Yonsei Med J.  2016 Nov;57(6):1494-1499. 10.3349/ymj.2016.57.6.1494.

Troglitazone Enhances the Apoptotic Response of DLD-1 Colon Cancer Cells to Photodynamic Therapy

• Park H
• Ko SH
• Lee JM
• Park JH
• Choi YH

• KMID: 2427170
• DOI: http://doi.org/10.3349/ymj.2016.57.6.1494

Abstract

PURPOSE
The aim of this study was to investigate whether the peroxisomal proliferator-activated receptor gamma (PPARγ) ligand troglitazone in combination with photodynamic therapy (PDT) enhances the apoptotic response of DLD-1 colon cancer cells.
MATERIALS AND METHODS
The effects of troglitazone, PDT, and troglitazone in combination with PDT on cell viability and apoptosis were assessed in DLD-1 cells. Cell viability and proliferation were evaluated using the tetrazolium-based MTT assay, and apoptosis was evaluated via cell staining with propidium iodide (PI) and annexin V-FITC. The levels of pro-caspase-3 were measured via Western blot analyses.
RESULTS
Treatment of troglitazone and PDT induced the growth retardation and cell death of DLD-1 cells in a dose-dependent manner, respectively. The combination treatment significantly suppressed cell growth and increased the apoptotic response of DLD-1 and resulted in apoptosis rather than necrosis, as shown by PI/annexin V staining and degradation of procaspase-3.
CONCLUSION
Conclusion: These results document the anti-proliferative and apoptotic activities of PDT in combination with the PPARγ ligand troglitazone and provide a strong rationale for testing the therapeutic potential of combination treatment in colon cancer.

Keyword

PDT; troglitazone; apoptosis

MeSH Terms

Antineoplastic Agents/pharmacology/therapeutic use
Apoptosis/*drug effects
Blotting, Western
Caspase 3
Cell Cycle/drug effects
Cell Line, Tumor
Cell Proliferation/*drug effects
Cell Survival/*drug effects
Chromans/*pharmacology
Colonic Neoplasms/metabolism/pathology
Dose-Response Relationship, Drug
Humans
PPAR gamma/metabolism/*pharmacology
*Photochemotherapy
Tetrazolium Salts
Thiazoles
Thiazolidinediones/*pharmacology/therapeutic use
Antineoplastic Agents
Chromans
PPAR gamma
Tetrazolium Salts
Thiazoles
Thiazolidinediones
Caspase 3

Figure

• 

  Fig. 1 Inhibition of DLD-1 proliferation by troglitazone. DLD-1 cells were seeded in a 96-well plate at a concentration of 1×104 cells/mL, and growth was analyzed at the indicated day via MTT assay. The small box represents the mRNA expression level of DLD-1, and preadipocytes were used as positive controls. Results are presented as the mean±SD of three independent experiments performed with triplicate samples. *p<0.001. D, DLD-1 cells; P, preadipocyte.

• 

  Fig. 2 Induction of DLD-1 cell death by PDT. (A) Cell death was induced by various doses of photosensitizer/PDT. DLD-1 cells (3×104 cells/mL) were placed in a 96-well plate, and PDT and MTT assays were performed as described in the Materials and Methods section. (B) Rapid uptake of photosensitizer in the cytoplasm of DLD-1. DLD-1 cells were loaded with 200 nM PPME for 1 h and subsequently detected using a fluorescent microscope at 650 nm. (C) Morphological change of DLD-1 after PDT was observed using a light microscope. *p<0.01, †p<0.001. M, medium only; DMF, N, N-dimethyl formamide; Control, DMF plus 120 mJ light; PDT, photosensitizer in DMF plus 120 mJ; PDT, photodynamic therapy; PPME, pyropheophorbide-a methyl ester.

• 

  Fig. 3 Enhancement of PDT-induced cell death by the pre-incubation of troglitazone. DLD-1, HCT-15, and HT-29 cells were treated with 20 µM troglitazone for 24 h and then treated to 0.4 µg/120 mJ PDT. After incubation for 24 h, cell viability was detected via MTT assay. *p<0.01, †p<0.001. M, solvent control only; Tro (T), troglitazone only; PDT (P), PDT only; Tro+PDT (T+P), combination of PDT and troglitazone, PDT, photodynamic therapy.

• 

  Fig. 4 Combination treatment induced apoptosis. After treatment as indicated, apoptotic cell death was detected with Annexin V/PI staining as described in the Materials and Methods section. The data represent three independent experiments. PDT, photodynamic therapy.

• 

  Fig. 5 Combination treatment induced procasepase-3 degradation after the pretreatment of troglitazone for 24 h, followed by incubation with or without PDT for an additional 24 h, using the same method as in Figs. 3 and 4. Whole cell lysate was compensated by BCA reagent, and an equal amount was analyzed using specific antibody as indicated. Approximately 34 KDa of caspase-3 precursor were detected. Nonspecific binding (NS) and total cytochrome C are shown as equivalent loading. Pro-caspase-3 levels were quantified densitometrically and normalized to the level of cytochrome C. The control level of procaspase-3 expression was assigned a value of 1. Data are expressed as mean values±SEM of two independent samples. *p<0.05 vs. untreated control. C, solvent control only; T, troglitazone only; P, PDT only; T+P, combination of PDT with troglitazone; PDT, photodynamic therapy.

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