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Lack of mitochondrial DNA enhances growth of hepatocellular carcinoma in vitro and in vivo
Hepatology Research 36 (2006) 209–216
Lack of mitochondrial DNA enhances growth of hepatocellular carcinoma in vitro and in vivo Anwarul Haque a , Manabu Nishikawa a,∗ , Wei Qian a , Masayuki Mashimo a , Masaki Hirose a , Shuhei Nishiguchi b , Masayasu Inoue a b
a Department of Biochemistry & Molecular Pathology, Osaka City University Medical School, Osaka 545-8585, Japan Division of Hepatobiliary and Pancreatic Diseases, Department of Internal Medicine, Hyogo College of Medicine, Hyogo 663-8131, Japan
Received 8 June 2006; received in revised form 3 July 2006; accepted 6 July 2006 Available online 21 August 2006
Abstract To elucidate the role of mitochondrial DNA (mtDNA) in determination of growth of hepatocellular carcinoma, we examined wild-type Hepa1-6 cells and their 0 cells with depleted mtDNA in vitro and in vivo. Cultured 0 cells grew more rapidly than did wild-type cells. Production of reactive oxygen species (ROS) was higher in wild-type cells than in 0 cells. Hypoxia inhibited the growth of wild-type cells more markedly than that of 0 cells. Resistance to mitochondrial respiratory inhibitor-induced cell death was stronger in 0 cells than in wild-type cells. 0 cells subcutaneously inoculated in the hind thigh of mice grew more rapidly and formed larger solid tumors. These findings indicate that lack of mtDNA increases growth of hepatocellular carcinoma by decreasing ROS production and increasing resistance to mitochondrial respiratory inhibition. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Hepatocellular carcinoma; Growth; Mitochondria; Mitochondrial DNA; Mutation; 0 ; Oxidative stress
1. Introduction Mitochondrial dysfunction is one of the most common and profound phenotypes of cancer, and is caused by accumulation of mitochondrial DNA (mtDNA) mutations in various types of cancers [1]. We recently reported that numerous mtDNA mutations accumulated in hepatocellular carcinoma and hepatic precancerous tissues in tight association with carcinogenesis [2–6]. Accumulation of mtDNA damage is due to the close proximity of mtDNA to the sites of reactive oxygen species (ROS) generation by the electron transport chain and lack of introns and protective histones, as a result of which ROS induce more extensive and more persistent damage to
Abbreviations: mtDNA, mitochondrial DNA; ROS, reactive oxygen species; DCFH-DA, 2,7-dichlorofluorescein diacetate; L-012, [8-amino5-chloro-7-phenylpyrido(3,4-d)pyridazine-1 and 4(2H,3H)dione]; H–E, hematoxylin and eosin; O2 − , superoxide; H2 O2 , hydrogen peroxide ∗ Corresponding author. Tel.: +81 6 6645 3722; fax: +81 6 6645 3721. E-mail address: [email protected] (M. Nishikawa). 1386-6346/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.hepres.2006.07.005
mtDNA than nuclear DNA [7,8], although repair of some forms of oxidative DNA damage occurs more efficiently in mitochondria than in the nucleus [9–11], suggesting that the mitochondrial capacity for base excision repair is comparable to that of the nucleus. In fact, the frequency of mtDNA mutations in cancer cells has been reported to be 10-fold that of nuclear DNA mutations [8,12]. It has been well documented that mitosis and growth of cells require large amounts of ATP. Hence, cells enriched in intact mitochondria might theoretically be expected to proliferate more rapidly than those with defective mitochondria, such as cancer cells with numerous mtDNA mutations. However, this appears inconsistent with the general finding that cancer cell growth is more rapid than that of normal cells. Any event decreasing oxidative phosphorylation, issuing either from genetic damage or from the cell environment, can provide a proliferative advantage to transformed or tumor cells [13]. The mitochondria of rapidly growing tumors tend to be fewer in number, and have smaller numbers of cristae than mitochondria in slowly growing tumors [14–16]. Given
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these findings, we used mtDNA-depleted 0 cells to explore the involvement of mitochondrial DNA in growth of hepatocellular carcinoma. 2. Materials and methods 2.1. Materials MitoTracker Red was purchased from Molecular Probes (Eugene, OR). Rotenone and antimycin A were obtained from Sigma Chemical Co. (St. Louis, MO). 2,2 (Hydroxynitrosohydrazono)bis-ethanamine (NOC-18), an NO donor, were obtained from Dojindo Chemical Co. (Kumamoto, Japan). Other reagents used were of the highest grade commercially available.
form highly fluorescent 2,7-dichlorofluorescein (DCF). Cultured cells were incubated with phenol red-free medium containing 20 M DCFH-DA at 37 ◦ C for 30 min. After washing three times with PBS (pH 7.4), cells were analyzed with a fluorescence microscope (OLYMPUS, Tokyo, Japan). Cultured cells (1 × 106 ) were also prepared and incubated in 0.5 ml of 0.9% NaCl solution containing 10 mM phosphate buffer (pH 7.4), 6 mM KCl, and 6 mM MgCl2 in the presence of 400 M [8-amino-5-chloro-7-phenylpyrido(3,4d)pyridazine-1 and 4(2H,3H)dione] (L-012), an extremely sensitive chemiluminescence probe for detection of ROS including superoxide (O2 − ) [18,19]. Chemiluminescence intensity was recorded and analyzed continuously for 30 min using a BLR-201 Luminescence Reader (Aloka, Tokyo, Japan). 2.6. Analysis of cultured cell growth in hypoxia
2.2. Cell preparation Hepa1-6 cells from mice were obtained from Riken Cell Bank (Tsukuba, Japan) and cultured at 37 ◦ C in 5% CO2 and 20% O2 in Dulbecco’s modified Eagle’s medium (Sigma, St. Louis, MO) supplemented with 10% heat-inactivated fetal calf serum, 50 units/ml of penicillin G, and 50 g/ml of streptomycin. MtDNA-depleted Hepa1-6 cells (0 cells) were established through long-term treatment of the cells with ethidium bromide (50 ng/ml) as described previously [17]. Depletion of mtDNA was confirmed by PCR amplification using several sets of mtDNA-specific primers. Reduction of mitochondrial density was confirmed by staining with MitoTracker Red, an indicator of mitochondria. Conventional fluorescence images were obtained with a fluorescence microscope (OLYMPUS, Tokyo, Japan). 2.3. Analysis of cultured cell growth Both wild-type and 0 cells were cultured in culture flasks. At the indicated times, numbers of cells were determined under a light microscope (OLYMPUS, Tokyo, Japan).
Both wild-type and 0 cells were cultured at 37 ◦ C in 5% CO2 and 20% or 1% O2 for 72 h. Numbers of cells were determined under a light microscope (OLYMPUS, Tokyo, Japan). 2.7. Cell death assay Cells were treated with mitochondrial respiratory inhibitor, 1 M rotenone and 0.1 M antimycin A, and with NO donor NOC-18 (10 M, 100 M, and 1 mM) for 24 h, and their mortality was determined by trypan blue exclusion assay performed as follows. Cells were suspended by gentle pipetting, and 50 l of 0.4% trypan blue solution was mixed with 200 l of the cell suspension (final concentration 0.08%) at room temperature. Stained cells were counted within 3 min after mixture with the trypan blue solution. One hundred cells were counted for each trypan blue exclusion assay. Mortality of cells was then determined as the percentage of trypan blue-stained cells. Cell mortality determined by this method thus represents the population of dead cells among all cells, including both adherent and floating cells, at the termination of experiments.
2.4. Analysis of apoptotic death of cultured cells
2.8. Animal experiments
The cells were cultured for 24, 48, and 72 h and then fixed with 4% paraformaldehyde. Apoptotic cell death was assessed by terminal deoxynucleotidyl transferase (TdT)mediated biotinylated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL)-staining using an in situ Apoptosis Detection Kit. The samples were examined under a fluorescence microscope (OLYMPUS, Tokyo, Japan).
All experiments were approved by the Animal Care and Use Committee of Osaka City University Medical School. C57BL/6J mice were obtained from SLC Co. (Shizuoka. Japan). Wild-type and 0 cells were inoculated subcutaneously in the hind thigh (106 cells), and allowed to grow in solid form. After cancer cell inoculation, mice were measured their wild-type and 0 tumor size every 2 days using a calipers, with tumor volume calculated using the formula volume = (length)2 × width × 0.52.
2.5. Analysis of ROS generation To detect ROS generation, cells were loaded with membrane-permeable 2,7-dichlorofluorescein diacetate (DCFH-DA). DCFH-DA is hydrolyzed to non-fluorescent DCFH, which mainly reacts with hydrogen peroxide to
2.9. Histological analysis On day 21 of cancer cell innoculation, the tumor tissues were quickly excised in anesthetized condition and 4–5 mm-
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thick slices were cut with a razor blade and fixed in 10% buffered formalin. The buffered formalin-fixed tumor tissue slices were routinely processed for embedding in paraffin. Thin sections (4 m) of tissue specimens were stained with hematoxylin and eosin (H–E). Apoptotic cell death in the tumor tissues was assessed by TUNEL-staining using an in situ Apoptosis Detection Kit. Tissue sections 4 m thick were deparaffinized with xylene and an alcohol series, and then treated with TdT Enzyme and the Labeling Safe Buffer. The specimens were examined under a fluorescence microscope (OLYMPUS, Tokyo, Japan).
3. Results
2.10. Statistics
3.2. Involvement of apoptotic cell death in cultured cell growth
Values are the mean ± S.D. The significance of differences were determined using Student’s t-test, with significance set at P < 0.05.
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3.1. Mitochondrial status and growth of cultured cells To determine the status of mitochondria, cells were stained with MitoTracker Red. The MitoTracker Red staining of mitochondria actually looks like accumulation of thread around nucleus. We found that mitochondria in 0 cells were not clearly stained, and were much smaller in number and size compared with those in wild-type cells (Fig. 1A). In addition, the rate of cultured 0 cell growth was twice that of wild-type cells (Fig. 1B).
To determine whether the enhanced rate of growth in vitro shown in Fig. 1B was due to suppression of apopto-
Fig. 1. Mitochondrial status, in vitro growth in wild-type and 0 cells. Reduction of mitochondrial density was confirmed by staining of cells with MitoTracker Red as described in the text (A). Numbers of cultured wild-type and 0 cells were counted at indicated times as described in the text (B). Wild-type cells: open circles; 0 cells: closed circles. Values are means ± S.D. (n = 10). * P < 0.05 vs. wild-type at 24 h. ** P < 0.005 vs. wild-type at 72 h.
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percentage between the groups (Fig. 2). Furthermore, almost no floating cells were found in culture medium before analysis in either group. Thus, the enhancement of growth rate in cultured 0 cells depended on enhancement of proliferation, and not on suppression of cell death. 3.3. ROS generation by cultured cells
Fig. 2. Apoptotic death of wild-type and 0 cultured cells. TUNEL-positive cultured cells were counted as described in the text. Wild-type cells: open circles; 0 cells: closed circles. Values are means ± S.D. (n = 7).
sis, enhanced proliferation, or a combination of both, we examined cultured cells with TUNEL-staining. We found extremely small percentages of TUNEL-positive cells among both wild-type and 0 cells, without significant difference in
Mitochondria are the main source of cellular production of ROS including O2 − and hydrogen peroxide. Production of O2 − and hydrogen peroxide was thus determined using two types of probes. The fluorescence intensity of DCF was significantly decreased in 0 cells compared with that in wildtype cells, suggesting reduced production of ROS including hydrogen peroxide in 0 cells (Fig. 3A). For quantitative analysis of ROS production, chemiluminescence intensity was determined with wild-type and 0 cells using L-012. The intensity in 0 cells was less than half that in wild-type cells, suggesting reduced production of ROS including O2 − in 0 cells (Fig. 3B). 3.4. Resistance to hypoxia-induced inhibition of cell growth To determine the effect of hypoxia on wild-type and 0 cell growth, both types of cells were also cultured in hypoxic
Fig. 3. ROS production in wild-type and 0 cells. DCF fluorescence was determined in cultured wild-type and 0 cells as described in the text (A). These experiments were repeated five times with similar results. Chemiluminescence intensity was also determined in cultured wild-type and 0 cells using L-012 probe as describes in the text (B). Wild-type cells: open columns; 0 cells: closed columns. Values are means ± S.D. (n = 5). * P < 0.005 vs. wild-type.
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inhibits mitochondrial electron transport and respiration in host defense system [20]. 0 cells were more resistant to high concentration of NO than wild-type cells, although the viability of both cell types was not affected by low concentrations of NO (Fig. 5). 3.6. Solid tumor growth in vivo
Fig. 4. Cell growth in hypoxic condition. Wild-type and 0 cells were cultured in 5% CO2 and 20% or 1% O2 for 72 h. Wild-type cells: open columns; 0 cells: closed colomns. Values are means ± S.D. (n = 10). * P < 0.01 vs. wild-type in 20% O2 at 72 h.
condition (1% O2 ) for 72 h. The growth of wild-type cells was significantly inhibited by hypoxia (Fig. 4). However, the 0 cell growth was not affected. 3.5. Resistance to mitochondrial respiratory inhibitors and nitric oxide To determine the effect of mitochondrial respiratory inhibition on survival of wild-type and 0 cells, both types of cells were cultured with mitochondrial respiratory inhibitors. 0 cells were more resistant to the mitochondrial respiratory inhibitors, rotenone and anitimycin A (Fig. 5). The rate of 0 cell survival after treatment with these agents was over twice that of wild-type cells. It is well known that nitric oxide (NO)
Fig. 5. Survival of mitochondrial respiratory inhibitor-treated cells. Wildtype and 0 cells were cultured in the presence of 1 M rotenone, 0.1 M antimycin A, NOC-18 (10 M, 100 M, and 1 mM) for 24 h. Cell survival were determined as described in the text. Values are means ± S.D. (n = 10). Wild-type cells: open columns; 0 cells: closed columns. * P < 0.005 vs. wildtype cells treated with rotenone. ** P < 0.005 vs. wild-type cells treated with antimycin A. † P < 0.005 vs. wild-type cells treated with 1 mM NOC-18.
To determine the in vivo growth of wild-type and 0 cells, both types of cells were inoculated in mice and tumor volumes were measured. The growth rate of subcutaneously inoculated 0 cells was about twice as rapid as that of wild-type cells (Fig. 6A), suggesting that 0 cells are much more tumorigenic than wild-type cells. Wild-type tumors were relatively enriched with stromal tissues (Fig. 6B). No significant differences were found in the histopathological findings between wild-type and 0 tumors other than stroma. 3.7. Apoptotic cell death in wild-type and 0 tumors To elucidate the role of mitochondrial status in the mechanism of apoptotic cell death in tumor growth, we counted the numbers of TUNEL-positive cells in wild-type and 0 tumors (Fig. 7). The number of cells that underwent apoptotic cell death was significantly higher in wild-type than in 0 tumors. 4. Discussion Our study yielded two major findings. First, cultured 0 cells grew more rapidly than did wild-type cells. Second, when inoculated subcutaneously into the hind thighs of mice, 0 cells grew more rapidly and formed larger solid tumors than did wild-type cells. The mitochondrial electron transport chain is both the cellular source of ATP generated via oxidative phosphorylation and the main source of ROS including O2 − [21,22]. Indeed, analysis using L-012 revealed that the generation of ROS including O2 − was larger in wild-type cells than 0 cells partly because of enrichment of mitochondria. Hydrogen peroxide, which is itself a dangerous oxidant due to its production of hydroxyl radicals via events such as the Fenton reaction, is generated both in the spontaneous dismutation of O2 − or in the catalytic dismutation of it by superoxide dismutase. In fact, analysis using DCF revealed that the amount of ROS including hydrogen peroxide was larger in wild-type cells than in 0 cells. In mitogenesis-regulating pathways, numerous positive and negative effectors of signaling are influenced by physiological fluctuations in oxidants, including receptor tyrosine kinases, small GTPases, mitogen-activated protein kinases, protein phosphatases, and transcription factors. In the present study, we found the enhanced 0 cell growth possibly because of modulation of signaling pathway by decreased ROS pro-
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Fig. 6. Growth of wild-type and 0 tumors. (A) Mice were inoculated subcutaneously with wild-type or 0 cells (106 cells/mouse, respectively) to allow growth as solid tumors. Tumor volumes were determined as described in the text. Wild-type-tumor: open circles; 0 tumor: closed circles. Values are mean tumor volume ± S.D. (n = 10). * P < 0.05 vs. wild-type on day 12, ** P < 0.01 vs. wild-type on day 21. (B) Wild-type and 0 tumors were examined by H–E staining on day 21 after cancer cell inoculation. Bar = 100 m.
duction. Furthermore, ROS trigger opening of the membrane permeability transition pores in mitochondria and induces apoptosis and/or necrosis [23–25]. It is also reported that a free radical scavenger attenuates hepatic cell death in acute liver injury [26]. In addition, lower amount of cytochrome c induced by decreased content of mitochondria in 0 cells appears to decrease the incidence of cell death, because release of cytochrome c from mitochondria to the cytosol activates the mitochondria-dependent pathway leading to apoptosis. In fact, the incidence of apoptotic cell death was much decreased in 0 tumors in the present in vivo study. Vaupel et al. [27] have produced a comprehensive compilation of findings on oxygen tensions in normal and tumor tissues. Compared to normal tissue, in human primary tumors the microcirculation is compromised, tissue oxygenation is not regulated by metabolic demand, and the pO2 of tissue
is markedly lower, linked to tumor size, and often close to 0 mmHg. Consistent with the concept of hypoxic cancer cells is the view that metabolism in cancer cells is more glycolytic than that in normal cells [28,29]. The glycolytic enzymes and glucose transporters are overexpressed in cancers [30–32]. Furthermore, cells with mitochondrial dysfunction due to loss of mtDNA (0 cells) compensate for this metabolic insult by increases in transcription of genes coding for glycolytic enzymes [33,34]. As a result of such compensation, 0 tumors may have a proliferative advantage compared with wild-type tumors depending on resistance to mitochondrial respiratory inhibition. Comparison between glycolytic system and mitochondrial status in wild-type and 0 tumors will give us an important information. Thus, the functional and morphological analysis of mitochondria related to the glycolytic system in wild-type and 0 tumors should be studied further.
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Fig. 7. Apoptotic cell death in wild-type and 0 tumors. TUNEL-positive cells were counted as described in the text. Wild-type tumor: open column; 0 tumor: closed column. Values are means ± S.D. (n = 5). * P < 0.005 vs. wild-type.
The present study thus suggests that mtDNA-depletion favors the acceleration of cell proliferation and the expansion of tumor mass. Findings regarding mitochondrial status may thus provide useful information for the prediction of the clinical course in individuals with cancer.
Acknowledgements This work was supported by the grant from the Ministry of Education, Science, Sports, and Culture, the special Coordination funds for Science and Technology from the Science and Technology Agency, and the National Institutes of Health of Japan.
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Lack of mitochondrial DNA enhances growth of hepatocellular carcinoma in vitro and in vivo Anwarul Haque a , Manabu Nishikawa a,∗ , Wei Qian a , Masayuki Mashimo a , Masaki Hirose a , Shuhei Nishiguchi b , Masayasu Inoue a b
a Department of Biochemistry & Molecular Pathology, Osaka City University Medical School, Osaka 545-8585, Japan Division of Hepatobiliary and Pancreatic Diseases, Department of Internal Medicine, Hyogo College of Medicine, Hyogo 663-8131, Japan
Received 8 June 2006; received in revised form 3 July 2006; accepted 6 July 2006 Available online 21 August 2006
Abstract To elucidate the role of mitochondrial DNA (mtDNA) in determination of growth of hepatocellular carcinoma, we examined wild-type Hepa1-6 cells and their 0 cells with depleted mtDNA in vitro and in vivo. Cultured 0 cells grew more rapidly than did wild-type cells. Production of reactive oxygen species (ROS) was higher in wild-type cells than in 0 cells. Hypoxia inhibited the growth of wild-type cells more markedly than that of 0 cells. Resistance to mitochondrial respiratory inhibitor-induced cell death was stronger in 0 cells than in wild-type cells. 0 cells subcutaneously inoculated in the hind thigh of mice grew more rapidly and formed larger solid tumors. These findings indicate that lack of mtDNA increases growth of hepatocellular carcinoma by decreasing ROS production and increasing resistance to mitochondrial respiratory inhibition. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Hepatocellular carcinoma; Growth; Mitochondria; Mitochondrial DNA; Mutation; 0 ; Oxidative stress
1. Introduction Mitochondrial dysfunction is one of the most common and profound phenotypes of cancer, and is caused by accumulation of mitochondrial DNA (mtDNA) mutations in various types of cancers [1]. We recently reported that numerous mtDNA mutations accumulated in hepatocellular carcinoma and hepatic precancerous tissues in tight association with carcinogenesis [2–6]. Accumulation of mtDNA damage is due to the close proximity of mtDNA to the sites of reactive oxygen species (ROS) generation by the electron transport chain and lack of introns and protective histones, as a result of which ROS induce more extensive and more persistent damage to
Abbreviations: mtDNA, mitochondrial DNA; ROS, reactive oxygen species; DCFH-DA, 2,7-dichlorofluorescein diacetate; L-012, [8-amino5-chloro-7-phenylpyrido(3,4-d)pyridazine-1 and 4(2H,3H)dione]; H–E, hematoxylin and eosin; O2 − , superoxide; H2 O2 , hydrogen peroxide ∗ Corresponding author. Tel.: +81 6 6645 3722; fax: +81 6 6645 3721. E-mail address: [email protected] (M. Nishikawa). 1386-6346/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.hepres.2006.07.005
mtDNA than nuclear DNA [7,8], although repair of some forms of oxidative DNA damage occurs more efficiently in mitochondria than in the nucleus [9–11], suggesting that the mitochondrial capacity for base excision repair is comparable to that of the nucleus. In fact, the frequency of mtDNA mutations in cancer cells has been reported to be 10-fold that of nuclear DNA mutations [8,12]. It has been well documented that mitosis and growth of cells require large amounts of ATP. Hence, cells enriched in intact mitochondria might theoretically be expected to proliferate more rapidly than those with defective mitochondria, such as cancer cells with numerous mtDNA mutations. However, this appears inconsistent with the general finding that cancer cell growth is more rapid than that of normal cells. Any event decreasing oxidative phosphorylation, issuing either from genetic damage or from the cell environment, can provide a proliferative advantage to transformed or tumor cells [13]. The mitochondria of rapidly growing tumors tend to be fewer in number, and have smaller numbers of cristae than mitochondria in slowly growing tumors [14–16]. Given
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these findings, we used mtDNA-depleted 0 cells to explore the involvement of mitochondrial DNA in growth of hepatocellular carcinoma. 2. Materials and methods 2.1. Materials MitoTracker Red was purchased from Molecular Probes (Eugene, OR). Rotenone and antimycin A were obtained from Sigma Chemical Co. (St. Louis, MO). 2,2 (Hydroxynitrosohydrazono)bis-ethanamine (NOC-18), an NO donor, were obtained from Dojindo Chemical Co. (Kumamoto, Japan). Other reagents used were of the highest grade commercially available.
form highly fluorescent 2,7-dichlorofluorescein (DCF). Cultured cells were incubated with phenol red-free medium containing 20 M DCFH-DA at 37 ◦ C for 30 min. After washing three times with PBS (pH 7.4), cells were analyzed with a fluorescence microscope (OLYMPUS, Tokyo, Japan). Cultured cells (1 × 106 ) were also prepared and incubated in 0.5 ml of 0.9% NaCl solution containing 10 mM phosphate buffer (pH 7.4), 6 mM KCl, and 6 mM MgCl2 in the presence of 400 M [8-amino-5-chloro-7-phenylpyrido(3,4d)pyridazine-1 and 4(2H,3H)dione] (L-012), an extremely sensitive chemiluminescence probe for detection of ROS including superoxide (O2 − ) [18,19]. Chemiluminescence intensity was recorded and analyzed continuously for 30 min using a BLR-201 Luminescence Reader (Aloka, Tokyo, Japan). 2.6. Analysis of cultured cell growth in hypoxia
2.2. Cell preparation Hepa1-6 cells from mice were obtained from Riken Cell Bank (Tsukuba, Japan) and cultured at 37 ◦ C in 5% CO2 and 20% O2 in Dulbecco’s modified Eagle’s medium (Sigma, St. Louis, MO) supplemented with 10% heat-inactivated fetal calf serum, 50 units/ml of penicillin G, and 50 g/ml of streptomycin. MtDNA-depleted Hepa1-6 cells (0 cells) were established through long-term treatment of the cells with ethidium bromide (50 ng/ml) as described previously [17]. Depletion of mtDNA was confirmed by PCR amplification using several sets of mtDNA-specific primers. Reduction of mitochondrial density was confirmed by staining with MitoTracker Red, an indicator of mitochondria. Conventional fluorescence images were obtained with a fluorescence microscope (OLYMPUS, Tokyo, Japan). 2.3. Analysis of cultured cell growth Both wild-type and 0 cells were cultured in culture flasks. At the indicated times, numbers of cells were determined under a light microscope (OLYMPUS, Tokyo, Japan).
Both wild-type and 0 cells were cultured at 37 ◦ C in 5% CO2 and 20% or 1% O2 for 72 h. Numbers of cells were determined under a light microscope (OLYMPUS, Tokyo, Japan). 2.7. Cell death assay Cells were treated with mitochondrial respiratory inhibitor, 1 M rotenone and 0.1 M antimycin A, and with NO donor NOC-18 (10 M, 100 M, and 1 mM) for 24 h, and their mortality was determined by trypan blue exclusion assay performed as follows. Cells were suspended by gentle pipetting, and 50 l of 0.4% trypan blue solution was mixed with 200 l of the cell suspension (final concentration 0.08%) at room temperature. Stained cells were counted within 3 min after mixture with the trypan blue solution. One hundred cells were counted for each trypan blue exclusion assay. Mortality of cells was then determined as the percentage of trypan blue-stained cells. Cell mortality determined by this method thus represents the population of dead cells among all cells, including both adherent and floating cells, at the termination of experiments.
2.4. Analysis of apoptotic death of cultured cells
2.8. Animal experiments
The cells were cultured for 24, 48, and 72 h and then fixed with 4% paraformaldehyde. Apoptotic cell death was assessed by terminal deoxynucleotidyl transferase (TdT)mediated biotinylated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL)-staining using an in situ Apoptosis Detection Kit. The samples were examined under a fluorescence microscope (OLYMPUS, Tokyo, Japan).
All experiments were approved by the Animal Care and Use Committee of Osaka City University Medical School. C57BL/6J mice were obtained from SLC Co. (Shizuoka. Japan). Wild-type and 0 cells were inoculated subcutaneously in the hind thigh (106 cells), and allowed to grow in solid form. After cancer cell inoculation, mice were measured their wild-type and 0 tumor size every 2 days using a calipers, with tumor volume calculated using the formula volume = (length)2 × width × 0.52.
2.5. Analysis of ROS generation To detect ROS generation, cells were loaded with membrane-permeable 2,7-dichlorofluorescein diacetate (DCFH-DA). DCFH-DA is hydrolyzed to non-fluorescent DCFH, which mainly reacts with hydrogen peroxide to
2.9. Histological analysis On day 21 of cancer cell innoculation, the tumor tissues were quickly excised in anesthetized condition and 4–5 mm-
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thick slices were cut with a razor blade and fixed in 10% buffered formalin. The buffered formalin-fixed tumor tissue slices were routinely processed for embedding in paraffin. Thin sections (4 m) of tissue specimens were stained with hematoxylin and eosin (H–E). Apoptotic cell death in the tumor tissues was assessed by TUNEL-staining using an in situ Apoptosis Detection Kit. Tissue sections 4 m thick were deparaffinized with xylene and an alcohol series, and then treated with TdT Enzyme and the Labeling Safe Buffer. The specimens were examined under a fluorescence microscope (OLYMPUS, Tokyo, Japan).
3. Results
2.10. Statistics
3.2. Involvement of apoptotic cell death in cultured cell growth
Values are the mean ± S.D. The significance of differences were determined using Student’s t-test, with significance set at P < 0.05.
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3.1. Mitochondrial status and growth of cultured cells To determine the status of mitochondria, cells were stained with MitoTracker Red. The MitoTracker Red staining of mitochondria actually looks like accumulation of thread around nucleus. We found that mitochondria in 0 cells were not clearly stained, and were much smaller in number and size compared with those in wild-type cells (Fig. 1A). In addition, the rate of cultured 0 cell growth was twice that of wild-type cells (Fig. 1B).
To determine whether the enhanced rate of growth in vitro shown in Fig. 1B was due to suppression of apopto-
Fig. 1. Mitochondrial status, in vitro growth in wild-type and 0 cells. Reduction of mitochondrial density was confirmed by staining of cells with MitoTracker Red as described in the text (A). Numbers of cultured wild-type and 0 cells were counted at indicated times as described in the text (B). Wild-type cells: open circles; 0 cells: closed circles. Values are means ± S.D. (n = 10). * P < 0.05 vs. wild-type at 24 h. ** P < 0.005 vs. wild-type at 72 h.
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percentage between the groups (Fig. 2). Furthermore, almost no floating cells were found in culture medium before analysis in either group. Thus, the enhancement of growth rate in cultured 0 cells depended on enhancement of proliferation, and not on suppression of cell death. 3.3. ROS generation by cultured cells
Fig. 2. Apoptotic death of wild-type and 0 cultured cells. TUNEL-positive cultured cells were counted as described in the text. Wild-type cells: open circles; 0 cells: closed circles. Values are means ± S.D. (n = 7).
sis, enhanced proliferation, or a combination of both, we examined cultured cells with TUNEL-staining. We found extremely small percentages of TUNEL-positive cells among both wild-type and 0 cells, without significant difference in
Mitochondria are the main source of cellular production of ROS including O2 − and hydrogen peroxide. Production of O2 − and hydrogen peroxide was thus determined using two types of probes. The fluorescence intensity of DCF was significantly decreased in 0 cells compared with that in wildtype cells, suggesting reduced production of ROS including hydrogen peroxide in 0 cells (Fig. 3A). For quantitative analysis of ROS production, chemiluminescence intensity was determined with wild-type and 0 cells using L-012. The intensity in 0 cells was less than half that in wild-type cells, suggesting reduced production of ROS including O2 − in 0 cells (Fig. 3B). 3.4. Resistance to hypoxia-induced inhibition of cell growth To determine the effect of hypoxia on wild-type and 0 cell growth, both types of cells were also cultured in hypoxic
Fig. 3. ROS production in wild-type and 0 cells. DCF fluorescence was determined in cultured wild-type and 0 cells as described in the text (A). These experiments were repeated five times with similar results. Chemiluminescence intensity was also determined in cultured wild-type and 0 cells using L-012 probe as describes in the text (B). Wild-type cells: open columns; 0 cells: closed columns. Values are means ± S.D. (n = 5). * P < 0.005 vs. wild-type.
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inhibits mitochondrial electron transport and respiration in host defense system [20]. 0 cells were more resistant to high concentration of NO than wild-type cells, although the viability of both cell types was not affected by low concentrations of NO (Fig. 5). 3.6. Solid tumor growth in vivo
Fig. 4. Cell growth in hypoxic condition. Wild-type and 0 cells were cultured in 5% CO2 and 20% or 1% O2 for 72 h. Wild-type cells: open columns; 0 cells: closed colomns. Values are means ± S.D. (n = 10). * P < 0.01 vs. wild-type in 20% O2 at 72 h.
condition (1% O2 ) for 72 h. The growth of wild-type cells was significantly inhibited by hypoxia (Fig. 4). However, the 0 cell growth was not affected. 3.5. Resistance to mitochondrial respiratory inhibitors and nitric oxide To determine the effect of mitochondrial respiratory inhibition on survival of wild-type and 0 cells, both types of cells were cultured with mitochondrial respiratory inhibitors. 0 cells were more resistant to the mitochondrial respiratory inhibitors, rotenone and anitimycin A (Fig. 5). The rate of 0 cell survival after treatment with these agents was over twice that of wild-type cells. It is well known that nitric oxide (NO)
Fig. 5. Survival of mitochondrial respiratory inhibitor-treated cells. Wildtype and 0 cells were cultured in the presence of 1 M rotenone, 0.1 M antimycin A, NOC-18 (10 M, 100 M, and 1 mM) for 24 h. Cell survival were determined as described in the text. Values are means ± S.D. (n = 10). Wild-type cells: open columns; 0 cells: closed columns. * P < 0.005 vs. wildtype cells treated with rotenone. ** P < 0.005 vs. wild-type cells treated with antimycin A. † P < 0.005 vs. wild-type cells treated with 1 mM NOC-18.
To determine the in vivo growth of wild-type and 0 cells, both types of cells were inoculated in mice and tumor volumes were measured. The growth rate of subcutaneously inoculated 0 cells was about twice as rapid as that of wild-type cells (Fig. 6A), suggesting that 0 cells are much more tumorigenic than wild-type cells. Wild-type tumors were relatively enriched with stromal tissues (Fig. 6B). No significant differences were found in the histopathological findings between wild-type and 0 tumors other than stroma. 3.7. Apoptotic cell death in wild-type and 0 tumors To elucidate the role of mitochondrial status in the mechanism of apoptotic cell death in tumor growth, we counted the numbers of TUNEL-positive cells in wild-type and 0 tumors (Fig. 7). The number of cells that underwent apoptotic cell death was significantly higher in wild-type than in 0 tumors. 4. Discussion Our study yielded two major findings. First, cultured 0 cells grew more rapidly than did wild-type cells. Second, when inoculated subcutaneously into the hind thighs of mice, 0 cells grew more rapidly and formed larger solid tumors than did wild-type cells. The mitochondrial electron transport chain is both the cellular source of ATP generated via oxidative phosphorylation and the main source of ROS including O2 − [21,22]. Indeed, analysis using L-012 revealed that the generation of ROS including O2 − was larger in wild-type cells than 0 cells partly because of enrichment of mitochondria. Hydrogen peroxide, which is itself a dangerous oxidant due to its production of hydroxyl radicals via events such as the Fenton reaction, is generated both in the spontaneous dismutation of O2 − or in the catalytic dismutation of it by superoxide dismutase. In fact, analysis using DCF revealed that the amount of ROS including hydrogen peroxide was larger in wild-type cells than in 0 cells. In mitogenesis-regulating pathways, numerous positive and negative effectors of signaling are influenced by physiological fluctuations in oxidants, including receptor tyrosine kinases, small GTPases, mitogen-activated protein kinases, protein phosphatases, and transcription factors. In the present study, we found the enhanced 0 cell growth possibly because of modulation of signaling pathway by decreased ROS pro-
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Fig. 6. Growth of wild-type and 0 tumors. (A) Mice were inoculated subcutaneously with wild-type or 0 cells (106 cells/mouse, respectively) to allow growth as solid tumors. Tumor volumes were determined as described in the text. Wild-type-tumor: open circles; 0 tumor: closed circles. Values are mean tumor volume ± S.D. (n = 10). * P < 0.05 vs. wild-type on day 12, ** P < 0.01 vs. wild-type on day 21. (B) Wild-type and 0 tumors were examined by H–E staining on day 21 after cancer cell inoculation. Bar = 100 m.
duction. Furthermore, ROS trigger opening of the membrane permeability transition pores in mitochondria and induces apoptosis and/or necrosis [23–25]. It is also reported that a free radical scavenger attenuates hepatic cell death in acute liver injury [26]. In addition, lower amount of cytochrome c induced by decreased content of mitochondria in 0 cells appears to decrease the incidence of cell death, because release of cytochrome c from mitochondria to the cytosol activates the mitochondria-dependent pathway leading to apoptosis. In fact, the incidence of apoptotic cell death was much decreased in 0 tumors in the present in vivo study. Vaupel et al. [27] have produced a comprehensive compilation of findings on oxygen tensions in normal and tumor tissues. Compared to normal tissue, in human primary tumors the microcirculation is compromised, tissue oxygenation is not regulated by metabolic demand, and the pO2 of tissue
is markedly lower, linked to tumor size, and often close to 0 mmHg. Consistent with the concept of hypoxic cancer cells is the view that metabolism in cancer cells is more glycolytic than that in normal cells [28,29]. The glycolytic enzymes and glucose transporters are overexpressed in cancers [30–32]. Furthermore, cells with mitochondrial dysfunction due to loss of mtDNA (0 cells) compensate for this metabolic insult by increases in transcription of genes coding for glycolytic enzymes [33,34]. As a result of such compensation, 0 tumors may have a proliferative advantage compared with wild-type tumors depending on resistance to mitochondrial respiratory inhibition. Comparison between glycolytic system and mitochondrial status in wild-type and 0 tumors will give us an important information. Thus, the functional and morphological analysis of mitochondria related to the glycolytic system in wild-type and 0 tumors should be studied further.
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Fig. 7. Apoptotic cell death in wild-type and 0 tumors. TUNEL-positive cells were counted as described in the text. Wild-type tumor: open column; 0 tumor: closed column. Values are means ± S.D. (n = 5). * P < 0.005 vs. wild-type.
The present study thus suggests that mtDNA-depletion favors the acceleration of cell proliferation and the expansion of tumor mass. Findings regarding mitochondrial status may thus provide useful information for the prediction of the clinical course in individuals with cancer.
Acknowledgements This work was supported by the grant from the Ministry of Education, Science, Sports, and Culture, the special Coordination funds for Science and Technology from the Science and Technology Agency, and the National Institutes of Health of Japan.
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