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RNA binding protein CUGBP2/CELF2 mediates curcumin-induced mitotic catastrophe of pancreatic cancer cells.

Subramaniam D, Ramalingam S, Linehan DC, Dieckgraefe BK, Postier RG, Houchen CW, Jensen RA, Anant S - PLoS ONE (2011)

Bottom Line: Curcumin inhibited the proliferation, while inducing G2-M arrest and apoptosis resulting in mitotic catastrophe of various pancreatic cancer cells.COX-2 and VEGF mRNA levels were reduced to control levels, while proteins levels were higher.These data suggest that translation inhibition is a novel mechanism of action for curcumin during the therapeutic intervention of pancreatic cancers.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas, United States of America. dsubramaniam@kumc.edu

ABSTRACT

Background: Curcumin inhibits the growth of pancreatic cancer tumor xenografts in nude mice; however, the mechanism of action is not well understood. It is becoming increasingly clear that RNA binding proteins regulate posttranscriptional gene expression and play a critical role in RNA stability and translation. Here, we have determined that curcumin modulates the expression of RNA binding protein CUGBP2 to inhibit pancreatic cancer growth.

Methodology/principal findings: In this study, we show that curcumin treated tumor xenografts have a significant reduction in tumor volume and angiogenesis. Curcumin inhibited the proliferation, while inducing G2-M arrest and apoptosis resulting in mitotic catastrophe of various pancreatic cancer cells. This was further confirmed by increased phosphorylation of checkpoint kinase 2 (Chk2) protein coupled with higher levels of nuclear cyclin B1 and Cdc-2. Curcumin increased the expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) mRNA, but protein levels were lower. Furthermore, curcumin increased the expression of RNA binding proteins CUGBP2/CELF2 and TIA-1. CUGBP2 binding to COX-2 and VEGF mRNA was also enhanced, thereby increasing mRNA stability, the half-life changing from 30 min to 8 h. On the other hand, silencer-mediated knockdown of CUGBP2 partially restored the expression of COX-2 and VEGF even with curcumin treatment. COX-2 and VEGF mRNA levels were reduced to control levels, while proteins levels were higher.

Conclusion/significance: Curcumin inhibits pancreatic tumor growth through mitotic catastrophe by increasing the expression of RNA binding protein CUGBP2, thereby inhibiting the translation of COX-2 and VEGF mRNA. These data suggest that translation inhibition is a novel mechanism of action for curcumin during the therapeutic intervention of pancreatic cancers.

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Curcumin inhibits COX-2 and VEGF protein expression in pancreatic cancer cells while inducing CUGBP2 and TIA-1.(A) mRNA expression. MiaPaCa-2 cells were treated with curcumin for 2 h. Curcumin treatment increased the levels of COX-2, VEGF, CUGBP2 and TIA-1 mRNA. There was no significant change of HuR mRNA expression (* p<0.05). Data from three independent experiments. (B) Western blot analyses demonstrate that lysates with curcumin treated MiaPaCa-2 cells have lower levels of COX-2 and VEGF proteins and increasing levels of CUGBP2 and TIA-1. Representative of three independent experiments. (C) (Left panel), Increased binding of CUGBP2 binding to COX-2 or VEGF mRNA following curcumin treatment. Whole cell extract (T) from curcumin-treated cells were immunoprecipitated with anti-CUGBP2 antibody, and RNA from the immunoprecipitates (P) and supernatant (S) were isolated and subjected to RT-PCR for COX-2 and VEGF mRNA. Data demonstrates increased COX-2 or VEGF mRNA in the pellet of curcumin-treated cells. (Right panel) Data was quantified and there was a clear increase in COX-2 and VEGF mRNA in the pellet of curcumin treated cells. (D) MiaPaCa-2 cells were treated with curcumin for 2 h and the stability of COX-2 and VEGF mRNA were determined following addition of actinomycin D (final concentration: 10 µg/ml). Curcumin increases the half-life of COX-2 mRNA from 30 min to 8 h. Similarly, curcumin increased the half-life of VEGF mRNA from 30 min to 8 h. Data demonstrates curcumin treatment significantly increased stability of COX-2 or VEGF mRNA.
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pone-0016958-g005: Curcumin inhibits COX-2 and VEGF protein expression in pancreatic cancer cells while inducing CUGBP2 and TIA-1.(A) mRNA expression. MiaPaCa-2 cells were treated with curcumin for 2 h. Curcumin treatment increased the levels of COX-2, VEGF, CUGBP2 and TIA-1 mRNA. There was no significant change of HuR mRNA expression (* p<0.05). Data from three independent experiments. (B) Western blot analyses demonstrate that lysates with curcumin treated MiaPaCa-2 cells have lower levels of COX-2 and VEGF proteins and increasing levels of CUGBP2 and TIA-1. Representative of three independent experiments. (C) (Left panel), Increased binding of CUGBP2 binding to COX-2 or VEGF mRNA following curcumin treatment. Whole cell extract (T) from curcumin-treated cells were immunoprecipitated with anti-CUGBP2 antibody, and RNA from the immunoprecipitates (P) and supernatant (S) were isolated and subjected to RT-PCR for COX-2 and VEGF mRNA. Data demonstrates increased COX-2 or VEGF mRNA in the pellet of curcumin-treated cells. (Right panel) Data was quantified and there was a clear increase in COX-2 and VEGF mRNA in the pellet of curcumin treated cells. (D) MiaPaCa-2 cells were treated with curcumin for 2 h and the stability of COX-2 and VEGF mRNA were determined following addition of actinomycin D (final concentration: 10 µg/ml). Curcumin increases the half-life of COX-2 mRNA from 30 min to 8 h. Similarly, curcumin increased the half-life of VEGF mRNA from 30 min to 8 h. Data demonstrates curcumin treatment significantly increased stability of COX-2 or VEGF mRNA.

Mentions: Previous studies have demonstrated increased levels of COX-2 mRNA and protein expression in pancreatic adenocarcinomas [38]. Therefore, we next determined the effects of curcumin treatment on COX-2 expression. Curcumin treatment significantly increased COX-2 mRNA levels in MiaPaCa-2 cells (Fig. 5A). However, there was a significant reduction in COX-2 protein levels (Fig. 5B). Similar results were observed on Pan02 cells (data not shown). We also determined the effect of curcumin on VEGF gene expression in the cells. Curcumin treatment significantly increased VEGF mRNA while inhibiting protein levels in MiaPaCa-2 cells (Fig. 5A,B). Similar results were observed in Pan02 cells (data not shown). On the other hand, curcumin significantly increased both CUGBP2 and TIA-1 mRNA and protein expression (Fig. 5A,B). However, there was no significant change observed with HuR expression (Fig. 5A,B). In previous studies, we have demonstrated that CUGBP2 binds to COX-2 3′UTR to inhibit its translation [28]. Given that COX-2 mRNA levels are higher, but protein levels are lower in response to curcumin treatment, we determined whether curcumin increases the binding of CUGBP2 to COX-2 mRNA. Immunoprecipitation coupled RT-PCR demonstrated that there was a higher level of CUGBP2 binding to COX-2 and VEGF mRNA when compared to control, untreated cells (Fig. 5C). Furthermore, actinomycin D experiments demonstrated that curcumin treatment resulted in significantly higher levels of COX-2 mRNA stability, the half-life increasing from 30 min in the untreated cells to ∼8 h (Fig. 5D). Similar results were observed with VEGF; the half-life of VEGF mRNA also increased from 30 min to >8 h in curcumin treated cells (Fig. 5D).


RNA binding protein CUGBP2/CELF2 mediates curcumin-induced mitotic catastrophe of pancreatic cancer cells.

Subramaniam D, Ramalingam S, Linehan DC, Dieckgraefe BK, Postier RG, Houchen CW, Jensen RA, Anant S - PLoS ONE (2011)

Curcumin inhibits COX-2 and VEGF protein expression in pancreatic cancer cells while inducing CUGBP2 and TIA-1.(A) mRNA expression. MiaPaCa-2 cells were treated with curcumin for 2 h. Curcumin treatment increased the levels of COX-2, VEGF, CUGBP2 and TIA-1 mRNA. There was no significant change of HuR mRNA expression (* p<0.05). Data from three independent experiments. (B) Western blot analyses demonstrate that lysates with curcumin treated MiaPaCa-2 cells have lower levels of COX-2 and VEGF proteins and increasing levels of CUGBP2 and TIA-1. Representative of three independent experiments. (C) (Left panel), Increased binding of CUGBP2 binding to COX-2 or VEGF mRNA following curcumin treatment. Whole cell extract (T) from curcumin-treated cells were immunoprecipitated with anti-CUGBP2 antibody, and RNA from the immunoprecipitates (P) and supernatant (S) were isolated and subjected to RT-PCR for COX-2 and VEGF mRNA. Data demonstrates increased COX-2 or VEGF mRNA in the pellet of curcumin-treated cells. (Right panel) Data was quantified and there was a clear increase in COX-2 and VEGF mRNA in the pellet of curcumin treated cells. (D) MiaPaCa-2 cells were treated with curcumin for 2 h and the stability of COX-2 and VEGF mRNA were determined following addition of actinomycin D (final concentration: 10 µg/ml). Curcumin increases the half-life of COX-2 mRNA from 30 min to 8 h. Similarly, curcumin increased the half-life of VEGF mRNA from 30 min to 8 h. Data demonstrates curcumin treatment significantly increased stability of COX-2 or VEGF mRNA.
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pone-0016958-g005: Curcumin inhibits COX-2 and VEGF protein expression in pancreatic cancer cells while inducing CUGBP2 and TIA-1.(A) mRNA expression. MiaPaCa-2 cells were treated with curcumin for 2 h. Curcumin treatment increased the levels of COX-2, VEGF, CUGBP2 and TIA-1 mRNA. There was no significant change of HuR mRNA expression (* p<0.05). Data from three independent experiments. (B) Western blot analyses demonstrate that lysates with curcumin treated MiaPaCa-2 cells have lower levels of COX-2 and VEGF proteins and increasing levels of CUGBP2 and TIA-1. Representative of three independent experiments. (C) (Left panel), Increased binding of CUGBP2 binding to COX-2 or VEGF mRNA following curcumin treatment. Whole cell extract (T) from curcumin-treated cells were immunoprecipitated with anti-CUGBP2 antibody, and RNA from the immunoprecipitates (P) and supernatant (S) were isolated and subjected to RT-PCR for COX-2 and VEGF mRNA. Data demonstrates increased COX-2 or VEGF mRNA in the pellet of curcumin-treated cells. (Right panel) Data was quantified and there was a clear increase in COX-2 and VEGF mRNA in the pellet of curcumin treated cells. (D) MiaPaCa-2 cells were treated with curcumin for 2 h and the stability of COX-2 and VEGF mRNA were determined following addition of actinomycin D (final concentration: 10 µg/ml). Curcumin increases the half-life of COX-2 mRNA from 30 min to 8 h. Similarly, curcumin increased the half-life of VEGF mRNA from 30 min to 8 h. Data demonstrates curcumin treatment significantly increased stability of COX-2 or VEGF mRNA.
Mentions: Previous studies have demonstrated increased levels of COX-2 mRNA and protein expression in pancreatic adenocarcinomas [38]. Therefore, we next determined the effects of curcumin treatment on COX-2 expression. Curcumin treatment significantly increased COX-2 mRNA levels in MiaPaCa-2 cells (Fig. 5A). However, there was a significant reduction in COX-2 protein levels (Fig. 5B). Similar results were observed on Pan02 cells (data not shown). We also determined the effect of curcumin on VEGF gene expression in the cells. Curcumin treatment significantly increased VEGF mRNA while inhibiting protein levels in MiaPaCa-2 cells (Fig. 5A,B). Similar results were observed in Pan02 cells (data not shown). On the other hand, curcumin significantly increased both CUGBP2 and TIA-1 mRNA and protein expression (Fig. 5A,B). However, there was no significant change observed with HuR expression (Fig. 5A,B). In previous studies, we have demonstrated that CUGBP2 binds to COX-2 3′UTR to inhibit its translation [28]. Given that COX-2 mRNA levels are higher, but protein levels are lower in response to curcumin treatment, we determined whether curcumin increases the binding of CUGBP2 to COX-2 mRNA. Immunoprecipitation coupled RT-PCR demonstrated that there was a higher level of CUGBP2 binding to COX-2 and VEGF mRNA when compared to control, untreated cells (Fig. 5C). Furthermore, actinomycin D experiments demonstrated that curcumin treatment resulted in significantly higher levels of COX-2 mRNA stability, the half-life increasing from 30 min in the untreated cells to ∼8 h (Fig. 5D). Similar results were observed with VEGF; the half-life of VEGF mRNA also increased from 30 min to >8 h in curcumin treated cells (Fig. 5D).

Bottom Line: Curcumin inhibited the proliferation, while inducing G2-M arrest and apoptosis resulting in mitotic catastrophe of various pancreatic cancer cells.COX-2 and VEGF mRNA levels were reduced to control levels, while proteins levels were higher.These data suggest that translation inhibition is a novel mechanism of action for curcumin during the therapeutic intervention of pancreatic cancers.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, Kansas, United States of America. dsubramaniam@kumc.edu

ABSTRACT

Background: Curcumin inhibits the growth of pancreatic cancer tumor xenografts in nude mice; however, the mechanism of action is not well understood. It is becoming increasingly clear that RNA binding proteins regulate posttranscriptional gene expression and play a critical role in RNA stability and translation. Here, we have determined that curcumin modulates the expression of RNA binding protein CUGBP2 to inhibit pancreatic cancer growth.

Methodology/principal findings: In this study, we show that curcumin treated tumor xenografts have a significant reduction in tumor volume and angiogenesis. Curcumin inhibited the proliferation, while inducing G2-M arrest and apoptosis resulting in mitotic catastrophe of various pancreatic cancer cells. This was further confirmed by increased phosphorylation of checkpoint kinase 2 (Chk2) protein coupled with higher levels of nuclear cyclin B1 and Cdc-2. Curcumin increased the expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) mRNA, but protein levels were lower. Furthermore, curcumin increased the expression of RNA binding proteins CUGBP2/CELF2 and TIA-1. CUGBP2 binding to COX-2 and VEGF mRNA was also enhanced, thereby increasing mRNA stability, the half-life changing from 30 min to 8 h. On the other hand, silencer-mediated knockdown of CUGBP2 partially restored the expression of COX-2 and VEGF even with curcumin treatment. COX-2 and VEGF mRNA levels were reduced to control levels, while proteins levels were higher.

Conclusion/significance: Curcumin inhibits pancreatic tumor growth through mitotic catastrophe by increasing the expression of RNA binding protein CUGBP2, thereby inhibiting the translation of COX-2 and VEGF mRNA. These data suggest that translation inhibition is a novel mechanism of action for curcumin during the therapeutic intervention of pancreatic cancers.

Show MeSH
Related in: MedlinePlus