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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 the promoter activity of the RET gene. a Effect of berberine on the luciferase expression in HEK293-WT and HEK293-MT1 cell lines following the treatment with berberine up to 24 h. Luciferase activity in cell lysates was measured as relative luminescence units (RLU) and normalized to the total protein content. Experiments were performed in triplicate. Error bar represent one s.d. above and below the mean % luciferase activity. b DMS foot printing on the RET-WT G-quadruplex forming sequence in the absence and in the presence of berberine (5 Equivalents) following 0.2 % DMS treatment (lanes C & B respectively). Purine & pyrimidine sequencing act as single base ladders to identify the protected and cleaved guanines after piperidine treatment (lanes 1 & 2 respectively). c Schematic models for the parallel G-quadruplexes formed by RET-WT (d) DMS foot printing on the RET-MT1 quadruplex forming sequence in the absence and in the presence of 5 equivalents of berberine following 0.2 % DMS treatment (lanes C & B respectively). e ChIP analysis to determine the effect of berberine in recruiting the SP1 and RNA pol II to the GC box region in the RET promoter region in TT cells following 48 h exposure at two different concentrations. Recruitment of SP1 and Pol II to the RET proximal promoter region was assessed by PCR using RET promoter specific primers. 1 % of the input DNA was used as internal control (Input) and isotype-matched IgG was used as a negative control for immunoprecipitation (Ig)
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Fig4: Effect of berberine on the promoter activity of the RET gene. a Effect of berberine on the luciferase expression in HEK293-WT and HEK293-MT1 cell lines following the treatment with berberine up to 24 h. Luciferase activity in cell lysates was measured as relative luminescence units (RLU) and normalized to the total protein content. Experiments were performed in triplicate. Error bar represent one s.d. above and below the mean % luciferase activity. b DMS foot printing on the RET-WT G-quadruplex forming sequence in the absence and in the presence of berberine (5 Equivalents) following 0.2 % DMS treatment (lanes C & B respectively). Purine & pyrimidine sequencing act as single base ladders to identify the protected and cleaved guanines after piperidine treatment (lanes 1 & 2 respectively). c Schematic models for the parallel G-quadruplexes formed by RET-WT (d) DMS foot printing on the RET-MT1 quadruplex forming sequence in the absence and in the presence of 5 equivalents of berberine following 0.2 % DMS treatment (lanes C & B respectively). e ChIP analysis to determine the effect of berberine in recruiting the SP1 and RNA pol II to the GC box region in the RET promoter region in TT cells following 48 h exposure at two different concentrations. Recruitment of SP1 and Pol II to the RET proximal promoter region was assessed by PCR using RET promoter specific primers. 1 % of the input DNA was used as internal control (Input) and isotype-matched IgG was used as a negative control for immunoprecipitation (Ig)

Mentions: To investigate whether berberine suppresses the RET gene expression by inhibiting its promoter activity through G-quadruplex stabilization, two isogenic cell lines HEK293-WT and HEK293-MT1 in which the expression of the luciferase reporter gene is under the control of wild-type and G4 knockout (KO) mutant RET promoter regions respectively were used [19]. As shown in Fig. 4a, approximately 60 % decrease in the luciferase expression was observed in both cell lines in the presence of berberine (10 μg/ml). This suggests that the inhibitory effect of berberine is independent of the G-quadruplex structure formed on the RET promoter region because the G4 KO sequence in the HEK293-MT1 cell line is unable to form this structure [19]. Based on this data we predicted that berberine could be able to bind the RET-MT1 sequence on the promoter region and might form an unknown structure, through which it inhibits RET gene promoter activity.Fig. 4


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 the promoter activity of the RET gene. a Effect of berberine on the luciferase expression in HEK293-WT and HEK293-MT1 cell lines following the treatment with berberine up to 24 h. Luciferase activity in cell lysates was measured as relative luminescence units (RLU) and normalized to the total protein content. Experiments were performed in triplicate. Error bar represent one s.d. above and below the mean % luciferase activity. b DMS foot printing on the RET-WT G-quadruplex forming sequence in the absence and in the presence of berberine (5 Equivalents) following 0.2 % DMS treatment (lanes C & B respectively). Purine & pyrimidine sequencing act as single base ladders to identify the protected and cleaved guanines after piperidine treatment (lanes 1 & 2 respectively). c Schematic models for the parallel G-quadruplexes formed by RET-WT (d) DMS foot printing on the RET-MT1 quadruplex forming sequence in the absence and in the presence of 5 equivalents of berberine following 0.2 % DMS treatment (lanes C & B respectively). e ChIP analysis to determine the effect of berberine in recruiting the SP1 and RNA pol II to the GC box region in the RET promoter region in TT cells following 48 h exposure at two different concentrations. Recruitment of SP1 and Pol II to the RET proximal promoter region was assessed by PCR using RET promoter specific primers. 1 % of the input DNA was used as internal control (Input) and isotype-matched IgG was used as a negative control for immunoprecipitation (Ig)
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Fig4: Effect of berberine on the promoter activity of the RET gene. a Effect of berberine on the luciferase expression in HEK293-WT and HEK293-MT1 cell lines following the treatment with berberine up to 24 h. Luciferase activity in cell lysates was measured as relative luminescence units (RLU) and normalized to the total protein content. Experiments were performed in triplicate. Error bar represent one s.d. above and below the mean % luciferase activity. b DMS foot printing on the RET-WT G-quadruplex forming sequence in the absence and in the presence of berberine (5 Equivalents) following 0.2 % DMS treatment (lanes C & B respectively). Purine & pyrimidine sequencing act as single base ladders to identify the protected and cleaved guanines after piperidine treatment (lanes 1 & 2 respectively). c Schematic models for the parallel G-quadruplexes formed by RET-WT (d) DMS foot printing on the RET-MT1 quadruplex forming sequence in the absence and in the presence of 5 equivalents of berberine following 0.2 % DMS treatment (lanes C & B respectively). e ChIP analysis to determine the effect of berberine in recruiting the SP1 and RNA pol II to the GC box region in the RET promoter region in TT cells following 48 h exposure at two different concentrations. Recruitment of SP1 and Pol II to the RET proximal promoter region was assessed by PCR using RET promoter specific primers. 1 % of the input DNA was used as internal control (Input) and isotype-matched IgG was used as a negative control for immunoprecipitation (Ig)
Mentions: To investigate whether berberine suppresses the RET gene expression by inhibiting its promoter activity through G-quadruplex stabilization, two isogenic cell lines HEK293-WT and HEK293-MT1 in which the expression of the luciferase reporter gene is under the control of wild-type and G4 knockout (KO) mutant RET promoter regions respectively were used [19]. As shown in Fig. 4a, approximately 60 % decrease in the luciferase expression was observed in both cell lines in the presence of berberine (10 μg/ml). This suggests that the inhibitory effect of berberine is independent of the G-quadruplex structure formed on the RET promoter region because the G4 KO sequence in the HEK293-MT1 cell line is unable to form this structure [19]. Based on this data we predicted that berberine could be able to bind the RET-MT1 sequence on the promoter region and might form an unknown structure, through which it inhibits RET gene promoter activity.Fig. 4

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