RESUMEN
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.
Asunto(s)
Berberina/farmacología , Productos Biológicos/farmacología , Carcinoma Neuroendocrino/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-ret/genética , Neoplasias de la Tiroides/genética , Antineoplásicos/farmacología , Berberina/análogos & derivados , Carcinoma Neuroendocrino/metabolismo , Línea Celular Tumoral , Expresión Génica , Genes Reporteros , Humanos , Regiones Promotoras Genéticas , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-ret/metabolismo , Neoplasias de la Tiroides/metabolismo , Activación Transcripcional/efectos de los fármacosRESUMEN
Rearranged during transfection kinase (RET) is a validated molecular target in medullary thyroid cancer (MTC), as activating mutations in RET are often associated with the development of MTC. The present study reports the first preclinical characterization of salinomycin and selected analogs as potent RET transcriptional inhibitors. Reverse transcriptionPCR and immunoblotting revealed that salinomycin profoundly decreased RET expression in the TT human MTC cell line by inhibiting RET transcription. Moreover, salinomycin resulted in remarkable antiproliferative activity against MTC that is driven by RET (gain of function mutation) by selectively inhibiting the intracellular PI3K/Akt/mTOR signaling pathway. Also, flow cytometry and fluorescenceactivated cell sorting showed that salinomycin induces G1 phase arrest and apoptosis by reducing the expression of retinoblastoma protein, E2F1, cyclin D and CDK4. The structureactivity relationship of salinomycin was investigated in this study. Some of the salinomycin derivatives showed the ability to reduce RET expression where others fail to alter RET expression. These results suggest that the RETsuppressing effect of salinomycin may be largely attributed to disruption of the Wnt pathway, presumably through interference with the ternary LRP6FrizzledWnt complex. Furthermore, these findings support the further preclinical evaluation of salinomycin and its analogs as a promising new class of therapeutic agents for the improved treatment of MTC.
Asunto(s)
Antibacterianos/farmacología , Biomarcadores de Tumor/antagonistas & inhibidores , Carcinoma Neuroendocrino/tratamiento farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-ret/antagonistas & inhibidores , Piranos/farmacología , Neoplasias de la Tiroides/tratamiento farmacológico , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma Neuroendocrino/metabolismo , Carcinoma Neuroendocrino/patología , Ciclo Celular , Proliferación Celular , Humanos , Proteínas Proto-Oncogénicas c-ret/genética , Proteínas Proto-Oncogénicas c-ret/metabolismo , Neoplasias de la Tiroides/metabolismo , Neoplasias de la Tiroides/patología , Células Tumorales CultivadasRESUMEN
Dominant-activating mutations in the RET (rearranged during transfection) proto-oncogene, which encodes a receptor tyrosine kinase, is often associated with the development of medullary thyroid carcinoma (MTC). The proximal promoter region of the RET gene consists of a guanine-rich sequence containing five runs of three consecutive guanine residues that serve as the binding site for transcriptional factors. As we have recently shown, this stretch of nucleotides in the promoter region is highly dynamic in nature and tend to form non-B DNA secondary structures called G-quadruplexes, which suppress the transcription of the RET gene. In the present study, ellipticine and its derivatives were identified as excellent RET G-quadruplex stabilizing agents. Circular dichroism (CD) spectroscopic studies revealed that the incorporation of a piperidine ring in an ellipticine derivative, NSC311153 improves its binding with the G-quadruplex structure and the stability induced by this compound is more potent than ellipticine. Furthermore, this compound also interfered with the transcriptional mechanism of the RET gene in an MTC derived cell line, TT cells and significantly decreased the endogenous RET protein expression. We demonstrated the specificity of NSC311153 by using papillary thyroid carcinoma (PTC) cells, the TPC1 cell line which lacks the G-quadruplex forming sequence in the promoter region due to chromosomal rearrangement. The RET downregulation selectively suppresses cell proliferation by inhibiting the intracellular Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways in the TT cells. In the present study, we also showed that the systemic administration of a water soluble NSC311153 analog in a mouse MTC xenograft model inhibited the tumor growth through RET downregulation.