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Abstract

Benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) is a highly reactive DNA damage agent and can induce cell death through both p53-independent and -dependent pathways. However, little is known about the molecular mechanisms of p53-independent pathways in BPDE-induced cell death. To understand the p53-independent mechanisms, we have now examined BPDE-induced cytotoxicity in p53-deficient baby mouse kidney (BMK) cells. The results showed that BPDE could induce Bax and Bak activation, cytochrome c release, caspases activation, and necrotic cell death in the BMK cells. Bax and Bak, two key molecules of mitochondrial permeability transition pore, were interdependently activated by BPDE, with Bax and Bak translocation to and Bax/Bak homo-oligomerization in mitochondria, release of cytochrome c was induced. Importantly, cytochrome c release and necrotic cell death were diminished in BMK cells (Bax^−/−), BMK cells (Bak^−/−), and BMK cells (Bax^−/−/Bak^−/−). Furthermore, overexpression of Bcl-2 could ameliorate BPDE-induced cytochrome c release and necrosis. Together the findings suggested that BPDE-induced necrosis was modulated by the p53-independent pathway, which was related to the translocation of Bax and Bak to mitochondria, release of cytochrome c, and activation of caspases.

Keywords

BPDE BMK cells Bax Bak necrosis

Introduction

Polycyclic aromatic hydrocarbons (PAH) are common environmental pollutants present in tobacco smoke and the diet from grilling or broiling of food and from atmospheric deposition.¹ Benzo(a)pyrene (BaP) is considered as a representative of PAH, which is known to be mutagenic and carcinogenic.² BaP exerts its toxic effect through transformation by cytochrome P450 enzymes in endoplasmic reticulum of the cell to become directly acting carcinogen benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE).³ BPDE can bind covalently to DNA and results in the activation of DNA damage checkpoints and/or the induction of cell death including apoptosis and necrosis.^4
–6 The BPDE can also act in synergy with other exposures (e.g. ultraviolet light), ultimately increasing the risk of DNA damage.⁷

Cell death including apoptosis and necrosis are key responses of cells that were exposed to BaP.⁸ Both apoptosis and necrosis are two distinct mechanisms of cell death. Apoptosis is a process of cell suicide that occurs in multicellular organisms in response to by various death stimuli, including death ligands such as tumor necrosis factor α (TNF-α), FasL/CD95/Apo1, and TNF-related apoptosis-inducing ligand (TRAIL), as well as chemotherapeutic or chemical agents such as cisplatin, doxorubicin, and 5-fluorouracil.^9,10 This programmed cell death involves extrinsic and/or intrinsic apoptotic pathways that converge in a series of biochemical events such as activation of caspases that leads to cell shrinkage and plasma membrane blebbing.^11,12 On the other hand, necrosis has long been described as uncontrolled and accidental cell death resulting from extremely harsh conditions, it is often caused by external factors, such as infection, toxins, trauma, or ischemia–reperfusion injury that leads to cell swelling and eventually rupture of the plasma membrane.¹³ However, accumulating evidence suggests that necrosis is also a programmed necrosis and can occur in a regulated manner.^8,14 The onset of mitochondrial permeability transition pore was involved in necrosis.¹⁵ Apoptosis and necrosis can occur concurrently and share some common pathways, which may be differentially induced by variable doses of pollutants.^16,17 However, the underlying molecular mechanisms still remain elusive.

p53 is a key regulator of DNA damage-induced apoptosis, which is central to its function as a tumor suppressor.^18,19 In response to DNA damage, p53 is rapidly phosphorylated by the ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) protein kinases, resulting in its activation.²⁰ Thereafter, a series of apoptosis-associated proteins including p53-upregulated modulator of apoptosis (PUMA), Bcl2-associated X protein (BAX), BCL-2 antagonist killer (BAK), and B-cell leukemia/lymphoma 2 (BCL-2) were induced by activated p53 protein.^21,22 Previous studies have also demonstrated that activation of p53 signaling pathway caused apoptosis after MCF-7 and T47-D cells exposure to BaP.²³ It was also reported that p53 is involved in regulating necrosis induced by BaP in HepG2 cells.⁶ Other studies also showed that necrotic cell death can occur in a concentration-dependent manner after exposure to BaP for 12 h, in human nonsmallcell lung carcinoma H1299 cells²⁴; moreover, early necrosis and late apoptosis were observed in p53 null liver cell line (Hep3B) after exposure to BaP for 12 and 24 h.⁸ Obviously, both p53-dependent and -independent mechanisms were involved in response to BaP-induced cell death. However, little is known about p53-independent mechanisms in BaP-induced cell death.

The current study sought to analyze the p53-independent mechanisms induced during BaP-induced cell death. Since BPDE is the ultimate metabolite of BaP, BPDE is selected at the concentration of 2.5 μM for treatment of p53-proficient BNL cells and p53-deficient BMK cells. Our results indicated that p53-independent cell necrosis during BPDE-induced cytotoxicity takes the mitochondrial-associated signaling pathways.

Materials and methods

Cell culture

The p53-deficient primary baby mouse kidney (BMK) cells, BMK cells (Bax^−/−), BMK cells (Bak^−/−), and BMK cells (Bax^−/−/Bak^−/−) were prepared as described by Ruley.²⁵ Mouse fetal hepatocyte line (BNL, wild-type (wt) p53) was obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Science (Shanghai, China). BMK cells and BNL cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco BRL, Gaithersburg, Maryland, USA) and in minimum essential medium (MEM) (Thermo Fisher Scientific, Beijing, China), respectively. Both media contained 10% (v/v) fetal bovine serum (FBS). All the cells were incubated at 37°C in a humidified atmosphere containing 5% carbon dioxide.

Reagents and antibodies

Antibodies used in this study were from the following sources: rabbit polyclonal anti-p53, rabbit polyclonal anti-BAX, rabbit polyclonal anti-BAK, and rabbit polyclonal anti-PUMA were supplied by Bioworld Technology (Beverly, Massachusetts, USA); rabbit polyclonal anti-BCL2 was from Santa Cruz Biotechnology (Santa Cruz, California, USA); monoclonal anti-cytochrome c was from Boster Bio-engineering Limited Company (Wuhan, China); monoclonal anti-cyclooxygenase IV was from Molecular Probes (Eugene, Oregon, USA); and all secondary antibodies were from Bioworld Technology. Chemical cross-linker bismaleimidohexane (BMH) was obtained from Pierce (Rockford, Illinois, USA); carbobenzoxy-Val-Ala-Aspfluoromethyl ketone (VAD), carbobenzoxy-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin (DEVD-AFC), and 7-amino-4-trifluoromethyl coumarin (AFC) were purchased from Enzyme Systems Products (Livermore, California, USA); and annexin V- fluorescein isothiocyanate (FITC) apoptosis detection kit was supplied by MultiSciences Biotech (Hangzhou, China); The mitochondria/mitochondria-free cytosol protein isolation kit was supplied by KeyGEN (Nanjing, China). Unless indicated, all other reagents including BPDE were supplied by Sigma (St Louis, Missouri, USA). Lipofectamine 2000 transfection reagent was supplied by Invitrogen (Carlsbad, California, USA).

Cytotoxic activity

BNL and BMK cells were seeded at a density of 5 × 10³ cells per well in 96-well plates for 24 h. BPDE dissolved in dimethyl sulffoxide (DMSO) was added to the cells at a final concentration of 2.5 μM in the culture medium. After incubation, the culture medium in each well was replaced with 90 μL of fresh serum-free medium and 10 μL of (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) (MTT) solution (5 mg/mL in 1× phosphate-buffered saline (PBS), pH=7.5) and incubated at 37°C for further 4 h. The liquid in each well was carefully aspirated, then 150 μL of DMSO was added. The optical density was measured at 570 nm using Multi-Detection Microplate Readers (Synergy 2, BioTek Instruments, Inc, Winooski, Vermont USA). Data are expressed as mean ± SD of three independent experiments performed in six parallel wells.

Flow cytometry

The cell death mode was analyzed by fluorescence-activated cell sorting after annexin V-FITC/propidium iodide (PI) staining. Briefly, the BMK cells were seeded at a density of 3 × 10⁵ cells per well in 6-well plates for 24 h. Then, BPDE dissolved in DMSO was added to the cells at a final concentration of 2.5 μM in the culture medium. After incubation, the harvested cells were trypsinized, washed twice with ice-cold PBS (pH 7.4), and resuspended cell pellet in 500 μL PBS (pH 7.4). The cells were incubated with 5 μL of annexin V-FITC and 10 μL of PI/100 μL PBS used in the dark for 20 min at room temperature. Finally, samples were analyzed with a FACSort flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) within 1 h. The cell lines without BPDE were used as negative controls and simultaneously measured. The percentage of apoptotic and necrotic cells was calculated with the ModFit software (Topsham, Maine, USA). For each sample, 10,000 cells were counted.

Caspase activity measurement

The activity of caspases was evaluated using DEVD-AFC, a fluorogenic peptide substrate. Briefly, the BMK cells were seeded at a density of 5 × 10⁵ cells per well in 6-well plates for 24 h. Then, BPDE dissolved in DMSO was added to the cells at a final concentration of 2.5 μM in the culture medium. After incubation, the cells were extracted with 1% Triton X-100. Then, 25 g protein of the lysates were added to enzymatic reactions containing 50 M DEVD-AFC. After 1 h incubation at 37°C, fluorescence at 360 nm (excitation)/530 nm (emission) was then monitored. For each measurement, a standard curve was constructed using free AFC. Based on the standard curve, the fluorescence intensity reading from each enzymatic reaction was converted into the nanomolar amount of liberated AFC per milligram protein to indicated caspase activity.

Western blotting analysis

To explore the underlying mechanisms of BPDE-induced cell death in this study, expression and or distribution of p53, BAX, BAK, PUMA, BCL-2, and cytochrome c proteins were measured by Western blotting analysis. Briefly, after treatment with the indicated concentrations of BPDE, 1 × 10⁷ BMK cells were harvested for protein extraction. Mitochondrial and mitochondria-free proteins were extracted according to the manufacturer’s protocols. The whole-cell lysate was extracted with a radioimmunoprecipitation assay buffer buffer (1 M Tris–hydrochloric acid, 5 M sodium chloride, 1% Nonidet P-40, 1% sodium deoxycholate, 0.05% sodium dodecyl sulfate (SDS), 1 mM phenylmethyl sulfonyl fluoride. The proteins were denatured at 96°C for 5 min after mixing with 5 μL SDS-loading buffer, separated by 12% SDS-polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, Massachusetts, USA). The membranes were then blocked with Tris-buffered saline containing 5% nonfat milk at 4°C for 1 h, incubated with the specific primary antibodies (1:400 dilution for all) at 4°C overnight, then with the corresponding IR dye-conjugated secondary antibodies (1:5000 dilution for all) at room temperature for 1 h. Membranes were visualized using the Odyssey Infrared Imaging System and Odyssey v1.2 (LI-COR, Lincoln, Nebraska, USA). The relative densities of the protein bands were analyzed using Quantity One software (Bio-Rad, Hercules, California, USA).

Analysis of Bax and Bak homo-oligomerization

BMH, a membrane-permeable, homobifunctional maleimide cross-linker, was used to analyze Bax/Bak oligomerization in this study. Briefly, after treatment with the indicated concentrations of BPDE, 1 × 10⁷ cells were harvested for protein extraction. The membrane fractions were collected by ultracentrifugation at 100,000g for 60 min at 4°C and resuspended in 250 μL of PBS. The resultant membrane fraction was then incubated with 0.2 mM BMH in PBS for 1 h at room temperature with gentle shaking. Finally, the cross-linking mixtures were dissolved in 4× SDS buffer and subjected to SDS-PAGE and analyzed by Western blotting with the antibodies.

Bcl-2-expressing plasmid transfection

To establish a Bcl-2 overexpression BMK cell model, a Bcl-2 express plasmid, pcDNA3-Bcl-2, was constructed.²⁶ For transfection of the plasmid, BMK cells were seeded at 3 × 10⁵ cells/well in 6-well plates. Then, the adherent cells were trypsinized by 0.25% trypsin and diluted in the DMEM. Lipofectamine 2000 transfection reagent was diluted into Opti-MEM I medium (final concentration: 5%) and incubated at room temperature for 10 min. The plasmid was also diluted into Opti-MEM I medium (final concentration: 50 nM). Diluted Lipofectamine 2000 transfection agent and diluted plasmid were combined and incubated at room temperature for an additional 20 min. The transfection mixture was added to six-well plates. Cell suspensions were overlaid onto the transfection complexes. After 6 h incubation, the medium was removed, and the cells were cultured in DMEM-containing 10% FBS.

Statistical analysis

All results are expressed as mean ± SD. All statistical analyses were performed using Statistical Package for Social Sciences version 12.0 for Windows (SPSS Inc., Chicago, Illinois, USA). The values of p < 0.05 were considered statistically significant.

Results

BPDE induced a p53-independent cell death

The cytotoxic effects of 2.5 μM BPDE on BNL and BMK cells were assessed using MTT assay at 2, 4, and 8 h posttreatment. First, the absence of p53 in the BMK cells was confirmed by detecting the expression of p53 and PUMA. The results are shown in Figure 1(a). BMK cells did not express p53 regardless of whether the cells were exposed to BPDE (lanes 3 and 4). As a positive control, p53 was also detected in BNL cells and induced by BPDE (lanes 1 and 2). Additionally, PUMA was also induced in BNL cells, but not in the BMK cells. These analyses confirmed the p53 deficiency in BMK cells, which were used in following experiments to analysis the p53-independent mechanism of BMK cell death during BPDE cytotoxicity. The MTT results are shown in Figure 1(b), in comparison with the control group, BPDE reduced proliferating ratios of the two cell lines in a time-dependent manner (p < 0.01); however, the cell viability in BNL cells were lower than in the p53-deficient BMK cells. For example, after 8 h of 2.5 μM BPDE treatment, the p53-proficient BNL and p53-deficient BMK cells showed 56 and 77% proliferating ratios, respectively. To further discriminate the cell death mode in the p53-deficient BMK cells, flow cytometry was performed. As shown in Figure 1(c) and (d), it was determined a time dependent-BPDE-induced necrosis in BMK cells. No significant cell death was detected during the first 2 h of BPDE treatment (p > 0.05). Significant necrosis was shown from 4 h and increased to 22% at 8 h (p < 0.01). However, there was no significant apoptosis detected during BPDE-induced cytotoxicity in BMK cells (p > 0.05).

Figure 1.

BNL and (or) BMK cells were treated with 2.5 μM BPDE for the indicated times. (a) Confirmation of the p53 deficiency in BMK cells using Western blotting. (b) Cell viability was determined by the MTT assay; mean absorption was normalized to control levels with controls being 100%. (c) Cell death mode of the BMK cell was analyzed by dual parameter flow cytometry utilizing annexin V-FITC and PI. (d) The results of three independent experiments of (c) were pooled and averaged values are shown graphically. The values represent average of three independent experiments with six replicate measurements (mean ± SD). **p < 0.01: compared with the control group. BNL: mouse fetal hepatocyte line; BMT: baby mouse kidney; MTT: (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide); FITC: fluorescein isothiocyanate; BPDE: Benzo(a)pyrene-7,8-diol-9,10-epoxide; PI: propium iodide.

The BPDE-induced necrotic cell death is mitochondria associated

To explore the underlying mechanisms of cell death in the BPDE-induced cytotoxicity, the protein levels and distribution of Bax and cytochrome c were examined by Western blotting. The results showed that Bax translocation to mitochondria and cytochrome c release from the mitochondria were induced in the two cell lines. The relative levels of Bax and cytochrome c were then quantified by densitometric analysis of the Western blotting (Figure 2(c) and (d)). Clearly, BPDE at the concentration of 2.5 μM significantly induced a time-dependent Bax translocation and cytochrome c release in both BNL and BMK cells (8 h: p < 0.01; 4 h: p < 0.05) but at a lower level in the latter, suggesting the BPDE-induced cell death in this study is mitochondria associated.

Figure 2.

BNL and BMK cells were treated with 2.5 μM BPDE for the indicated times. (a) BNL and BMK cells were also fractionated into cytosolic and mitochondrial fractions for analysis distribution of Bax and cytochrome c by Western blotting. (b) Relative level of mitochondrial Bax and cytosolic Bax. (c) Relative level of cytosolic cytochrome c and mitochondrial cytochrome c. Data are presented as mean ± SD. *p < 0.05; **p < 0.01: compared with the control group. BNL: mouse fetal hepatocyte line; BMK: baby mouse kidney; BPDE: Benzo(a)pyrene-7,8-diol-9,10-epoxide.

Caspase activation involved in the BPDE-induced necrotic cell death

To explore the influence of caspases activity on the BPDE-induced necrotic cell death, the activation of caspases were detected. As shown in Figure 3(a), it showed that BPDE induced time-dependent caspase activation in the BMK cells. Caspase activation was significantly induced after 4 h of BPDE treatment and increased more obvious after 8 h of BPDE treatment (p < 0.01). To further determine the involvement of caspases in BMK cell necrosis, we tested the influence of VAD, a peptide inhibitor of caspases with fluoromethyl ketone moiety using flow cytometry. As shown in Figure 3(b) and summarized in Figure 3(c), compared with the control cells (without BPDE treatment), after treatment with BPDE for 8 h, increased necrotic cells (BPDE) were observed (p < 0.01), which were almost completely inhibited by 200 μM VAD (p > 0.05), indicating the BPDE-induced BMK cell necrosis is caspase dependent.

Figure 3.

BMK cells were treated with 2.5 μM BPDE in the absence or presence of 100 μM VAD. (a) Caspase activity during BPDE-treated BMK cells for 0–8 h. (b) Necrotic cell death ratio of the BMK cell was analyzed by dual-parameter flow cytometry utilizing annexin V-FITC and PI. (c) The results of three independent experiments of (b) were pooled and averaged values are shown graphically. Data are presented as mean ± SD. **p < 0.01: compared with untreated control group. VAD: Val-Ala-Aspfluoromethyl ketone; BMK: baby mouse kidney; BPDE: Benzo(a)pyrene-7,8-diol-9,10-epoxide; FITC: fluorescein isothiocyanate; PI: propium iodide.

Oligomerization of Bax and Bak during BPDE-induced BMK cell necrosis

To analyze the molecular oligomerization of Bax and Bak in p53-deficient BMK cells, we extracted mitochondrial lysates for chemical cross-linking to stabilize the oligomers for Western blotting. As shown in Figure 4(a), Bax oligomers were not increased at the first 2 h of BPDE treatment (Bax, lanes 1 and 2). BPDE treatment for 4–8 h increased Bax oligomerization with appearance of a dimer (Bax, lanes 3 and 4). In the same sample, the amount of Bax monomer also increased. These results confirmed the previous results of Bax translocation to mitochondria. Similarly, as shown in Figure 4(a), Bak oligomerized were also increased after 4–8 h of BPDE treatment (Bak, lanes 3 and 4). To further examine the role of Bax and Bak in this experimental model, we compared BPDE-induced necrosis of BMK cells (wt), BMK cells (Bax^−/−), BMK cells (Bak^−/−), and BMK cells (Bax^−/−/Bak^−/−). First, the presence or absence of Bax or (and) Bak in the above cells were confirmed by Western blotting analysis (Figure 4(b)). When the cells were subjected to BPDE treatment, compared with the corresponding control group (without BPDE group), significant necrosis was induced in BMK cells (wt), BMK cells (Bax^−/−), and BMK cells (Bak^−/−; p < 0.01), but not in BMK cells (Bax^−/−/Bak^−/−; p > 0.05); and interestingly, the ratio of necrosis in the BMK cells (wt) is significantly higher than the Bax or (and) Bak-deficient cells (p < 0.01; Figure 4(c)). The necrosis was further confirmed by caspase activity analysis, compared with the corresponding control group (without BPDE group), BPDE significantly increased the caspase activation in BMK cells (wt), BMK cells (Bax^−/−), and BMK cells (Bak^−/− p < 0.01), but not in BMK cells (Bax^−/−/Bak^−/−; p > 0.05); however, compared with the BMK cells (wt), the BPDE-induced caspase activation was significantly reduced in BMK cells (Bax^−/−) or BMK cells (Bak^−/−) and almost completely diminished in BMK cells (Bax^−/−/Bak^−/−; p < 0.01). Taken together, the results suggest that Bax and Bak are interdependent and both were involved in the BPDE-induced BMK cell necrosis.

Figure 4.

(a) Bax and Bak oligomerization in BMK cells (wt) after treatment with 2.5 μM BPDE for 0–8 h. (b) Bax and Bak protein levels in BMK cells (wt; lane 1), BMK cells (Bax^−/−; lane 2), BMK cells (Bak^−/−; lane 3), and BMK cells (Bax^−/−/Bak^−/−; lane 4). (c) Necrotic cell death ratio of BMK cells (wt), BMK cells (Bax^−/−), BMK cells (Bak^−/−), and BMK cells (Bax^−/−/Bak^−/−) after treatment with or without 2.5 μM BPDE for 8 h. (d) Caspase activity of BMK cells (wt), BMK cells (Bax^−/−), BMK cells (Bak^−/−), and BMK cells (Bax^−/−/Bak^−/−) after treatment with or without 2.5 μM BPDE for 8 h. ^## p < 0.01: compared with the corresponding cells without BPDE treatment; **p < 0.01: compared with BPDE-treated BMK cells (wt). BMK: baby mouse kidney; BPDE: Benzo(a)pyrene-7,8-diol-9,10-epoxide; wt: wild-type.

Effects of Bax and Bak on cytochrome c release

We further analyzed the effects of Bax or (and) Bak deficiency on cytochrome c release. As shown in Figure 5(a), cytochrome c was mainly located in mitochondria of the control cells irrespective of their Bax or Bak status (lanes 1, 3, 5, and 7). After BPDE treatment, increased level of cytochrome c was released into the cytosol in BMK cells (wt; lane 2), and the released level was markedly reduced in BMK cells (Bax^−/−), BMK cells (Bak^−/−), and BMK cells (Bax^−/−/Bak^−/−; lanes 4, 6, and 8). We also noticed differences in cytochrome c expression levels in these cell lines. Analysis of the whole-cell lysate showed that BMK cells (Bax^−/−/Bak^−/−) had a lower cytochrome c expression level than other cell types (Figure 5(b)), whereas cyclooxygenase IV, an integral protein of mitochondria, did not show any obvious difference. To semiquantify cytochrome c release, we analyzed the Western blotting by densitometry to calculate the release ratio. As shown in Figure 5(c), BPDE significantly increased the released level of cytochrome c in BMK cells (wt), BMK cells (Bax^−/−), and BMK cells (Bak^−/−; p < 0.01), but not in BMK cells (Bax^−/−/Bak^−/−; p > 0.05); and when compared with the BMK cells (wt), Bax or (and) Bak deficiency significantly reduced BPDE-induced cytochrome c release (p < 0.01).

Figure 5.

BMK cells (wt), BMK cells (Bax^−/−), BMK cells (Bak^−/−), and BMK cells (Bax^−/−/Bak^−/−) were treated with or without 2.5 μM BPDE for 8 h. (a) The four genotype BMK cells were fractionated into cytosolic and mitochondrial fractions for analysis distribution cytochrome c by Western blotting. (b) Whole-cell lysates were also collected to analysis cytochrome c expression level in the four genotype BMK cells. (c) Relative level of cytosolic cytochrome c and mitochondrial cytochrome c. ^## p < 0.01: compared with the corresponding cells without BPDE treatment; **p < 0.01: compared with BPDE-treated BMK cells (wt). BMK: baby mouse kidney; BPDE: Benzo(a)pyrene-7,8-diol-9,10-epoxide.

Effects of Bcl-2 overexpression on BPDE-induced cytochrome c release and necrosis in BMK cells

Finally, the effects of overexpression antagonist of Bax and Bak, Bcl-2, on BPDE-induced necrosis in BMK cells were determined. The transfection efficiency in BMK cells is shown in Figure 6(a), the levels in the transfected cells were fourfold than the control cells (data not shown). The transfected cells were then subjected to BPDE treatment, and the cell viability was revealed by MTT assay. As shown in Figure 6(b), compared with the nontreated control group, cell viabilities in wild-type and Bcl-2-transfected BMK cells were both significantly reduced after BPDE treatment (p < 0.01), but the cell viability in the Bcl-2-transfected BMK cells was significant higher than in the BMK cells (wt; p < 0.05). The above result was further confirmed by flow cytometry after the cells were stained with annexin V and PI staining, as shown in Figure 6(c), and when compared with the control group, BPDE significantly induced 22% necrosis in the BMK cells (wt; p < 0.01); also, the BPDE-induced necrosis was significantly reduced to 13% in Bcl-2 overexpression BMK cells (p < 0.01); these results confirm that Bcl-2 protected the BMK cells from BPDE-induced necrosis in this experimental model.

Figure 6.

Bcl-2 overexpression BMK cell (wt) model was established. (a) The Bcl-2 overexpression in the BMK cells (wt) was confirmed. (b) Cell viability in the BMK cells was determined by the MTT assay after treatment with or without BPDE for 8 h, mean absorption was normalized to control levels with controls being 100%. (c) Necrotic cell death ratio of the BMK cells was analyzed after treatment with or without BPDE for 8 h by dual-parameter flow cytometry utilizing annexin V-FITC and PI. ^## p < 0.01: compared with the corresponding cells without BPDE treatment; **p < 0.01: compared with BPDE-treated BMK cells (wt). BMK: baby mouse kidney; MTT: (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide); BPDE: Benzo(a)pyrene-7,8-diol-9,10-epoxide; wt: wild-type.

Discussion

BaP is a typical DNA damage agent that has been reported to induce cell death through the activation of p53-dependent and/or -independent pathways.^8,27 In agreement with these studies, as the final reactive metabolite of BaP, BPDE, at concentrations of 2.5 µM significantly reduced the viability of the p53-proficient BNL cells and p53-deficient BMK cells 4–8 h posttreatment. The BPDE-induced p53-independent cell death result was further confirmed by flow cytometry, which demonstrated that BPDE at concentrations of 2.5 µM induced necrotic cell death in BMK cells after 4–8 h of treatment. Our results also show that, in the absence of p53, BPDE induces BMK necrosis via the mitochondrial-associated pathway. Under this condition, both Bax and Bak are activated, leading to cytochrome c release from mitochondria and subsequent caspase activation. Moreover, it is shown that BPDE-induced necrosis is attenuated in Bax or/and Bak-deficient cells and also by Bcl-2 overexpression. These findings have provided insights into the p53-independent mechanism of BPDE-induced cell necrosis.

p53 plays pivotal roles in DNA damage response (DDR).²⁸ An early DDR has been revealed, in which ATR and Chk2 signaling has been shown to contribute to p53 activation.²⁹ Among the numerous downstream effectors of p53, several effectors have been suggested to be transcriptionally activated by p53, including PUMA, p21, and others.^30,31 Among them, PUMA have been shown to interact with the prosurvival effector, Bcl-2, at mitochondria to activate Bax/Bak and induce cytochrome c release.³² Of interest, the current study indicates that Bax and Bak can also be activated by BPDE independently of p53, forming oligomers at mitochondria; the latter interacts with the permeability transition pore to induce permeability transition and cytochrome c release. Apparently, both p53-dependent and -independent mechanisms of cell death mode converge at mitochondria during BPDE cytotoxicity. These results are important for understanding the complexity and integration of multiple signaling pathways under p53-deficient or mutant conditions. Together these results suggest that p53-dependent and -independent mechanisms might work in parallel or synergistically to induce cell death.

Bax/Bak and Bcl-2 have proapoptotic or prosurvival activities. They regulate the mitochondrial apoptosis or/and necroptosis pathways through controlling permeabilization of the mitochondrial membrane.^33,34 However, it is unknown how the cell death signal during BPDE cytotoxicity is transferred to Bax/Bak to provoke mitochondrial injury and necrosis in the absence of p53. In this regard, previous studies have suggested several possibilities. In p53-deficient Saos-2 cells, overexpression of p73, a member of the p53 family of proteins, leading to cell death, and interestingly, the p73-induced apoptosis was shown to be mediated by PUMA transactivation, which can activate Bax mitochondrial translocation and cytochrome c release.^35,36 In addition, several protein kinases, such as MEK/ERK, JNK, and PKC (mitogen-activated protein/extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, protein kinase C) were also shown to be provoked in response to DNA damage in p53-mutated or -deficient tumor cells, inducing cell death through the mitochondrial-associated pathways.^37
–39 These results combined with our results indicated that the regulatory mechanisms of p53 dependent and -independent could contribute to the simultaneous activations of Bax/Bak, which may be associated with cell-type specificity and specificity of cell death inducer. And further research should test these possibilities.

Another observation of this study is the interdependence between Bax and Bak for their activation during BPDE-induced necrosis. While Bax and Bak are simultaneously activated in BMK cells (wt), Bax translocation and oligomerization are significantly suppressed in the BMK cells (Bak^−/−), at the same time, Bak translocation and oligomerization are significantly suppressed in the BMK cells (Bax^−/−). And similar interactions were also observed in renal tubular epithelial cells during adenosine triphosphate exhaust and KB-3 human carcinoma cells treated with DNA damage agents.^40,41 Furthermore, the anti-survival protein, Bcl-2, was shown to prevent cell necrosis via blocking the Bax–Bak interaction and cytochrome c release. These findings raised the questions of how the interdependence takes place and whether it is related with their function on mitochondria permeabilization and cell death. A few studies suggested several possibilities to answer the above questions, for instance, Bax and Bak were initially thought to be functionally redundant; several further studies have suggested the possible functional cooperation between Bax and Bak in response to cell death stimuli^40
–42; in another case, TNF-α-induced cytochrome c release and apoptosis in Hela cells were shown to be related not only to homo-oligomerization but also to hetero-oligomerization of Bax and Bak.⁴² These results suggested that Bax and Bak may function cooperatively and interdependently to facilitate mitochondrial membrane disruption, resulting in the release of cell death effectors during BPDE cytotoxicity.

Release of cytochrome c from mitochondria triggers activation of caspase proteases, the latter then participates the ordered dismantling of vital cellular structures, disruption of cellular metabolism, inactivation of cell death-inhibitory proteins, and the activation of other apoptotic effectors; thus, the caspase activation is the key event of mitochondria-dependent apoptosis.⁴³ Multiple lines of evidence suggested that execution of necrosis is a caspase-independent cell death.^44
–46 However, in this study, the activities of caspases were increased during the BPDE-induced BMK cell necrosis. These results strongly suggest that apoptosis and necrosis may share some common effectors. The detailed mechanisms of discrimination between apoptosis and necrosis during BPDE cytotoxicity are needed for further investigation.

In conclusion, the findings demonstrated that BPDE can induce necrosis in p53-deficient BMK cells via mitochondria-associated pathways. In concert with the p53-dependent mechanism, the p53-independent signaling may also activate Bax/Bak to permeabilize the membrane of mitochondria, unleashing the necrotic cascade during BPDE cytotoxicity. Further studies need to be conducted to characterize the p53-independent signaling pathways in response to BPDE-induced necrosis.

Footnotes

Authors’ Note

These authors WZ and NL contributed equally to this work.

Funding

This work was supported by grants from the National Natural Science Foundation of China (No. 82172752); Ministry of Science and Technology, China (No. 2012DFB34320), and National Clinical Key Subject Construction Project.

References

Stahl

Heudorf

Moriske

Polycyclic aromatic hydrocarbons (PAH) in an indoor environment. Sources, exposure and risk evaluation. Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz 2004 47(9): 868–81.

Darwish

Ikenaka

Eldaly

Mutagenic activation and detoxification of benzo[a]pyrene in vitro by hepatic cytochrome P450 1A1 and phase II enzymes in three meat-producing animals. Food Chem Toxicol 2011; 48(8–9): 2526–2531.

Montero

Serrano

Davila

Infection of rats with Taenia taeniformis metacestodes increases hepatic CYP450, induces the activity of CYP1A1, CYP2B1 and COH isoforms and increases the genotoxicity of the procarcinogens benzo[a]pyrene, cyclophosphamide and aflatoxin B(1). Mutagenesis 2003; 18(2): 211–216.

Said

Ross

Salib

Modulation of DNA adduct formation by successive exposures of DNA to small and bulky chemical carcinogens. Carcinogenesis 1995; 16(12): 3057–3062.

Pliskova

Vondracek

Vojtesek

Deregulation of cell proliferation by polycyclic aromatic hydrocarbons in human breast carcinoma MCF-7 cells reflects both genotoxic and nongenotoxic events. Toxicol Sci 2005; 83(2): 246–256.

Lin

Yang

. Benzo[a]pyrene-induced necrosis in the HepG(2) cells via PARP-1 activation and NAD(+) depletion. Toxicology 2008; 245(1–2): 147–153.

Burke

Wei

. Synergistic damage by UVA radiation and pollutants. Toxicol Ind Health 2009; 25(4–5): 219–224.

Jiang

Zhou

Chen

Benzo(a)pyrene-induced mitochondrial dysfunction and cell death in p53-null Hep3B cells. Mutat Res 2012; 726(1): 75–83.

Houghton

. Apoptosis and drug response. Curr Opin Oncol 1999; 11(6): 475–481.

10.

Vaithinathan

Saradha

Mathur

. Methoxychlor induces apoptosis via mitochondria- and FasL-mediated pathways in adult rat testis. Chem Biol Interact 2012; 185(2): 110–118.

11.

Yaoxian

Hui

Yunyan

Emodin induces apoptosis of human cervical cancer hela cells via intrinsic mitochondrial and extrinsic death receptor pathway. Canc Cell Int 2013; 13(1): 71.

12.

Darzynkiewicz

Zhao

Halicka

DNA damage signaling assessed in individual cells in relation to the cell cycle phase and induction of apoptosis. Crit Rev Clin Lab Sci 2012; 49(5–6): 199–217.

13.

Messner

Frotschnig

Steinacher-Nigisch

Apoptosis and necrosis: two different outcomes of cigarette smoke condensate-induced endothelial cell death. Cell Death Dis 2012; 3: e424.

14.

Wochna

Niemczyk

Kurono

A possible role of oxidative stress in the switch mechanism of the cell death mode from apoptosis to necrosis--studies on rho0 cells. Mitochondrion 2007; 7(1–2): 119–124.

15.

Karch

Kwong

Burr

Bax and Bak function as the outer membrane component of the mitochondrial permeability pore in regulating necrotic cell death in mice. Elife 2012; 2: e00772.

16.

Gao

Effect of spider venom on cell apoptosis and necrosis rates in MCF-7 cells. DNA Cell Biol 2007; 26(7): 485–489.

17.

Leung

Redlak

Miller

. Oxygen radical induced gastric mucosal cell death: apoptosis or necrosis? Dig Dis Sci 2008; 53(9): 2429–2435.

18.

Callen

Jankovic

Wong

Essential role for DNA-PKcs in DNA double-strand break repair and apoptosis in ATM-deficient lymphocytes. Mol Cell 2009; 34(3): 285–297.

19.

Elias

Dimitrio

Clairambault

The p53 protein and its molecular network: modelling a missing link between DNA damage and cell fate. Biochim Biophys Acta 2014; 1844: 232–247.

20.

Biton

Gropp

Itsykson

ATM-mediated response to DNA double strand breaks in human neurons derived from stem cells. DNA Repair (Amst) 2007; 6(1): 128–134.

21.

Galluzzi

Morselli

Kepp

Targeting p53 to mitochondria for cancer therapy. Cell Cycle 2008; 7(13): 1949–1955.

22.

Ding

Chen

A coordinated action of Bax, PUMA, and p53 promotes MG132-induced mitochondria activation and apoptosis in colon cancer cells. Mol Canc Ther 2007; 6(3): 1062–1069.

23.

Sadikovic

Rodenhiser

. Benzopyrene exposure disrupts DNA methylation and growth dynamics in breast cancer cells. Toxicol Appl Pharmacol 2006; 216(3): 458–468.

24.

Jiang

Rao

Yang

Benzo(a)pyrene induces p73 mRNA expression and necrosis in human lung adenocarcinoma H1299 cells. Environ Toxicol 2011; 27(4): 202–210.

25.

Ruley

. Adenovirus early region 1A enables viral and cellular transforming genes to transform primary cells in culture. Nature 1983; 304(5927): 602–606.

26.

Guo

. Bcl-2 over-expression and activation of protein kinase C suppress the TRAIL-induced apoptosis in Jurkat T cells. Cell Res 2001; 11(2): 101–106.

27.

Lin

Mak

Yang

. MAPK regulate p53-dependent cell death induced by benzo[a]pyrene: involvement of p53 phosphorylation and acetylation. Toxicology 2008; 247(2–3): 145–153.

28.

Meek

Anderson

. Posttranslational modification of p53: cooperative integrators of function. Cold Spring Harb Perspect Biol 2009; 1(6): a000950.

29.

Efeyan

Serrano

. p53: guardian of the genome and policeman of the oncogenes. Cell Cycle 2007; 6(9): 1006–1010.

30.

Wang

Zhang

Both p53-PUMA/NOXA-Bax-mitochondrion and p53-p21cip1 pathways are involved in the CDglyTK-mediated tumor cell suppression. Biochem Biophys Res Commun 2009; 386(4): 607–611.

31.

Lee

Kim

Zhang

Role of p53, PUMA, and Bax in wogonin-induced apoptosis in human cancer cells. Biochem Pharmacol 2008; 75(10): 2020–2033.

32.

Gallenne

Gautier

Oliver

Bax activation by the BH3-only protein Puma promotes cell dependence on antiapoptotic Bcl-2 family members. J Cell Biol 2009; 185(2): 279–290.

33.

Fletcher

Meusburger

Hawkins

Apoptosis is triggered when prosurvival Bcl-2 proteins cannot restrain Bax. Proc Natl Acad Sci USA 2008; 105(47): 18081–18087.

34.

Bossowski

Analysis of intracellular proapoptotic (Bax, Bak) and antiapoptotic (Bcl-2, Bcl-XL) proteins expression in thyrocytes from young patients with immune and non-immune thyroid disorders. Pediatr Endocrinol Diabetes Metab 2007; 13(2): 63–70.

35.

Nicotera

Melino

. Regulation of the apoptosis-necrosis switch. Oncogene 2004; 23(16): 2757–2765.

36.

Ramadan

Terrinoni

Catani

p73 induces apoptosis by different mechanisms. Biochem Biophys Res Commun 2005; 331(3): 713–717.

37.

Bae

Kang

Yoon

Cellular components involved in the cell death induced by cisplatin in the absence of p53 activation. Oncol Rep 2006; 15(5): 1175–1180.

38.

Lasfer

Protein kinase PKC delta and c-Abl are required for mitochondrial apoptosis induction by genotoxic stress in the absence of p53, p73 and Fas receptor. FEBS Lett 2006; 580(11): 2547–2552.

39.

Park

De Leon

Devarajan

. Cisplatin induces apoptosis in LLC-PK1 cells via activation of mitochondrial pathways. J Am Soc Nephrol 2002; 13(4): 858–865.

40.

Mikhailov

Degenhardt

Association of Bax and Bak homo-oligomers in mitochondria. Bax requirement for Bak reorganization and cytochrome c release. J Biol Chem 2003; 278(7): 5367–5376.

41.

Upreti

Chu

Galitovskaya

Key role for Bak activation and Bak-Bax interaction in the apoptotic response to vinblastine. Mol Cancer Ther 2008; 7(7): 2224–2232.

42.

Sundararajan

Cuconati

Nelson

Tumor necrosis factor-alpha induces Bax-Bak interaction and apoptosis, which is inhibited by adenovirus E1B 19K. J Biol Chem 2001; 276(48): 45120–45127.

43.

Martinou

Desagher

Antonsson

. Cytochrome c release from mitochondria: all or nothing. Nat Cell Biol 2000; 2(3): E41–E43.

44.

Wilson

Browning

. Death of HT29 adenocarcinoma cells induced by TNF family receptor activation is caspase-independent and displays features of both apoptosis and necrosis. Cell Death Differ 2002; 9(12): 1321–1333.

45.

Iwasaki

Ito

Ishida

Catechin, green tea component, causes caspase-independent necrosis-like cell death in chronic myelogenous leukemia. Canc Sci 2009; 100(2): 349–356.

46.

Artus

Boujrad

Bouharrour

AIF promotes chromatinolysis and caspase-independent programmed necrosis by interacting with histone H2AX. Embo J 2012; 29(9): 1585–1599.

Benzo(a)pyrene-7,8-diol-9,10-epoxide induced p53-independent necrosis via the mitochondria-associated pathway involving Bax and Bak activation

Abstract

Keywords

Introduction

Materials and methods

Cell culture

Reagents and antibodies

Cytotoxic activity

Flow cytometry

Caspase activity measurement

Western blotting analysis

Analysis of Bax and Bak homo-oligomerization

Bcl-2-expressing plasmid transfection

Statistical analysis

Results

BPDE induced a p53-independent cell death

The BPDE-induced necrotic cell death is mitochondria associated

Caspase activation involved in the BPDE-induced necrotic cell death

Oligomerization of Bax and Bak during BPDE-induced BMK cell necrosis

Effects of Bax and Bak on cytochrome c release

Effects of Bcl-2 overexpression on BPDE-induced cytochrome c release and necrosis in BMK cells

Discussion

Footnotes

Authors’ Note

Funding

References