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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, VEGF and Cyclin D1 expression in the tumors while inducing CUGBP2 and TIA-1.(A) Total RNA from Pan02 tumor xenografts were subjected to Real time PCR. Curcumin treatment resulted in reduced COX-2, VEGF and cyclin D1 mRNA levels when compared to controls. On the other hand, CUGBP2 and TIA-1 mRNA expression was increased (*p<0.05). Data is an average from xenografts in 5 mice. (B) Western blot analysis demonstrated that tissue lysates from the curcumin treated animals have significantly lower levels of COX-2, VEGF, and cyclin D1 proteins but increased levels of CUGBP2 and TIA-1 proteins. (C) Immunohistochemistry demonstrates that curcumin treatment significantly reduced the expression of COX-2 and VEGF. (D) Immunohistochemistry demonstrates increased expression of CUGBP2 and TIA-1 in the tumor xenografts.
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pone-0016958-g004: Curcumin inhibits COX-2, VEGF and Cyclin D1 expression in the tumors while inducing CUGBP2 and TIA-1.(A) Total RNA from Pan02 tumor xenografts were subjected to Real time PCR. Curcumin treatment resulted in reduced COX-2, VEGF and cyclin D1 mRNA levels when compared to controls. On the other hand, CUGBP2 and TIA-1 mRNA expression was increased (*p<0.05). Data is an average from xenografts in 5 mice. (B) Western blot analysis demonstrated that tissue lysates from the curcumin treated animals have significantly lower levels of COX-2, VEGF, and cyclin D1 proteins but increased levels of CUGBP2 and TIA-1 proteins. (C) Immunohistochemistry demonstrates that curcumin treatment significantly reduced the expression of COX-2 and VEGF. (D) Immunohistochemistry demonstrates increased expression of CUGBP2 and TIA-1 in the tumor xenografts.

Mentions: COX-2 plays a significant role in carcinogenesis, including increased invasiveness, promotion of angiogenesis and resistance to apoptosis [38]. We therefore determined its expression in tumor xenografts. Real time PCR analyses demonstrated that COX-2 mRNA expression was significantly lower in curcumin treated tumor xenografts when compared to the control tumors (Fig. 4A). This was further confirmed by western blot and immunohistochemistry analyses, where lower levels of COX-2 protein were observed in the curcumin-treated tissues (Fig. 4B,C). Prostaglandins and the other tumor promoters are known to induce the expression of VEGF in epithelial cells which promotes angiogenesis and thereby tumor growth [39]. VEGF mRNA and protein expression was analyzed and was also found to be significantly lower in curcumin treated tumor xenografts when compared to the control tumors (Fig. 4A,B). Immunohistochemistry also demonstrated that curcumin treatment significantly reduced the VEGF staining when compared to control tumor (Fig. 4C). Cyclin D1 is a cell cycle regulatory protein that regulates the G1 to S-phase transition and functions as a cofactor for several transcription factors [40]. Cyclin D1 overexpression has also been linked to the development and progression of cancer [34]. Curcumin treatment inhibited cyclin D1 expression in the tumor xenografts suggesting that it inhibits cancer cell proliferation (Fig. 4A,B). A number of RNA-binding proteins have been identified to recognize ARE-containing sequences and regulate mRNA stability. Members of the CELF (CUGBP2) and ELAV (HuR) family of RNA binding proteins are implicated in various cellular processes including mRNA splicing, editing, stability and translation [23], [41]. We have previously demonstrated that CUGBP2 is induced in cells undergoing apoptosis and functions to inhibit COX-2 mRNA translation [28], [42]. T-cell- restricted intracellular antigen-1 (TIA-1) is also an RNA binding protein that functions as a translational suppressor, especially under conditions of stress [43]. Here we observed that curcumin treatment resulted in a significant increase in both CUGBP2 and TIA-1 mRNA and protein expression in the tumor xenografts (Fig. 4A, B, D). On the other hand, there was no significant change observed in both HuR mRNA and protein levels in response to curcumin (Fig. 3A, B, D).


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, VEGF and Cyclin D1 expression in the tumors while inducing CUGBP2 and TIA-1.(A) Total RNA from Pan02 tumor xenografts were subjected to Real time PCR. Curcumin treatment resulted in reduced COX-2, VEGF and cyclin D1 mRNA levels when compared to controls. On the other hand, CUGBP2 and TIA-1 mRNA expression was increased (*p<0.05). Data is an average from xenografts in 5 mice. (B) Western blot analysis demonstrated that tissue lysates from the curcumin treated animals have significantly lower levels of COX-2, VEGF, and cyclin D1 proteins but increased levels of CUGBP2 and TIA-1 proteins. (C) Immunohistochemistry demonstrates that curcumin treatment significantly reduced the expression of COX-2 and VEGF. (D) Immunohistochemistry demonstrates increased expression of CUGBP2 and TIA-1 in the tumor xenografts.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3037932&req=5

pone-0016958-g004: Curcumin inhibits COX-2, VEGF and Cyclin D1 expression in the tumors while inducing CUGBP2 and TIA-1.(A) Total RNA from Pan02 tumor xenografts were subjected to Real time PCR. Curcumin treatment resulted in reduced COX-2, VEGF and cyclin D1 mRNA levels when compared to controls. On the other hand, CUGBP2 and TIA-1 mRNA expression was increased (*p<0.05). Data is an average from xenografts in 5 mice. (B) Western blot analysis demonstrated that tissue lysates from the curcumin treated animals have significantly lower levels of COX-2, VEGF, and cyclin D1 proteins but increased levels of CUGBP2 and TIA-1 proteins. (C) Immunohistochemistry demonstrates that curcumin treatment significantly reduced the expression of COX-2 and VEGF. (D) Immunohistochemistry demonstrates increased expression of CUGBP2 and TIA-1 in the tumor xenografts.
Mentions: COX-2 plays a significant role in carcinogenesis, including increased invasiveness, promotion of angiogenesis and resistance to apoptosis [38]. We therefore determined its expression in tumor xenografts. Real time PCR analyses demonstrated that COX-2 mRNA expression was significantly lower in curcumin treated tumor xenografts when compared to the control tumors (Fig. 4A). This was further confirmed by western blot and immunohistochemistry analyses, where lower levels of COX-2 protein were observed in the curcumin-treated tissues (Fig. 4B,C). Prostaglandins and the other tumor promoters are known to induce the expression of VEGF in epithelial cells which promotes angiogenesis and thereby tumor growth [39]. VEGF mRNA and protein expression was analyzed and was also found to be significantly lower in curcumin treated tumor xenografts when compared to the control tumors (Fig. 4A,B). Immunohistochemistry also demonstrated that curcumin treatment significantly reduced the VEGF staining when compared to control tumor (Fig. 4C). Cyclin D1 is a cell cycle regulatory protein that regulates the G1 to S-phase transition and functions as a cofactor for several transcription factors [40]. Cyclin D1 overexpression has also been linked to the development and progression of cancer [34]. Curcumin treatment inhibited cyclin D1 expression in the tumor xenografts suggesting that it inhibits cancer cell proliferation (Fig. 4A,B). A number of RNA-binding proteins have been identified to recognize ARE-containing sequences and regulate mRNA stability. Members of the CELF (CUGBP2) and ELAV (HuR) family of RNA binding proteins are implicated in various cellular processes including mRNA splicing, editing, stability and translation [23], [41]. We have previously demonstrated that CUGBP2 is induced in cells undergoing apoptosis and functions to inhibit COX-2 mRNA translation [28], [42]. T-cell- restricted intracellular antigen-1 (TIA-1) is also an RNA binding protein that functions as a translational suppressor, especially under conditions of stress [43]. Here we observed that curcumin treatment resulted in a significant increase in both CUGBP2 and TIA-1 mRNA and protein expression in the tumor xenografts (Fig. 4A, B, D). On the other hand, there was no significant change observed in both HuR mRNA and protein levels in response to curcumin (Fig. 3A, B, D).

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