J Korean Med Sci.  2013 Apr;28(4):527-533. 10.3346/jkms.2013.28.4.527.

The Synergistic Apoptotic Interaction of Indole-3-Carbinol and Genistein with TRAIL on Endometrial Cancer Cells

• Parajuli B
• Shin SJ
• Kwon SH
• Cha SD
• Lee HG
• Bae I
• Cho CH

• KMID: 1786959
• DOI: http://doi.org/10.3346/jkms.2013.28.4.527

Abstract

Induction of apoptosis in target cells is a key mechanism by which chemotherapy promotes cell killing. The purpose of this study was to determine whether Indole-3-Carbinol (I3C) and Genistein in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induce apoptosis in endometrial cancer cell (Ishikawa) and to assess apoptotic mechanism. The MTT assay and flow cytometry were performed to determine cell viability and cell cycle. The induction of apoptosis was measured by caspase-3 activity test, DNA fragmentation assay, annexin V binding assay and western blot analysis. There was no effect in cell growth inhibition and cell cycle progression alone or in two-combination. However, the treatment of I3C and Genistein followed by TRAIL showed significant cell death and marked increase in sub-G1 arrest. Three-combination treatment revealed elevated expression of DR4, DR5 and cleaved forms of caspase-3, caspase-8, PARP. The Flip was found down regulated. Moreover, increase in caspase-3 activity and DNA fragmentation indicated the induction of apoptosis. The results indicate that I3C and Genistein with TRAIL synergistically induced apoptosis via death receptor dependent pathway. Our findings might provide a new insight into the development of novel combination therapies against endometrial cancer.

Keyword

Apoptosis; Indole-3-Carbinol; Genistein; TRAIL; Endometrial Neoplasms

MeSH Terms

Anticarcinogenic Agents/*pharmacology
Apoptosis/*drug effects
Caspase 3/metabolism
Caspase 8/metabolism
Cell Line, Tumor
Drug Synergism
Endometrial Neoplasms/metabolism/pathology
Female
G1 Phase Cell Cycle Checkpoints/drug effects
Genistein/*pharmacology
Humans
Indoles/*pharmacology
Poly(ADP-ribose) Polymerases/metabolism
Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism
TNF-Related Apoptosis-Inducing Ligand/*pharmacology
Anticarcinogenic Agents
Indoles
Receptors, TNF-Related Apoptosis-Inducing Ligand
TNF-Related Apoptosis-Inducing Ligand
Genistein
Poly(ADP-ribose) Polymerases
Caspase 3
Caspase 8

Figure

• 

  Fig. 1 Growth inhibitory effect of I3C, Genistein (G) and TRAIL (T) in Ishikawa cells. Cells were treated with DMSO (control) or I3C (50 µM) and Genistein (20 µM) and TRAIL (10, 20, and 50 ng/mL) alone or in combination for 24 hr. Cell viability was measured by MTT assay and the results were expressed in percentage of viable cells. Values are means ± SD. *P < 0.05.

• 

  Fig. 2 Effect of I3C, Genistein (G) and TRAIL (T) in cell cycle profile and apoptosis. (A) After treatment with DMSO (control) or I3C (50 µM), Genistein (20 µM) and TRAIL (10, 20, and 50 ng/mL) alone or in combination, cultured endometrial cancer cells were harvested, fixed, stained with PI and analyzed by flow cytometric analysis. The values represent the number of cells in different phases of the cell cycle progression as a percentage of total cells. (B) The dual parameter dots combining annexin V-FITC and PI fluorescence showed apoptotic cells in the lower right quadrant (annexin V+PI-) and necrotic cells in the upper left quadrant (annexin V-PI+).

• 

  Fig. 3 Effect of I3C, Genistein (G) and TRAIL (T) on caspase-3 activity and DNA fragmentation. Cells were treated with DMSO (control) or I3C (50 µM), Genistein (20 µM) and TRAIL (10, 20, and 50 ng/mL) alone or in combination for 24 hr. (A) Cell lysates were prepared and used to profile caspase-3 activity. (B) DNA fragmentation was analyzed using an ELISA and (C) agarose gel electrophoresis. Values are means ± SD. *P < 0.05.

• 

  Fig. 4 Effect of I3C and Genistein followed by TRAIL on apoptosis in Ishikawa cells. Cells were exposed to I3C (50 µM), Genistein (20 µM) and TRAIL (10, 20, and 50 ng/mL) alone or in combination for 24 hr. After treatment, total protein was isolated. Expression of DR4, DR5 cleaved caspase-3, caspase-8, PARP and Flip proteins were analyzed by western blotting. Beta-actin was used as an internal loading control. Each band was quantified by densitometric analysis and presented in a bar graph.

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