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Oncogene. Author manuscript; available in PMC 2008 March 17.
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Oncogene. 2008 January 31; 27(6): 831–838.
Published online 2007 July 16. doi: 10.1038/sj.onc.1210681.
PMCID: PMC2268644
NIHMSID: NIHMS37214
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Increased Mitochondrial DNA Induces Acquired Docetaxel Resistance in Head and Neck Cancer Cells
T Mizumachi,1 S Suzuki,1,2 A Naito,1 J Carcel-Trullols,1 TT Evans,1 PM Spring,3 N Oridate,2 Y Furuta,2 S Fukuda,2 and M Higuchi1
1 Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA
2 Department of Otolaryngology-Head and Neck Surgery, Hokkaido University Graduate School of Medicine, Sapporo, Japan
3 Department of Otolaryngology-Head and Neck Surgery, University of Arkansas for Medical Sciences, Little Rock, AR, USA
Correspondence: Assistant Professor M Higuchi, Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, 4301 W. Markham St., slot 516 Little Rock, AR 72205, USA, Phone: 501-526-7520, Fax: 501-686-8169, E-mail: mhiguchi/at/uams.edu
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Abstract
Docetaxel is one of the most effective chemotherapeutic agents against cancer; nevertheless, some patients develop resistance. Unfortunately, their causes and mechanisms remain unknown. We created docetaxel-resistant DRHEp2 from human laryngeal cancer HEp2 and investigated the roles of mitochondrial DNA (mtDNA) and ROS on docetaxel resistance. DRHEp2 had greatly increased mtDNA content. Reduction of mtDNA content in DRHEp2 by ethidium bromide treatment reduced the resistance. These results indicate the possible roles of mtDNA-coded enzymes in mitochondrial respiratory chain (MRC) in resistant mechanisms. Oligomycin A, an Fo-ATPase inhibitor, eliminated docetaxel resistance in DRHEp2. In contrast, inhibitors of other MRC did not. RNA interference targeted to Fo-ATPase d-subunit restored docetaxel-induced cytotoxicity to DRHEp2. These results indicate the roles of Fo-ATPase for resistant mechanisms. Docetaxel induced ROS generation in HEp2 but not in DRHEp2 and antioxidant pyrrolidine dithiocarbamate eliminated docetaxel-induced cytotoxicity, suggesting roles of ROS in docetaxel-induced cell death. Furthermore, inhibition of Fo-ATPase by Oligomycin A induced docetaxel–mediated ROS generation in DRHEp2. Taken together, DRHEp2 acquired docetaxel resistance through increasing Fo-ATPase, which led to diminish docetaxel-induced ROS generation and subsequently inhibited cell death. In conclusion, mtDNA plays an important role in developing docetaxel resistance through the reduction of ROS generation by regulating Fo-ATPase.
Keywords: docetaxel, drug resistance, head and neck cancer, mitochondrial DNA, Fo-ATPase
Widely used in treating breast cancer, non-small-cell lung cancer, ovarian cancer, and head and neck cancer, docetaxel is one of the most effective chemotherapeutic agents in clinical use (Crown and O’leary, 2000; Schrijvers and Vermorken, 2005). Cytotoxicity stems from its ability to promote tubulin polymerization and formation of stable microtubles. The stabilized microtubules are resistant to disassembly by physiologic stimuli, so cells accumulate disorganized arrays of microtubules. This results in arresting the cell cycle in G2-M phase, which interferes with mitosis and, ultimately, results in apoptosis.
Despite its utility, development of acquired resistance to docetaxel often occurs and is a notable clinical problem. Several possible mechanisms for the acquired resistance have been suggested, including altered expression of mRNA β-tubulin isotypes (Shalli et al., 2005), modulation of β-tubulin protein levels (Shalli et al., 2005), high expression of P-glycoprotein (Bissery et al., 1995; Liu et al., 2001), reduced expression of p27 (Brown et al., 2004), and high expression of thioredoxin (Kim et al., 2005). However, the mechanism is not yet completely understood. Understanding docetaxel resistance is essential to design strategies to overcome the resistance.
The mitochondria play important roles in cellular energy metabolism, reactive oxygen species (ROS) generation, and apoptosis. Besides nucleus, mitochondria are the only other organelles in mammalian cells to carry a genome. Human mitochondrial DNA (mtDNA) is a 16.6-kb, circular, double-stranded DNA molecule that is present in a high number of copies per cell; the number varies widely with cell type. mtDNA encodes 13 polypeptides involved in respiration and oxidative phosphorylation, 2 rRNAs, and a set of 22 tRNAs essential for protein synthesis within mitochondria.
Alterations in mtDNA are reported in various types of cancer (Carew and Huang, 2002), and several reports (Higuchi et al., 1997; Higuchi et al., 2006; Lievre et al., 2005; Qian et al., 2005) showed that mtDNA plays an important role in cellular sensitivity to cancer therapeutic agents. The mitochondrial respiratory chain (MRC) is a major source of ROS. Recent studies (Caporossi et al., 2003; Schaaf et al., 2002) revealed that a variety of anticancer agents induce cell death by modifying the signaling pathways involving ROS. When cells were exposed to chemotherapeutic agents, the rate of ROS generation increased in and around mitochondria (Schaaf et al., 2002; Suzuki et al., 1999). To examine the mechanisms of docetaxel resistance, we used human laryngeal cancer (HEp2) cells to create docetaxel-resistant cells (DRHEp2) and investigated the roles of mtDNA and mitochondrial ROS on docetaxel resistance.
Establishing docetaxel-resistant DRHEp2 by treatment with docetaxel
Docetaxel-resistant cells were selected by a dose-escalating exposure to docetaxel as described in Material and Methods. We investigated the cytotoxic effect of docetaxel on HEp2 and DRHEp2. The dose–response curve of docetaxel indicates that DRHEp2 cells were more than 75 times more resistant to docetaxel than HEp2 (Figure 1Figure 1).
Figure 1
Figure 1
Figure 1
Effects of docetaxel on HEp2 and DRHEp2. HEp2 (●) and DRHEp2 (■) were treated with indicated concentrations of docetaxel for 72 hours. Cell survival was estimated as described in Material and Methods. For each cell line, 100% cell survival (more ...)
DRHEp2 has increased amounts of mtDNA
To evaluate the roles of mtDNA in the mechanisms of acquired docetaxel resistance, we used PCR amplification of full-length mitochondrial genomes to examine mtDNA from single cells of HEp2 and DRHEp2. We extracted mtDNA from 1 cell and from a pool of 50 cells of HEp2 and DRHEp2, and examined the amounts of mtDNA. Wild-type mtDNA (16-kb band) from one cell (Figure 2aFigure 2) was detected in DRHEp2 but not in HEp2. We could detect small amount of deleted form of mtDNA in both cell lines but ratio of the deleted form to wild-type mtDNA did have not changed. These results suggest that DRHEp2 has increased amounts of mtDNA compared with HEp2.
Figure 2
Figure 2
Figure 2
Increased mtDNA content in DRHEp2. (a) DNA from individual cells was extracted and subjected to long-distance nested PCR. Wild-type mtDNA is visible as 16-kb band. (b) Total DNA (1 μg) from HEp2 and DRHEp2 was subjected to Southern blot analysis (more ...)
We then performed Southern blot analysis using 1 μg of DNA derived from HEp2 and DRHEp2. Bands corresponding to wild-type mtDNA were significantly increased in samples from DRHEp2 compared with those from HEp2 (Figure 2bFigure 2). We could detect deleted form of mtDNA in both cell lines but ratio of the deleted form to wild-type mtDNA did not change, confirming the results from PCR.
Then, mitochondrial respiratory function was detected by measuring oxygen consumption. The oxygen consumption in DRHEp2 was approximately 2.3-fold increased compared with that in HEp2 (Figure 2cFigure 2) possibly through the enhancement of MRC enzymatic activities by the increase in mtDNA content.
Furthermore, we examined whether reduction of the enhanced mtDNA content in DRHEp2 could eliminate the resistance to docetaxel. We treated DRHEp2 with ethidium bromide (EtBr) which is well known to reduce mtDNA content (King and Attardi, 1989). PCR products from 50 cells of HEp2, DRHEp2, and EtBr treated DRHEp2 were examined and the band intensity of wild-type mtDNA was compared. The wild-type mtDNA band intensity in DRHEp2 was 6.4-fold higher than in HEp2, and that in HEp2 and EtBr treated DRHEp2 were almost the same (Figure 3aFigure 3). As shown in Figure 3bFigure 3, EtBr treatment increased sensitivity to docetaxel in DRHEp2. These results indicate that mtDNA increase in DRHEp2 is responsible for docetaxel-resistant phenotype.
Figure 3
Figure 3
Figure 3
Reduction of mtDNA content and docetaxel resistance in DRHEp2. (a) Long-distance nested PCR products from 50 cells of HEp2, DRHEp2, and EtBr treated DRHEp2 were separated in the same agarose gel and stained with ethidium bromide. Densitometric analysis (more ...)
Next, we analyzed multidrug-resistant (MDR) 1 transcripts from HEp2, DRHEp2, and EtBr treated DRHEp2 because docetaxel was demonstrated to be a substrate of human P-glycoprotein (P-gp) (Wils et al., 1994) and one of the mechanisms for docetaxel resistance is an overexpression of P-gp in cancer cells (Bissery et al., 1995). Denstiometric analysis of mRNA transcripts showed 2.0-fold of MDR1 mRNA expression (after normalizing to glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) (Figure 3cFigure 3). In contrast, there was no difference between DRHEp2 and EtBr treated DRHEp2 in the expression level of MDR1 mRNA (Figure 3cFigure 3).
Docetaxel resistance in DRHEp2 is overcome by Fo-ATPase inhibitor oligomycin A but not by inhibitors of other mitochondrial respiratory chain (MRC)
We hypothesized that the increased mtDNA content in DRHEp2 leads to increased expression of mtDNA-encoded MRC proteins and that the resultant increased enzymatic activities of MRC are responsible for the docetaxel-resistant phenotype of DRHEp2.
We tested this hypothesis by investigating whether inhibition of specific MRC abrogates docetaxel resistance in DRHEp2. DRHEp2 was treated with MRC inhibitors or uncoupler in the presence or absence of docetaxel for 3 days, and their cytotoxic effects was measured. Concentration of all inhibitors used showed inhibitory effect on oxygen consumption, indicating that each inhibitor inhibits their specific site in MRC (Supplemental Table 1). Uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) increased oxygen consumption, suggesting that FCCP induced uncoupling (Supplemental Table 1). Oligomycin A, an inhibitor of Fo-ATPase (MRC V) (Adam et al., 1996), increased sensitivity of DRHEp2 to docetaxel in a dose-dependent manner (Figure 4aFigure 4). In contrast, inhibitors of other MRC and uncoupler did not abrogate docetaxel resistance of DRHEp2 (Figure 4aFigure 4). Next, we analyzed the expression level of ATPase6 and ATPase8, which are components of Fo-ATPase and encoded by mtDNA (Anderson et al., 1981), in HEp2, DRHEp2, and EtBr treated DRHEp2. As shown in Figure 4bFigure 4, DRHEp2 had 2.8-fold increased ATPase6 and 1.5-fold increased ATPase8 in mRNA levels compared with HEp2 (after normalizing to GAPDH). In contrast, we could not detect the transcripts of ATPase6 and ATPase8 in EtBr treated DRHEp2 (Figure 4bFigure 4). These results indicate that Fo-ATPase, proton-specific channels, specifically plays an important role in the mechanism of docetaxel resistance in DRHEp2.
Figure 4
Figure 4
Figure 4
Roles of Fo-ATPase in docetaxel-resistance in DRHEp2. (a) DRHEp2 were treated with various concentrations of docetaxel (0–160 nM) and either 80 nM rotenone (■), 500 μM theonyltrifluoroacetone (TTFA) (▲), 50 ng/ml antimycin (more ...)
Suppression of Fo-ATPase d-subunit expression increased docetaxel-induced cytotoxicity in DRHEp2
To determine whether Fo-ATPase plays a causative role in docetaxel resistance, DRHEp2 was transfected with siRNA specific to the d-subunit of the Fo-ATPase protein complex. Transfection of the Fo-ATPase d-subunit small interfering RNA (siRNA) greatly reduced Fo-ATPase d-subunit expression 24–96 hours after transfection (Figure 5aFigure 5). We then investigated whether this reduction in expression affected docetaxel-induced cytotoxicity in DRHEp2. Cells transfected with Fo-ATPase d-subunit siRNA were more sensitive to docetaxel than cells transfected with negative control RNAi (Figure 5bFigure 5). These results indicate that Fo-ATPase plays a role in mechanisms of docetaxel resistance in DRHEp2.
Figure 5
Figure 5
Figure 5
Effects of siRNA transfection on the expression of Fo-ATPase d-subunit and docetaxel resistance in DRHEp2. (a) DRHEp2 was transfected with Stealth RNAi specific for Fo-ATPase d-subunit or nonsilencing negative control. Cells were harvested after 24, 48, (more ...)
Roles of ROS in resistant mechanisms to docetaxel-induced cytotoxicity
Several reports show that mitochondrial ROS play a critical role in the cytotoxicity of anticancer agents; therefore, we first tested whether docetaxel increases ROS generation in HEp2 and DRHEp2. Treating HEp2 with 50 nM docetaxel resulted in 1.9-fold increased ROS generation (Figure 6aFigure 6). In contrast, docetaxel slightly increased ROS generation in DRHEp2 (Figure 6bFigure 6). Background level of ROS generation in DRHEp2 was 26% lower than that in HEp2 (Figure 6cFigure 6).
Figure 6
Figure 6
Figure 6
Effects of docetaxel on generation of superoxide in HEp2 and DRHEp2. HEp2 (a) and DRHEp2 (b) were treated with or without 10 or 50 nM docetaxel for 24 hours. (c) Reactive oxygen species (ROS) generation in untreated HEp2 and DRHEp2. (d) DRHEp2 was treated (more ...)
To investigate whether docetaxel-mediated cell death was caused by the induced ROS generation by docetaxel in HEp2 but not in DRHEp2, HEp2 was treated with docetaxel in the presence or absence of antioxidant pyrrolidine dithiocarbamate (PDTC) for 24 hours and evaluated for cell survival. Using spectroscopic analysis of crystal violet staining, we found that docetaxel induced dose-dependent cytotoxicity on HEp2, and PDTC nearly completely eliminated the cytotoxic effect (Figure 7aFigure 7). To confirm this visually, cells treated with docetaxel and PDTC were fixed and stained with crystal violet solution and observed with inverted microscopy (Figure 7bFigure 7). PDTC greatly eliminated docetaxel-induced cell death in HEp2. These results demonstrate that docetaxel-induced cell death results from increased ROS generation in HEp2, and reduction of ROS generation in DRHEp2 might be the cause for docetaxel resistance.
Figure 7
Figure 7
Figure 7
Pyrrolidine dithiocarbamate (PDTC) inhibits docetaxel-induced cell death in HEp2. (a) HEp2 was treated with indicated concentrations of docetaxel alone (●) or with 200 μM PDTC (■) for 24 hours. For each sample, OD550 of cells without (more ...)
Synergistic ROS generation by docetaxel and oligomycin A in DRHEp2
Our results suggested that docetaxel-induced ROS caused cell death in HEp2 and that the ROS generation was inhibited by the increase in Fo-ATPase coded in mtDNA in DRHEp2. Therefore, we investigated whether inhibition of Fo-ATPase by oligomycin A induces high protonic potential and enhance ROS generation in the presence of docetaxel in DRHEp2.
To test this hypothesis, we investigated whether docetaxel treatment induces ROS generation in DRHEp2 in the presence of oligomycin A. Treating DRHEp2 with docetaxel or oligomycin A alone resulted in slightly increased ROS generation. In contrast, treating DRHEp2 with both docetaxel and oligomycin A resulted in 2.3-fold increased ROS generation (Figure 6dFigure 6). These results indicate that enhancement of mtDNA might increase Fo-ATPase activity, which reduces docetaxel-induced ROS generation through regulating protonic potential and ultimately leads to acquired resistance to docetaxel.
This study provides the first evidence that enhancing mtDNA levels in cancer cells induces mechanisms that facilitate resistance to cytotoxicity of chemotherapeutic reagents. We demonstrated that DRHEp2 acquired docetaxel resistance through increases in Fo-ATPase activity, which led to reduction of docetaxel-induced ROS generation. The role of Fo-ATPase in mechanisms of docetaxel resistance in DRHEp2 is supported by data demonstrating the increased levels of mtDNA in DRHEp2 and the increased cytotoxicity of docetaxel in DRHEp2 as a result of treatment with oligomycin A or suppression of Fo-ATPase d-subunit expression.
Here, we report the effect of enhancing amount of mtDNA, rather than depleting mtDNA, on drug-resistant mechanisms. Using long-distance nested PCR from individual cells and Southern blot analysis, we showed that DRHEp2 has more mtDNA than HEp2. Furthermore, we confirmed that reduction of mtDNA in DRHEp2 increased the sensitivity to docetaxel. For PCR studies, we amplified full-length mitochondrial genomes from single cells. The method we used to measure the amount of mtDNA in individual cells was established to detect all possible deletions in mtDNA of human cardiomyocytes (Khrapko et al., 1999). Unlike the cardiomyocytes study, we did not detect the deleted form of mtDNA in single HEp2 and DRHEp2.
We showed that mtDNA content was enhanced in DRHEp2. We also showed that reduced mtDNA content by treating DRHEp2 with EtBr for 5 days resulted in increased sensitivity to docetaxel. It is unlikely that 5 day EtBr treatment induced mutation of nuclear DNA leading to the change in phenotype of most of the cells to docetaxel-sensitive because of the limited mutation rate and the speed of the selection to docetaxel-sensitive phenotype. We also showed that there were no differences in the expression of MDR1 transcripts in DRHEp2 and EtBr treated DRHEp2. These results were consistent with the report of Cavalli et al., showing that the expression of the drug resistance genes MDR1, multidrug resistance protein, or O6-alkyltransferase were not affected by reducing mtDNA content (Cavalli et al., 1997). These results indicate that mtDNA plays an important role in docetaxel resistant mechanism independent of P-gp mechanisms, even though increased MDR1 expression in DRHEp2 might contribute to drug resistance. Further studies need to be performed to know the relations of mtDNA content and other resistant mechanisms.
To investigate how increased mtDNA content in DRHEp2 is responsible for the docetaxel-resistant phenotype of DRHEp2, we tested whether docetaxel resistance was affected by addition of specific MRC inhibitors. Our results showed that treating DRHEp2 with oligomycin A, an Fo-ATPase inhibitor, reduced docetaxel resistance, whereas inhibitors targeting other MRC and an MRC uncoupler did not reverse docetaxel resistance. Several lines of evidence indicate that oligomycin A is a promoter of apoptosis (Li et al., 2004; Wolvetang et al., 1994); however, other reports suggest oligomycin A has an inhibitory effect on apoptosis (Matsuyama et al., 1998; Santamaria et al., 2006). Our results indicate that oligomycin A promotes docetaxel-induced cell death in DRHEp2 and that docetaxel resistance is linked with Fo-ATPase activity level.
Moreover, transfecting DRHEp2 with siRNA targeting Fo-ATPase d-subunit efficiently suppressed protein expression and eliminated resistance. These findings provide evidence that Fo-ATPase may play a role in docetaxel-resistant mechanisms. On the other hand, Shin et al. reported that resistance to 5-fluorouracil (5-FU) in colon cancer cells is linked to downregulation of MRC V. They showed that oligomycin A antagonized 5-FU-induced suppression of cell proliferation and that suppressed Fo-ATPase d-subunit expression, resulting from siRNA transfection, increased cell viability in the presence of 5-FU (Shin et al., 2005). This discrepancy might come from the difference in the cell death mechanisms induced by 5-FU and docetaxel but further analysis is necessary.
We showed that combining docetaxel and oligomycin A increased mitochondrial generation of ROS in DRHEp2. These results suggest that enhancing the amount of mtDNA may increase the activity of Fo-ATPase, thereby inhibiting docetaxel-induced ROS generation, leading to acquired resistance. The mechanism that oligomycin A increases ROS generation can be explained as follows (Kirkland and Franklin, 2007); oligomycin A blocks passage of protons through the mitochondrial Fo-F1 ATPase. This block hyperpolarizes mitochondrial membrane potential by increasing the proton gradient across the inner membrane. Hyperpolarization of mitochondrial membrane potential can increase leakage of electrons from the mitochondrial electron transport chain and then these electrons reduce molecular oxygen to superoxide. Our results are supported by the finding that docetaxel resistance in breast cancer can be mediated by activating several genes controlling the cellular redox environment (Iwao-Koizumi et al., 2005). Furthermore, Alexandre et al. showed that ROS participate in paclitaxel cytotoxicity (Alexandre et al., 2006).
The mechanisms responsible for the increased amount of mtDNA in DRHEp2 are unknown. Lee et al. reported that the amount of mtDNA was increased by treatment with paclitaxel or a low dose of hydrogen peroxide in human osteosarcoma cells (Lee et al., 2005). They also showed that mitochondria abnormally proliferated in cells. Collectively, the data suggest that docetaxel treatment of HEp2 induces ROS, and this oxidative stress increases mtDNA replication during the process of acquiring docetaxel resistance.
In conclusion, our study demonstrates that mtDNA plays a critical role in development of docetaxel resistance through diminished ROS generation from MRC, and Fo-ATPase could be a novel target for strategies to overcome docetaxel resistance.
Materials
Details of materials are provided in Supplementary information.
Cell culture
HEp2 cells were kindly provided by Nippon Kayaku Co. (Tokyo, Japan). Cells were maintained in Dulbecco’s modified Eagle medium (DMEM, Gibco, Grand Island, NY) supplemented with 5% heat-inactivated fetal bovine serum (Hyclone, Logan, UT) at 37°C with 5% CO2. Docetaxel-resistant DRHEp2 were selected by a dose-escalating exposure to docetaxel. HEp2 were initially exposed to 10 nM docetaxel continuously. Initially, cell growth was inhibited and gradually restored. Subsequently, the cells were exposed to 20 nM docetaxel, increasing to 160 nM. DRHEp2 were maintained in medium supplemented with 160 nM docetaxel.
Cell survival assay
Details of cell survival assay are provided in Supplementary information.
Isolation and amplification of mtDNA from individual cells
Details of isolation and amplification of mtDNA from individual cells are provided in Supplementary information.
Southern blot analysis
Details of Southern blot analysis are provided in Supplementary Information.
Measurement of oxygen consumption
Details of oxygen consumption are provided in Supplementary Information.
EtBr treatment in DRHEp2
DRHEp2 was incubated in Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% FBS, 50 μg/ml uridine and 100 μg/ml sodium pyruvate in the presence of 25.6 μg/ml EtBr for 5 days to reduce mtDNA content.
Semiquantitative reverse transcription-PCR (RT-PCR)analysis
Details of RT-PCR are provided in Supplementary information.
RNA interference
Details of RNA interference are provided in Supplementary information.
Western blot analysis
Details of Western blot analysis are provided in Supplementary information.
Measurement of ROS generation
Details of measurement of ROS generation are provided in Supplementary information.
Statistical analysis
Two-tailed unpaired Student’s t test was used to evaluate statistical significance. P < .01 was considered statistically significant.
Supplementary Material
Material-Metho
Supplementary information is available at Oncogene’s website.
Click here to view.(49K, doc)
Supp Table 1
Click here to view.(38K, doc)
Acknowledgments
The authors thank Sanae Yamaguchi (Department of Otolaryngology-Head and Neck Surgery, Hokkaido University Graduate School of Medicine) for their technical assistance. This work was supported by Taiho Pharmaceutical Co. Ltd, Tobacco Settlement at State of Arkansas and NIH grant RO1 CA100846 to MH.
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