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Selective repression of RET proto-oncogene in medullary thyroid carcinoma by a natural alkaloid berberine.

Kumarasamy VM, Shin YJ, White J, Sun D - BMC Cancer (2015)

Bottom Line: Berberine suppressed the RET expression by more than 90 % in MTC TT cells at a concentration of 2.5 μg/ml with minimal effect on the TPC1 cells.Canadine, which is a structural analogue of berberine, showed little interaction with RET G-quadruplex and also had no effect on RET expression in MTC TT cells.The down-regulation of RET with berberine further inhibited the cell proliferation through cell cycle arrest and activation of apoptosis in TT cells, which was confirmed by a 2-fold increase in the caspase-3 activity and the down-regulation of cell-cycle regulatory proteins.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, University of Arizona, Tucson, Arizona, 85721. vishnuk@email.arizona.edu.

ABSTRACT

Background: The gain-of-function mutation of the RET proto-oncogene, which encodes a receptor tyrosine kinase, is strongly associated with the development of several medullary thyroid carcinomas (MTCs). Thus, the RET protein has been explored as an excellent target for progressive and advanced MTC. In this study we have demonstrated a therapeutic strategy for MTC by suppressing the transcription of RET proto-oncogene though the stabilization of G-quadruplex structure formed on the promoter region of this gene using a natural product berberine.

Methods: Medullary thyroid carcinoma (MTC) TT cell line has been used to evaluate the effects of berberine on RET expression and its downstream signaling pathways. The specificity of berberine was demonstrated by using the papillary thyroid carcinoma TPC1 cell line, which lacks the G-quadruplex forming sequence on the RET promoter region due to chromosomal rearrangement.

Results: Berberine suppressed the RET expression by more than 90 % in MTC TT cells at a concentration of 2.5 μg/ml with minimal effect on the TPC1 cells. Canadine, which is a structural analogue of berberine, showed little interaction with RET G-quadruplex and also had no effect on RET expression in MTC TT cells. The down-regulation of RET with berberine further inhibited the cell proliferation through cell cycle arrest and activation of apoptosis in TT cells, which was confirmed by a 2-fold increase in the caspase-3 activity and the down-regulation of cell-cycle regulatory proteins.

Conclusion: Our data strongly suggest that the G-quadruplex forming region and the stabilization of this structure play a critical role in mediating the repressive effect of berberine on RET transcription.

No MeSH data available.


Related in: MedlinePlus

Effect of berberine on RET expression in both the TT and TPC1 cell lines. a Effect of berberine on the RET mRNA expression in TT cells after 24 and 48 h treatments at various concentrations. b RET protein expression in TT cells were determined by western blotting after the 24 and 48 h exposure to increasing concentrations of berberine. c Effect of berberine on the RET/PTC1 mRNA expression in TPC1 cells was determined following 24 h exposure with various concentrations of berberine. d Protein expression levels of VEGF and c-MYC in TT cells in the presence of berberine at various concentrations and exposed up to 48 h
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Fig3: Effect of berberine on RET expression in both the TT and TPC1 cell lines. a Effect of berberine on the RET mRNA expression in TT cells after 24 and 48 h treatments at various concentrations. b RET protein expression in TT cells were determined by western blotting after the 24 and 48 h exposure to increasing concentrations of berberine. c Effect of berberine on the RET/PTC1 mRNA expression in TPC1 cells was determined following 24 h exposure with various concentrations of berberine. d Protein expression levels of VEGF and c-MYC in TT cells in the presence of berberine at various concentrations and exposed up to 48 h

Mentions: Since our in vitro studies revealed that berberine is a potential small molecule that interacts with and stabilizes the G-quadruplex structure formed on the RET promoter region, we next examined the effect of berberine on the RET expression using in vitro cell-based assays. The MTC TT cell line was chosen in our study because the expression of the RET proto-oncogene in this cell line is under the control of the promoter region that contains the G-rich sequence, which adopts G-quadruplex structure [19]. As shown in Fig. 3a, berberine decreased the RET gene expression by more than 50 % and 90 % after 24 and 48 h exposure respectively at a non-toxic concentration of 2.5 μg/ml. The RET protein expression was also diminished based on the western blot analysis (Fig. 3b).Fig. 3


Selective repression of RET proto-oncogene in medullary thyroid carcinoma by a natural alkaloid berberine.

Kumarasamy VM, Shin YJ, White J, Sun D - BMC Cancer (2015)

Effect of berberine on RET expression in both the TT and TPC1 cell lines. a Effect of berberine on the RET mRNA expression in TT cells after 24 and 48 h treatments at various concentrations. b RET protein expression in TT cells were determined by western blotting after the 24 and 48 h exposure to increasing concentrations of berberine. c Effect of berberine on the RET/PTC1 mRNA expression in TPC1 cells was determined following 24 h exposure with various concentrations of berberine. d Protein expression levels of VEGF and c-MYC in TT cells in the presence of berberine at various concentrations and exposed up to 48 h
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4549123&req=5

Fig3: Effect of berberine on RET expression in both the TT and TPC1 cell lines. a Effect of berberine on the RET mRNA expression in TT cells after 24 and 48 h treatments at various concentrations. b RET protein expression in TT cells were determined by western blotting after the 24 and 48 h exposure to increasing concentrations of berberine. c Effect of berberine on the RET/PTC1 mRNA expression in TPC1 cells was determined following 24 h exposure with various concentrations of berberine. d Protein expression levels of VEGF and c-MYC in TT cells in the presence of berberine at various concentrations and exposed up to 48 h
Mentions: Since our in vitro studies revealed that berberine is a potential small molecule that interacts with and stabilizes the G-quadruplex structure formed on the RET promoter region, we next examined the effect of berberine on the RET expression using in vitro cell-based assays. The MTC TT cell line was chosen in our study because the expression of the RET proto-oncogene in this cell line is under the control of the promoter region that contains the G-rich sequence, which adopts G-quadruplex structure [19]. As shown in Fig. 3a, berberine decreased the RET gene expression by more than 50 % and 90 % after 24 and 48 h exposure respectively at a non-toxic concentration of 2.5 μg/ml. The RET protein expression was also diminished based on the western blot analysis (Fig. 3b).Fig. 3

Bottom Line: Berberine suppressed the RET expression by more than 90 % in MTC TT cells at a concentration of 2.5 μg/ml with minimal effect on the TPC1 cells.Canadine, which is a structural analogue of berberine, showed little interaction with RET G-quadruplex and also had no effect on RET expression in MTC TT cells.The down-regulation of RET with berberine further inhibited the cell proliferation through cell cycle arrest and activation of apoptosis in TT cells, which was confirmed by a 2-fold increase in the caspase-3 activity and the down-regulation of cell-cycle regulatory proteins.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, University of Arizona, Tucson, Arizona, 85721. vishnuk@email.arizona.edu.

ABSTRACT

Background: The gain-of-function mutation of the RET proto-oncogene, which encodes a receptor tyrosine kinase, is strongly associated with the development of several medullary thyroid carcinomas (MTCs). Thus, the RET protein has been explored as an excellent target for progressive and advanced MTC. In this study we have demonstrated a therapeutic strategy for MTC by suppressing the transcription of RET proto-oncogene though the stabilization of G-quadruplex structure formed on the promoter region of this gene using a natural product berberine.

Methods: Medullary thyroid carcinoma (MTC) TT cell line has been used to evaluate the effects of berberine on RET expression and its downstream signaling pathways. The specificity of berberine was demonstrated by using the papillary thyroid carcinoma TPC1 cell line, which lacks the G-quadruplex forming sequence on the RET promoter region due to chromosomal rearrangement.

Results: Berberine suppressed the RET expression by more than 90 % in MTC TT cells at a concentration of 2.5 μg/ml with minimal effect on the TPC1 cells. Canadine, which is a structural analogue of berberine, showed little interaction with RET G-quadruplex and also had no effect on RET expression in MTC TT cells. The down-regulation of RET with berberine further inhibited the cell proliferation through cell cycle arrest and activation of apoptosis in TT cells, which was confirmed by a 2-fold increase in the caspase-3 activity and the down-regulation of cell-cycle regulatory proteins.

Conclusion: Our data strongly suggest that the G-quadruplex forming region and the stabilization of this structure play a critical role in mediating the repressive effect of berberine on RET transcription.

No MeSH data available.


Related in: MedlinePlus