Cancer Res Treat.  2011 Jun;43(2):124-130.

Up-regulation of the DR5 Expression by Proteasome Inhibitor MG132 Augments TRAIL-Induced Apoptosis in Soft Tissue Sarcoma Cell Lines

• Cheong HJ
• Lee KS
• Woo IS
• Won JH
• Byun JH

Abstract

PURPOSE
Current chemotherapeutics for treating locally advanced or metastatic soft tissue sarcomas (STS) are limited. Accordingly, the present in vitro study was conducted to evaluate the effects of treatment of STS cells with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) applied as a single agent or in combination with a proteasome inhibitor, MG132.
MATERIALS AND METHODS
Sensitivity to TRAIL and activity of TRAIL-induced apoptotic pathways were analyzed in four STS cell lines: HTB-82 (rhabdomyosarcoma), HT-1080 (fibrosarcoma), HTB-93 (synovial sarcoma), and HTB-94 (chondrosarcoma). Reduction of the dye dimethylthiazolyl 2,5 diphenyltetrazolium bromide (MTT) was used to evaluate cytotoxic activity; western blots were used to evaluate TRAIL-induced apoptosis.
RESULTS
TRAIL induced apoptosis in HTB-93 cells, but had little effect in HTB-82, HT-1080, or HTB-94 cells. Expression of TRAIL receptor-1 and -2 did not correlate with sensitivity to TRAIL. Co-incubation of cells with TRAIL and a proteasome inhibitor, MG132, augmented the apoptotic effect of TRAIL in both TRAIL-sensitive and TRAIL-resistant cells. This effect was due to up-regulation of TRAIL receptors and members of the pro-apoptotic BCL-2 family by MG132.
CONCLUSION
These data show that combining TRAIL with MG132 enhances apoptosis and overcomes TRAIL resistance. This restoration of TRAIL sensitivity occurs through an increase in the expression of death receptor 5 and of pro-apoptotic BCL-2 family members such as BAX.

Keyword

TNF-related apoptosis-inducing ligand; MG 132; Soft tissue sarcoma; Apoptosis

MeSH Terms

Apoptosis
Blotting, Western
Cell Line
Humans
Leupeptins
Necrosis
Proteasome Endopeptidase Complex
Proteasome Inhibitors
Receptors, TNF-Related Apoptosis-Inducing Ligand
Sarcoma
TNF-Related Apoptosis-Inducing Ligand
Up-Regulation
Leupeptins
Proteasome Endopeptidase Complex
Proteasome Inhibitors
Receptors, TNF-Related Apoptosis-Inducing Ligand
TNF-Related Apoptosis-Inducing Ligand

Figure

• 

  Fig. 1 Cell viability of soft tissue sarcomas (STS) cell lines treated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), MG132 and a combination of both agents. (A, B) Cells were incubated for 24 hr in the presence of different concentrations of TRAIL and/or MG132. *p<0.05, compared to control or lower dose of each treatment. Cell viability was quantified by MTT assay; each treatment was performed in triplicate. (C) Adding MG132 to TRAIL augmented the apoptosis compared to exposure to a single agent, TRAIL or MG132, in TRAIL-sensitive cells (HTB-93) and TRAIL-resistant cells (HTB-82, HT-1080, and HTB-94). *p<0.05, compared to TRAIL 10 ng/mL, MG132 1 µM, and MG132 2 µM. (T10/M1: 10 ng/mL TRAIL+1 µM MG132; T10/M2: 10 ng/mL TRAIL+2 µM MG132).

• 

  Fig. 2 Evaluation of apoptosis. The broad-spectrum caspase inhibitor z-VAD-fmk or the caspase-8 inhibitor z-IETD-fmk was applied for 1 hr before treatment of soft tissue sarcomas (STS) cells with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and MG132 for 24 hr. (A, B) The decrease in cell viability induced by combined treatment with MG132 and TRAIL and measured by MTT assay was caspase dependent. Similar results were obtained in three separate experiments. *p<0.05 compared to the combination of TRAIL and MG132.

• 

  Fig. 3 Expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors and apoptotic molecules in soft tissue sarcomas (STS) cells. (A) After incubating each of the four STS cell lines for 24 hr with different concentrations of MG132, expression levels of TRAIL receptors and apoptotic molecules were determined by western blot analysis. (B) Analysis of the surface expression of death receptor (DR)4 and DR5 was determined by flow cytometry in HT-1080 and HTB-82 cells. C, control; M1, 1 µM MG132; M2, 2 µM MG132; M10, 10 µM MG132.

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