Dichloroacetate reduces cisplatin-induced apoptosis by inhibiting the JNK/14-3-3/Bax/caspase-9 pathway and suppressing caspase-8 activation via cFLIP in murine tubular cells

Figures

• 

  Fig. 1. DCA reduced cisplatin-induced apoptosis by suppressing caspase-3 and -8 activation in mProx cells. mProx cells were incubated for 24 h with growth buffer containing cisplatin (0 or 25 µM) in the presence or absence of DCA (A: 1, 3, 5, 10, and 20 mM; B–E: 10 mM). Cleaved caspase-3 (A, B) and caspase-8 (E) levels in cell lysates were determined by immunoblot analyses in mProx cells treated as indicated. A representative blot is shown in the upper panel (A, B, E). (C–D) Harvested cells were stained with Annexin V-FITC and propidium iodide (PI), and analyzed by flow cytometry using fluorescence-activated cell sorting scan. Percentages of Q2 and Q4 were calculated based on the data from each well sample. Individual data are expressed as dot plots with the mean ± SEM of a representative experiment (n = 3). *, P < 0.05 and **, P < 0.01 compared with cisplatin treatment alone (A) or under the indicated conditions (B, D, E), according to one-way ANOVA with Scheffe’s post hoc comparisons.

• 

  Fig. 2. DCA reduced cisplatin-induced caspase-3 activation by inhibiting the JNK/14-3-3/Bax/caspase-9 pathway. mProx cells were treated for 16–24 h with growth buffer containing cisplatin (0 or 25 µM) in the presence or absence of DCA (10 mM). Whole cell lysates and cytosolic and mitochondrial fractions were prepared as described in the Materials and Methods. Levels of p53 and phosphorylated p53 (p-p53) (A), mitochondrial Bax and Cox IV (B), cleaved caspase-9 and β-actin (C), JNK and phosphorylated JNK (p-JNK) (E), 14-3-3 and phosphorylated 14-3-3 (p-14-3-3) (F), and PDH and phosphorylated PDH (p-PDH) (G) were determined by immunoblotting. The amount of each protein was normalized to that of the indicated antigen. Representative blots are shown in the upper panels (A-C and E–G). Bax mRNA expression (D) was determined by real-time PCR assay and normalized to that of β-microglobulin mRNA. The average Bax mRNA level in untreated cells was set to 1.0. Individual data are expressed as dot plots with the mean ± SEM of a representative experiment (n = 3). NS, not significant; *, P < 0.05; **, P < 0.01 according to one-way ANOVA with Scheffe’s post hoc comparisons.

• 

  Fig. 3. DCA did not inhibit cisplatin-enhanced inflammation or expression of death receptors and ligands but reduced Bid degradation induced by caspase-8 activation. mProx cells were treated for 16, 18, or 24 h with growth buffer containing cisplatin (0 or 25 µM) in the presence or absence of DCA (10 mM). The levels of p38 and phosphorylated p38 (p-p38) (A), NF-B and phosphorylated NF-B (p-NF-B) (B), ERK and phosphorylated ERK(p-ERK) (C), Bid (h), and β-actin were determined by immunoblotting using whole cell lysates. The amount of each phosphorylated protein was normalized to that of the corresponding protein. Representative blots are shown in the upper panels (A–C, H). The levels of TNF-α (D), TL1A (E), FasL (F), and Fas (G) mRNA were determined by real-time PCR assay and normalized to that of β-microglobulin mRNA. The average mRNA level of each gene in untreated cells was set to 1.0. Individual data are expressed as dot plots with the mean ± SEM of a representative experiment (n = 3). NS, not significant; *, P < 0.05; **, P < 0.01 according to one-way ANOVA with Scheffe’s post hoc comparisons.

• 

  Fig. 4. Whole transcriptome sequencing revealed that DCA increases gene expression of the anti-apoptotic protein cFLIP in cisplatin-treated and -untreated mProx cells. mProx cells were treated for 24 h with growth buffer containing cisplatin (0 or 25 µM) in the presence or absence of DCA (10 mM). Total RNA was extracted from the cells and used for whole-transcriptome sequencing. Significant differential expression of anti-apoptotic protein genes (n = 9) was detected in mProx cells subjected to different treatments (P < 0.05 and q value < 0.05, one-way ANOVA; Supplementary Table S4d online). The heat map shows the scaled average expression of the detected genes. Blue and yellow colors indicate low and high expression, respectively.

• 

  Fig. 5. Whole transcriptome sequencing showed that DCA markedly reduced the expression of oxidative phosphorylation-associated genes in cisplatin-treated mProx cells. mProx cells were treated for 24 h with growth buffer containing cisplatin (0 or 25 µM) in the presence or absence of DCA (10 mM). Total RNA was extracted from the cells used for whole-transcriptome sequencing. Significant differential expression of oxidative phosphorylation-associated genes (n = 114) was detected in mProx cells subjected to different treatments (n = 3 per group) (P < 0.05 and q value < 0.05, one-way ANOVA). The heat map shows the scaled average expression of the detected genes. Blue and yellow colors indicate low and high expression, respectively.

• 

  Fig. 6. DCA increased cFLIP expression at mRNA and antigen levels in mProx treated with and without cisplatin. mProx cells were treated for 6 h, 13 h, 16 h, 18 h, or 24 h with growth buffer containing cisplatin (0 or 25 µM) in the presence or absence of DCA (10 mM). The levels of cFLIP mRNA at 6 h (A), 13 h (B), 18 h (C), and 24 h (D) were determined using real-time PCR assay and normalized that of β-microglobulin mRNA. The average cFLIP mRNA level in the untreated cells was set to 1.0. The amounts of cFLIP antigen and β−actin at 16 h (E) and 24 h (F) were determined by immunoblotting using whole cell lysates. The amount of cFLIP was normalized to that of corresponding β-actin. Individual data are expressed as dot plots with the mean ± SEM of a representative experiment (n = 3). NS, not significant; *, P < 0.05; ** P < 0.01 according to one-way ANOVA with Scheffe’s post hoc comparisons. #, P < 0.01 according to Student’s t-test.

• 

  Fig. 7. cFLIP or PDK4 deficiency modulated cisplatin-induced activation of caspase-3 and -8, while p62 deficiency did not influence cisplatin-induced caspase-8 activation. mProx cells were transfected for 40 h with negative control siRNA (A–J), siRNA against cFLIP (A–D), p62 (E, F), or PDK4 (G–J). The cells refreshed with modified K-1 medium were then incubated in K-1 medium containing cisplatin (0 or 25 µM) or DCA (0 or 10 mM) for an additional 24 h. The levels of cFLIP (a) and p62 (E) mRNA were determined using real-time PCR assays and normalized to that of β2-microglobulin mRNA. The average mRNA levels in control siRNA-treated mProx cells was set to 1.0. The amounts cFLIP (B), cleaved caspase-3 (C, G), p62 (E), cleaved caspase-8 (D, F), PDH and p-PDH (H), JNK and p-JNK (I), 14-3-3 and p-14-3-3 (J), and β-actin were determined by immunoblotting. The ratio of cleaved caspase-3 (C) and caspase-8 (D) to β-actin in cFLIP siRNA-transfected mProx cells treated with cisplatin alone and ratio of the cleaved caspase-3 to β-actin in control siRNA-transfected mProx cells treated with cisplatin alone (G) was set to 1.0. Individual data are expressed as dot plots with the mean ± SEM of a…

• 

  Fig. 8. DCA reduced cisplatin-induced acute kidney injury in mice by suppressing caspase-3 and -8 activation. Male S129 mice (8–10 weeks old) were pre-treated with daily intra-peritoneal administration of DCA (250 mg/kg) or saline for two days, intra-peritoneally administered a single dose of cisplatin (20 mg/kg) or saline, followed by daily DCA or saline treatment for three days, and then sacrificed. (A) Serum creatinine and blood urea nitrogen (BUN) levels were measured in the four groups described above. (B) Periodic acid–Schiff (PAS) staining of the kidney tissues of mice. (Original magnification, ×100; *, damaged tubules). (C) Tubular injury score was determined for 10 random fields from each mouse kidney. The amounts of cleaved caspase-3 and caspase-8 (D), p-53 and Bax (E), p-JNK and JNK (F), p-14-3-3 and 14-3-3 (F), cFLIP (K) and β-actin in the kidneys of mice from each group were measured by immunoblotting and normalized to that of the indicated proteins. The levels of TNF-α (G), TNFR1 (H), Fas (I), FasL (J), and cFLIP (K) mRNA were determined using real-time PCR assays and normalized to that of β2-microglobulin mRNA. The average mRNA level of each gene in untreated cells was set…