Ann Surg Treat Res.  2014 Feb;86(2):68-75. 10.4174/astr.2014.86.2.68.

Antichemosensitizing effect of resveratrol in cotreatment with oxaliplatin in HCT116 colon cancer cell

• Park DG

• KMID: 1908500
• DOI: http://doi.org/10.4174/astr.2014.86.2.68

Abstract

PURPOSE
Although oxaliplatin is one of the most widely used chemotherapeutic agents for the treatment of advanced stages of colorectal cancers in clinic, cancer cells often develop oxaliplatin drug resistance. Thus, overcoming oxaliplatin drug resistance is a major issue in the successful treatment for advanced stages of colorectal malignancy. In order to maximize oxaliplatin therapy, we examined whether resveratrol, a natural phytochemical known to have chemopreventive effects on cancers, can have a chemosensitizing effect upon cotreatment with oxaliplatin. Survivin, a small inhibitor of apoptosis protein (IAP), expression is examined using HCT116 colon cancer cells.
METHODS
In order to examine resveratrol chemosensitizing effect upon oxaliplatin cotreatment, survivin transcripts and protein expression, cell proliferation, and apoptotic responses were evaluated using HCT116 cells. Reverse transcription polymerase chain reaction (RT-PCR), Western blot, crystal violet staining analyses were performed. For survivin specific inhibition, YM155 molecule was used.
RESULTS
Although oxaliplatin significantly suppressed survivin transcripts and protein expression level in HCT116 cells, resveratrol cotreatment induced restoration of survivin expression level of both transcripts and protein. Apoptotic induction by oxaliplatin only treatment was nullified upon resveratrol cotreatment. Induction of survivin restoration upon resveratrol cotreatment also occurred when survivin specific inhibitor, YM155, was used. In addition to survivin restoration, resveratrol cotreatment also induced restoration of Bcl-2/caspase-3 expression suppressed by oxaliplatin only treatment.
CONCLUSION
Resveratrol has an antichemosensitizing effect upon cotreatment with oxaliplatin in HCT colon cancer cells. This antichemosensitizing effect of resveratrol can be cell-type specific. However, clinical use of resveratrol cotreatment with oxaliplatin should be approached cautiously.

Keyword

Resveratrol; Survivin; Oxaliplatin; Chemoresistance; Colorectal neoplasms

MeSH Terms

Blotting, Western
Cell Proliferation
Colon*
Colonic Neoplasms*
Colorectal Neoplasms
Drug Resistance
Gentian Violet
HCT116 Cells
Inhibitor of Apoptosis Proteins
Polymerase Chain Reaction
Reverse Transcription
Gentian Violet
Inhibitor of Apoptosis Proteins

Figure

• 

  Fig. 1 Resveratrol posttreatment nullifies oxaliplatin pretreatment induced survivin suppression. (A) HCT116 cells were incubated with oxaliplatin (2, 5 µM), resveratrol (30, 50 µM). Expression of survivin mRNA and its alternative splicing variants (Delta Ex3, 2B) was measured by reverse transcription-polymerase chain reaction (RT-PCR). The primers used were described in materials and methods. (B) Surviving cells pretreated with oxaliplatin (5 µM for 24 hours) were collected and posttreated with oxaliplatin (5 µM) and resveratrol (5 µM) for 24 hours additionally. And expression of survivin mRNA and its alternative splicing variants (Delta Ex3, 2B) was measured by RT-PCR. (C) Surviving cells pretreated with resveratrol (50 µM for 24 hours) were collected and treated with oxaliplatin (5 µM) and resveratrol (50 µM) for 24 hours additionally. And the expression of survivin mRNA and its alternative splicing variants (Delta Ex3, 2B) was measured by RT-PCR.

• 

  Fig. 2 Resveratrol post-treatment nullifies YM155 and oxaliplatin pre-treatment induced cell proliferation suppression. HCT116 cells were pre-treated with YM155 (survivin inhibitor) for 1 hour. Cells were, then, treated with oxaliplatin and posttreated with resveratrol as described in METHODS section. Cell proliferation activity was measured by crystal violet staining analysis as described in METHODS section. Cell proliferation activity was additively suppressed by oxaliplatin and YM155 copretreatment. The suppressive effect by copretreatment nullified by resveratrol post-treatment (*P < 0.05. **P < 0.05).

• 

  Fig. 3 Resveratrol post-treatment nullifies YM155 and oxaliplatin pretreatment induced apoptotic activity. (A) HCT116 cells were pretreated with YM155 (survivin inhibitor) for 1 hour. Cells were, then, treated with oxaliplatin and posttreated with resveratrol as described in METHODS section. Cells were stained with Hoechest 33258 and photos were taken for morphological analysis as described in METHODS section. (B) Resveratrol posttreatment nullified oxaliplatin pretreatment induced apoptotic activity (*P < 0.005). (C) Resveratrol post-treatment nullified YM155 and oxaliplatin copretreatment induced apoptotic activity (*P < 0.05. **P < 0.005. ***P < 0.005). The results were analyzed using analysis of variance followed by Tukey multiple comparisons of means (95% family-wise confidence level). A P-value of <0.05 was designated as the level of significance.

• 

  Fig. 4 Resveratrol posttreatment nullifies YM155 and oxaliplatin pretreatment induced protein expression. HCT116 cells were pretreated with YM155 (survivin inhibitor) for 1 hour. Cells were, then, treated with oxaliplatin and posttreated with resveratrol as described in METHODS section. Protein expression of survivin, Bcl-2, and caspase-3 was examined by Western blot analysis. Protein expression levels of survivin, Bcl-2, and caspase-3 were additively suppressed by oxaliplatin and YM155 co-pretreatment. The suppressive effect by co-pretreatment was nullified by resveratrol posttreatment.

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