Depletion of cytosolic or mitochondrial thioredoxin increases CYP2E1-induced oxidative stress via an ASK-1-JNK1 pathway in HepG2 cells

Abstract

Thioredoxin is an important reducing molecule in biological systems. Increasing CYP2E1 activity induces oxidative stress and cell toxicity. However, whether thioredoxin protects cells against CYP2E1-induced oxidative stress and toxicity is unknown. SiRNA were used to knockdown either cytosolic (TRX-1) or mitochondrial thioredoxin (TRX-2) in HepG2 cells expressing CYP2E1 (E47 cells) or without expressing CYP2E1 (C34 cells). Cell viability decreased 40-60% in E47 but not C34 cells with 80-90% knockdown of either TRX-1 or TRX-2. Depletion of either thioredoxin also potentiated the toxicity produced either by a glutathione synthesis inhibitor or by TNFα in E47 cells. Generation of reactive oxygen species and 4-HNE protein adducts increased in E47 but not C34 cells with either thioredoxin knockdown. GSH was decreased and adding GSH completely blocked E47 cell death induced by either thioredoxin knockdown. Lowering TRX-1 or TRX-2 in E47 cells caused an early activation of ASK-1, followed by phosphorylation of JNK1 after 48 h of siRNA treatment. A JNK inhibitor caused a partial recovery of E47 cell viability after thioredoxin knockdown. In conclusion, knockdown of TRX-1 or TRX-2 sensitizes cells to CYP2E1-induced oxidant stress partially via ASK-1 and JNK1 signaling pathways. Both TRX-1 and TRX-2 are important for defense against CYP2E1-induced oxidative stress.

Fig. 1. Knockdown of cytosolic or mitochondrial thioredoxin by either TRX-1 or TRX-2 siRNA

C34 and E47 cells were treated with TRX-1 siRNA, or TRX-2 siRNA, or both TRX-1 plus TRX-2 siRNA, or control siRNA for 72 hrs. Cells were harvested and lysed. SDS-PAGE was performed to detect the expression of TRX-1, TRX-2, and CYP2E1.

Fig. 2. Effect of thioredoxin knockdown on E47 and C34 cell viability

C34 and E47 cells were treated with TRX-1 siRNA, or TRX-2 siRNA, or both TRX-1 and TRX-2 siRNA, or control siRNA for 72 hours. Cell viability was detected by MTT assay and by cell morphology. A. Cell viability was expressed as the percentage of viable cells for the specific siRNA treatment to the control siRNA group in C34 and E47 cells respectively. * p<0.05 comparing specific siRNA treatment groups to control siRNA group in C34 and E47 cells respectively, n=3. B. Images were taken with the light microscope at 10× magnitude. C. PI staining of E47 cells under fluorescence and light microscopy. Images were merged from light and fluorescence microscopy. PI staining positive cells were shown as white dots. D. Annexin V staining of E47 cells by flow cytometry. E47 cells were treated with specific siRNA for 72 hrs, trypsinized from the plate and stained with Annexin V. Fluorescence was analyzed by flow cytometry under FITC channel.

Fig. 2. Effect of thioredoxin knockdown on E47 and C34 cell viability

C34 and E47 cells were treated with TRX-1 siRNA, or TRX-2 siRNA, or both TRX-1 and TRX-2 siRNA, or control siRNA for 72 hours. Cell viability was detected by MTT assay and by cell morphology. A. Cell viability was expressed as the percentage of viable cells for the specific siRNA treatment to the control siRNA group in C34 and E47 cells respectively. * p<0.05 comparing specific siRNA treatment groups to control siRNA group in C34 and E47 cells respectively, n=3. B. Images were taken with the light microscope at 10× magnitude. C. PI staining of E47 cells under fluorescence and light microscopy. Images were merged from light and fluorescence microscopy. PI staining positive cells were shown as white dots. D. Annexin V staining of E47 cells by flow cytometry. E47 cells were treated with specific siRNA for 72 hrs, trypsinized from the plate and stained with Annexin V. Fluorescence was analyzed by flow cytometry under FITC channel.

Fig. 3. Thioredoxin knockdown potentiated E47 cell toxicity by BSO and TNFα

After 24 hrs of TRX-1 siRNA, or TRX-2 siRNA, or control siRNA treatment, C34 (A and C) and E47 (B and D) cells were treated with either 200µM of BSO (A and B) or 2ng/ml TNFα plus 10ng/ml cycloheximide (C and D) along with the appropriate siRNA for another 48 hrs. Cell viability was expressed as the percentage of viable cells for the specific siRNA treatment to the control siRNA group in C34 and E47 cells respectively. * p<0.05 comparing either plus BSO or plus TNFα treatment group to either minus BSO or minus TNFα group with corresponding siRNA treatment, n=3. & p<0.05 represents TRX-1 or TRX-2 siRNA treatment compared to control siRNA treatment in minus BSO or TNFα treatment group in corresponding cell type, n=3. # p<0.05 represents TRX-1 or TRX-2 siRNA treatment compared to control siRNA treatment in plus BSO or TNFα treatment group in corresponding cell type, n=3.

Fig. 4. Effect of thioredoxin knockdown on total glutathione levels. A

C34 and E47 cells were treated with TRX-1 siRNA, or TRX-2 siRNA, or both, or control siRNA for 72 hours. Cells were trypsinized and sonicated and assayed for total glutathione. Glutathione level in each group was expressed as the value relative to that of the control siRNA treatment group in E47 cells. * p<0.05 comparing specific siRNA group to control siRNA in E47 cells, n=3. B. E47 cells grown on 96-well plates were treated with either TRX-1, or TRX-2 siRNA, or both, or control siRNA for 72 hours. At 24 hrs, 5mM glutathione ethyl ester was added into the culture medium and the cells were incubated along with the appropriate siRNAs for 48 hrs before MTT assay was performed. Cell viability was expressed as viability in each thioredoxin siRNA treatment group relative to that in the control siRNA group. * p<0.05 comparing specific siRNA group to control siRNA group, n=3. & p<0.05 comparing the plus GSSE group to the minus GSSE group in the corresponding siRNA treatment, n=3.

Fig. 5. Effect of thioredoxin knockdown on ROS production in E47 cells and C34 cells

C34 and E47 cells were treated with TRX-1 siRNA, or TRX-2 siRNA, or both, or control siRNA for 72 hours. Cells were grown on glass cover slides for fluorescence microscopy, and on 6-well plates for flow cytometry or for spectrofluorometry assay. Total ROS was detected with a Total ROS Detection Kit as described in ‘Methods’. A. Total ROS detection by fluorescence microscopy. B. total ROS detected by flow cytometry. B1–B3: C34 cells; B4–B6: E47 cells. C. Spectrofluorimetry of ROS. Arbitrary units of fluorescence produced by E47 and C34 cells in the presence of control, TRX-1, TRX-2, and TRX-1 plus TRX-2 siRNA. P<0.05 compared to control siRNA. D. Dihydroethidium and E. mitoSOX fluorescence as an assay for cytosolic and mitochondrial production of superoxide, respectively. C34 and E47 cells were treated with control siRNA or TRX-1 siRNA or TRX-2 siRNA or TRX-1 plus TRX-2 siRNA for 72 hrs followed by the addition of either 40µM DHE or 5µM mitoSOX. Images of mitoSOX were the merged ones of light microscopy and fluorescence microscopy. Examples of mitoSOX red positive cells were pointed with white arrow.

Fig. 5. Effect of thioredoxin knockdown on ROS production in E47 cells and C34 cells

C34 and E47 cells were treated with TRX-1 siRNA, or TRX-2 siRNA, or both, or control siRNA for 72 hours. Cells were grown on glass cover slides for fluorescence microscopy, and on 6-well plates for flow cytometry or for spectrofluorometry assay. Total ROS was detected with a Total ROS Detection Kit as described in ‘Methods’. A. Total ROS detection by fluorescence microscopy. B. total ROS detected by flow cytometry. B1–B3: C34 cells; B4–B6: E47 cells. C. Spectrofluorimetry of ROS. Arbitrary units of fluorescence produced by E47 and C34 cells in the presence of control, TRX-1, TRX-2, and TRX-1 plus TRX-2 siRNA. P<0.05 compared to control siRNA. D. Dihydroethidium and E. mitoSOX fluorescence as an assay for cytosolic and mitochondrial production of superoxide, respectively. C34 and E47 cells were treated with control siRNA or TRX-1 siRNA or TRX-2 siRNA or TRX-1 plus TRX-2 siRNA for 72 hrs followed by the addition of either 40µM DHE or 5µM mitoSOX. Images of mitoSOX were the merged ones of light microscopy and fluorescence microscopy. Examples of mitoSOX red positive cells were pointed with white arrow.

Fig. 6. Effect of thioredoxin knockdown on 4-HNE expression in E47 cells and C34 cells

Cells grown on cover slides were treated with TRX-1 siRNA, or TRX-2 siRNA, or both, or control siRNA for 72 hrs. Cells were fixed with 10% formalin and stained for 4-HNE as described in ‘Methods’. Images were taken with the light microscope at 10× magnitude.

Fig. 7. Effect of thioredoxin knockdown on ASK-1 phosphorylation in E47 cells

E47 cells grown on cover slides or on 6-well plates were treated with TRX-1 siRNA, or TRX-2 siRNA, or both, or control siRNA for 5, 24, 48 and 72 hrs. A. Cells were fixed with 10% formalin and stained for p-ASK-1 as described in ‘Methods’. Images were taken using a fluorescence microscope at 20× magnitude. B. Cell lysates were prepared and western blots were carried out to detect p-ASK-1 and ASK-1 levels. Numbers under the blots refer to the ratio of p-ASK-1/ASK-1. Images were quantified with the Image J program.

Fig. 7. Effect of thioredoxin knockdown on ASK-1 phosphorylation in E47 cells

E47 cells grown on cover slides or on 6-well plates were treated with TRX-1 siRNA, or TRX-2 siRNA, or both, or control siRNA for 5, 24, 48 and 72 hrs. A. Cells were fixed with 10% formalin and stained for p-ASK-1 as described in ‘Methods’. Images were taken using a fluorescence microscope at 20× magnitude. B. Cell lysates were prepared and western blots were carried out to detect p-ASK-1 and ASK-1 levels. Numbers under the blots refer to the ratio of p-ASK-1/ASK-1. Images were quantified with the Image J program.

Fig. 8. Effect of thioredoxin knockdown on the activation of JNK in E47 cells

E47 cells grown on 6-well plates were treated with TRX-1 siRNA or TRX-2 siRNA separately or both together, or control siRNA for 5, 24, 48 and 72 hrs. Cells were lysed and western blot was performed for the analysis of A: pJNK, JNK; B: pP38, and p38 MAPK as described in ‘Methods’. Images were quantified with the Image J program. The ratio of the phosphorylated JNK1 to the total JNK1 was expressed relative to the value of the control siRNA group at the 48 hrs time point as shown in the bar graph. * p<0.05 comparing the ratio of pJNK1/JNK1 in specific siRNA to that in control siRNA group after 48 hrs siRNA treatment, n=4. C: Western blot for p-cJUN and cJUN was carried out at 72 hrs after siRNA treatment. Numbers under the blots refer to the p-cJUN/cJUN ratio.

Fig. 9. A JNK inhibitor partially protects E47 cells from loss of viability from thioredoxin knockdown

E47 cells were treated with 5 µM of the JNK inhibitor- L-JNKI1 for 3 hours, then cells were treated with TRX-1 siRNA, TRX-2 siRNA or both or control siRNA for another 72 hours. E47 cells were also treated in parallel in the absence of L-JNKI1. A: Cell viability was performed with the MTT assay. Cell viability was expressed as the percentage of each siRNA in the absence or presence of the JNK inhibitor with the control siRNA taken as 100% viability. * p<0.05 comparing the specific siRNA treatment group to the control siRNA group without L-JNKI1 treatment, n=3. & p<0.05 comparing the plus L-JNKI1 group to the minus L-JNKI1 group with corresponding siRNA treatment, n=3. # p<0.05 comparing the specific siRNA treatment group to the control siRNA group with L-JNKI1 treatment, n=3. B: Inhibition of JNK activiation by L-JNKI1. Immunoblots for p-JNK and JNK in the absence or presence of L-JNKI1 were carried out 48 hrs after thioredoxin knockdown. Numbers under the blots refer to the p-JNK/JNK ratio. The minus JNKi results are repeated from Fig. 8 48 hrs data in order to show the effectiveness of the JNK inhibitor in blunting JNK activation.

Fig. 10. Effect of thioredoxin knockdown on the expression of anti-oxidant enzymes

C34 and E47 cells were treated with TRX-1 siRNA, or TRX-2 siRNA, or both, or control siRNA for 72 hrs. Cells were lysed and Western Blots to detect SOD-1, SOD-2, GPX4, and catalase were performed as described in ‘Methods’. Images were quantified with the Image J program. Only the SOD-1/β-actin ratios showed some difference between the thioredoxin knockdown versus the control siRNA (bar graph). * p<0.05 comparing specific siRNA treatment to control siRNA group, n=4.

Fig. 11. Effect of thioredoxin knockdown on autophagy and ER stress

E47 cells were treated with control siRNA, or TRX-1 siRNA, or TRX-2 siRNA or TRX-1 plus TRX-2 siRNA for 72 hrs. Cell lysates were prepared and immunoblots for detection of autophagy markers LC3-I and LC3-II or ER stress markers PDI, p-EIF2α, and GRP-78 were carried out. There was no effect of thioredoxin knockdown on the LC3-II/LC3-I ratio or on level of PDI, p-EIF2α, or GRP-78.