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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.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.

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

Cellular effects mediated by RET expression in TT cells. a MTT assay for TT cells, treated with an increasing concentration of berberine for 96 h to determine the cell viability. b The phosphorylation status of Akt, MEK1/2 and ERK1/2 were determined in TT cells following the exposure with different concentrations of berberine. c The Bcl-2 and the Capsase-3 activity in TT cells were determined in the presence of increasing concentrations of berberine. d FACS analysis of TT cells treated with various concentrations of berberine for 48 h. Data are mean +/−s.e.m of three separate experiments. e Western Blot analysis to determine the protein expression of pRb, p-pRb, E2F1, Cyclin E in TT cells
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Fig6: Cellular effects mediated by RET expression in TT cells. a MTT assay for TT cells, treated with an increasing concentration of berberine for 96 h to determine the cell viability. b The phosphorylation status of Akt, MEK1/2 and ERK1/2 were determined in TT cells following the exposure with different concentrations of berberine. c The Bcl-2 and the Capsase-3 activity in TT cells were determined in the presence of increasing concentrations of berberine. d FACS analysis of TT cells treated with various concentrations of berberine for 48 h. Data are mean +/−s.e.m of three separate experiments. e Western Blot analysis to determine the protein expression of pRb, p-pRb, E2F1, Cyclin E in TT cells

Mentions: Since RET is a growth factor receptor and mainly involved in mediating the cell proliferation and viability, we examined the effect of berberine on the growth of TT cell line by MTT assay. As shown in Fig. 6a, the viability of TT cells decreased with increasing concentrations of berberine and the IC50 was found to be approximately 1 μg/ml after 96 h exposure. This clearly shows that the RET expression plays a major role in regulating the cell proliferation and viability.Fig. 6


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)

Cellular effects mediated by RET expression in TT cells. a MTT assay for TT cells, treated with an increasing concentration of berberine for 96 h to determine the cell viability. b The phosphorylation status of Akt, MEK1/2 and ERK1/2 were determined in TT cells following the exposure with different concentrations of berberine. c The Bcl-2 and the Capsase-3 activity in TT cells were determined in the presence of increasing concentrations of berberine. d FACS analysis of TT cells treated with various concentrations of berberine for 48 h. Data are mean +/−s.e.m of three separate experiments. e Western Blot analysis to determine the protein expression of pRb, p-pRb, E2F1, Cyclin E in TT cells
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: Cellular effects mediated by RET expression in TT cells. a MTT assay for TT cells, treated with an increasing concentration of berberine for 96 h to determine the cell viability. b The phosphorylation status of Akt, MEK1/2 and ERK1/2 were determined in TT cells following the exposure with different concentrations of berberine. c The Bcl-2 and the Capsase-3 activity in TT cells were determined in the presence of increasing concentrations of berberine. d FACS analysis of TT cells treated with various concentrations of berberine for 48 h. Data are mean +/−s.e.m of three separate experiments. e Western Blot analysis to determine the protein expression of pRb, p-pRb, E2F1, Cyclin E in TT cells
Mentions: Since RET is a growth factor receptor and mainly involved in mediating the cell proliferation and viability, we examined the effect of berberine on the growth of TT cell line by MTT assay. As shown in Fig. 6a, the viability of TT cells decreased with increasing concentrations of berberine and the IC50 was found to be approximately 1 μg/ml after 96 h exposure. This clearly shows that the RET expression plays a major role in regulating the cell proliferation and viability.Fig. 6

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.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.

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