Intest Res.  2014 Jan;12(1):34-41. 10.5217/ir.2014.12.1.34.

Parthenolide Sensitizes Human Colorectal Cancer Cells to Tumor Necrosis Factor-related Apoptosis-inducing Ligand through Mitochondrial and Caspase Dependent Pathway

• Trang KT
• Kim SL
• Park SB
• Seo SY
• Choi CH
• Park JK
• Moon JC
• Lee ST
• Kim SW

• KMID: 2174335
• DOI: http://doi.org/10.5217/ir.2014.12.1.34

Abstract

BACKGROUND/AIMS
Combination therapy utilizing tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in conjunction with other anticancer agents, is a promising strategy to overcome TRAIL resistance in malignant cells. Recently, parthenolide (PT) has proved to be a promising anticancer agent, and several studies have explored its use in combination therapy. Here, we investigated the molecular mechanisms by which PT sensitizes colorectal cancer (CRC) cells to TRAIL-induced apoptosis.
METHODS
HT-29 cells (TRAIL-resistant) were treated with PT and/or TRAIL for 24 hours. The inhibitory effect on proliferation was detected using the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Annexin V staining, cell cycle analysis, and Hoechst 33258 staining were used to assess apoptotic cell death. Activation of an apoptotic pathway was confirmed by Western blot.
RESULTS
Treatment with TRAIL alone inhibited the proliferation of HCT 116 cells in a dose-dependent manner, whereas proliferation was not affected in HT-29 cells. Combination PT and TRAIL treatment significantly inhibited cell growth and induced apoptosis of HT-29 cells. We observed that the synergistic effect was associated with misregulation of B-cell lymphoma 2 (Bcl-2) family members, release of cytochrome C to the cytosol, activation of caspases, and increased levels of p53.
CONCLUSION
Combination therapy using PT and TRAIL might offer an effetive strategy to overcome TRAIL resistance in certain CRC cells.

Keyword

Parthenolide; TRAIL; Colorectal neoplasm; TRAIL resistance; Apoptosis

MeSH Terms

Annexin A5
Antineoplastic Agents
Apoptosis
Bisbenzimidazole
Blotting, Western
Caspases
Cell Cycle
Cell Death
Colorectal Neoplasms*
Cytochromes c
Cytosol
HCT116 Cells
HT29 Cells
Humans*
Lymphoma, B-Cell
Necrosis*
Tumor Necrosis Factor-alpha
Annexin A5
Antineoplastic Agents
Bisbenzimidazole
Caspases
Cytochromes c
Tumor Necrosis Factor-alpha

Figure

• 

  Fig. 1 The inhibitory effect of combined parthenolide (PT) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) treatment on cell proliferation. (A) HT-29 and HCT 116 cells were treated with TRAIL for 24 hours, at the concentrations indicated, before being analyzed for viability by 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Data represent the mean±SE of at least three independent experiments. (B) Cells were co-treated with PT (10 µM) and TRAIL (25 ng/mL) for 24 hours. The data represent the mean±SE of three independent experiments. *P<0.05, compared to control.

• 

  Fig. 2 The apoptotic effect of combined parthenolide (PT) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) treatment. (A) Apoptotic cell death induced by combination treatment. After treatment with TRAIL and/or 5-fluorouracil (5-FU) for 24 hours, cells were harvested and stained with annexin V-fluorescein isothiocyanate (FITC), to show that apoptosis is induced by combination treatment. (B) Cell cycle modification induced by combination treatment. After treatment with PT and/or TRAIL for 24 hours, cells were harvested and stained with propidium iodide (PI). The percentage of sub-G1 cells within the population is shown in each histogram. The total number of cells analyzed for each condition was 10,000. (C) DNA condensation and fragmentation induced by combination treatment. Apoptosis-associated DNA condensation was examined using Hoechst 33258 (1 µg/mL) staining. Apoptotic nuclei show intense fluorescence, corresponding to chromatin condensation (arrowhead) and fragmentation.

• 

  Fig. 3 Regulation of caspase and converting enzyme-inhibitory protein (cFLIP) levels by treatment with parthenolide (PT) and/or tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). (A) Cell lysates were prepared after treatment for 24 hours and then analyzed by western blot. The levels of full-length caspase-3 and -9 proteins were decreased by combination treatment. Caspase-3 and -9 levels, however, were not altered when cells were pre-treated with the pan-caspase inhibitor Z-VAD-FMK. Actin was used as a loading control. (B) The protein level of cFLIP was decreased by combination treatment. Actin was used as a loading control.

• 

  Fig. 4 The effect of combined parthenolide (PT) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) treatment on apoptosis-associated protein expression. Cell lysates were prepared after treatment for 24 hours and analyzed by western blots. The following proteins were probed: B-cell lymphoma 2 (Bcl-2), Bid, Bax, cytochrome C, and p53. The level of the anti-apoptotic protein Bcl-2 was significantly decreased by combined treatment with PT and TRAIL. Conversely, pro-apoptotic proteins, cleaved-Bid and Bax, were increased under combined treatment conditions. The levels of cytochrome C and p53 were increased after combination treatment. Actin was used as a loading control.

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