Proteasome inhibitors-mediated TRAIL resensitization and Bik accumulation - PubMed (original) (raw)

Proteasome inhibitors-mediated TRAIL resensitization and Bik accumulation

Hongbo Zhu et al. Cancer Biol Ther. 2005 Jul.

Abstract

Proteasome inhibitors can resensitize cells that are resistant to tumor necrosis factor-related apoptotic-inducing ligand (TRAIL)-mediated apoptosis. However, the underlying mechanisms of this effect are unclear. To characterize the mechanisms of interaction between proteasome inhibitors and TRAIL protein, we evaluated the effects of combined treatment with the proteasome inhibitors bortezomib and MG132 and TRAIL protein on two TRAIL-resistant human colon cancer cell lines, DLD1-TRAIL/R and LOVO-TRAIL/R. Both bortezomib and MG132 in combination with TRAIL enhanced apoptotosis induction in these cells, as evidenced by enhanced cleavage of caspases 8, 9, and 3, Bid, poly(ADP-ribose) polymerase and by the release of cytochrome C and Smac. Subsequent studies showed that combined treatment with bortezomib or MG132 resulted in an increase of death receptor (DR) 5 and Bik at protein levels but had no effects on protein levels of DR4, Bax, Bak, Bcl-2, Bcl-XL or Flice-inhibitory protein (FLIP). Moreover, c-Jun N-terminal kinase (JNK) is activated by these proteasome inhibitors. Blocking JNK activation with the JNK inhibitor SP600125 attenuated DR5 increase, but enhancement of apoptosis induction and increase of Bik protein were not affected. However, bortezomib-mediated TRAIL sensitization was partially blocked by using siRNA to knockdown Bik. Thus, our data suggests that accumulation of Bik may be critical for proteasome inhibitor-mediated resensitization of TRAIL.

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Figures

FIG. 1

FIG. 1

Combined effects of proteasome inhibitors and TRAIL protein in TRAIL-resistant DLD1-TRAIL/R cells. DLD1-TRAIL/R cells were treated with bortezomib or MG132 for 2 h, followed by 20 ng/ml of TRAIL protein for 4 h. (A) Cell viability was determined by XTT assay and (B) proportion of apoptotic cells determined by FACS analysis. (C). Cell viability 24 h after addition of TRAIL protein. Each assay was performed in quadruplicate. The data presented are means + SD. * P < 0.05 compared with the proteasome inhibitor alone.

FIG. 1

FIG. 1

Combined effects of proteasome inhibitors and TRAIL protein in TRAIL-resistant DLD1-TRAIL/R cells. DLD1-TRAIL/R cells were treated with bortezomib or MG132 for 2 h, followed by 20 ng/ml of TRAIL protein for 4 h. (A) Cell viability was determined by XTT assay and (B) proportion of apoptotic cells determined by FACS analysis. (C). Cell viability 24 h after addition of TRAIL protein. Each assay was performed in quadruplicate. The data presented are means + SD. * P < 0.05 compared with the proteasome inhibitor alone.

FIG. 1

FIG. 1

Combined effects of proteasome inhibitors and TRAIL protein in TRAIL-resistant DLD1-TRAIL/R cells. DLD1-TRAIL/R cells were treated with bortezomib or MG132 for 2 h, followed by 20 ng/ml of TRAIL protein for 4 h. (A) Cell viability was determined by XTT assay and (B) proportion of apoptotic cells determined by FACS analysis. (C). Cell viability 24 h after addition of TRAIL protein. Each assay was performed in quadruplicate. The data presented are means + SD. * P < 0.05 compared with the proteasome inhibitor alone.

FIG. 2

FIG. 2

Combined effect of proteasome inhibitors and TRAIL protein in TRAIL-resistant LOVO-TRAIL/R cells. LOVO-TRAIL/R cells were treated with bortezomib or MG132 for 2 h, followed by 20 ng/ml of TRAIL protein for 24 h. Cell viability was then determined by XTT assay. The data presented are mean + SD of triplicate assays. * P < 0.05 compared with the proteasome inhibitor alone.

FIG. 3

FIG. 3

Apoptosis profiles of DLD1-TRAIL/R cells treated with bortezomib (1 μM) or MG132 (5 μM) for 2 h, followed by 20 ng/ml of TRAIL protein for 4 h. Cell lysates were then subjected to Western blot analysis. (A) cleavage of caspases. (B) Release of cytochrome C and Smac. The mitochondrial fraction (Mito) was used as a positive control. (C) Caspase activation 24 h after addition of TRAIL protein. The data presented were from one of two independent experiments with similar results.

FIG. 3

FIG. 3

Apoptosis profiles of DLD1-TRAIL/R cells treated with bortezomib (1 μM) or MG132 (5 μM) for 2 h, followed by 20 ng/ml of TRAIL protein for 4 h. Cell lysates were then subjected to Western blot analysis. (A) cleavage of caspases. (B) Release of cytochrome C and Smac. The mitochondrial fraction (Mito) was used as a positive control. (C) Caspase activation 24 h after addition of TRAIL protein. The data presented were from one of two independent experiments with similar results.

FIG. 3

FIG. 3

Apoptosis profiles of DLD1-TRAIL/R cells treated with bortezomib (1 μM) or MG132 (5 μM) for 2 h, followed by 20 ng/ml of TRAIL protein for 4 h. Cell lysates were then subjected to Western blot analysis. (A) cleavage of caspases. (B) Release of cytochrome C and Smac. The mitochondrial fraction (Mito) was used as a positive control. (C) Caspase activation 24 h after addition of TRAIL protein. The data presented were from one of two independent experiments with similar results.

FIG. 4

FIG. 4

DR5 and Bik upregulation by proteasome inhibitors. DLD1-TRAIL/R TRAIL-resistant cells treated with bortezomib (1 μM) or MG132 (5 μM) for 2 h followed by 20 ng/ml of TRAIL protein for 4 h. The cell lysates were then subjected to Western blot analysis. The data presented are from one of two independent experiments with similar results.

FIG. 5

FIG. 5

JNK pathway activation by proteasome inhibitors. (A). JNK activation detected in TRAIL-resistant DLD1-TRAIL/R cells treated as described in the Fig. 4. The data presented are from one of two independent experiments with similar results. (B) Effect of JNK inhibitor. DLD1-TRAIL/R cells were treated with 50-μM in SP600125 for 2 h, followed by the combination treatment mentioned in Fig. 4. The cell lysates were then subjected to Western blot analysis. The data presented one of two independent experiments with similar results. (C) Apoptosis in cells treated as in B and determined by FACS analysis. The data represented means + SD of a triplicate assays. (D) Effect of Bik siRNA on proteasome inhibitor mediated TRAIL re-sensitization. DLD1-TRAIL/R cells were treated with 100 nM of Bik siRNA or Luciferase siRNA as indicated. 24 hours later, 1 μM of bortezomib was added for 2 hours, followed by 20ng/ml of TRAIL protein for another 4 hours. Apoptosis was then determined by FACS. The value represents mean + SD of a triplicate assay. * represents P<0.05.

FIG. 5

FIG. 5

JNK pathway activation by proteasome inhibitors. (A). JNK activation detected in TRAIL-resistant DLD1-TRAIL/R cells treated as described in the Fig. 4. The data presented are from one of two independent experiments with similar results. (B) Effect of JNK inhibitor. DLD1-TRAIL/R cells were treated with 50-μM in SP600125 for 2 h, followed by the combination treatment mentioned in Fig. 4. The cell lysates were then subjected to Western blot analysis. The data presented one of two independent experiments with similar results. (C) Apoptosis in cells treated as in B and determined by FACS analysis. The data represented means + SD of a triplicate assays. (D) Effect of Bik siRNA on proteasome inhibitor mediated TRAIL re-sensitization. DLD1-TRAIL/R cells were treated with 100 nM of Bik siRNA or Luciferase siRNA as indicated. 24 hours later, 1 μM of bortezomib was added for 2 hours, followed by 20ng/ml of TRAIL protein for another 4 hours. Apoptosis was then determined by FACS. The value represents mean + SD of a triplicate assay. * represents P<0.05.

FIG. 5

FIG. 5

JNK pathway activation by proteasome inhibitors. (A). JNK activation detected in TRAIL-resistant DLD1-TRAIL/R cells treated as described in the Fig. 4. The data presented are from one of two independent experiments with similar results. (B) Effect of JNK inhibitor. DLD1-TRAIL/R cells were treated with 50-μM in SP600125 for 2 h, followed by the combination treatment mentioned in Fig. 4. The cell lysates were then subjected to Western blot analysis. The data presented one of two independent experiments with similar results. (C) Apoptosis in cells treated as in B and determined by FACS analysis. The data represented means + SD of a triplicate assays. (D) Effect of Bik siRNA on proteasome inhibitor mediated TRAIL re-sensitization. DLD1-TRAIL/R cells were treated with 100 nM of Bik siRNA or Luciferase siRNA as indicated. 24 hours later, 1 μM of bortezomib was added for 2 hours, followed by 20ng/ml of TRAIL protein for another 4 hours. Apoptosis was then determined by FACS. The value represents mean + SD of a triplicate assay. * represents P<0.05.

FIG. 5

FIG. 5

JNK pathway activation by proteasome inhibitors. (A). JNK activation detected in TRAIL-resistant DLD1-TRAIL/R cells treated as described in the Fig. 4. The data presented are from one of two independent experiments with similar results. (B) Effect of JNK inhibitor. DLD1-TRAIL/R cells were treated with 50-μM in SP600125 for 2 h, followed by the combination treatment mentioned in Fig. 4. The cell lysates were then subjected to Western blot analysis. The data presented one of two independent experiments with similar results. (C) Apoptosis in cells treated as in B and determined by FACS analysis. The data represented means + SD of a triplicate assays. (D) Effect of Bik siRNA on proteasome inhibitor mediated TRAIL re-sensitization. DLD1-TRAIL/R cells were treated with 100 nM of Bik siRNA or Luciferase siRNA as indicated. 24 hours later, 1 μM of bortezomib was added for 2 hours, followed by 20ng/ml of TRAIL protein for another 4 hours. Apoptosis was then determined by FACS. The value represents mean + SD of a triplicate assay. * represents P<0.05.

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