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, 7 (7), 646-57

Interactive Effects of HDAC Inhibitors and TRAIL on Apoptosis Are Associated With Changes in Mitochondrial Functions and Expressions of Cell Cycle Regulatory Genes in Multiple Myeloma

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Interactive Effects of HDAC Inhibitors and TRAIL on Apoptosis Are Associated With Changes in Mitochondrial Functions and Expressions of Cell Cycle Regulatory Genes in Multiple Myeloma

Tamer E Fandy et al. Neoplasia.

Abstract

In this study, we have evaluated the cytotoxic effect of combining two HDAC inhibitors, SAHA and TSA, with TRAIL in human multiple myeloma cell lines. Low doses of SAHA or TSA enhanced the cytotoxic and apoptotic effects of TRAIL and upregulated the surface expression of TRAIL death receptors (DR4 and/or DR5). SAHA and TSA induced G1 phase cell cycle growth arrest by upregulating p21(WAF1) and p27(Kip1) expression and by inhibiting E2F transcriptional activity. The enhanced TRAIL effect after pretreatment with HDAC inhibitors was consistent with the upregulation of the proapoptotic Bcl-2 family members (Bim, Bak, Bax, Noxa, and PUMA), the downregulation of the anti-apoptotic members of the Bcl-2 family (Bcl-2 and Bcl-X(L)), and IAPs. SAHA and TSA dissipated the mitochondrial membrane potential and enhanced the release of Omi/HtrA2 and AIF from the mitochondria to the cytosol. The cytotoxic effect of both SAHA and TSA was caspase- and calpain-independent. Inhibition of NF(kappa)B activation by the proteasome inhibitor, MG132, enhanced the apoptotic effect of TSA. Our study demonstrated the enhancing effects of HDAC inhibitors on apoptosis when combined with TRAIL and, for the first time, emphasized the role of AIF in mediating the cytotoxic effects of HDAC inhibitors.

Figures

Figure 1
Figure 1
SAHA and TSA induce the accumulation of acetylated histones in ARP-1 cells. ARP-1 cells were treated with SAHA (500 nM) or TSA (50 nM) for 6 and 12 hours followed by extraction of cellular histones and Western blot analysis to detect the levels of expression of acetylated H3 (Ac-H3) and acetylated H4 (Ac-H4). Ponceau S staining was used to confirm the proper loading of histones. Ctrl represents the control lane.
Figure 2
Figure 2
SAHA and TSA augment TRAIL cytotoxic and apoptotic effects. (A) ARP-1 cells were pretreated with different TSA concentrations for 24 hours followed by treatment with different TRAIL concentrations (50 and 100 ng/ml) for another 24 hours. XTT cell proliferation assay was used to estimate cell survival. Percentage of survival was estimated as a percentage of the value of the untreated control. Data represent the mean for four replicates ± SD. (B) ARP-1 cells were treated with different concentrations of SAHA for 24 hours followed by treatment with 100 ng/ml TRAIL for another 24 hours and the percentage of survival was determined by XTT assay as described above. Data represent the mean for four replicates ± SD. (C) ARP-1 cells were treated with different concentrations of SAHA and TSA, alone or in combination with 50 ng/ml TRAIL, for 24 hours and the percentage of apoptotic cells was determined by Annexin V staining. Data represent the mean for four replicates ± SD. *Significant difference from the respective control at P < .05. (D) ARP-1 cells were treated with SAHA (500 nM) or TSA (50 nM) for 24 and 48 hours and the ∼85 kDa cleavage product of PARP was detected by Western blot analysis. β-Actin was used as a loading control. Ctrl represents the control lane.
Figure 3
Figure 3
SAHA and TSA upregulate the surface expression and transcription of DR4/DR5 in ARP-1 cells. ARP-1 cells were treated with SAHA (500 nM) for 48 hours and the surface expression of DR4 (A) and DR5 (B) was measured as described under the Materials and Methods section. ARP-1 cells were treated with TSA (50 nM) for 48 hours, and the surface expression of DR4 (C) and DR5 (D) was measured. (E) ARP-1 cells were treated with SAHA (500 nM) or TSA (50 nM) for 12 hours; total RNA was extracted to perform RPA (hAPO-3d template set) as described under the Materials and Methods section. GAPDH was used as a loading control. Ctrl represents the control lane.
Figure 4
Figure 4
HDAC inhibitors induce G1 phase growth arrest and decrease the binding and transcriptional activity of E2F-1. (A) Synchronized ARP-1 cells were treated with SAHA (500 nM) or TSA (50 nM) for 6, 12, and 24 hours. The expressions of p21WAF1, p27Kip1, p53, and phosphorylated Rb were determined at each time point by immunoblotting. β-Actin was used as a loading control. Ctrl represents the control lane. (B) Nuclear proteins were extracted from ARP-1 cells after treatment with TSA (50 nM) for 30 and 120 minutes, and EMSA was performed as described under the Materials and Methods section. Cold E2F lane represents excess unlabelled E2F1 consensus sequence. (C) ARP-1 cells were transfected with E2F1 luciferase reporter plasmid and CMV 4 promoter-drivenβ -gal expression plasmid to normalize the transfection efficiency. Cells were treated with TSA (50 nM) for different time intervals (30, 60, 120, and 300 min) and the relative luciferase activity was determined according to the manufacturer's instructions. Data represent the mean for three replicates ± SD. *Significant difference from the control at P < .05.
Figure 5
Figure 5
Differential modulation of the expression of the pro-apoptotic and anti-apoptotic members of the Bcl-2 family and IAPs by SAHA and TSA. (A) ARP1 cells were treated with SAHA (500 nM) or TSA (50 nM) for 12, 24, and 48 hours and the protein expression of Bim, Bak, Noxa, Bax, and PUMA β/δ was determined by immunoblotting at each time point. (B) ARP-1 cells were treated with SAHA (500 nM) or TSA (50 nM) for 12, 24, and 48 hours and the protein expression of Bcl-XL and Bcl-2 was determined by immunoblotting. β-Actin was used as a loading control. Ctrl represents the control lane. (C) ARP-1 cells were treated with SAHA 500 nM or TSA 50 nM for 12, 24, and 48 hours, and the protein expression of cIAP1, cIAP2, and XIAP was determined by immunoblotting. β-Actin was used as a loading control. Ctrl represents the control lane.
Figure 6
Figure 6
Involvement of the mitochondria in SAHA- and TSA-induced apoptosis. (A) ARP-1 cells were treated with SAHA (500 nM) or TSA (50 nM) for 1, 2, 4, and 12 hours followed by staining with JC-1 dye and measurement of the mitochondrial membrane potential (ΔΨm) as described under the Materials and Methods section. The untreated cells (control) were assigned an arbitrary value and the value of the other treatments was estimated relative to the control. Data represent the mean for four replicates ± SD. *Significant difference from the control at P < .05. (B) ARP-1 cells were treated with SAHA (500 nM) or TSA (50 nM) for 12 and 24 hours, the cytosolic fraction was isolated, and the expression of AIF and Omi/HtrA2 was determined at each time point by immunoblotting. β-Actin was used as a loading control. (C) ARP-1 cells were treated with SAHA (500 nM) or TSA (50 nM) for 12 hours followed by double immunofluorescence staining of the nucleus (blue color) and AIF (green) as described under the Materials and Methods section. (D) ARP-1 cells were either pretreated with TPCK (50 µM) for 2 hours or left untreated, followed by treatment with SAHA (3 µM) or TSA (200 nM) for 24 hours. The percentage of survival was determined by XTT assay as described previously.
Figure 7
Figure 7
SAHA and TSA cytotoxic effects are caspase- and calpain-independent. (A) ARP-1 cells were either pretreated with 30 µM Z-VAD-FMK (VAD) for 2 hours or left untreated, then different doses of SAHA, TSA, or TRAIL (positive control) were added to both the treated and untreated cells for 36 hours. XTT cell proliferation assay was used to estimate cell survival and percentage of survival was estimated as a percentage of the value of the untreated control. Pretreatment with Z-VAD-FMK. Data represent the mean for four replicates ± SD. There was no significant difference at P < .05 between the Z-VAD-FMK-pretreated and -untreated cells after TSA or SAHA treatment. (B) ARP-1 cells were either pretreated with 30 µM calpeptin for 2 hours or left untreated, then different doses of SAHA or TSA were added to both the treated and untreated cells for 36 hours. XTT cell proliferation assay was used to assess cell survival. Data represent the mean for four replicates ± SD. No significant difference was observed between the control and calpeptin-treated cells at P < .05.
Figure 8
Figure 8
TSA increases the binding activity of NF-κB in ARP-1 myeloma cells. (A) ARP-1 cells were treated with 50 nM TSA for 0, 40, 60, and 120 minutes followed by extraction of the nuclear proteins and EMSA. The control lane represents the negative control (no nuclear proteins) and the cold NF-κB lane contains excess unlabeled NF-κB (x 100) to compete with the labeled NF-κB for binding to nuclear proteins. (B) ARP-1 cells were treated with MG132 (10 µM) or TSA (100 nM) alone for 24 hours or pretreated with MG132 (10 µM) for 2 hours followed by TSA treatment (100 nM) for 24 hours. DAPI staining was used to assess apoptosis and the percentage of apoptosis was estimated as a percentage of the value of the untreated control. Data represent the mean for four replicates ± SD. *Significant difference from the respective controls at P < .05.

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