Limits...
The role of apoptotic cell death in the radiosensitising effect of gemcitabine.

Pauwels B, Vermorken JB, Wouters A, Ides J, Van Laere S, Lambrechts HA, Pattyn GG, Vermeulen K, Meijnders P, Lardon F - Br. J. Cancer (2009)

Bottom Line: When cell-cycle distribution returned to pre-treatment levels, an increased induction of apoptosis was observed with activation of caspase 8 and 9 and a reduction of the mitochondrial membrane potential.A role for the cell-cycle perturbations and the induction of apoptosis could be attributed to the radiosensitising effect of gemcitabine.Apoptosis induction was comparable with the apoptotic pathway observed after the TRAIL treatment, that is the involvement of the extrinsic apoptosis pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cancer Research and Clinical Oncology, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium. bea.pauwels@ua.ac.be

ABSTRACT

Background: The aim of this study was to evaluate the radiosensitising effect of gemcitabine, in terms of cell-cycle progression, induction of apoptosis, and to investigate the molecular events regulating apoptosis.

Methods: Tumour cells were treated with gemcitabine, radiation, or the combination. 0-72 h after treatment, cells were collected for cell-cycle analysis and apoptosis determination. Caspase 8 and 9, Bid and tBid expression were determined by western blot. The mitochondrial membrane potential was determined using flow cytometry. An RT(2) Profiler PCR Array for human apoptotic genes was performed after the combination or TRAIL treatment.

Results: Gemcitabine and radiation resulted in an early S-phase block immediately after treatment, after which the cells moved synchronously through the cell cycle. When cell-cycle distribution returned to pre-treatment levels, an increased induction of apoptosis was observed with activation of caspase 8 and 9 and a reduction of the mitochondrial membrane potential. Gene expression after treatment with radiosensitising conditions was comparable with expression after the TRAIL treatment.

Conclusion: A role for the cell-cycle perturbations and the induction of apoptosis could be attributed to the radiosensitising effect of gemcitabine. Apoptosis induction was comparable with the apoptotic pathway observed after the TRAIL treatment, that is the involvement of the extrinsic apoptosis pathway.

Show MeSH

Related in: MedlinePlus

(A) TUNEL-positive ECV304 cells 72 h after treatment with gemcitabine, radiotherapy, or the combination of gemcitabine and radiotherapy. Dot plot from a representative experiment (R2=normal cell population, R3=apoptotic cell population). (B) Caspase 3 activity of ECV304 and H292 cells 72 h after treatment with gemcitabine, radiotherapy, or the combination of gemcitabine and radiotherapy. *P<0.05 vs 0 nM–0 Gy, †P<0.05 vs 0 nM–6 Gy, §P<0.05 vs 8 nM–0 Gy. (C) Caspase 8, caspase 9, Bid and tBid expression of H292 cells 72 h after gemcitabine (IC25, IC90) and/or radiotherapy. Similar results were observed with ECV304 cells. (D) Annexin V-positive cells with reduced TMRM fluorescence (loss of Δψm) and TMRM-positive cells after treatment with gemcitabine, radiotherapy, or the combination. *P<0.05 vs 0 nM–0 Gy, †P<0.05 vs 2 nM–0 Gy, §P<0.05 vs 0 nM–2 Gy, ∣∣P<0.05 vs 0 nM–6 Gy, **P<0.05 vs 8 nM–0 Gy, ††P<18 nM–0 Gy.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2736812&req=5

fig3: (A) TUNEL-positive ECV304 cells 72 h after treatment with gemcitabine, radiotherapy, or the combination of gemcitabine and radiotherapy. Dot plot from a representative experiment (R2=normal cell population, R3=apoptotic cell population). (B) Caspase 3 activity of ECV304 and H292 cells 72 h after treatment with gemcitabine, radiotherapy, or the combination of gemcitabine and radiotherapy. *P<0.05 vs 0 nM–0 Gy, †P<0.05 vs 0 nM–6 Gy, §P<0.05 vs 8 nM–0 Gy. (C) Caspase 8, caspase 9, Bid and tBid expression of H292 cells 72 h after gemcitabine (IC25, IC90) and/or radiotherapy. Similar results were observed with ECV304 cells. (D) Annexin V-positive cells with reduced TMRM fluorescence (loss of Δψm) and TMRM-positive cells after treatment with gemcitabine, radiotherapy, or the combination. *P<0.05 vs 0 nM–0 Gy, †P<0.05 vs 2 nM–0 Gy, §P<0.05 vs 0 nM–2 Gy, ∣∣P<0.05 vs 0 nM–6 Gy, **P<0.05 vs 8 nM–0 Gy, ††P<18 nM–0 Gy.

Mentions: The amount of apoptotic cells was determined using Annexin V staining and caspase 3 activity assay. For ECV304 cells, apoptosis induction was also evaluated by TUNEL assay. In Table 1, the amount of Annexin V-positive cells is summarised. For both ECV304 and H292 cells, the amount of early apoptotic cells increased with the combination gemcitabine and radiation. More apoptotic cells were observed with a higher concentration gemcitabine or higher radiation dose. Similar results were observed with TUNEL assay for ECV304 cells (Figure 3A). In fact, treatment with 8 nM of gemcitabine and 6 Gy radiation resulted in more than 50% TUNEL-stained cells 72 h after treatment.


The role of apoptotic cell death in the radiosensitising effect of gemcitabine.

Pauwels B, Vermorken JB, Wouters A, Ides J, Van Laere S, Lambrechts HA, Pattyn GG, Vermeulen K, Meijnders P, Lardon F - Br. J. Cancer (2009)

(A) TUNEL-positive ECV304 cells 72 h after treatment with gemcitabine, radiotherapy, or the combination of gemcitabine and radiotherapy. Dot plot from a representative experiment (R2=normal cell population, R3=apoptotic cell population). (B) Caspase 3 activity of ECV304 and H292 cells 72 h after treatment with gemcitabine, radiotherapy, or the combination of gemcitabine and radiotherapy. *P<0.05 vs 0 nM–0 Gy, †P<0.05 vs 0 nM–6 Gy, §P<0.05 vs 8 nM–0 Gy. (C) Caspase 8, caspase 9, Bid and tBid expression of H292 cells 72 h after gemcitabine (IC25, IC90) and/or radiotherapy. Similar results were observed with ECV304 cells. (D) Annexin V-positive cells with reduced TMRM fluorescence (loss of Δψm) and TMRM-positive cells after treatment with gemcitabine, radiotherapy, or the combination. *P<0.05 vs 0 nM–0 Gy, †P<0.05 vs 2 nM–0 Gy, §P<0.05 vs 0 nM–2 Gy, ∣∣P<0.05 vs 0 nM–6 Gy, **P<0.05 vs 8 nM–0 Gy, ††P<18 nM–0 Gy.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2736812&req=5

fig3: (A) TUNEL-positive ECV304 cells 72 h after treatment with gemcitabine, radiotherapy, or the combination of gemcitabine and radiotherapy. Dot plot from a representative experiment (R2=normal cell population, R3=apoptotic cell population). (B) Caspase 3 activity of ECV304 and H292 cells 72 h after treatment with gemcitabine, radiotherapy, or the combination of gemcitabine and radiotherapy. *P<0.05 vs 0 nM–0 Gy, †P<0.05 vs 0 nM–6 Gy, §P<0.05 vs 8 nM–0 Gy. (C) Caspase 8, caspase 9, Bid and tBid expression of H292 cells 72 h after gemcitabine (IC25, IC90) and/or radiotherapy. Similar results were observed with ECV304 cells. (D) Annexin V-positive cells with reduced TMRM fluorescence (loss of Δψm) and TMRM-positive cells after treatment with gemcitabine, radiotherapy, or the combination. *P<0.05 vs 0 nM–0 Gy, †P<0.05 vs 2 nM–0 Gy, §P<0.05 vs 0 nM–2 Gy, ∣∣P<0.05 vs 0 nM–6 Gy, **P<0.05 vs 8 nM–0 Gy, ††P<18 nM–0 Gy.
Mentions: The amount of apoptotic cells was determined using Annexin V staining and caspase 3 activity assay. For ECV304 cells, apoptosis induction was also evaluated by TUNEL assay. In Table 1, the amount of Annexin V-positive cells is summarised. For both ECV304 and H292 cells, the amount of early apoptotic cells increased with the combination gemcitabine and radiation. More apoptotic cells were observed with a higher concentration gemcitabine or higher radiation dose. Similar results were observed with TUNEL assay for ECV304 cells (Figure 3A). In fact, treatment with 8 nM of gemcitabine and 6 Gy radiation resulted in more than 50% TUNEL-stained cells 72 h after treatment.

Bottom Line: When cell-cycle distribution returned to pre-treatment levels, an increased induction of apoptosis was observed with activation of caspase 8 and 9 and a reduction of the mitochondrial membrane potential.A role for the cell-cycle perturbations and the induction of apoptosis could be attributed to the radiosensitising effect of gemcitabine.Apoptosis induction was comparable with the apoptotic pathway observed after the TRAIL treatment, that is the involvement of the extrinsic apoptosis pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cancer Research and Clinical Oncology, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium. bea.pauwels@ua.ac.be

ABSTRACT

Background: The aim of this study was to evaluate the radiosensitising effect of gemcitabine, in terms of cell-cycle progression, induction of apoptosis, and to investigate the molecular events regulating apoptosis.

Methods: Tumour cells were treated with gemcitabine, radiation, or the combination. 0-72 h after treatment, cells were collected for cell-cycle analysis and apoptosis determination. Caspase 8 and 9, Bid and tBid expression were determined by western blot. The mitochondrial membrane potential was determined using flow cytometry. An RT(2) Profiler PCR Array for human apoptotic genes was performed after the combination or TRAIL treatment.

Results: Gemcitabine and radiation resulted in an early S-phase block immediately after treatment, after which the cells moved synchronously through the cell cycle. When cell-cycle distribution returned to pre-treatment levels, an increased induction of apoptosis was observed with activation of caspase 8 and 9 and a reduction of the mitochondrial membrane potential. Gene expression after treatment with radiosensitising conditions was comparable with expression after the TRAIL treatment.

Conclusion: A role for the cell-cycle perturbations and the induction of apoptosis could be attributed to the radiosensitising effect of gemcitabine. Apoptosis induction was comparable with the apoptotic pathway observed after the TRAIL treatment, that is the involvement of the extrinsic apoptosis pathway.

Show MeSH
Related in: MedlinePlus