Increased mitochondrial DNA induces acquired docetaxel resistance in head and neck cancer cells

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 reactive oxygen species (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.

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 was set as OD₅₅₀ of cells cultured without docetaxel. Results are presented as the mean ± standard error of six replicate wells. *, P < .001 as determined using two-tailed unpaired Student’s t test when treated cells were compared with untreated.

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 and probed for mtDNA. (c) Oxygen consumption in HEp2 and DRHEp2 was detected as described in Material and Methods. Results are presented as the mean ± standard deviation.

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 was done using Image J Software. Bar graph is presented as the mean ± standard deviation of three samples. (b) EtBr treated DRHEp2 was treated with indicated concentrations of docetaxel for 72 hours. For each sample, OD₅₅₀ of cells without docetaxel treatment was set as 0% death. Results are presented as the mean ± standard error of six replicate wells. *, P < .01 as determined using two-tailed unpaired Student’s t test when EtBr-treated DRHEp2 was compared with DRHEp2. (c) RT-PCR analysis of MDR1 mRNA in HEp2, DRHEp2, and EtBr treated DRHEp2. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was analyzed as respective controls.

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 A (◆), 800 μM sodium azide (○), 2 μg/ml oligomycin A (□), 200 nM aurovertin B (△), or 1 μM carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) (◇) for 72 hours; control cells (●) were treated with docetaxel alone. OD₅₅₀ of untreated control cells was calculated as 100% cell survival. Results are presented as the mean ± standard error of six replicate wells. *, P < .001 as determined using two-tailed unpaired Student’s t test when cells treated with docetaxel and mitochondrial respiratory chain (MRC) inhibitors or uncoupler were compared with cells treated with MRC inhibitors or uncoupler alone. (b) RT-PCR analysis of ATPase6 and ATPase8 mRNA in HEp2, DRHEp2, and EtBr treated DRHEp2. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was analyzed as respective controls.

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, 72, and 96 hours, and cell lysates were subjected to Western blot analysis using the antibodies indicated (right). (b) After 24 hours, DRHEp2 (●) and DRHEp2 transfected with Stealth RNAi specific for Fo-ATPase d-subunit (▲) or nonsilencing negative control (■) were treated with docetaxel for 72 hours. For each sample, 100% cell survival was set as OD₅₅₀ of docetaxel-untreated cells. Results are presented as the mean ± standard error of triplicate experiments. *, P < .001 as determined using two-tailed unpaired Student’s t test when cells transfected with Stealth RNAi specific for Fo-ATPase d-subunit and then treated with docetaxel were compared with cells treated with docetaxel alone.

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 with either 50 nM docetaxel, 2 μg/ml oligomycin A, or both.

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 docetaxel treatment was set as 100% survival. Results are presented as the mean ± standard error of six replicate wells. *, P < .001 as determined using two-tailed unpaired Student’s t test when cells treated with docetaxel and PDTC were compared with cells treated with docetaxel alone. (b) HEp2 treated with docetaxel or with docetaxel and PDTC were fixed and stained with crystal violet solution and then photographed under inverted microscopy. Scale bar = 50 μm.