plays a critical role in the pathogenesis of Hep-G2 cell injury induced by rhein, and imply that MPT may contribute to the anti-cancer activity of rhein. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In China, during 2009, liver cancer ranked second in males and third in females among cancer mortality [1]. The therapies
Abbreviations: CsA, Cyclosporine A; Cyto c, Cytochrome c; DAPI 4′, 6-Diamidino-2-phenylindole; FITC, Fluorescein isothiocyanate; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; HEPES, 2-[4-(2-Hydroxyethyl)1-piperazine] ethanesulfonic acid; HRP, Horseradish peroxidase; MPT, Mitochondrial permeability transition; MTP, Mitochondrial transmembrane potential; PBS, Phosphate-buffered saline; ROS, Reactive oxygen species; SDS–PAGE, Sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TBS, Tris-buffered saline. ⁎ Correspondence to: B. Yu, Department of Pharmacy, Fudan University Shanghai Cancer Center, 270 Dong-An Road, Shanghai 200032, China. Tel.: +86 15821279851. ⁎⁎ Correspondence to: Q. Zhai, Department of Pharmacy, Fudan University Shanghai Cancer Center, 270 Dong-An Road, Shanghai 200032, China. E-mail addresses: [email protected] (B. Yu), [email protected] (Q. Zhai). 1 These authors contributed equally to this work. 0367-326X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2013.08.008
for this high-mortality cancer have included surgery, chemical therapy, and targeted therapy, but the 5-year survival rate remains only between 3% and 67% [2]. An alternative therapy for liver cancer is certainly needed. Previous studies have shown that rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid), a compound found in the free state as a glucoside in the root of rhubarb (Rheum palmatum L. or Rheum tanguticum Maxim) [3], inhibited the growth of many cancer cells, including human breast cancer [4], cervical cancer [5], and gastric cancer [6], as well as human hepatocellular carcinomas [7]; however, the mechanism involving mitochondrial pathways in rhein-induced cell death remains unknown. Ca2+ overload, oxidative stress, hypoxia, and cytotoxic drugs may all lead to mitochondrial permeability transition (MPT) [8]. Furthermore, MPT occurs along with the loss of mitochondrial transmembrane potential (MTP), which is significant for mitochondrial ATP production [9], and plays an important role in drug-induced necrosis and apoptosis [10,11]. MPT is defined as an increase of the mitochondrial inner membrane permeability
Q. Du et al. / Fitoterapia 91 (2013) 68–73
to allow ions and solutes with molecular masses up to 1500 Da to leak from the mitochondria leading to matrix swelling. The subsequent breaking of the mitochondrial outer membrane will lead to the release of proteins into the inter-membrane space, such as cytochrome c (Cyto c) and other factors that play a critical role in apoptotic cell death [12]. The present study was undertaken to identify the contribution of mitochondrial components to rhein-induced tumor cell death by using the human hepatocellular carcinoma cell line, Hep-G2, and to investigate the possible mechanisms involved. In this study, we focused on rhein-induced MPT and its role on human hepatocellular carcinoma Hep-G2 cell death. 2. Materials and methods
69
peroxidase (HRP)-labeled secondary antibodies were the products of Cell Signaling Technology (Danvers, MA, USA). 2.2. Preparation of rat liver mitochondria and induction of MPT Hepatic mitochondria were isolated from male Sprague– Dawley rats in a sucrose- 2-[4-(2-Hydroxyethyl)-1-piperazine] ethanesulfonic acid (HEPES) buffer by differential centrifugation [13]. Mitochondria (75 μg mitochondrial protein per ml) were suspended in 1 ml buffer (2 mM HEPES, pH 7.5, 0.25 M sucrose, 10 mM succinate, 1 mM potassium phosphate). MPT was initiated by the addition of rhein with calcium (20 μM final concentration) as indicated in Fig. 1. The progression of the MPT was monitored by the change in absorbance at 540 nm at room temperature.
2.1. Materials 2.3. Measurement of mitochondrial Ca2+ release All chemicals and solvents used were purchased from Sigma (St. Louis, MO, USA) and were of analytical grade unless otherwise stated. RPMI-1640 medium and fetal calf serum were purchased from GIBCO/BRL (Grand Island, NY, USA). Monoclonal antibodies against human glyceraldehyde3-phosphate dehydrogenase (GAPDH), Cyto c, and horseradish
Ca2+ release from the mitochondria was measured as described [14]. Briefly, Arsenazo III (50 μM) was added to the medium as mentioned above. Absorbance of the medium was monitored at the 675/685 nm wavelength pair at room temperature. Rhein was added where indicated.
Fig. 1. Induction of mitochondrial swelling (A) and calcium release (B) by rhein in isolated hepatic mitochondria. (A) Swelling effect was monitored as the decrease of absorbance at 540 nm in energized mitochondria, and rhein was added where arrows indicated. Ca2+ (20 μM final concentration) was used as the control. Note that 20 μM Ca2+ alone could not induce MPT. Mitochondrial swelling was inhibited by adding 1 μM CsA. The data represented a typical experiment conducted at least 3 times with similar results. (B) Induction of calcium release by rhein in isolated mitochondria. Calcium release was monitored as the difference of absorbance at 675 nm and 685 nm in mitochondria energized by succinate. Rhein was added where indicated. Note that 20 μM Ca2+ alone could not induce calcium release. This effect was inhibited by adding 1 μM CsA. The data represent 6 independent tests with similar results.
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2.4. Cell culture Hep-G2 cells were cultured in RPMI-1640 medium containing 10% fetal calf serum, 1% penicillin (100 IU/mL), and 1% streptomycin (100 μg/mL), in a 5% CO2 atmosphere, at 37 °C. 2.5. Cell viability Cells were treated with different concentrations of rhein, and the general viabilities of cultured cells were determined with the Cell Counting Kit-8 (Dojindo Laboratories, Tokyo, Japan). 2.6. Measurement of MTP MTP was evaluated as the accumulation of rhodamine 123 (final concentration of 1.5 μM) according to the methods described by Wu [15]. CsA (1 μM) was used in this procedure as an inhibitor of MPT. Fluorescence readings were taken on a fluorimeter (NOVOstar, BMG LABTECH, Offenburg, Germany) with the excitation wavelength at 485 nm and the emission wavelength at 520 nm.
and removed at 6000 ×g for 25 min. The supernatant was further subjected to centrifugation at 20000 ×g for 60 min, and the final supernatant was used as the cytosolic fraction for the detection of released Cyto c. Between 20 and 40 μg of protein from each sample were loaded on 5% stacking gels and separated by SDS–PAGE, using 12% separating gels, and then analyzed as indicated above. 2.11. Caspase 3 activity assay Caspase 3 activity was measured as described [24]. Briefly, Caspase 3 assay kit (BD Biosciences Pharmingen, San Diego, CA, USA) was used and fluorescence was measured in the fluorimeter (NOVOstar, BMG LABTECH, Offenburg, Germany) with excitation at 380 nm and emission at 460 nm. 2.12. Statistical analyses Results were expressed as means ± SD of six independent experiments, unless otherwise indicated. Statistical analyses were performed using Student's t-test as appropriate, with the level of significance set at p b 0.05.
2.7. Measurement of cellular ATP content
3. Results
Intracellular ATP levels in the presence or absence of CsA (1 μM) were determined using CellTiter-GloTM Luminescent Cell Viability Assay kit according to manufacturer's instructions (Promega, Madison, WI, USA). Bioluminescence was measured with Novostar (BMG LABTECH, Offenburg, Germany).
3.1. Rhein-induced mitochondrial swelling
2.8. TUNEL assay After the induction of apoptosis, Hep-G2 cells were collected and apoptosis was evaluated using the In Situ Cell Death Detection Kit (Roche Applied Science, Penzberg, Germany).
First, we subjected isolated hepatic mitochondria to rhein acute stimuli and monitored the apparent decrease in the absorbance of the mitochondria suspension at 540 nm. As shown in Fig. 1A, rhein-induced mitochondrial swelling in a dose-dependent manner with a quick onset and large magnitude. This swelling effect was fully blocked by CsA, a specific blocker of MPT. This result occurred simultaneously with the Ca2+ release assay (Fig. 1B). These results suggest that rhein targets the MPT complex and might induce the breakdown of MTP.
2.9. Western blotting 3.2. Rhein-induced mitochondrial dysfunction Between 20 and 40 μg of protein from each sample were loaded on 5% stacking gels and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) using 12% separating gels. Proteins were transferred onto nitrocellulose paper and blocked for 1 h with 5% skim milk in Trisbuffered saline (TBS) containing 0.01% NaN3 and 0.1% Tween 20, and then incubated overnight with the primary antibody in TBS/0.1% Tween 20 at 4 °C. After three washes with TBS/0.1% Tween 20, membranes were incubated with HRP-conjugated secondary antibody at 1:5000 dilution for 1 h. Bands were visualized by enhanced chemiluminescence.
To further define MPT induced by rhein in Hep-G2 cells, the membrane potential-sensitive probe, rhodamine 123, was used to detect MTP. Significant reductions in MTP were observed after rhein treatment, indicating mitochondrial dysfunction upon rhein stimuli. Meanwhile, co-treatment of 1 μM CsA blocked the decrease in MTP (Fig. 2A). The loss of MTP may only occur when supplies of ATP are depleted [15]. In this study, the production of ATP was decreased upon rhein treatment (Fig. 2B) and restored by CsA. These data imply the importance of MPT in rhein-induced mitochondrial dysfunction.
2.10. Determination of translocation by Western blotting
3.3. Rhein-induced Cyto c release and caspase 3 activation
The cells were washed twice with ice-cold phosphatebuffered saline (PBS) and then resuspended in isotonic homogenization buffer (250 mM sucrose, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTANa2, 1 mM EGTA-Na2, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 10 μg/mL pepstatin A, 10 mM Tris-HCl, pH 7.4). After 80 strokes in a Dounce homogenizer, the unbroken cells were spun down at 30 ×g for 10 min. The nuclei and mitochondria fractions were fractionated
Cyto c release and caspase 3 enzyme activation are connected to the breakdown of the mitochondrial membrane. We further investigated the subcellular distribution of Cyto c from mitochondria and the activation of caspase 3. A significant increase of Cyto c release is shown in Fig. 3A. At the same time, activation of caspase 3 (Fig. 3B) was detected in cells treated with rhein. Meanwhile, all of these effects could be blocked by adding CsA to the culture medium.
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Fig. 2. Effects of rhein on (A) MTP decrease and (B) ATP depletion of Hep-G2 cells. Samples were taken after incubation with indicated concentrations of rhein for 48 h. Results are presented as means ± SD, n = 6. Significantly different from the control group at each time point: *p b 0.05, **p b 0.01 or from groups treated with the same concentration of rhein without CsA: # p b 0.05, ##p b 0.01.
3.4. Rhein-induced apoptosis of Hep-G2 cells A significant increase in the percentage of apoptotic cells was detected after rhein treatment, and the addition of CsA in the culture medium alleviated Hep-G2 cell apoptosis induced by rhein (Fig. 4). 4. Discussion The study confirmed that rhein caused damage to Hep-G2 cells through a mitochondria-dependent mechanism and showed, for the first time, that MPT played a key role in this toxic process. Several lines of evidence support this conclusion. First, rhein-induced mitochondrial swelling as well as the release of Ca2+ from isolated mitochondria (Fig. 1), causing
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Fig. 3. (A) Western blot analysis of Cyto c release, and (B) activation of caspase 3 in Rhein-treated Hep-G2 cells after indicated time periods. The insert in (A) is a representative result of 3 independent tests with similar results. Results are presented as the mean ± SD, n = 3. Significantly different from control at each time point: *p b 0.05 and **p b 0.01 or from groups treated with the same concentration of rhein without CsA: #p b 0.05, ## p b 0.01.
membrane depolarization and a decrease in ATP production in Hep-G2 cells (Fig. 2). Second, rhein-induced the release of Cyto c and activated caspase 3 from Hep-G2 cells (Fig. 3). All of these toxic effects could be inhibited by CsA. Finally, CsA protected the cells against rhein-induced apoptosis (Fig. 4). Our results thus suggest that MPT activated by rhein is responsible for rhein-induced mitochondrial and cellular damage. It has been reported that Ca2+ levels are enhanced in cancer cells treated with rhein [16,17]. As a key regulator of mitochondria, Ca2+ combined with other stimuli may cause MPT [18]. Loss of MTP induced by rhein, which represents the damaged mitochondria, has also been reported in other cancer cells [19,20]. However, these phenomena firstly joined each other in the present study. Rhein can act directly on mitochondria, resulting in MPT and the release of Ca2+ and
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Acknowledgements The current study is supported partially by the National Natural Science Foundation of China (No. NSFC81201807 to BY). We thank Sonya Vargulick for excellent comments on manuscript preparation. References
Fig. 4. TUNEL assay for Hep-G2 cell apoptosis. Hep-G2 cells were treated with normal medium (A, D, and G), 100 μM rhein (B, E, and H) and 100 μM rhein + 1 μM CsA (C, F, and I) for 72 h. Cells were stained with 4′,6diamidino-2-phenylindole (DAPI) (A-C), fluorescein isothiocyanate (FITC) (D–F), or merge (G–I). Scale bar is 100 μm. A–I: representative data of 3 similar results. The values shown in J are mean ± SD, n = 3. Significantly different from the control group at each time point: *p b 0.05, **p b 0.01 and ## p b 0.01.
Cyto c into the cytosol; also triggering caspase 3 activation and the subsequent process of cell death. Oxidative injury induced by rhein, is considered a crucial modulator of MPT [21], but was not investigated in the present study. There are reports on the role of reactive oxygen species (ROS) in rhein-induced cancer cell death [20,22,23]; interestingly, however, MPT mediated cell death does not necessarily lead to ROS production [24] and increased ROS can lead to tumorigenesis [25,26]. To disclose the role of ROS in MPT and Hep-G2 cell death induced by rhein, further research should be conducted. Overall, the most important finding of the current study was that MPT plays a critical role in rhein-induced cell toxicity.
Conflict of interest The authors declare that there are no conflicts of interest.
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