Technical Note pubs.acs.org/ac
Identification of Mitochondria-Targeting Anticancer Compounds by an in Vitro Strategy Xiang Zhang,† Shuyue Zhang,† Shaobin Zhu,† Sha Chen,† Jinyan Han,† Kaimin Gao,† Jin-zhang Zeng,‡ and Xiaomei Yan*,† †
The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, P. R. China ‡ School of Pharmaceutical Sciences and Institute for Biomedical Research, Xiamen University, Xiamen, Fujian 361005, P. R. China S Supporting Information *
ABSTRACT: Mitochondria play a pivotal role in determining the point-of-no-return of the apoptotic process. Therefore, anticancer drugs that directly target mitochondria hold great potential to evade resistance mechanisms that have developed toward conventional chemotherapeutics. In this study, we report the development of an in vitro strategy to quickly identify the therapeutic agents that induce apoptosis via directly affecting mitochondria. This result is achieved by treating isolated mitochondria with potential anticancer compounds, followed by simultaneously measuring the side scatter and mitochondrial membrane potential (Δψm) fluorescence of individual mitochondria using a laboratory-built high-sensitivity flow cytometer. The feasibility of this method was tested with eight widely used anticarcinogens. Dose-dependent Δψm losses were observed for paclitaxel, antimycin A, betulinic acid, curcumin, ABT-737, and triptolide, but not for cisplatin or actinomycin D, which agrees well with their mechanisms of apoptosis induction reported in the literature. The as-developed method offers an effective approach to identify mitochondria-targeting anticancer compounds.
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the new conjugate is able to overcome multidrug resistance mechanisms, such as P-glycoprotein-mediated efflux, which strongly proves that drugs directly acting on mitochondria represent an attractive strategy for cancer therapy. Many of the known mitochondria-targeting anticancer drugs are derived from natural compounds and have been identified by serendipity rather than by systematic screening.7 In this report, by employing Δψm loss as an early apoptotic marker, we attempt to develop a systematic approach to specifically identify mitochondria-targeting anticancer compounds. The following describes the experimental design used in this study. The anticancer agent to be examined was used to treat isolated mitochondria. After fluorescent labeling with DiOC6(3), a lipophilic cationic probe specific for assessing Δψm,10,11 the mitochondria were passed individually through the tightly focused laser beam of a laboratory-built high sensitivity flow cytometer (HSFCM). The bursts of photons that were detected on both the side scatter (SS) and the fluorescence (FL) channels were recorded simultaneously. The SS signal delineates the mitochondrial refractive index and internal complexity,12,13 wherease the FL intensity reflects the extent of Δψm via DiOC6(3) accumulation in the mitochondrial matrix space in inverse proportion to Δψm, according to the
ost classical anticancer drugs induce apoptosis by activating signaling pathways that lie upstream of mitochondria. Unfortunately, cancer cells have developed numerous strategies to evade apoptosis by blocking these upstream events, such as reduced expression of death receptors, aberrant expression of decoy receptors, overexpression of antiapoptotic Bcl-2 family proteins, underexpression or inactivation of proapoptotic proteins (such as Bax and BH3only proteins), and inhibition of p53 translocation, which make the established therapeutic regimens lose efficacy.1−3 It has been well recognized that both extrinsic and intrinsic signaling pathways converge on mitochondria, and mitochondrial membrane permeabilization (MMP) is the decisive point of the irreversible process of apoptosis.4,5 With the onset of MMP, mitochondrial membrane potential (Δψm) is lost, and cytochrome c and other pro-apoptotic proteins are released from the mitochondrial intermembrane space into the cytosol where a caspase cascade is initiated and the cell is disassembled into apoptotic bodies. Therefore, mitochondria have been considered one of the most promising targets for anticancer drug development because compounds that can directly simulate MMP hold great potential to circumvent the resistance mechanisms that have developed toward conventional chemotherapeutics.6−8 Thus, it is interesting to note the work of Kelley et al., who engineered the first mitochondrially targeted version of doxorubicin by attaching it to a mitochondriapenetrating peptide (MPP).9 In contrast to doxorubicin itself, © 2014 American Chemical Society
Received: March 12, 2014 Accepted: May 13, 2014 Published: May 13, 2014 5232
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Figure 1. Flow cytometric analysis of the unstained and 500 nM DiOC6(3)-stained mitochondria before and after CCCP treatment on the laboratory-built HSFCM: (A-1 and A-2) Representative side scatter (SS) and green fluorescence (FL) burst traces of unstained mitochondria. (B-1 and B-2) Representative side scatter and green fluorescence burst traces of DiOC6(3)-stained mitochondria. (C-1 and C-2) Representative side scatter and green fluorescence burst traces of DiOC6(3)-stained mitochondria treated with 200 μM CCCP. (A-3, B-3, and C-3) Bivariate dot-plots of green fluorescence burst area versus side scatter burst area derived from 60 s of data for the unstained and stained mitochondria without or with CCCP treatment, respectively. The signal saturation voltage is 10 V for both detection channels.
Nernst equation.11 Therefore, depolarized (less negative) mitochondria will have lower cationic dye concentrations and lower fluorescence. Compared to in vivo approaches, in vitro assays using isolated mitochondria in a cell-free system can eliminate potential interferences from other subcellular structures. Although the fluorescence microtiter plate assay allows for high throughput Δψ m analysis of isolated mitochondria, the results are an average of a large number of mitochondria.14 Because mitochondria within an individual cell are highly heterogeneous with respect to their morphological and biochemical properties,15 single mitochondria analysis is required to reveal the large intrinsic heterogeneity of a mitochondrial population and to make data interpretation more conclusive.16−18 Moreover, with simultaneous light scatter and fluorescence analysis of single mitochondria, Δψm can be correlated with physical properties (e.g., size, inner composition) at the single organelle level to facilitate highly relevant systematic studies. Although flow cytometry is a well-established technique for the rapid, quantitative, and multiparameter analysis of single cells and microscopic particles,19,20 sensitive detection of submicrometer-sized polymer particles smaller than 200 nm in diameter is typically hindered by background from impurity particles in the sheath fluid and from optical and electronic noise.21−23 We have previously reported that, despite their relatively large particle sizes (0.5−1.0 μm), mitochondria scatter less incident light than polystyrene nanoparticles of the same size because of their smaller refractive index. It was determined that the side scatter intensity of mitochondria is broadly distributed and mainly falls in the intensity range of 110
and 200 nm polystyrene nanospheres.18 Therefore, the assistance of a strong fluorescence signal is generally required for the accurate discrimination of mitochondria from debris and background noise by conventional flow cytometry. For example, although forward scatter (FSC) and side scatter (SSC) parameters were used to establish the gating via analyzing the Δψm of isolated mitochondria on a FACSCalibur flow cytometer, the validity of the gating needed to be systematically confirmed via Δψm-insensitive MitoTracker or NAO labeling.12,24 To meet the demand for enabling sensitive light scatter and fluorescence detection of nanosized biological particles, we have developed a HSFCM that is tens to hundreds fold more sensitive than conventional flow cytometry.25,26 Particularly, the multiparameter detection of side scatter, cardiolipin, and mitochondrial DNA and the simultaneous measurement of side scatter, cytochrome c, and porin protein of individual mitochondria have been demonstrated.18 In the present study, treating isolated mitochondria with mitochondria-targeting compounds will result in the collapse of Δψm and the release of inter membrane space contents, which could lead to much weaker signals in both the fluorescence and the side scatter of mitochondria. These reduced signals can be easily interfered or completely buried in the background noise when these samples are analyzed by conventional flow cytometry. Thus, the high sensitivity of HSFCM is exploited for the sensitive side scatter and Δψm fluorescence detection of individual mitochondria upon treatment with mitochondriatargeting compounds. 5233
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Figure 2. Flow cytometric analysis of mitochondria treated without and with different concentrations of anticancer drugs on the laboratory-built HSFCM. Panels A-1−A-5 display bivariate dot-plots of the DiOC6(3) green fluorescence burst area versus the side scatter burst area for mitochondria treated with 0, 10, 50, 100, and 200 μM paclitaxel, respectively. Panels B-1−B-5 and C-1−C-5 display the same bivariate dot-plots for mitochondria treated with 0, 10, 50, 100, and 200 μM betulinic acid (B) or cisplatin (C). These figures were all derived from 60 s of data, and only events with correlated SS and FL signals are plotted.
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Western Blot Analysis of Cytochrome c Release. Isolated mitochondria were treated with anticancer drugs (200 μM, 2 h), and the supernatant was mixed with Laemmli’s loading buffer, boiled for 10 min, and subjected to SDS/12% PAGE at 120 V, followed by electroblotting on a polyvinylidene fluoride membrane for 90 min at 230 mA. The membrane was blocked for 1 h with 5% nonfat milk in TBST at room temperature and subsequently probed overnight with anticytochrome c antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, CA). The membrane was rinsed and incubated with a horseradish-peroxidase-conjugated secondary antibody (1:5000, Abcam, Cambridge, MA) for 1 h. Finally, the membrane was rinsed and detected by using enhanced chemiluminescence according to the manufacturer’s instructions.
EXPERIMENTAL SECTION Reagents and Chemicals. An Alexa Fluor 488 Annexin V/ Dead Cell Apoptosis Kit was purchased from Molecular Probes (Eugene, OR, USA). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), betulinic acid, antimycin A, curcumin, ABT-737, triptolide, cisplatin, actinomycin D, paclitaxel, and DiOC6(3) were obtained from Sigma (St. Louis, MO, USA). Anticancer compounds were dissolved in 100 mM dimethyl sulfoxide (DMSO) and stored in aliquots at −20 °C. DiOC6(3) was dissolved in DMSO (10 mM) and stored in aliquots at −20 °C. A mitochondrial buffer (MT buffer) containing 250 mM sucrose, 10 mM HEPES, 1 mM EGTA, 4.2 mM sodium succinate hexahydrate, and 1 mM potassium dihydrogen phosphate and adjusted to pH 7.2 with 1 M potassium hydroxide was used for mitochondria washing, stimulation, and staining. Distilled, deionized water supplied by a Milli-Q RG unit was filtered through a 0.22 μm filter and used for the buffer preparation and served as the sheath fluid of the HSFCM. Drug Treatment of Isolated Mitochondria. Mitochondria were isolated from HeLa cells following the same procedure as described previously.18 Three hundred microliters of isolated mitochondria suspension (∼5 × 107/mL) was treated with different drugs at indicated concentrations for 2 h at 37 °C. A potent mitochondrial membrane uncoupling agent, CCCP, was used as the positive control. Mitochondria incubated with 0.2% DMSO (the highest concentration of drug solvent) served as the solvent control. Next, the mitochondrial samples were washed once with MT buffer, and the pellets were resuspended in 200 μL of DiOC6(3) and incubated for 15 min at 37 °C. The optimal DiOC6(3) staining concentration was identified to be 500 nM (data not shown). After washing once with MT buffer, the DiOC6(3)-stained mitochondria were resuspended in 50 μL of MT buffer for HSFCM analysis.
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RESULTS AND DISCUSSION The representative side scatter and fluorescence burst traces that were obtained for the unstained and 500 nM DiOC6(3)stained mitochondria before and after CCCP treatment are displayed in Figure 1. The excitation and emission spectra of DiOC6(3)-stained mitochondria are presented in Figure S1 of the Supporting Information. Clearly, the side scattering signal of the individual mitochondria can be easily detected above the background. Upon staining with DiOC6(3), bright fluorescence signals were observed for single mitochondria concurrently with their corresponding side scatter bursts (Figure 1B-2). For each burst, the integrated number of detected photons was stored as the burst area. As seen in the bivariate dot-plots of the green fluorescence burst area versus the side scatter burst area, over 3000 individual mitochondria can be detected in 1 min and distinct separation between the unstained and the DiOC6(3)stained mitochondria was observed in the green fluorescence signal (Panels A-3 and B-3). Mitochondria treated with CCCP 5234
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Figure 3. Drug concentration effect on Δψm of isolated mitochondria upon treatment with paclitaxel (A), betulinic acid (B), and cisplatin (C).
concentration, from nanomolar to micromolar levels.29 Given that some compounds exhibit high levels of intracellular accumulation, the concentration required to cause dissipation of Δψm in isolated mitochondria may reflect the true intracellular concentration needed to induce MMP. Paclitaxel is a widely used chemotherapeutic agent with clinical efficacy in a number of human cancers.30 It has been reported that paclitaxel can act directly on mitochondria and 10−100 μM paclitaxel could induce a 27−72% release of cytochrome c from isolated neuroblastoma mitochondria.31 Figures 2A and 3A indicate that, when the isolated mitochondria were stimulated with 1−200 μM paclitaxel for 2 h, the median fluorescence of the Δψm signal decreased 33− 76% whereas the side scatter signal barely changed. These data suggest that paclitaxel can induce mitochondrial permeabilization but with a marginal effect on the structural integrity of mitochondria. Betulinic acid is a natural compound that can directly trigger MMP of isolated mitochondria in association with Δψm dissipation and cytochrome c release.32 The potent antitumor activity of betulinic acid has been linked to its ability to interact with the antiapoptotic protein, Bcl-xL, and trigger the mitochondrial pathway of apoptosis in cancer cells.33 Figures 2B and 3B show that 10 μM betulinic acid resulted in an approximately 50% decrease of the Δψm signal with nearly no change in the side scatter of mitochondria. When the concentration was increased to 100 μM, the median fluorescence of the Δψm signal decreased to approximately 1/ 4 of its initial state. It is interesting to note that a 200 μM stimulation concentration resulted in not only a further decrease of Δψm (median fluorescence reduced to 1/15 of its initial state) but also a severe disruption of the mitochondria. Compared to the 3029 correlated events (side scatter and fluorescence were detected concurrently) detected for the untreated mitochondria in 1 min, only 190 correlated events were detected upon 200 μM treatment. These data suggest that, accompanying the excessive dissipation of Δψm, the mitochondria also exhibited an irreversible and uncontrolled breakdown process. Although larger pieces of debris can still be detected on the side scatter channel (1168eVents in 1 min), the complete loss of Δψm rendered the fluorescence signal undetectable. Clearly, betulinic acid exhibited a more violent effect in MMP and mitochondria disruption when compared to paclitaxel. In contrast to betulinic acid and paclitaxel, cisplatin did not cause any notable change in the mitochondrial morphology or in the Δψm, even under a high concentration of treatment (Figures 2C and 3C). These data suggest that cisplatin cannot act on mitochondria directly. Apoptosis triggered by cisplatin generally results from its binding to nuclear DNA.34,35 Kelley et al. synthesized a mitochondrial-localized platinum complex by
was used as the positive control and a series of CCCP concentrations ranging from 1 to 200 μM were tested (37 °C, 2 h). We found that, when the isolated mitochondria were stimulated with 1−20 μM of CCCP, the Δψm fluorescence signal rarely changed. When the CCCP concentration was increased to 50−200 μM, a 21%−75% decrease of the Δψm signal was observed (see Figure S3 of the Supporting Information). After the Δψm-affecting experimental treatment with 200 μM CCCP, the median fluorescence intensity of the individual mitochondria decreased significantly from 12 516 to 3107 (Panels B-3 and C-3), although the side scatter signal remained nearly unchanged. CCCP treatment is known to transport protons across phospholipid membranes and abolish the mitochondrial Δψm gradient. Because CCCP treatment does not induce cytochrome c release,27 no remarkable change in the side scatter signal should be expected. These experiments demonstrate that HSFCM is sensitive enough to detect the side scatter and membrane potential fluorescence of individual mitochondria. Paclitaxel, antimycin A, betulinic acid, curcumin, ABT-737, triptolide, cisplatin, and actinomycin D are widely used anticancer drugs. Annexin V/PI apoptosis analysis confirms that all of these drugs can effectively induce apoptosis in HeLa cells with apoptotic rates ranging from 30% to 80% upon 24 h of treatment (see Figure S2 of the Supporting Information). As for their modes of action to trigger apoptosis, some of them have been proven to undergo the death receptor pathway or the mitochondrial pathway, whereas others are still under discussion. To identify which compound can directly act on mitochondria, mitochondria isolated from HeLa cells were incubated with each of these anticancer drugs for 2 h, stained with DiOC6(3), and then analyzed on the HSFCM to measure the Δψm change upon drug treatment. Figures 2 and 3 show the results obtained with paclitaxel, betulinic acid, and cisplatin. The bivariate dot-plots of the green fluorescence versus the side scatter for each treatment provide a detailed examination of both the internal structure and the Δψm of mitochondria. By plotting the median fluorescence intensity against the drug concentration, a dose-dependent Δψm decrease was observed for mitochondria treated with paclitaxel (Figure 3A) and betulinic acid (Figure 3B), whereas even 200 μM cisplatin failed to cause any notable change in Δψm (Figure 3C). The same study was conducted for the other five drugs, the drug concentration effects of which are displayed in Figure S4 of the Supporting Information. It is worth noting that much higher drug concentrations are needed to induce a Δψm decrease of isolated mitochondria compared to those (e.g., 50 nM) required to trigger cell apoptosis.28 This observation is most likely because the drug molecules in cells can be concentrated several hundred fold over the extracellular 5235
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attaching an MPP to the cis-{Pt(NH3)2}2+ DNA-binding unit of cisplatin.36 They found that this conjugation was able to enter mitochondria, damage mtDNA, and lead to apoptosis of both wild-type and cisplatin-resistant ovarian cancer cells. Given that the resistance to cisplatin, either acquired or inherent, is a common problem, the design of such a mitochondria-targeting cisplatin adduct could be a powerful approach to avoid cisplatin resistance. It would be interesting to determine whether these MPP-conjugated complexes function by directly acting on mitochondria by testing them with isolated mitochondria using the present in vitro approach. To further validate the applicability of our method in identifying mitochondria-targeting drugs, five other commonly used anticancer compounds were tested. The results of curcumin, triptolide, ABT-737, antimycin A, and actinomycin D are shown in Figure S3 of the Supporting Information. Dosedependent Δψm loss was observed for curcumin, triptolide, ABT-737, and antimycin A but not for actinomycin D. These data suggest that the first four drugs may induce apoptosis via a direct effect on mitochondria. Curcumin is a naturally occurring compound isolated from turmeric and exhibits potent antitumor properties which have been ascribed to its downregulation of Bcl-2, up-regulation of Bax, and generation of ROS.37 Apoptosis of human leukemia cells induced by triptolide involves translocation of Bax, loss of the mitochondrial membrane potential, and activation of caspase-3, which suggest that apoptosis proceeds through the mitochondrial pathway.38 In light of the fact that few studies have been reported on the direct interaction between these two drugs and isolated mitochondria, our results may contribute to the further interpretation of their signaling pathways leading to apoptosis. ABT-737 and antimycin A have been reported to exhibit a direct effect on mitochondria. ABT-737 is a mitochondriatargeting anticancer agent and has been considered as a Badlike BH3 mimetic that binds and neutralizes the pro-survival proteins Bcl-2/Bcl-xL and enhances apoptotic signals.39 Antimycin A is an inhibitor of mitochondrial electron transport, and the generation of reactive oxygen species leads to mitochondrial membrane depolarization, Bcl-2 down-regulation, and Bax up-regulation.40 Actinomycin D has been used for the treatment of some highly malignant tumors. It could downregulate rRNA synthesis, increase the level of p53, and finally trigger a mitochondria-dependent apoptosis.41 For an easy comparison, the median fluorescence signals of mitochondria untreated or treated with 200 μM of each of the eight widely used anticarcinogens tested are plotted together in Figure 4. Western blot analyses of cytochrome c release performed in parallel with Δψm measurements show that triptolide, antimycin A, paclitaxel, betulinic acid, ABT-737, and curcumin induced cytochrome c release from isolated mitochondria, whereas cisplatin and actinomycin D did not. Moreover, more cytochrome c release from betulinic acidtreated mitochondria was observed compared to that of paclitaxel-treated mitochondria. These data are in excellent agreement with the Δψm results obtained at the single mitochondria level by HSFCM analysis.
Figure 4. Comparison of results between the HSFCM and Western blot analyses. (A) HSFCM analysis of Δψm fluorescence signals before and after treatment with different drugs. (B) Western blotting analysis of cytochrome c release from mitochondria upon drug treatment. Untreated mitochondria were analyzed as a control.
detection can reveal the change of internal contents upon drug treatment at the single-organelle level with high resolution. For the eight widely used anticarcinogens tested, it was found that triptolide, antimycin A, paclitaxel, betulinic acid, ABT-737, and curcumin exhibited direct action on mitochondria, whereas actinomycin D and cisplatin did not. These results agree well with the literature reports and were confirmed with a Western blot analysis. This new method could provide a powerful tool for anticancer drug discovery by quickly identifying compounds that induce apoptosis via directly targeting mitochondria, which is important for avoiding resistance mechanisms. Moreover, the rapid data acquisition provided by the HSFCM would make it possible to follow the kinetics of membrane potential loss upon compound challenge in isolated mitochondria. We could first stain mitochondria, then apply the compound, and subsequently determine the velocity of membrane loss. The possibility to discriminate strong versus weak or intermediate mitochondrial aggressors through kinetic measurements will be examined in the near future.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: 86-592-2184519. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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CONCLUSIONS We have established an effective method for the systematic identification of mitochondria-targeting anticancer compounds by measuring the Δψm decrease of isolated mitochondria. On a laboratory-built high sensitivity flow cytometer, the analysis of mitochondria is nearly background free, and side scatter
ACKNOWLEDGMENTS This work was supported by the National Key Basic Research Program of China (Grant 2013CB933703), the National Natural Science Foundation of China (Grants 91313302, 90913015, 21225523, 21027010, and 20975087), the Key project of Fujian science and technology plan (2012I0010), the 5236
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(35) Cheung-Ong, K.; Song, K. T.; Ma, Z. D.; Shabtai, D.; Lee, A. Y.; Gallo, D.; Heisler, L. E.; Brown, G. W.; Bierbach, U.; Giaever, G.; Nislow, C. ACS Chem. Biol. 2012, 7, 1892−1901. (36) Wisnovsky, S. P.; Wilson, J. J.; Radford, R. J.; Pereira, M. P.; Chan, M. R.; Laposa, R. R.; Lippard, S. J.; Kelley, S. O. Chem. Biol. 2013, 20, 1323−1328. (37) Reuter, S.; Eifes, S.; Dicato, M.; Aggarwal, B. B.; Diederich, M. Biochem. Pharmacol. 2008, 76, 1340−1351. (38) Liu, L.; Li, G.; Li, Q.; Jin, Z.; Zhang, L.; Zhou, J.; Hu, X.; Zhou, T.; Chen, J.; Gao, N. Cell Death Dis. 2013, 4, No. e941. (39) Touzeau, C.; Dousset, C.; Bodet, L.; Gomez-Bougie, P.; Bonnaud, S.; Moreau, A.; Moreau, P.; Pellat-Deceunynk, C.; Amiot, M.; Le Gouill, S. Clin. Cancer Res. 2011, 17, 5973−5981. (40) Park, W. H.; Han, Y. W.; Kim, S. H.; Kim, S. Z. J. Cell Biochem. 2007, 102, 98−109. (41) Kalousek, I.; Brodska, B.; Otevrelova, P.; Roselova, P. AntiCancer Drugs 2007, 18, 763−772.
Research Funds for the Doctoral Program of Higher Education of China (20090121110009), the Program for Changjiang Scholars and Innovative Research Team in University (IRT13036), and the NFFTBS (Grant J1310024), for which we are most grateful.
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Identification of Mitochondria-Targeting Anticancer Compounds by an in Vitro Strategy Xiang Zhang,† Shuyue Zhang,† Shaobin Zhu,† Sha Chen,† Jinyan Han,† Kaimin Gao,† Jin-zhang Zeng,‡ and Xiaomei Yan*,† †
The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, P. R. China ‡ School of Pharmaceutical Sciences and Institute for Biomedical Research, Xiamen University, Xiamen, Fujian 361005, P. R. China S Supporting Information *
ABSTRACT: Mitochondria play a pivotal role in determining the point-of-no-return of the apoptotic process. Therefore, anticancer drugs that directly target mitochondria hold great potential to evade resistance mechanisms that have developed toward conventional chemotherapeutics. In this study, we report the development of an in vitro strategy to quickly identify the therapeutic agents that induce apoptosis via directly affecting mitochondria. This result is achieved by treating isolated mitochondria with potential anticancer compounds, followed by simultaneously measuring the side scatter and mitochondrial membrane potential (Δψm) fluorescence of individual mitochondria using a laboratory-built high-sensitivity flow cytometer. The feasibility of this method was tested with eight widely used anticarcinogens. Dose-dependent Δψm losses were observed for paclitaxel, antimycin A, betulinic acid, curcumin, ABT-737, and triptolide, but not for cisplatin or actinomycin D, which agrees well with their mechanisms of apoptosis induction reported in the literature. The as-developed method offers an effective approach to identify mitochondria-targeting anticancer compounds.
M
the new conjugate is able to overcome multidrug resistance mechanisms, such as P-glycoprotein-mediated efflux, which strongly proves that drugs directly acting on mitochondria represent an attractive strategy for cancer therapy. Many of the known mitochondria-targeting anticancer drugs are derived from natural compounds and have been identified by serendipity rather than by systematic screening.7 In this report, by employing Δψm loss as an early apoptotic marker, we attempt to develop a systematic approach to specifically identify mitochondria-targeting anticancer compounds. The following describes the experimental design used in this study. The anticancer agent to be examined was used to treat isolated mitochondria. After fluorescent labeling with DiOC6(3), a lipophilic cationic probe specific for assessing Δψm,10,11 the mitochondria were passed individually through the tightly focused laser beam of a laboratory-built high sensitivity flow cytometer (HSFCM). The bursts of photons that were detected on both the side scatter (SS) and the fluorescence (FL) channels were recorded simultaneously. The SS signal delineates the mitochondrial refractive index and internal complexity,12,13 wherease the FL intensity reflects the extent of Δψm via DiOC6(3) accumulation in the mitochondrial matrix space in inverse proportion to Δψm, according to the
ost classical anticancer drugs induce apoptosis by activating signaling pathways that lie upstream of mitochondria. Unfortunately, cancer cells have developed numerous strategies to evade apoptosis by blocking these upstream events, such as reduced expression of death receptors, aberrant expression of decoy receptors, overexpression of antiapoptotic Bcl-2 family proteins, underexpression or inactivation of proapoptotic proteins (such as Bax and BH3only proteins), and inhibition of p53 translocation, which make the established therapeutic regimens lose efficacy.1−3 It has been well recognized that both extrinsic and intrinsic signaling pathways converge on mitochondria, and mitochondrial membrane permeabilization (MMP) is the decisive point of the irreversible process of apoptosis.4,5 With the onset of MMP, mitochondrial membrane potential (Δψm) is lost, and cytochrome c and other pro-apoptotic proteins are released from the mitochondrial intermembrane space into the cytosol where a caspase cascade is initiated and the cell is disassembled into apoptotic bodies. Therefore, mitochondria have been considered one of the most promising targets for anticancer drug development because compounds that can directly simulate MMP hold great potential to circumvent the resistance mechanisms that have developed toward conventional chemotherapeutics.6−8 Thus, it is interesting to note the work of Kelley et al., who engineered the first mitochondrially targeted version of doxorubicin by attaching it to a mitochondriapenetrating peptide (MPP).9 In contrast to doxorubicin itself, © 2014 American Chemical Society
Received: March 12, 2014 Accepted: May 13, 2014 Published: May 13, 2014 5232
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Figure 1. Flow cytometric analysis of the unstained and 500 nM DiOC6(3)-stained mitochondria before and after CCCP treatment on the laboratory-built HSFCM: (A-1 and A-2) Representative side scatter (SS) and green fluorescence (FL) burst traces of unstained mitochondria. (B-1 and B-2) Representative side scatter and green fluorescence burst traces of DiOC6(3)-stained mitochondria. (C-1 and C-2) Representative side scatter and green fluorescence burst traces of DiOC6(3)-stained mitochondria treated with 200 μM CCCP. (A-3, B-3, and C-3) Bivariate dot-plots of green fluorescence burst area versus side scatter burst area derived from 60 s of data for the unstained and stained mitochondria without or with CCCP treatment, respectively. The signal saturation voltage is 10 V for both detection channels.
Nernst equation.11 Therefore, depolarized (less negative) mitochondria will have lower cationic dye concentrations and lower fluorescence. Compared to in vivo approaches, in vitro assays using isolated mitochondria in a cell-free system can eliminate potential interferences from other subcellular structures. Although the fluorescence microtiter plate assay allows for high throughput Δψ m analysis of isolated mitochondria, the results are an average of a large number of mitochondria.14 Because mitochondria within an individual cell are highly heterogeneous with respect to their morphological and biochemical properties,15 single mitochondria analysis is required to reveal the large intrinsic heterogeneity of a mitochondrial population and to make data interpretation more conclusive.16−18 Moreover, with simultaneous light scatter and fluorescence analysis of single mitochondria, Δψm can be correlated with physical properties (e.g., size, inner composition) at the single organelle level to facilitate highly relevant systematic studies. Although flow cytometry is a well-established technique for the rapid, quantitative, and multiparameter analysis of single cells and microscopic particles,19,20 sensitive detection of submicrometer-sized polymer particles smaller than 200 nm in diameter is typically hindered by background from impurity particles in the sheath fluid and from optical and electronic noise.21−23 We have previously reported that, despite their relatively large particle sizes (0.5−1.0 μm), mitochondria scatter less incident light than polystyrene nanoparticles of the same size because of their smaller refractive index. It was determined that the side scatter intensity of mitochondria is broadly distributed and mainly falls in the intensity range of 110
and 200 nm polystyrene nanospheres.18 Therefore, the assistance of a strong fluorescence signal is generally required for the accurate discrimination of mitochondria from debris and background noise by conventional flow cytometry. For example, although forward scatter (FSC) and side scatter (SSC) parameters were used to establish the gating via analyzing the Δψm of isolated mitochondria on a FACSCalibur flow cytometer, the validity of the gating needed to be systematically confirmed via Δψm-insensitive MitoTracker or NAO labeling.12,24 To meet the demand for enabling sensitive light scatter and fluorescence detection of nanosized biological particles, we have developed a HSFCM that is tens to hundreds fold more sensitive than conventional flow cytometry.25,26 Particularly, the multiparameter detection of side scatter, cardiolipin, and mitochondrial DNA and the simultaneous measurement of side scatter, cytochrome c, and porin protein of individual mitochondria have been demonstrated.18 In the present study, treating isolated mitochondria with mitochondria-targeting compounds will result in the collapse of Δψm and the release of inter membrane space contents, which could lead to much weaker signals in both the fluorescence and the side scatter of mitochondria. These reduced signals can be easily interfered or completely buried in the background noise when these samples are analyzed by conventional flow cytometry. Thus, the high sensitivity of HSFCM is exploited for the sensitive side scatter and Δψm fluorescence detection of individual mitochondria upon treatment with mitochondriatargeting compounds. 5233
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Figure 2. Flow cytometric analysis of mitochondria treated without and with different concentrations of anticancer drugs on the laboratory-built HSFCM. Panels A-1−A-5 display bivariate dot-plots of the DiOC6(3) green fluorescence burst area versus the side scatter burst area for mitochondria treated with 0, 10, 50, 100, and 200 μM paclitaxel, respectively. Panels B-1−B-5 and C-1−C-5 display the same bivariate dot-plots for mitochondria treated with 0, 10, 50, 100, and 200 μM betulinic acid (B) or cisplatin (C). These figures were all derived from 60 s of data, and only events with correlated SS and FL signals are plotted.
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Western Blot Analysis of Cytochrome c Release. Isolated mitochondria were treated with anticancer drugs (200 μM, 2 h), and the supernatant was mixed with Laemmli’s loading buffer, boiled for 10 min, and subjected to SDS/12% PAGE at 120 V, followed by electroblotting on a polyvinylidene fluoride membrane for 90 min at 230 mA. The membrane was blocked for 1 h with 5% nonfat milk in TBST at room temperature and subsequently probed overnight with anticytochrome c antibody (1:500, Santa Cruz Biotechnology, Santa Cruz, CA). The membrane was rinsed and incubated with a horseradish-peroxidase-conjugated secondary antibody (1:5000, Abcam, Cambridge, MA) for 1 h. Finally, the membrane was rinsed and detected by using enhanced chemiluminescence according to the manufacturer’s instructions.
EXPERIMENTAL SECTION Reagents and Chemicals. An Alexa Fluor 488 Annexin V/ Dead Cell Apoptosis Kit was purchased from Molecular Probes (Eugene, OR, USA). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), betulinic acid, antimycin A, curcumin, ABT-737, triptolide, cisplatin, actinomycin D, paclitaxel, and DiOC6(3) were obtained from Sigma (St. Louis, MO, USA). Anticancer compounds were dissolved in 100 mM dimethyl sulfoxide (DMSO) and stored in aliquots at −20 °C. DiOC6(3) was dissolved in DMSO (10 mM) and stored in aliquots at −20 °C. A mitochondrial buffer (MT buffer) containing 250 mM sucrose, 10 mM HEPES, 1 mM EGTA, 4.2 mM sodium succinate hexahydrate, and 1 mM potassium dihydrogen phosphate and adjusted to pH 7.2 with 1 M potassium hydroxide was used for mitochondria washing, stimulation, and staining. Distilled, deionized water supplied by a Milli-Q RG unit was filtered through a 0.22 μm filter and used for the buffer preparation and served as the sheath fluid of the HSFCM. Drug Treatment of Isolated Mitochondria. Mitochondria were isolated from HeLa cells following the same procedure as described previously.18 Three hundred microliters of isolated mitochondria suspension (∼5 × 107/mL) was treated with different drugs at indicated concentrations for 2 h at 37 °C. A potent mitochondrial membrane uncoupling agent, CCCP, was used as the positive control. Mitochondria incubated with 0.2% DMSO (the highest concentration of drug solvent) served as the solvent control. Next, the mitochondrial samples were washed once with MT buffer, and the pellets were resuspended in 200 μL of DiOC6(3) and incubated for 15 min at 37 °C. The optimal DiOC6(3) staining concentration was identified to be 500 nM (data not shown). After washing once with MT buffer, the DiOC6(3)-stained mitochondria were resuspended in 50 μL of MT buffer for HSFCM analysis.
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RESULTS AND DISCUSSION The representative side scatter and fluorescence burst traces that were obtained for the unstained and 500 nM DiOC6(3)stained mitochondria before and after CCCP treatment are displayed in Figure 1. The excitation and emission spectra of DiOC6(3)-stained mitochondria are presented in Figure S1 of the Supporting Information. Clearly, the side scattering signal of the individual mitochondria can be easily detected above the background. Upon staining with DiOC6(3), bright fluorescence signals were observed for single mitochondria concurrently with their corresponding side scatter bursts (Figure 1B-2). For each burst, the integrated number of detected photons was stored as the burst area. As seen in the bivariate dot-plots of the green fluorescence burst area versus the side scatter burst area, over 3000 individual mitochondria can be detected in 1 min and distinct separation between the unstained and the DiOC6(3)stained mitochondria was observed in the green fluorescence signal (Panels A-3 and B-3). Mitochondria treated with CCCP 5234
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Figure 3. Drug concentration effect on Δψm of isolated mitochondria upon treatment with paclitaxel (A), betulinic acid (B), and cisplatin (C).
concentration, from nanomolar to micromolar levels.29 Given that some compounds exhibit high levels of intracellular accumulation, the concentration required to cause dissipation of Δψm in isolated mitochondria may reflect the true intracellular concentration needed to induce MMP. Paclitaxel is a widely used chemotherapeutic agent with clinical efficacy in a number of human cancers.30 It has been reported that paclitaxel can act directly on mitochondria and 10−100 μM paclitaxel could induce a 27−72% release of cytochrome c from isolated neuroblastoma mitochondria.31 Figures 2A and 3A indicate that, when the isolated mitochondria were stimulated with 1−200 μM paclitaxel for 2 h, the median fluorescence of the Δψm signal decreased 33− 76% whereas the side scatter signal barely changed. These data suggest that paclitaxel can induce mitochondrial permeabilization but with a marginal effect on the structural integrity of mitochondria. Betulinic acid is a natural compound that can directly trigger MMP of isolated mitochondria in association with Δψm dissipation and cytochrome c release.32 The potent antitumor activity of betulinic acid has been linked to its ability to interact with the antiapoptotic protein, Bcl-xL, and trigger the mitochondrial pathway of apoptosis in cancer cells.33 Figures 2B and 3B show that 10 μM betulinic acid resulted in an approximately 50% decrease of the Δψm signal with nearly no change in the side scatter of mitochondria. When the concentration was increased to 100 μM, the median fluorescence of the Δψm signal decreased to approximately 1/ 4 of its initial state. It is interesting to note that a 200 μM stimulation concentration resulted in not only a further decrease of Δψm (median fluorescence reduced to 1/15 of its initial state) but also a severe disruption of the mitochondria. Compared to the 3029 correlated events (side scatter and fluorescence were detected concurrently) detected for the untreated mitochondria in 1 min, only 190 correlated events were detected upon 200 μM treatment. These data suggest that, accompanying the excessive dissipation of Δψm, the mitochondria also exhibited an irreversible and uncontrolled breakdown process. Although larger pieces of debris can still be detected on the side scatter channel (1168eVents in 1 min), the complete loss of Δψm rendered the fluorescence signal undetectable. Clearly, betulinic acid exhibited a more violent effect in MMP and mitochondria disruption when compared to paclitaxel. In contrast to betulinic acid and paclitaxel, cisplatin did not cause any notable change in the mitochondrial morphology or in the Δψm, even under a high concentration of treatment (Figures 2C and 3C). These data suggest that cisplatin cannot act on mitochondria directly. Apoptosis triggered by cisplatin generally results from its binding to nuclear DNA.34,35 Kelley et al. synthesized a mitochondrial-localized platinum complex by
was used as the positive control and a series of CCCP concentrations ranging from 1 to 200 μM were tested (37 °C, 2 h). We found that, when the isolated mitochondria were stimulated with 1−20 μM of CCCP, the Δψm fluorescence signal rarely changed. When the CCCP concentration was increased to 50−200 μM, a 21%−75% decrease of the Δψm signal was observed (see Figure S3 of the Supporting Information). After the Δψm-affecting experimental treatment with 200 μM CCCP, the median fluorescence intensity of the individual mitochondria decreased significantly from 12 516 to 3107 (Panels B-3 and C-3), although the side scatter signal remained nearly unchanged. CCCP treatment is known to transport protons across phospholipid membranes and abolish the mitochondrial Δψm gradient. Because CCCP treatment does not induce cytochrome c release,27 no remarkable change in the side scatter signal should be expected. These experiments demonstrate that HSFCM is sensitive enough to detect the side scatter and membrane potential fluorescence of individual mitochondria. Paclitaxel, antimycin A, betulinic acid, curcumin, ABT-737, triptolide, cisplatin, and actinomycin D are widely used anticancer drugs. Annexin V/PI apoptosis analysis confirms that all of these drugs can effectively induce apoptosis in HeLa cells with apoptotic rates ranging from 30% to 80% upon 24 h of treatment (see Figure S2 of the Supporting Information). As for their modes of action to trigger apoptosis, some of them have been proven to undergo the death receptor pathway or the mitochondrial pathway, whereas others are still under discussion. To identify which compound can directly act on mitochondria, mitochondria isolated from HeLa cells were incubated with each of these anticancer drugs for 2 h, stained with DiOC6(3), and then analyzed on the HSFCM to measure the Δψm change upon drug treatment. Figures 2 and 3 show the results obtained with paclitaxel, betulinic acid, and cisplatin. The bivariate dot-plots of the green fluorescence versus the side scatter for each treatment provide a detailed examination of both the internal structure and the Δψm of mitochondria. By plotting the median fluorescence intensity against the drug concentration, a dose-dependent Δψm decrease was observed for mitochondria treated with paclitaxel (Figure 3A) and betulinic acid (Figure 3B), whereas even 200 μM cisplatin failed to cause any notable change in Δψm (Figure 3C). The same study was conducted for the other five drugs, the drug concentration effects of which are displayed in Figure S4 of the Supporting Information. It is worth noting that much higher drug concentrations are needed to induce a Δψm decrease of isolated mitochondria compared to those (e.g., 50 nM) required to trigger cell apoptosis.28 This observation is most likely because the drug molecules in cells can be concentrated several hundred fold over the extracellular 5235
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attaching an MPP to the cis-{Pt(NH3)2}2+ DNA-binding unit of cisplatin.36 They found that this conjugation was able to enter mitochondria, damage mtDNA, and lead to apoptosis of both wild-type and cisplatin-resistant ovarian cancer cells. Given that the resistance to cisplatin, either acquired or inherent, is a common problem, the design of such a mitochondria-targeting cisplatin adduct could be a powerful approach to avoid cisplatin resistance. It would be interesting to determine whether these MPP-conjugated complexes function by directly acting on mitochondria by testing them with isolated mitochondria using the present in vitro approach. To further validate the applicability of our method in identifying mitochondria-targeting drugs, five other commonly used anticancer compounds were tested. The results of curcumin, triptolide, ABT-737, antimycin A, and actinomycin D are shown in Figure S3 of the Supporting Information. Dosedependent Δψm loss was observed for curcumin, triptolide, ABT-737, and antimycin A but not for actinomycin D. These data suggest that the first four drugs may induce apoptosis via a direct effect on mitochondria. Curcumin is a naturally occurring compound isolated from turmeric and exhibits potent antitumor properties which have been ascribed to its downregulation of Bcl-2, up-regulation of Bax, and generation of ROS.37 Apoptosis of human leukemia cells induced by triptolide involves translocation of Bax, loss of the mitochondrial membrane potential, and activation of caspase-3, which suggest that apoptosis proceeds through the mitochondrial pathway.38 In light of the fact that few studies have been reported on the direct interaction between these two drugs and isolated mitochondria, our results may contribute to the further interpretation of their signaling pathways leading to apoptosis. ABT-737 and antimycin A have been reported to exhibit a direct effect on mitochondria. ABT-737 is a mitochondriatargeting anticancer agent and has been considered as a Badlike BH3 mimetic that binds and neutralizes the pro-survival proteins Bcl-2/Bcl-xL and enhances apoptotic signals.39 Antimycin A is an inhibitor of mitochondrial electron transport, and the generation of reactive oxygen species leads to mitochondrial membrane depolarization, Bcl-2 down-regulation, and Bax up-regulation.40 Actinomycin D has been used for the treatment of some highly malignant tumors. It could downregulate rRNA synthesis, increase the level of p53, and finally trigger a mitochondria-dependent apoptosis.41 For an easy comparison, the median fluorescence signals of mitochondria untreated or treated with 200 μM of each of the eight widely used anticarcinogens tested are plotted together in Figure 4. Western blot analyses of cytochrome c release performed in parallel with Δψm measurements show that triptolide, antimycin A, paclitaxel, betulinic acid, ABT-737, and curcumin induced cytochrome c release from isolated mitochondria, whereas cisplatin and actinomycin D did not. Moreover, more cytochrome c release from betulinic acidtreated mitochondria was observed compared to that of paclitaxel-treated mitochondria. These data are in excellent agreement with the Δψm results obtained at the single mitochondria level by HSFCM analysis.
Figure 4. Comparison of results between the HSFCM and Western blot analyses. (A) HSFCM analysis of Δψm fluorescence signals before and after treatment with different drugs. (B) Western blotting analysis of cytochrome c release from mitochondria upon drug treatment. Untreated mitochondria were analyzed as a control.
detection can reveal the change of internal contents upon drug treatment at the single-organelle level with high resolution. For the eight widely used anticarcinogens tested, it was found that triptolide, antimycin A, paclitaxel, betulinic acid, ABT-737, and curcumin exhibited direct action on mitochondria, whereas actinomycin D and cisplatin did not. These results agree well with the literature reports and were confirmed with a Western blot analysis. This new method could provide a powerful tool for anticancer drug discovery by quickly identifying compounds that induce apoptosis via directly targeting mitochondria, which is important for avoiding resistance mechanisms. Moreover, the rapid data acquisition provided by the HSFCM would make it possible to follow the kinetics of membrane potential loss upon compound challenge in isolated mitochondria. We could first stain mitochondria, then apply the compound, and subsequently determine the velocity of membrane loss. The possibility to discriminate strong versus weak or intermediate mitochondrial aggressors through kinetic measurements will be examined in the near future.
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ASSOCIATED CONTENT
S Supporting Information *
Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: 86-592-2184519. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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CONCLUSIONS We have established an effective method for the systematic identification of mitochondria-targeting anticancer compounds by measuring the Δψm decrease of isolated mitochondria. On a laboratory-built high sensitivity flow cytometer, the analysis of mitochondria is nearly background free, and side scatter
ACKNOWLEDGMENTS This work was supported by the National Key Basic Research Program of China (Grant 2013CB933703), the National Natural Science Foundation of China (Grants 91313302, 90913015, 21225523, 21027010, and 20975087), the Key project of Fujian science and technology plan (2012I0010), the 5236
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(35) Cheung-Ong, K.; Song, K. T.; Ma, Z. D.; Shabtai, D.; Lee, A. Y.; Gallo, D.; Heisler, L. E.; Brown, G. W.; Bierbach, U.; Giaever, G.; Nislow, C. ACS Chem. Biol. 2012, 7, 1892−1901. (36) Wisnovsky, S. P.; Wilson, J. J.; Radford, R. J.; Pereira, M. P.; Chan, M. R.; Laposa, R. R.; Lippard, S. J.; Kelley, S. O. Chem. Biol. 2013, 20, 1323−1328. (37) Reuter, S.; Eifes, S.; Dicato, M.; Aggarwal, B. B.; Diederich, M. Biochem. Pharmacol. 2008, 76, 1340−1351. (38) Liu, L.; Li, G.; Li, Q.; Jin, Z.; Zhang, L.; Zhou, J.; Hu, X.; Zhou, T.; Chen, J.; Gao, N. Cell Death Dis. 2013, 4, No. e941. (39) Touzeau, C.; Dousset, C.; Bodet, L.; Gomez-Bougie, P.; Bonnaud, S.; Moreau, A.; Moreau, P.; Pellat-Deceunynk, C.; Amiot, M.; Le Gouill, S. Clin. Cancer Res. 2011, 17, 5973−5981. (40) Park, W. H.; Han, Y. W.; Kim, S. H.; Kim, S. Z. J. Cell Biochem. 2007, 102, 98−109. (41) Kalousek, I.; Brodska, B.; Otevrelova, P.; Roselova, P. AntiCancer Drugs 2007, 18, 763−772.
Research Funds for the Doctoral Program of Higher Education of China (20090121110009), the Program for Changjiang Scholars and Innovative Research Team in University (IRT13036), and the NFFTBS (Grant J1310024), for which we are most grateful.
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