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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

CD spectroscopic studies to validate the stabilization of RET G-quadruplex with berberine. a CD titration spectra for the RET-WT (5 μM) in the absence and presence of increasing concentrations of berberine (b) CD titration spectra for the RET-WT (5 μM) in the absence and in the presence of berberine (5 Equivalents) with increasing temperatures from 20 °C to 90 °C. c Melting curve of the RET-WT G-quadruplex in the absence and in the presence of berberine (5 Equivalents)
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Fig2: CD spectroscopic studies to validate the stabilization of RET G-quadruplex with berberine. a CD titration spectra for the RET-WT (5 μM) in the absence and presence of increasing concentrations of berberine (b) CD titration spectra for the RET-WT (5 μM) in the absence and in the presence of berberine (5 Equivalents) with increasing temperatures from 20 °C to 90 °C. c Melting curve of the RET-WT G-quadruplex in the absence and in the presence of berberine (5 Equivalents)

Mentions: To further validate the stabilization of the RET G-quadruplex with berberine, CD spectroscopic analysis was performed for the RET-WT sequence in the absence and presence of berberine. First, we determined whether the binding of berberine changes the structural configuration of the RET G-quadruplex structure by monitoring the CD spectra in the absence and presence of increasing concentrations of berberine. As shown in the Fig. 2a the titration of RET-WT sequence (5 μM) with increasing concentrations of berberine did not affect the peak at 262 nm suggesting that berberine does not alter the parallel configuration of the RET G-quadruplex structure [35]. Next, we determined whether the binding of berberine stabilizes the G-quadruplex structure by monitoring the CD spectra in the absence and presence of berberine (5 Equivalents) at increasing temperatures. As shown in Fig. 2b, the intensity of the maximum peak at 262 nm gradually decreased as the temperature increases from 20 °C to 90 °C and a 50 % decrease was observed at ~72 °C, which corresponds to its melting temperature (Tm) (Fig. 2b, c). In the presence of berberine (5 Equivalents) the Tm was increased to ~85 °C (Fig. 2b, c), indicating that the RET G-quadruplex could be stabilized with berberine.Fig. 2


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)

CD spectroscopic studies to validate the stabilization of RET G-quadruplex with berberine. a CD titration spectra for the RET-WT (5 μM) in the absence and presence of increasing concentrations of berberine (b) CD titration spectra for the RET-WT (5 μM) in the absence and in the presence of berberine (5 Equivalents) with increasing temperatures from 20 °C to 90 °C. c Melting curve of the RET-WT G-quadruplex in the absence and in the presence of berberine (5 Equivalents)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: CD spectroscopic studies to validate the stabilization of RET G-quadruplex with berberine. a CD titration spectra for the RET-WT (5 μM) in the absence and presence of increasing concentrations of berberine (b) CD titration spectra for the RET-WT (5 μM) in the absence and in the presence of berberine (5 Equivalents) with increasing temperatures from 20 °C to 90 °C. c Melting curve of the RET-WT G-quadruplex in the absence and in the presence of berberine (5 Equivalents)
Mentions: To further validate the stabilization of the RET G-quadruplex with berberine, CD spectroscopic analysis was performed for the RET-WT sequence in the absence and presence of berberine. First, we determined whether the binding of berberine changes the structural configuration of the RET G-quadruplex structure by monitoring the CD spectra in the absence and presence of increasing concentrations of berberine. As shown in the Fig. 2a the titration of RET-WT sequence (5 μM) with increasing concentrations of berberine did not affect the peak at 262 nm suggesting that berberine does not alter the parallel configuration of the RET G-quadruplex structure [35]. Next, we determined whether the binding of berberine stabilizes the G-quadruplex structure by monitoring the CD spectra in the absence and presence of berberine (5 Equivalents) at increasing temperatures. As shown in Fig. 2b, the intensity of the maximum peak at 262 nm gradually decreased as the temperature increases from 20 °C to 90 °C and a 50 % decrease was observed at ~72 °C, which corresponds to its melting temperature (Tm) (Fig. 2b, c). In the presence of berberine (5 Equivalents) the Tm was increased to ~85 °C (Fig. 2b, c), indicating that the RET G-quadruplex could be stabilized with berberine.Fig. 2

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