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. 2013;8(3):e59330.
doi: 10.1371/journal.pone.0059330. Epub 2013 Mar 22.

Endoplasmic reticulum stress signaling is involved in mitomycin C (MMC)-induced apoptosis in human fibroblasts via PERK pathway

Kun Shi  1 , Daode Wang, Xiaojian Cao, Yingbin Ge
Affiliations

Endoplasmic reticulum stress signaling is involved in mitomycin C (MMC)-induced apoptosis in human fibroblasts via PERK pathway

Kun Shi et al. PLoS One. 2013.

Abstract

Endoplasmic reticulum (ER) stress-mediated cell apoptosis has been implicated in various cell types, including fibroblasts. Previous studies have shown that mitomycin C (MMC)-induced apoptosis occurs in fibroblasts, but the effects of MMC on ER stress-mediated apoptosis in fibroblasts have not been examined. Here, MMC-induced apoptosis in human primary fibroblasts was investigated by exposing cells to a single dose of MMC for 5 minutes. Significant inhibition of cell proliferation and increased apoptosis were observed using a cell viability assay, Annexin V/propidium iodide double staining, cell cycle analysis, and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining. Upregulation of proapoptotic factors, including cleaved caspase-3 and poly ADP-ribose polymerase (PARP), was detected by Western blotting. MMC-induced apoptosis was correlated with elevation of 78-kDa glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP), which are hallmarks of ER stress. Three unfolded protein response (UPR) sensors (inositol-requiring enzyme 1, IRE1; activating transcription factor 6, ATF6; and PKR-like ER kinase, PERK) and their downstream signaling pathways were also activated. Knockdown of CHOP attenuated MMC-induced apoptosis by increasing the ratio of BCL-2/BAX and decreasing BIM expression, suggesting that ER stress is involved in MMC-induced fibroblast apoptosis. Interestingly, knockdown of PERK significantly decreased ER stress-mediated apoptosis by reducing the expression of CHOP, BIM and cleaved caspase-3. Reactive oxygen species (ROS) scavenging also decreased the expression of GRP78, phospho-PERK, CHOP, and BIM. These results demonstrate that MMC-induced apoptosis is triggered by ROS generation and PERK activation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Application of MMC for 5 minutes induces apoptosis in human fibroblasts.
(A) Cell viability after different treatments was detected by using the CCK-8 assay. MMC-induced growth inhibition occurred in a dose- and time-dependent manner. (B) Apoptosis analysis was assessed via Annexin V/propidium iodide double staining. Cells were exposed to 0.4 mg/ml MMC for 5 minutes and then incubated for the corresponding indicated times. Apoptosis rates were determined via flow cytometry analysis. The cells shown in the bottom right quadrant were Annexin V-FITC positive and propidium iodide negative, indicating an early stage of apoptosis. The cells in the top right quadrant stained positively for Annexin V-FITC and propidium iodide, indicating that they consisted of secondary late apoptotic/necrotic cells. The total percentage of cell death is shown in bold. Statistical analysis of the total recorded apoptotic cells was performed, and the results are shown in the bar graphs. (C) Cell cycle analysis was conducted to detect the changes in the cell cycle at different time points after MMC treatment (0.4 mg/ml, 5 minutes). (D) Cells were TUNEL stained 48 hours after MMC treatment and then observed using a fluorescence microscope. Nuclei are shown in blue, and TUNEL staining is shown in green. (E) Western blots revealed that MMC induces cleavage of caspase-3 from a size of 35 kDa to 17 kDa and the degradation of poly ADP-ribose polymerase (PARP) by the cleaved caspase-3 into 85 to 90 kDa fragments. β-actin was included as a control. Gels were run in triplicate. The histograms in panels A, B, C and D represent the mean ±SD of three independent experiments. *P<0.05, **P<0.01 versus 0 or the control group.
Figure 2
Figure 2. MMC induces ER stress and the UPR.
(A) Relative CHOP and GRP78 mRNA levels were detected by quantitative real-time PCR and normalized to GAPDH mRNA levels. These values were compared with the 0 hour group. The values represent the mean ± SD of three separate experiments. *P<0.05, **P<0.01. (B-C) The doses- and time-dependent effects of MMC on ER stress-associated proteins. Cells were treated with different concentrations of MMC, respectively, for 5 minutes and then cultured for 24 hours (phospho-IRE1 and IRE1 were detected at 9 hours) or treated with 0.4 mg/ml MMC for 5 minutes and cultured for different time intervals. The expression of GRP-78, P-IRE1, IRE1, P-PERK, PERK, P-eIF2α, eIF2α, CHOP, ATF6 and β-actin (loading control) was analyzed by Western blotting. (D) MMC-induced alternative splicing of XBP1 mRNA was examined via RT-PCR. The gel data shown in panels B, C and D are from experiments performed in triplicate with similar results (P- represents phospho-).
Figure 3
Figure 3. CHOP is essential for MMC-induced apoptosis.
Fibroblasts were transfected with CHOP or control siRNA (non-targeting siRNA). After treatment with 0.4 mg/ml MMC for 48 hours, the cells were analyzed in a number of assays. (A) Cell viability was assessed with the CCK-8 assay. (B) Annexin V/propidium iodide double staining was performed to detect the apoptosis rate. (C) TUNEL-stained cells were observed under a fluorescence microscope. (D) Whole-cell lysates were used for Western blotting with antibodies specific for CHOP, BAX, BCL-2, BIM and β-actin (loading control). This experiment was performed in triplicate. (E) The band intensities for CHOP, BCL-2/BAX and BIM were expressed as a histogram relative to β-actin. The control group (no MMC treatment) was normalized to a value of 1.0-fold. The data in panels A, B, C and E are the mean ± SD of at least three independent experiments. *P<0.05, **P<0.01 versus the control group (with MMC treatment).
Figure 4
Figure 4. The role of three UPR sensors in MMC-induced apoptosis in fibroblasts.
Fibroblasts were transfected with the PERK, ATF6, IRE1 or non-targeting lentiviral-mediated shRNAs. (A) The expression levels of the targeted transcripts were determined by Western blotting with PERK, ATF6, IRE1 and β-actin antibodies. The presented data represents the results of two independent experiments. (B) Transfected cells were treated with MMC as described in the text and then cell viability was measured after 48 hours. (C) Apoptosis rates were determined via Annexin V/propidium iodide double staining and are shown in the bar graph. (D) After MMC treatment (0.4 mg/ml, 5 minutes) and incubation for 48 hours, equal amounts of the whole cell lysates were analyzed by Western blotting with antibodies specific for CHOP, BIM, cleaved caspase-3, and β-actin (loading control). This experiment was performed in triplicate. The data presented in panels B and C are the mean ± SD of three independent experiments, *P<0.05, ***P<0.001.
Figure 5
Figure 5. The IRE1 pathway is not necessary for MMC-induced JNK activation.
(A) JNK activation in response to MMC treatment (0.4 mg/ml, 5 minutes) was detected at various times by Western blotting. (B) Fibroblasts were pretreated with a 5 µM concentration of the JNK inhibitor SP600125, then treated with MMC (0.4 mg/ml, 5 minutes). The expression of P-JNK, JNK, and β-actin (loading control) was measured by Western blotting 24 hours after MMC treatment. (C) Cell viability and (D) apoptosis rates were quantified using CCK-8 kit assays and Annexin V/propidium iodide double staining, 48 hours after MMC treatment. The histograms represent the mean ± SD of three independent experiments. **P<0.01. (E) Fibroblasts were transfected with encoded IRE1 shRNA or non-targeting shRNA (control shRNA) and then treated with MMC (0.4 mg/ml, 5 minutes). Whole cell lysates were prepared 24 h after MMC treatment and subjected to Western blotting to detect P-JNK, JNK and β-actin (loading control). The results from the gels shown in panels A, B and E are from experiments performed in triplicate with similar results.
Figure 6
Figure 6. Increases in ROS induced by MMC triggers ER stress that can be blocked by antioxidants.
(A) ROS levels were detected by using DCFH-DA and observed via fluorescence microscopy (200x magnification) 48 hours after MMC treatment. Nuclei are shown in blue and ROS staining in green. The fibroblasts were pretreated with 10 mM NAC, 10 mM GSH and 10 µM edaravone for 2 hours, respectively, then treated with MMC (0.4 mg/ml, 5 minutes) and incubated for 48 hours. (B) MDA generation was measured and is shown in the histogram. (C) The mean of DCF fluorescence intensity, which is indicative of ROS generation, was measured via flow cytometry and is depicted graphically as the relative fold increase of the control. (D) Cell viability and (E) apoptosis rates were examined 48 hours after MMC treatment using CCK-8 assays or Annexin V/propidium iodide double staining. (F) Western blot analysis of related proteins in fibroblasts 24 hours after MMC treatment. The expression of GRP-78, P-PERK, PERK, CHOP, BIM, cleaved caspase-3, PARP, cleaved PARP and β-actin (loading control) was analyzed. (G) The band intensities for GRP-78, P-PERK, PERK, CHOP, BIM, cleaved caspase-3, and cleaved PARP are shown as a histogram. The control group without MMC treatment was normalized to a value of 1.0-fold. The gel data results from the gels come from experiments performed in triplicate with similar results. The presented bar graphs shown in panels B, C, D and E are the average results from three different experiments. *P<0.05, **P<0.01, ***P<0.001 versus the control group (without MMC), #P<0.05, ##P<0.01, ###P<0.001 versus the MMC treatment group (without antioxidants).
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This work was supported by the National Natural Science Foundation of China, grants #30973058 and #81171694 (http://www.nsfc.gov.cn/Portal0/default166.htm, http://nsfc.pubmed.cn/b7c13b133, http://nsfc.pubmed.cn/042d8caa1); Jiangsu Province Nature Science Foundation (BE2010743), http://www.jsnsf.gov.cn/Index.aspxl; Program for Development of Innovative Research Team in the First Affiliated Hospital of NJMU (No. IRT-015), http://www.jsph.org.cn/art/2011/3/8/art_674_3373.html, http://www.jsph.net/; A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions(JX10231801). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.