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Polyphyllin D induces mitochondrial fragmentation and acts directly on the mitochondria to induce apoptosis in drug-resistant HepG2 cells
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Cancer Letters 261 (2008) 158–164 www.elsevier.com/locate/canlet
Polyphyllin D induces mitochondrial fragmentation and acts directly on the mitochondria to induce apoptosis in drug-resistant HepG2 cells Rose C.Y. Ong a, Jin Lei b, Rebecca K.Y. Lee a, Jenny Y.N. Cheung a, K.P. Fung a,c, Chinlon Lin b, H.P. Ho b, Biao Yu d, Ming Li d, S.K. Kong b
a,*
a Department of Biochemistry, The Chinese University of Hong Kong, Shatin, Hong Kong, China Biophotonic Laboratory, Department of Electronic Engineering, The Chinese University of Hong Kong, Shatin, Hong Kong, China c Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, China d State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China
Received 25 September 2007; received in revised form 7 November 2007; accepted 7 November 2007
Abstract We previously showed that polyphyllin D (PD) produced a stronger apoptotic effect in R-HepG2 with multi-drug resistance (MDR) than that in its parent HepG2 cells without MDR. In this study, PD was found to elicit mitochondrial fragmentation in live cells by using total internal reflection fluorescence microscopy (TIRFM). When mitochondria were isolated and treated directly with PD, a stronger swelling, deeper transmembrane depolarization, and more apoptosisinducing factor (AIF) release were observed from the mitochondria of R-HepG2 than that of HepG2. These observations suggest that PD is a potent anti-cancer agent that bypasses MDR and elicits apoptosis via mitochondrial injury. 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Polyphyllin D; Apoptosis; Multi-drug resistance; Mitochondria; Total internal reflection fluorescence; R-HepG2
1. Introduction Abbreviations: AIF, apoptosis-inducing factor; AMC, 7-amino-4methylcoumarin; Dox, doxorubicin; Grp, glucose regulatory protein; JC-1, 5,5 0 ,6,6 0 -tetrachloro-1,1 0 ,3,3 0 -tetraethyl-benzimidazolcarbocyanine iodide; MDR, multi-drug resistance; PARP, poly (ADP-ribose) polymerase; PD, polyphyllin D; DWm, mitochondrial transmembrane potential; TIRF, total internal reflection fluorescence. * Corresponding author. Tel.: +86 852 2609 6799; fax: +86 852 2603 5123. E-mail address: [email protected] (S.K. Kong).
Apoptosis is a tightly regulated process that enables a cell to self-degrade in a controlled and organized manner [1]. This universal cellular suicide program is central to various physiological processes and the maintenance of homeostasis in multi-cellular organisms. Apoptosis can be induced by two major pathways, the extrinsic pathway through the binding of death ligands to death receptors and the activation of the intrinsic pathway with
0304-3835/$ - see front matter 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2007.11.005
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the involvement of mitochondria [2]. Dysregulation in apoptosis is always associated with tumorigenesis which is often a result of the over-expression of antiapoptotic proteins [3]. For example, over-expression of Bcl-2, although originally discovered in B cell lymphoma, is commonly found in different types of cancers of both hematological and non-hematological origin [4]. Also, many alterations were found in the extrinsic and intrinsic apoptotic pathway leading to the development of MDR in cancer chemotherapy [5]. With this understanding, releasing the apoptotic brakes represent attractive targets for the development of novel anti-cancer agents to overcome chemotherapy resistance in cancer cells [6]. Since mitochondria are the key players that control and regulate apoptosis, a direct action on mitochondria might therefore be a good strategy to elicit death in cancer cells in which upstream apoptotic signals have been disabled [7]. In this connection, a number of small mitochondrial toxins such as CD437 and lonidamine have been identified [6,8]. Polyphyllin D (PD) is a steroidal saponin (Fig. 1) found in a traditional Chinese medical herb P. polyphylla. The systematic name of polyphyllin D is diosgenyl a-L-rhamnopyranosyl-(1!2)-(b-L-arabinofuranosyl-(1!4)-b-D-glucopyranoside) with a molecular weight of 855.02. In China, the rhizome of P. polyphylla, known as Chong-Lou, is prescribed by herbal practitioners to treat a number of tumors including pancreas, urinary bladder and liver tumor [9]. Our recent findings indicate that PD is a potent anti-cancer agent that can overcome the MDR and elicit programmed cell death in drug-resistant RHepG2 cells [10]. In this study, we tried to elucidate the action mechanism how PD kills the R-HepG2 cells. We showed here that PD was able to induce the activation of caspase-3 without affecting the activity of caspase-8. With the use of TIRFM, PD O
O
OH O O
O
O
HO O
OH HO
Me OH HO HO
O OH
Fig. 1. The chemical structure of polyphyllin D.
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was found to be able to induce mitochondrial fragmentation. When isolated mitochondria were treated directly with PD, PD could depolarize the mitochondrial transmembrane potential (DWm), release AIF and elicit mitochondrial swelling. These results suggest that PD is a potent anti-cancer agent that bypasses the MDR with a direct effect on mitochondria to trigger the apoptotic pathway in RHepG2 cells. 2. Materials and methods 2.1. Materials JC-1 (5,5 0 ,6,6 0 -tetrachloro-1,1 0 ,3,3 0 -tetraethylbenzimidazolcarbocyanine iodide) was purchased from Molecular Probes. Antibodies against pro-caspase-3, glucose regulatory protein (Grp) 75, b-tubulin, lamin B, poly (ADP-ribose) polymerase (PARP), AIF and the secondary antibody with horseradish peroxidase were obtained from Santa Cruz. Valinomycin and z-DEVD-fmk were bought from Calbiochem. Kits for mitochondria isolation were obtained from BioVision. All other reagents were from Sigma. PD and dansyl-PD was synthesized by Prof. Biao Yu as previously mentioned [11–13]. Dansyl-PD can be excited at 360 nm and emits fluorescence at 530 nm. 2.2. Cell culture HepG2 hepatocellular carcinoma cells, obtained from American Type Culture Collection, were cultured in RPMI 1640 medium (Sigma) supplemented with 10% (v/ v) fetal calf serum (FCS) (Gibco) at 37 C, 5% CO2. For the development of Doxorubicin (Dox)-resistant cells, HepG2 cells were cultured in the presence of Dox and survival cells were treated stepwise with a higher concentration of Dox from 0.1 to 100 lM during cell passages. After more than 10 rounds of selection, a clone R-HepG2 with Dox resistance was obtained. To maintain the Doxresistance, R-HepG2 cells were cultured with 1.2 lM Dox during passages. From time to time, the sensitivity of cells to Dox and other anti-cancer agents was analyzed to confirm their resistance to Dox. 2.3. Mitochondria isolation HepG2 or R-HepG2 cells after washing with PBS were re-suspended in cytosol extraction buffer from the Mitochondria/Cytosol Fractionation Kit (BioVision) on ice for 30 min. Subsequently, cell suspension was added to a Dounce homogenizer and homogenized 30 strokes on ice. Cell lysate was then centrifuged at 700g for 10 min to pellet the nucleus and cell debris. The supernatant was collected and centrifuged again at 12,000g for 3 min
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2.5. Detection of mitochondrial transmembrane potential (DWm)
to pellet mitochondria. The mitochondria pellet was then re-suspended in buffer (10 mM HEPES, 10 mM succinate, 215 mM mannitol, 220 mM sucrose, 0.05% (w/v) BSA, 0.8 mM potassium phosphate, 1 mM EGTA, pH 7.4) for further analysis. Mitochondria were kept in ice bath throughout the isolation process.
The DWm was analyzed by JC-1 [14]. JC-1 is capable of selectively entering mitochondria, where it forms monomers and emits green fluorescence when DWm is relatively low. At high DWm, JC-1 aggregates and gives red fluorescence. The ratio between green and red fluorescence provides an estimate of DWm that is independent of the mitochondrial mass. Briefly, isolated mitochondria were incubated with JC-1 (1 lg/ml) for 10 min at room temperature in darkness. After PD treatment, changes in JC-1 signals were analyzed on a flow cytometer (FACSort, Becton Dickinson). Valinomycin (500 nM) was used as a positive control to depolarize the DWm for the calculation of the relative change of DWm.
2.4. Western blot analysis Cells were lysed in 1% (w/v) SDS, 1 mM Na3VO4, 10 mM Tris, 5 mM MgCl2 with protease inhibitors 21 lg/ml aprotinin, 5 lg/ml leupeptin and 1 mM PMSF at pH 7.4 for 1 h at room temperature. Samples were then boiled for 10 min and then centrifuged at 20,000g at 4 C. Subsequently, 25 lg of protein extracts were analyzed by 12% SDS–PAGE and blotted to PVDF (polyvinylidene fluoride) membrane. Membranes were incubated with mouse anti-human caspase-3 monoclonal antibody (for pro-caspase-3) (Santa Cruz) (antibody dilution: 1:1000), rabbit polyclonal anti-PARP antibody (Santa Cruz) (dilution: 1:1000), goat anti-human Grp 75 (Santa Cruz) (dilution: 1:1000), goat anti-human lamin B (Santa Cruz) (dilution: 1:1000), mouse anti-human AIF (Santa Cruz) (dilution: 1:1000), or mouse anti-human b-tubulin monoclonal antibody (Boehringer) (dilution: 1:2000) followed by horseradish peroxidase-conjugated secondary antibody (Santa Cruz) (dilution: 1:2000) and developed with ECL reagent (Amersham).
2.6. Caspase-3 activity determination Activation of caspase-3 activity was determined with fluorescent synthetic substrates [15]. Briefly, cells after treatments were lysed by lysis buffer (1% Igepal CA-630, 150 mM NaCl, 50 mM Tris–HCl, 1 mM EDTA, 5 lg/ml leupeptin, 21 lg/ml aprotinin, pH 7.5). Protein concentration was determined by bicinchoninic acid assay. Lysate containing 50 lg of protein in 90 ll was mixed with a caspase-3 fluorescent synthetic substrate Ac-DEVD (final
a
b
N O
O
S
N H
O
O O NH O O O OH HO
HO
OHMe HO
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OH OH
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c HepG2, PD (μM) Solvent Control Control 2.5
R-HepG2, PD (μM) 5
Solvent Control Control
2.5
5
Pro-Caspase-8 (50/55kDa Doublet) Grp75 (75kDa)
Fig. 2. Polyphyllin D did not activate caspase-8. The chemical structure of dansyl-polyphyllin D (a). HepG2 cells (1.5 · 105/ml) were incubated with dansyl-PD (10 lM) for 30 min at 37 C, 5% CO2 in dark. After washing, cells were observed under light (upper) and fluorescent (lower panel) microscope. The scale bar represents the cell dimension in lm (b). HepG2 (1.5 · 105/ml) and R-HepG2 (2.0 · 105/ml) cells were treated with PD, solvent control (0.01% DMSO) or medium alone as indicated for 24 h at 37 C, 5% CO2. After incubation, proteins were extracted and analyzed with Western blot with anti-caspase-8 and Grp 75 antibody (c).
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concentration 10 lM) conjugated with AMC (7-amino-4methylcoumarin) in the presence or absence of caspase-3 inhibitor z-DEVD-fmk (final concentration 10 lM). After incubation at 37 C for 1 h, fluorescence from the AMC released from the substrate by the action of caspase-3 was determined at 460 nm with an excitation at 390 nm by a fluorescence plate reader (CytoFluo, Millipore).
2.7. Total internal reflection fluorescence microscopy (TIRFM) TIRFM was used to study mitochondrial fragmentation near the plasma membrane [16,17]. TIRFM selectively reveals fluorescent molecules near the membrane of adherent cells through the use of evanescent excitation light that decays exponentially in intensity along the direction perpendicular to the liquid–glass interface to which the cells adhere [16]. Briefly, cells labeled with JC-1 were treated with PD and the spatial and temporal change in the JC-1 fluorescence near the plasma membrane was acquired by the TIRFM (Nikon C1 Plus) with an excitation at 488 nm. Both the green and red fluorescence were acquired to give a brighter image. Data were then processed and analyzed by software EZ-C1 3.5 (Nikon).
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pase-8 when compared to control without PD treatment, suggesting that PD is not a death ligand analogue acting on the extrinsic pathway. 3.1. Polyphyllin D induced caspase-3 activation and PARP degradation in HepG2 and R-HepG2 cells Next, we evaluated the effect of PD on the activation of caspase-3, one of the important effector caspases in the downstream execution apoptotic pathway [18]. As shown in the Western blot analysis (Fig. 3a), incubation of HepG2 and R-HepG2 cells with PD cleaved the zymogen pro-caspase-3 in a dose-dependent manner. Furthermore, the level of caspase-3 specific substrate PARP (116 kDa) was reduced with a concomitant increase in the amount of cleaved PARP product (85 kDa). Similar activity was obtained when caspase-3 specific synthetic fluorescent substrates were incubated with the lysates of cells after PD treatments (Fig. 3b). However, in the presence of z-DEVD-fmk, a caspase-3 inhibitor, such PD-mediated caspase-3 activity was reduced. These observations therefore suggest that PD is an apoptosis-causing agent that elicits cell death with the involvement of caspase-3.
a
HepG2, PD (μM) Control
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5
R-HepG2, PD (μM) 7
Control
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5
7
3. Results and discussion
Pro-caspase-3 (35kDa)
Our previous work shows that PD is able to kill RHepG2 cells through the induction of apoptosis [10]. In an attempt to investigate the molecular basis of the observed cytotoxicity of PD in HepG2 and R-HepG2 cells, we asked whether PD elicits its apoptotic effects through the extrinsic pathway, intrinsic pathway or both. To answer this question, we prepared a fluorescent PD that consisted of a PD molecule and a fluorescent dansyl group which was attached to the polysaccharide moiety of the PD (Fig. 2a). We reasoned that if dansyl-PD is a death ligand analogue, a short incubation of HepG2 cells with dansyl-PD will give fluorescent signals on the cell surface when dansyl-PD binds to the death receptors. As shown in Fig. 2b, the fluorescence of dansyl-PD was not exclusively found on the plasma membrane but distributed throughout the cytoplasm. These results suggest (1) dansyl-PD could cross the plasma membrane and diffuse into the cytosol to interact with the molecules and organelles inside, and (2) dansyl-PD might be unable to bind to the death receptors to trigger the extrinsic pathway. To confirm the latter point, we tried to detect the changes, if any, at the level of the initiator caspase, the caspase-8, of the extrinsic pathway in the PD-treated cells. As shown in Fig. 2c, incubation of HepG2 or R-HepG2 with PD did not alter the level of pro-cas-
PARP (116kDa) PARP fragment (85kDa)
Caspase-3 Activity (% of Control)
b
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-z-DEVD-fmk +z-DEVD-fmk
800 600 400 200 0 Control
12h
24h
Control
12h
24h
Fig. 3. Polyphyllin D activated caspase-3. HepG2 and R-HepG2 (1 · 105/ml) were treated with PD at the concentration as indicated for 24 h at 37 C, 5% CO2. After cell lysis, lysates containing equal amount of proteins per lane were subject to standard SDS–PAGE and Western blot analysis with antibodies for pro-caspase-3 (35 kDa), PARP (116 kDa) and cleaved PARP (85 kDa) (a). Caspase-3 activities were also determined with the cell lysates from cells treated with PD (5 lM) for 12 or 24 h by Ac-DEVD-AMC in the presence or absence of caspase-3 specific inhibitor z-DEVD-fmk (10 lM). Results are mean ± SD for three determinations (b).
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3.2. Polyphyllin D elicited mitochondrial fragmentation, swelling, and aggregation in living cells Our previous work using flow cytometry demonstrates that PD reduces the DWm in a dose-dependent manner in both HepG2 and R-HepG2 cells. In this study, we employed TIRFM to examine the spatial and temporal changes in mitochondria. TIRFM is a technique using evanescent waves to selectively excite fluorescent molecules in a very thin optical layer near the plasma membrane of cells that are grown on glass coverslips. Evanescent waves are created only when an incident light is totally reflected at the cell–glass interface. The evanescent wave decays exponentially from the interface, and thus penetrates to a depth of only 100 nm into the sample and this provides a high signal-to-noise resolution unmatched by any other techniques using living cells [16,17]. To visualize the changes in the mitochondrial morphology before and after PD treatment, mitochondria were labeled with JC-1. In the untreated HepG2 cells, mitochondria were normal and abundant throughout the cytoplasm (Fig. 4, upper panel). Also, the mitochondria appeared tubular and interconnected in a filamentous network. In the R-HepG2 cells, the mitochondria were shorter, less elongated and more rounded (Fig. 4, lower panel). When cells were challenged with PD, the reticular mitochondria of HepG2 cells became fragmented, swollen and aggregated in circular forms. Similar phenomenon was found in R-HepG2 cells. Yet, the effect of PD in RHepG2 cells was faster. For example, 6 min after the
PD treatment, almost all the mitochondria in R-HepG2 cells were rounded up while the same incident in HepG2 cells required 9 min incubation (Fig. 4). For the mitochondrial fragmentation, it is generally believed that it is a result of altered morphological dynamics possibly through an increase in mitochondrial-fission and a decrease in mitochondrial-fusion. Importantly, this process seems to contribute to mitochondrial injury and leads to the release of apoptogenic factors from mitochondria during apoptosis [19–21]. In our case, we previously showed that PD is able to release AIF from the mitochondria to the cytosol in both cell types [10]. Now, our results further suggest that PD is able to provoke mitochondrial fragmentation in HepG2 and R-HepG2 cells and this striking event occurs early during apoptosis. 3.3. Polyphyllin D elicited mitochondrial swelling, loss of DWm, and release of AIF in isolated mitochondria Next, we isolated mitochondria from HepG2 and RHepG2 cells to further examine whether PD directly acts on mitochondria. As can be seen in Fig. 5a, our preparation for isolated mitochondria was relatively pure since mitochondrial marker protein Grp 75 was mainly found in the mitochondrial fraction; neither lamin B (nucleus marker protein) nor b-tubulin (cytosol marker) was detected. Mitochondria were then treated with PD for 3 h and change in mitochondrial size was analyzed by flow cytometry using the forward scatter detector [22]. It is clear in Fig. 5b that PD caused mitochondrial swelling
Fig. 4. Polyphyllin D induced mitochondrial fragmentation. HepG2 and R-HepG2 (1 · 105/ml) were seeded on glass coverslips overnight at 37 C, 5% CO2. After washing, cells were incubated with JC-1 (1 lg/ml) for 30 min. Fluorescence signals were captured by TIRFM at the time as indicated. PD (10 lM) was added at time zero. The scale bar represents the cell dimension in lm and the pseudo-color palette represents the fluorescence intensity. Insets show the enlargement of the selected areas. Images shown here are typical results in three trials with >50 cells.
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2
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3
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Grp 75 (75kDa) β Tubulin (50kDa)
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No. of Mitochondria
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Control 9.4% Treated 35.5%
R-HepG2 Control 14.2% Treated 49.5%
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Valinomycin
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Band density
(100%) (12%) (23%) (31%) (100%) (39%) (55%) (82%) (% of total release)
Fig. 5. Polyphyllin D induced injury in isolated mitochondria. Mitochondria from HepG2 cells and R-HepG2 were isolated with mitochondrial isolation kits according to the supplier’s instructions. Cell fractions of the same amount of protein were analyzed with Western blot with antibodies targeting different markers. Lanes 1 and 2, mitochondrial fractions from two separate preparations; lane 3, whole cell lysate (a). Mitochondria isolated from both HepG2 and R-HepG2 cells were loaded with JC-1 (1 lg/ml) at 37 C for 10 min and then treated with PD at 5 lM for 3 h at 37 C. Subsequently, the isolated mitochondria were analyzed by flow cytometry for forward scatter signals (b), fluorescence at 530 nm and beyond 650 nm with an excitation at 488 nm. For the positive controls, isolated mitochondria with JC-1 were treated with valinomycin (500 nM) for 30 min (c). Mitochondrial proteins AIF released from the isolated mitochondria to the buffer after PD treatments were examined by Western blot analysis. AIF in total lysates (T) were also prepared from the isolated mitochondria with digitonin (8 lg/ml) for comparison (d). Figures below the protein bands are the relative density measured by software ImageJ and % of total release of each protein band were normalized by corresponding control. Data shown here are typical results from at least two trials.
and the effect was again stronger in the mitochondria isolated from R-HepG2 (from 14.2% (control) to 49.5% (PDtreated)) than that in HepG2 (from 9.4% (control) to 35.5% (PD-treated)). Also, PD elicited a dose-dependent
disruption of DWm in the isolated mitochondria as measured by JC-1, which was validated by valinomycin, a K+ ionophore, as a positive control (Fig. 5c). Again, the net change in the DWm depolarization in isolated mito-
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chondria was stronger in R-HepG2 cells after PD treatment. Moreover, AIF was released from the isolated mitochondria after incubation with PD in a dose-dependent manner (Fig. 5d). Similar to our pervious observations [10], more AIF was released from the R-HepG2 mitochondria by PD as evidenced by the band density and percentage of total release. As DWm depolarization and release of AIF and cytochrome c have been thought to represent a point of no return in the apoptotic process [23], our results therefore clearly indicate that PD is an agent that triggers apoptosis in HepG2 and R-HepG2 cells through mitochondrial injury. Also, our recent in vivo study shows that administration of PD (2.73 mg/kg body weight) through intravenous injection for 10 days in nude mice bearing MCF-7 cells effectively reduced tumor growth by 50% in terms of tumor weight and size, and gave no significant hepatic and cardiac toxicity [24]. These observations suggest that PD is a promising anti-cancer drug candidate without severe toxicity in animals. In summary, we have demonstrated here for the first time using TIRFM that PD is able to induce mitochondrial fragmentation. Also, PD is able to kill more cancer cells with MDR and act on mitochondria directly to release apoptogenic protein AIF to the cytosol. Taken together, PD is a novel and safe anticancer agent which can bypass MDR and elicit apoptosis through mitochondrial injury.
Acknowledgement This study was supported by a direct Grant and a Scheme C support (RAC/2006/136-7) from CUHK and grants from Shanghai-Hong Kong Anson Research Foundation and Shun Hing Institute of Advanced Engineering. References [1] D.R. Green, G. Kroemer, The pathophysiology of mitochondrial cell death, Science 305 (2004) 626–629. [2] M.D. Jacobson, M. Weil, M.C. Raff, Programmed cell death in animal development, Cell 88 (1997) 34–54. [3] R.W. Johnstone, A.A. Ruefli, S.W. Lowe, Apoptosis: a link between cancer genetics and chemotherapy, Cell 108 (2002) 153–264. [4] V. Kirkin, S. Joos, M. Zornig, The role of Bcl-2 family members in tumorigenesis, Biochim. Biophys. Acta 1644 (2004) 229–249. [5] A.A. Stavrovskaya, Cellular mechanisms of multidrug resistance of tumor cells, Biochemistry (Mosc.) 65 (2000) 95–106. [6] J.S. Armstrong, Mitochondria: a target for cancer therapy, Br. J. Pharmacol. 147 (2006) 239–248. [7] K.M. Debatin, D. Poncet, G. Kroemer, Chemotherapy: targeting the mitochondrial cell death pathway, Oncogene 21 (2002) 8786–8803.
[8] Y.C. Li, K.P. Fung, T.T. Kwok, C.Y. Lee, Y.K. Suen, S.K. Kong, Mitochondrial targeting drug lonidamine triggered apoptosis in doxorubicin-resistant HepG2 cells, Life Sci. 71 (2002) 2240–2729. [9] Pharmacopoeia Commission of the Ministry of Public Health, The Pharmacopoeia of People’s Republic of China, China, 1990. [10] J.Y. Cheung, R.C. Ong, Y.K. Suen, V. Ooi, H.N. Wong, T.C. Mak, K.P. Fung KP, B. Yu, S.K. Kong, Polyphyllin D is a potent apoptosis inducer in drug-resistant HepG2 cells, Cancer Lett. 217 (2005) 203–211. [11] S. Deng, B. Yu, Y. Hui, H. Yu, X. Han, Synthesis of three diosgenyl saponins: dioscin, polyphyllin D, and balanitin 7, Carbohydr. Res. 317 (1999) 53–62. [12] B. Li, B. Yu, Y. Hui, M. Li, X. Han, K.P. Fung, An improved synthesis of the saponin, polyphyllin D, Carbohydr. Res. 331 (2001) 1–7. [13] Z. Yang, E.L. Wong, T.Y. Shum, C.M. Che, Y.Y. Hui, Fluorophore-appended steroidal saponin (dioscin and polyphyllin D) derivatives, Org. Lett. 7 (2005) 669–672. [14] A. Cossarizza, M. Baccarani-Contri, G. Kalashnikova, C. Franceschi, A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5 0 ,6,6 0 -tetrachloro-1,1 0 ,3,3 0 -tetraethylbenzimidazolcarbocyanine iodide (JC-1), Biochem. Biophys. Res. Commun. 197 (1993) 40–45. [15] C. Kohler, S. Orrenius, B. Zhivotovsky, Evaluation of caspase activity in apoptotic cells, J. Immunol. Methods 265 (2002) 97–110. [16] D. Axelrod, Total internal reflection fluorescence microscopy in cell biology, Methods Enzymol. 361 (2003) 1–33. [17] H. Schneckenburger, Total internal reflection fluorescence microscopy: technical innovations and novel applications, Curr. Opin. Biotechnol. 16 (2005) 13–18. [18] G.S. Salvesen, J.M. Abrams, Caspase activation – stepping on the gas or releasing the brakes? Lessons from humans and flies, Oncogene 23 (2004) 2774–2784. [19] R.J. Youle, M. Karbowski, Mitochondrial fission in apoptosis, Nat. Rev. Mol. Cell Biol. 6 (2005) 657–663. [20] R. Jagasia, P. Grote, B. Westermann, B. Conradt, DRP-1mediated mitochondrial fragmentation during EGL-1induced cell death in C. elegans, Nature 433 (2005) 754– 760. [21] C. Brooks, Q. Wei, L. Feng, G. Dong, Y. Tao, L. Mei, Z.J. Xie, Z. Dong, Bak regulates mitochondrial morphology and pathology during apoptosis by interacting with mitofusins, Proc. Natl. Acad. Sci. USA 104 (2007) 11649–11654. [22] H. Lecoeur, A. Langonne, L. Baux, D. Rebouillat, P. Rustin, M.C. Prevost, C. Brenner, L. Edelman, E. Jacotot, Real-time flow cytometry analysis of permeability transition in isolated mitochondria, Exp. Cell Res. 294 (2004) 106–117. [23] S.J. Riedl, G.S. Salvesen, The apoptosome: signalling platform of cell death, Nat. Rev. Mol .Cell Biol. 8 (2007) 405–413. [24] M.S. Lee, J.Y.W. Chan, S.K. Kong SK, B. Yu, V.E.C. Ooi, H.N.C. Wong, T.C.W. Mak, K.P. Fung, Effects of polyphyllin D, a steroidal saponin in Paris polyphylla, in growth inhibition of human breast cancer cells and in xenograft, Cancer Biol. Ther. 4 (2005) 1248–1254.
Cancer Letters 261 (2008) 158–164 www.elsevier.com/locate/canlet
Polyphyllin D induces mitochondrial fragmentation and acts directly on the mitochondria to induce apoptosis in drug-resistant HepG2 cells Rose C.Y. Ong a, Jin Lei b, Rebecca K.Y. Lee a, Jenny Y.N. Cheung a, K.P. Fung a,c, Chinlon Lin b, H.P. Ho b, Biao Yu d, Ming Li d, S.K. Kong b
a,*
a Department of Biochemistry, The Chinese University of Hong Kong, Shatin, Hong Kong, China Biophotonic Laboratory, Department of Electronic Engineering, The Chinese University of Hong Kong, Shatin, Hong Kong, China c Institute of Chinese Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, China d State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China
Received 25 September 2007; received in revised form 7 November 2007; accepted 7 November 2007
Abstract We previously showed that polyphyllin D (PD) produced a stronger apoptotic effect in R-HepG2 with multi-drug resistance (MDR) than that in its parent HepG2 cells without MDR. In this study, PD was found to elicit mitochondrial fragmentation in live cells by using total internal reflection fluorescence microscopy (TIRFM). When mitochondria were isolated and treated directly with PD, a stronger swelling, deeper transmembrane depolarization, and more apoptosisinducing factor (AIF) release were observed from the mitochondria of R-HepG2 than that of HepG2. These observations suggest that PD is a potent anti-cancer agent that bypasses MDR and elicits apoptosis via mitochondrial injury. 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Polyphyllin D; Apoptosis; Multi-drug resistance; Mitochondria; Total internal reflection fluorescence; R-HepG2
1. Introduction Abbreviations: AIF, apoptosis-inducing factor; AMC, 7-amino-4methylcoumarin; Dox, doxorubicin; Grp, glucose regulatory protein; JC-1, 5,5 0 ,6,6 0 -tetrachloro-1,1 0 ,3,3 0 -tetraethyl-benzimidazolcarbocyanine iodide; MDR, multi-drug resistance; PARP, poly (ADP-ribose) polymerase; PD, polyphyllin D; DWm, mitochondrial transmembrane potential; TIRF, total internal reflection fluorescence. * Corresponding author. Tel.: +86 852 2609 6799; fax: +86 852 2603 5123. E-mail address: [email protected] (S.K. Kong).
Apoptosis is a tightly regulated process that enables a cell to self-degrade in a controlled and organized manner [1]. This universal cellular suicide program is central to various physiological processes and the maintenance of homeostasis in multi-cellular organisms. Apoptosis can be induced by two major pathways, the extrinsic pathway through the binding of death ligands to death receptors and the activation of the intrinsic pathway with
0304-3835/$ - see front matter 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2007.11.005
R.C.Y. Ong et al. / Cancer Letters 261 (2008) 158–164
the involvement of mitochondria [2]. Dysregulation in apoptosis is always associated with tumorigenesis which is often a result of the over-expression of antiapoptotic proteins [3]. For example, over-expression of Bcl-2, although originally discovered in B cell lymphoma, is commonly found in different types of cancers of both hematological and non-hematological origin [4]. Also, many alterations were found in the extrinsic and intrinsic apoptotic pathway leading to the development of MDR in cancer chemotherapy [5]. With this understanding, releasing the apoptotic brakes represent attractive targets for the development of novel anti-cancer agents to overcome chemotherapy resistance in cancer cells [6]. Since mitochondria are the key players that control and regulate apoptosis, a direct action on mitochondria might therefore be a good strategy to elicit death in cancer cells in which upstream apoptotic signals have been disabled [7]. In this connection, a number of small mitochondrial toxins such as CD437 and lonidamine have been identified [6,8]. Polyphyllin D (PD) is a steroidal saponin (Fig. 1) found in a traditional Chinese medical herb P. polyphylla. The systematic name of polyphyllin D is diosgenyl a-L-rhamnopyranosyl-(1!2)-(b-L-arabinofuranosyl-(1!4)-b-D-glucopyranoside) with a molecular weight of 855.02. In China, the rhizome of P. polyphylla, known as Chong-Lou, is prescribed by herbal practitioners to treat a number of tumors including pancreas, urinary bladder and liver tumor [9]. Our recent findings indicate that PD is a potent anti-cancer agent that can overcome the MDR and elicit programmed cell death in drug-resistant RHepG2 cells [10]. In this study, we tried to elucidate the action mechanism how PD kills the R-HepG2 cells. We showed here that PD was able to induce the activation of caspase-3 without affecting the activity of caspase-8. With the use of TIRFM, PD O
O
OH O O
O
O
HO O
OH HO
Me OH HO HO
O OH
Fig. 1. The chemical structure of polyphyllin D.
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was found to be able to induce mitochondrial fragmentation. When isolated mitochondria were treated directly with PD, PD could depolarize the mitochondrial transmembrane potential (DWm), release AIF and elicit mitochondrial swelling. These results suggest that PD is a potent anti-cancer agent that bypasses the MDR with a direct effect on mitochondria to trigger the apoptotic pathway in RHepG2 cells. 2. Materials and methods 2.1. Materials JC-1 (5,5 0 ,6,6 0 -tetrachloro-1,1 0 ,3,3 0 -tetraethylbenzimidazolcarbocyanine iodide) was purchased from Molecular Probes. Antibodies against pro-caspase-3, glucose regulatory protein (Grp) 75, b-tubulin, lamin B, poly (ADP-ribose) polymerase (PARP), AIF and the secondary antibody with horseradish peroxidase were obtained from Santa Cruz. Valinomycin and z-DEVD-fmk were bought from Calbiochem. Kits for mitochondria isolation were obtained from BioVision. All other reagents were from Sigma. PD and dansyl-PD was synthesized by Prof. Biao Yu as previously mentioned [11–13]. Dansyl-PD can be excited at 360 nm and emits fluorescence at 530 nm. 2.2. Cell culture HepG2 hepatocellular carcinoma cells, obtained from American Type Culture Collection, were cultured in RPMI 1640 medium (Sigma) supplemented with 10% (v/ v) fetal calf serum (FCS) (Gibco) at 37 C, 5% CO2. For the development of Doxorubicin (Dox)-resistant cells, HepG2 cells were cultured in the presence of Dox and survival cells were treated stepwise with a higher concentration of Dox from 0.1 to 100 lM during cell passages. After more than 10 rounds of selection, a clone R-HepG2 with Dox resistance was obtained. To maintain the Doxresistance, R-HepG2 cells were cultured with 1.2 lM Dox during passages. From time to time, the sensitivity of cells to Dox and other anti-cancer agents was analyzed to confirm their resistance to Dox. 2.3. Mitochondria isolation HepG2 or R-HepG2 cells after washing with PBS were re-suspended in cytosol extraction buffer from the Mitochondria/Cytosol Fractionation Kit (BioVision) on ice for 30 min. Subsequently, cell suspension was added to a Dounce homogenizer and homogenized 30 strokes on ice. Cell lysate was then centrifuged at 700g for 10 min to pellet the nucleus and cell debris. The supernatant was collected and centrifuged again at 12,000g for 3 min
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2.5. Detection of mitochondrial transmembrane potential (DWm)
to pellet mitochondria. The mitochondria pellet was then re-suspended in buffer (10 mM HEPES, 10 mM succinate, 215 mM mannitol, 220 mM sucrose, 0.05% (w/v) BSA, 0.8 mM potassium phosphate, 1 mM EGTA, pH 7.4) for further analysis. Mitochondria were kept in ice bath throughout the isolation process.
The DWm was analyzed by JC-1 [14]. JC-1 is capable of selectively entering mitochondria, where it forms monomers and emits green fluorescence when DWm is relatively low. At high DWm, JC-1 aggregates and gives red fluorescence. The ratio between green and red fluorescence provides an estimate of DWm that is independent of the mitochondrial mass. Briefly, isolated mitochondria were incubated with JC-1 (1 lg/ml) for 10 min at room temperature in darkness. After PD treatment, changes in JC-1 signals were analyzed on a flow cytometer (FACSort, Becton Dickinson). Valinomycin (500 nM) was used as a positive control to depolarize the DWm for the calculation of the relative change of DWm.
2.4. Western blot analysis Cells were lysed in 1% (w/v) SDS, 1 mM Na3VO4, 10 mM Tris, 5 mM MgCl2 with protease inhibitors 21 lg/ml aprotinin, 5 lg/ml leupeptin and 1 mM PMSF at pH 7.4 for 1 h at room temperature. Samples were then boiled for 10 min and then centrifuged at 20,000g at 4 C. Subsequently, 25 lg of protein extracts were analyzed by 12% SDS–PAGE and blotted to PVDF (polyvinylidene fluoride) membrane. Membranes were incubated with mouse anti-human caspase-3 monoclonal antibody (for pro-caspase-3) (Santa Cruz) (antibody dilution: 1:1000), rabbit polyclonal anti-PARP antibody (Santa Cruz) (dilution: 1:1000), goat anti-human Grp 75 (Santa Cruz) (dilution: 1:1000), goat anti-human lamin B (Santa Cruz) (dilution: 1:1000), mouse anti-human AIF (Santa Cruz) (dilution: 1:1000), or mouse anti-human b-tubulin monoclonal antibody (Boehringer) (dilution: 1:2000) followed by horseradish peroxidase-conjugated secondary antibody (Santa Cruz) (dilution: 1:2000) and developed with ECL reagent (Amersham).
2.6. Caspase-3 activity determination Activation of caspase-3 activity was determined with fluorescent synthetic substrates [15]. Briefly, cells after treatments were lysed by lysis buffer (1% Igepal CA-630, 150 mM NaCl, 50 mM Tris–HCl, 1 mM EDTA, 5 lg/ml leupeptin, 21 lg/ml aprotinin, pH 7.5). Protein concentration was determined by bicinchoninic acid assay. Lysate containing 50 lg of protein in 90 ll was mixed with a caspase-3 fluorescent synthetic substrate Ac-DEVD (final
a
b
N O
O
S
N H
O
O O NH O O O OH HO
HO
OHMe HO
O
O O
OH OH
30
c HepG2, PD (μM) Solvent Control Control 2.5
R-HepG2, PD (μM) 5
Solvent Control Control
2.5
5
Pro-Caspase-8 (50/55kDa Doublet) Grp75 (75kDa)
Fig. 2. Polyphyllin D did not activate caspase-8. The chemical structure of dansyl-polyphyllin D (a). HepG2 cells (1.5 · 105/ml) were incubated with dansyl-PD (10 lM) for 30 min at 37 C, 5% CO2 in dark. After washing, cells were observed under light (upper) and fluorescent (lower panel) microscope. The scale bar represents the cell dimension in lm (b). HepG2 (1.5 · 105/ml) and R-HepG2 (2.0 · 105/ml) cells were treated with PD, solvent control (0.01% DMSO) or medium alone as indicated for 24 h at 37 C, 5% CO2. After incubation, proteins were extracted and analyzed with Western blot with anti-caspase-8 and Grp 75 antibody (c).
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concentration 10 lM) conjugated with AMC (7-amino-4methylcoumarin) in the presence or absence of caspase-3 inhibitor z-DEVD-fmk (final concentration 10 lM). After incubation at 37 C for 1 h, fluorescence from the AMC released from the substrate by the action of caspase-3 was determined at 460 nm with an excitation at 390 nm by a fluorescence plate reader (CytoFluo, Millipore).
2.7. Total internal reflection fluorescence microscopy (TIRFM) TIRFM was used to study mitochondrial fragmentation near the plasma membrane [16,17]. TIRFM selectively reveals fluorescent molecules near the membrane of adherent cells through the use of evanescent excitation light that decays exponentially in intensity along the direction perpendicular to the liquid–glass interface to which the cells adhere [16]. Briefly, cells labeled with JC-1 were treated with PD and the spatial and temporal change in the JC-1 fluorescence near the plasma membrane was acquired by the TIRFM (Nikon C1 Plus) with an excitation at 488 nm. Both the green and red fluorescence were acquired to give a brighter image. Data were then processed and analyzed by software EZ-C1 3.5 (Nikon).
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pase-8 when compared to control without PD treatment, suggesting that PD is not a death ligand analogue acting on the extrinsic pathway. 3.1. Polyphyllin D induced caspase-3 activation and PARP degradation in HepG2 and R-HepG2 cells Next, we evaluated the effect of PD on the activation of caspase-3, one of the important effector caspases in the downstream execution apoptotic pathway [18]. As shown in the Western blot analysis (Fig. 3a), incubation of HepG2 and R-HepG2 cells with PD cleaved the zymogen pro-caspase-3 in a dose-dependent manner. Furthermore, the level of caspase-3 specific substrate PARP (116 kDa) was reduced with a concomitant increase in the amount of cleaved PARP product (85 kDa). Similar activity was obtained when caspase-3 specific synthetic fluorescent substrates were incubated with the lysates of cells after PD treatments (Fig. 3b). However, in the presence of z-DEVD-fmk, a caspase-3 inhibitor, such PD-mediated caspase-3 activity was reduced. These observations therefore suggest that PD is an apoptosis-causing agent that elicits cell death with the involvement of caspase-3.
a
HepG2, PD (μM) Control
3
5
R-HepG2, PD (μM) 7
Control
3
5
7
3. Results and discussion
Pro-caspase-3 (35kDa)
Our previous work shows that PD is able to kill RHepG2 cells through the induction of apoptosis [10]. In an attempt to investigate the molecular basis of the observed cytotoxicity of PD in HepG2 and R-HepG2 cells, we asked whether PD elicits its apoptotic effects through the extrinsic pathway, intrinsic pathway or both. To answer this question, we prepared a fluorescent PD that consisted of a PD molecule and a fluorescent dansyl group which was attached to the polysaccharide moiety of the PD (Fig. 2a). We reasoned that if dansyl-PD is a death ligand analogue, a short incubation of HepG2 cells with dansyl-PD will give fluorescent signals on the cell surface when dansyl-PD binds to the death receptors. As shown in Fig. 2b, the fluorescence of dansyl-PD was not exclusively found on the plasma membrane but distributed throughout the cytoplasm. These results suggest (1) dansyl-PD could cross the plasma membrane and diffuse into the cytosol to interact with the molecules and organelles inside, and (2) dansyl-PD might be unable to bind to the death receptors to trigger the extrinsic pathway. To confirm the latter point, we tried to detect the changes, if any, at the level of the initiator caspase, the caspase-8, of the extrinsic pathway in the PD-treated cells. As shown in Fig. 2c, incubation of HepG2 or R-HepG2 with PD did not alter the level of pro-cas-
PARP (116kDa) PARP fragment (85kDa)
Caspase-3 Activity (% of Control)
b
HepG2 1000
R-HepG2 -z-DEVD-fmk +z-DEVD-fmk
-z-DEVD-fmk +z-DEVD-fmk
800 600 400 200 0 Control
12h
24h
Control
12h
24h
Fig. 3. Polyphyllin D activated caspase-3. HepG2 and R-HepG2 (1 · 105/ml) were treated with PD at the concentration as indicated for 24 h at 37 C, 5% CO2. After cell lysis, lysates containing equal amount of proteins per lane were subject to standard SDS–PAGE and Western blot analysis with antibodies for pro-caspase-3 (35 kDa), PARP (116 kDa) and cleaved PARP (85 kDa) (a). Caspase-3 activities were also determined with the cell lysates from cells treated with PD (5 lM) for 12 or 24 h by Ac-DEVD-AMC in the presence or absence of caspase-3 specific inhibitor z-DEVD-fmk (10 lM). Results are mean ± SD for three determinations (b).
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3.2. Polyphyllin D elicited mitochondrial fragmentation, swelling, and aggregation in living cells Our previous work using flow cytometry demonstrates that PD reduces the DWm in a dose-dependent manner in both HepG2 and R-HepG2 cells. In this study, we employed TIRFM to examine the spatial and temporal changes in mitochondria. TIRFM is a technique using evanescent waves to selectively excite fluorescent molecules in a very thin optical layer near the plasma membrane of cells that are grown on glass coverslips. Evanescent waves are created only when an incident light is totally reflected at the cell–glass interface. The evanescent wave decays exponentially from the interface, and thus penetrates to a depth of only 100 nm into the sample and this provides a high signal-to-noise resolution unmatched by any other techniques using living cells [16,17]. To visualize the changes in the mitochondrial morphology before and after PD treatment, mitochondria were labeled with JC-1. In the untreated HepG2 cells, mitochondria were normal and abundant throughout the cytoplasm (Fig. 4, upper panel). Also, the mitochondria appeared tubular and interconnected in a filamentous network. In the R-HepG2 cells, the mitochondria were shorter, less elongated and more rounded (Fig. 4, lower panel). When cells were challenged with PD, the reticular mitochondria of HepG2 cells became fragmented, swollen and aggregated in circular forms. Similar phenomenon was found in R-HepG2 cells. Yet, the effect of PD in RHepG2 cells was faster. For example, 6 min after the
PD treatment, almost all the mitochondria in R-HepG2 cells were rounded up while the same incident in HepG2 cells required 9 min incubation (Fig. 4). For the mitochondrial fragmentation, it is generally believed that it is a result of altered morphological dynamics possibly through an increase in mitochondrial-fission and a decrease in mitochondrial-fusion. Importantly, this process seems to contribute to mitochondrial injury and leads to the release of apoptogenic factors from mitochondria during apoptosis [19–21]. In our case, we previously showed that PD is able to release AIF from the mitochondria to the cytosol in both cell types [10]. Now, our results further suggest that PD is able to provoke mitochondrial fragmentation in HepG2 and R-HepG2 cells and this striking event occurs early during apoptosis. 3.3. Polyphyllin D elicited mitochondrial swelling, loss of DWm, and release of AIF in isolated mitochondria Next, we isolated mitochondria from HepG2 and RHepG2 cells to further examine whether PD directly acts on mitochondria. As can be seen in Fig. 5a, our preparation for isolated mitochondria was relatively pure since mitochondrial marker protein Grp 75 was mainly found in the mitochondrial fraction; neither lamin B (nucleus marker protein) nor b-tubulin (cytosol marker) was detected. Mitochondria were then treated with PD for 3 h and change in mitochondrial size was analyzed by flow cytometry using the forward scatter detector [22]. It is clear in Fig. 5b that PD caused mitochondrial swelling
Fig. 4. Polyphyllin D induced mitochondrial fragmentation. HepG2 and R-HepG2 (1 · 105/ml) were seeded on glass coverslips overnight at 37 C, 5% CO2. After washing, cells were incubated with JC-1 (1 lg/ml) for 30 min. Fluorescence signals were captured by TIRFM at the time as indicated. PD (10 lM) was added at time zero. The scale bar represents the cell dimension in lm and the pseudo-color palette represents the fluorescence intensity. Insets show the enlargement of the selected areas. Images shown here are typical results in three trials with >50 cells.
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1
2
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3
a
Grp 75 (75kDa) β Tubulin (50kDa)
b
No. of Mitochondria
Lamin B (70kDa)
HepG2
Control 9.4% Treated 35.5%
R-HepG2 Control 14.2% Treated 49.5%
Size of Mitochondria (FSC) PD (μM)
c
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20
Valinomycin
HepG2
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36.6%
50.0%
25.5%
42.2%
57.8%
54.1%
62.4%
74.4%
55.2%
69.8%
R-HepG2
JC-1 Red Fluorescence
Control
JC-1 Green Fluorescence
d
HepG2 T
0
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R-HepG2 10
T
0
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PD (μM) AIF (57 kDa)
100
12
23
31
83
32
46
68
Band density
(100%) (12%) (23%) (31%) (100%) (39%) (55%) (82%) (% of total release)
Fig. 5. Polyphyllin D induced injury in isolated mitochondria. Mitochondria from HepG2 cells and R-HepG2 were isolated with mitochondrial isolation kits according to the supplier’s instructions. Cell fractions of the same amount of protein were analyzed with Western blot with antibodies targeting different markers. Lanes 1 and 2, mitochondrial fractions from two separate preparations; lane 3, whole cell lysate (a). Mitochondria isolated from both HepG2 and R-HepG2 cells were loaded with JC-1 (1 lg/ml) at 37 C for 10 min and then treated with PD at 5 lM for 3 h at 37 C. Subsequently, the isolated mitochondria were analyzed by flow cytometry for forward scatter signals (b), fluorescence at 530 nm and beyond 650 nm with an excitation at 488 nm. For the positive controls, isolated mitochondria with JC-1 were treated with valinomycin (500 nM) for 30 min (c). Mitochondrial proteins AIF released from the isolated mitochondria to the buffer after PD treatments were examined by Western blot analysis. AIF in total lysates (T) were also prepared from the isolated mitochondria with digitonin (8 lg/ml) for comparison (d). Figures below the protein bands are the relative density measured by software ImageJ and % of total release of each protein band were normalized by corresponding control. Data shown here are typical results from at least two trials.
and the effect was again stronger in the mitochondria isolated from R-HepG2 (from 14.2% (control) to 49.5% (PDtreated)) than that in HepG2 (from 9.4% (control) to 35.5% (PD-treated)). Also, PD elicited a dose-dependent
disruption of DWm in the isolated mitochondria as measured by JC-1, which was validated by valinomycin, a K+ ionophore, as a positive control (Fig. 5c). Again, the net change in the DWm depolarization in isolated mito-
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chondria was stronger in R-HepG2 cells after PD treatment. Moreover, AIF was released from the isolated mitochondria after incubation with PD in a dose-dependent manner (Fig. 5d). Similar to our pervious observations [10], more AIF was released from the R-HepG2 mitochondria by PD as evidenced by the band density and percentage of total release. As DWm depolarization and release of AIF and cytochrome c have been thought to represent a point of no return in the apoptotic process [23], our results therefore clearly indicate that PD is an agent that triggers apoptosis in HepG2 and R-HepG2 cells through mitochondrial injury. Also, our recent in vivo study shows that administration of PD (2.73 mg/kg body weight) through intravenous injection for 10 days in nude mice bearing MCF-7 cells effectively reduced tumor growth by 50% in terms of tumor weight and size, and gave no significant hepatic and cardiac toxicity [24]. These observations suggest that PD is a promising anti-cancer drug candidate without severe toxicity in animals. In summary, we have demonstrated here for the first time using TIRFM that PD is able to induce mitochondrial fragmentation. Also, PD is able to kill more cancer cells with MDR and act on mitochondria directly to release apoptogenic protein AIF to the cytosol. Taken together, PD is a novel and safe anticancer agent which can bypass MDR and elicit apoptosis through mitochondrial injury.
Acknowledgement This study was supported by a direct Grant and a Scheme C support (RAC/2006/136-7) from CUHK and grants from Shanghai-Hong Kong Anson Research Foundation and Shun Hing Institute of Advanced Engineering. References [1] D.R. Green, G. Kroemer, The pathophysiology of mitochondrial cell death, Science 305 (2004) 626–629. [2] M.D. Jacobson, M. Weil, M.C. Raff, Programmed cell death in animal development, Cell 88 (1997) 34–54. [3] R.W. Johnstone, A.A. Ruefli, S.W. Lowe, Apoptosis: a link between cancer genetics and chemotherapy, Cell 108 (2002) 153–264. [4] V. Kirkin, S. Joos, M. Zornig, The role of Bcl-2 family members in tumorigenesis, Biochim. Biophys. Acta 1644 (2004) 229–249. [5] A.A. Stavrovskaya, Cellular mechanisms of multidrug resistance of tumor cells, Biochemistry (Mosc.) 65 (2000) 95–106. [6] J.S. Armstrong, Mitochondria: a target for cancer therapy, Br. J. Pharmacol. 147 (2006) 239–248. [7] K.M. Debatin, D. Poncet, G. Kroemer, Chemotherapy: targeting the mitochondrial cell death pathway, Oncogene 21 (2002) 8786–8803.
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