Adefovir Dipivoxil as a Therapeutic Candidate for Medullary Thyroid Carcinoma: Targeting RET and STAT3 Proto-Oncogenes.

Aberrant gene expression is often linked to the progression of various cancers, making the targeting of oncogene transcriptional activation a potential strategy to control tumor growth and development. The RET proto-oncogene's gain-of-function mutation is a major cause of medullary thyroid carcinoma (MTC), which is part of multiple endocrine neoplasia type 2 (MEN2) syndrome. In this study, we used a cell-based bioluminescence reporter system driven by the RET promoter to screen for small molecules that potentially suppress the RET gene transcription. We identified adefovir dipivoxil as a transcriptional inhibitor of the RET gene, which suppressed endogenous RET protein expression in MTC TT cells. Adefovir dipivoxil also interfered with STAT3 phosphorylation and showed high affinity to bind to STAT3. Additionally, it inhibited RET-dependent TT cell proliferation and increased apoptosis. These results demonstrate the potential of cell-based screening assays in identifying transcriptional inhibitors for other oncogenes.

Selective Antitumor Activity of Datelliptium toward Medullary Thyroid Carcinoma by Downregulating RET Transcriptional Activity.

Medullary thyroid carcinoma (MTC) is a rare aggressive form of thyroid cancer with high rates of metastasis. Sporadic and hereditary MTC are strongly driven by somatic and germline mutations, respectively, in the transmembrane REarranged during Transfection (RET) proto-oncogene, which encodes a receptor tyrosine kinase. Our previous study identified datelliptium as a novel RET transcription inhibitor, which stabilizes the RET G-quadruplex structures and suppresses RET oncogene transcription. The present study aimed to elucidate the effect of datelliptium on the suppression of epithelial-to-mesenchymal transition (EMT) and metastasis-related behaviors of MTC cells, including cell migration and formation of cancer stem cells (CSCs). Our results demonstrated that datelliptium downregulated the expression of the mesenchymal markers, including N-cadherin, vimentin, slug, snail, and claudin-1. Compared to untreated cells, datelliptium significantly decreased the migration of TT cells in a dose-dependent manner in a wound healing assay. Additionally, datelliptium significantly reduced the size of preformed spheroids from TT cells over the time course. Finally, datelliptium inhibited approximately 75% of MTC xenograft growth with minimal systemic toxicity. In conclusion, datelliptium exerts its antitumor activity against MTC cells by reducing the EMT program, migratory ability, and self-renewal capacity of TT cells, thus preventing invasive and metastatic behavior of MTC.

Salinomycin and its derivatives as potent RET transcriptional inhibitors for the treatment of medullary thyroid carcinoma.

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.

Electrophoretic Mobility Shift Assay and Dimethyl Sulfate Footprinting for Characterization of G-Quadruplexes and G-Quadruplex-Protein Complexes.

DNA G-quadruplexes are globular nucleic acid secondary structures which occur throughout the human genome under physiological conditions. There is accumulating evidence supporting G-quadruplex involvement in a number of important aspects of genome functions, including transcription, replication, and genomic stability, and that protein and enzyme recognition of G-quadruplexes may represent a key event to regulate physiological or pathological pathways. Two important techniques to study G-quadruplexes and their protein interactions are the electrophoretic mobility shift assay (EMSA) and dimethyl sulfate (DMS) footprinting assay. EMSA, one of the most sensitive and robust methods for studying the DNA-protein interactions, can be used to determine the binding parameters and relative affinities of a protein for the G-quadruplex. DMS footprinting is a powerful assay for the initial characterization of G-quadruplexes, which can be used to deduce the guanine bases involved in the formation of G-tetrads under physiological salt conditions. DMS footprinting can also reveal important information in G-quadruplex-protein complexes on protein contacts and regional changes in DNA G-quadruplex upon protein binding. In this paper, we will provide a detailed protocol for the EMSA and DMS footprinting assays for characterization of G-quadruplexes and G-quadruplex-protein complexes. Expected outcomes and references to extensions of the method will be further discussed.

Chromatin Immunoprecipitation Assay to Analyze the Effect of G-Quadruplex Interactive Agents on the Binding of RNA Polymerase II and Transcription Factors to a Target Promoter Region.

Growing evidence suggests the existence of G-quadruplexes and their involvement in transcriptional regulation of many human genes, including VEGF. These studies also provide strong evidence that G-quadruplex structures are stabilized by binding to small molecules, resulting in the modulation of the transcription of genes whose promoters form G-quadruplexes. Here, we describe a chromatin immunoprecipitation (ChIP) assay to determine whether G-quadruplex-interactive agents influence the recruitment of cellular transcription factors, such as Sp1, nucleolin, or hnRNP-K to target genes that contain potential G-quadruplex (G4)-forming sequences in their promoters, subsequently modulating the occupancy of RNA Pol II on the same promoter region.

Demonstration of a potent RET transcriptional inhibitor for the treatment of medullary thyroid carcinoma based on an ellipticine derivative.

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.

Synthesis of Constrained Heterocycles Employing Two Post-Ugi Cyclization Methods for Rapid Library Generation with In Cellulo Activity.

Benzimidazoles and quinoxalinones are present in the core of many pharmacologically relevant compounds. Several combinatorial methods have been developed to attach ring systems to both scaffolds for derivatization at select positions. Herein, we describe the development of novel constrained heterocyclic compounds attached to the N1 position of both benzimidazole and quinoxalinone scaffolds. Utilizing robust post-Ugi cyclization methods, including the Ugi-deprotection-cyclization (UDC) methodology, allows for efficient access to a new area of chemical space. Additionally, molecular modeling and in cellulo screening was employed to therapeutically validate the compounds formed with this method.

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

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.

Synthesis and characterization of a small analogue of the anticancer natural product leinamycin.

Leinamycin (1) is a Streptomyces-derived natural product that displays nanomolar IC(50) values against human cancer cell lines. In the work described here, we report the synthesis and characterization of a small leinamycin analogue 19 that closely resembles the 'upper-right quadrant' of the natural product, consisting of an alicyclic 1,2-dithiolan-3-one 1-oxide heterocycle connected to an alkene by a two-carbon linker. The results indicate that this small analogue contains the core set of functional groups required to enable thiol-triggered generation of both redox active polysulfides and an episulfonium ion intermediate via the complex reaction cascade first seen in the natural product leinamycin. The small leinamycin analogue 19 caused thiol-triggered oxidative DNA strand cleavage in a manner similar to the natural product, but did not alkyate duplex DNA effectively. This highlights the central role of the 18-membered macrocycle of leinamycin in driving efficient DNA alkylation by the natural product.

DNA cleavage induced by antitumor antibiotic leinamycin and its biological consequences.

The natural product leinamycin has been found to produce abasic sites in duplex DNA through the hydrolysis of the glycosidic bond of guanine residues modified by this drug. In the present study, using a synthetic oligonucleotide duplex, we demonstrate spontaneous DNA strand cleavage at leinamycin-induced abasic sites through a ß-elimination reaction. However, methoxyamine modification of leinamycin-induced abasic sites was found to be refractory to the spontaneous ß-elimination reaction. Furthermore, this complex was even resistant to the δ-elimination reaction with hot piperidine treatment. Bleomycin and methyl methanesulfonate also induced strand cleavage in a synthetic oligonucleotide duplex even without thermal treatment. However, methoxyamine has a negligible effect on DNA strand cleavage induced by both drugs, suggesting that the mechanism of DNA cleavage induced by leinamycin might be different from those induced by bleomycin or methyl methanesulfonate. In this study, we also assessed the cytotoxicity of leinamycin against a collection of mammalian cell lines defective in various repair pathways. The mammalian cell line defective in the nucleotide excision repair (NER) or base excision repair (BER) pathways was about 3 to 5 times more sensitive to leinamycin as compared to the parental cell line. In contrast, the radiosensitive mutant xrs-5 cell line deficient in V(D)J recombination showed similar sensitivity towards leinamycin compared to the parental cell line. Collectively, our findings suggest that both NER and BER pathways play an important role in the repair of DNA damage caused by leinamycin.

The macrocycle of leinamycin imparts hydrolytic stability to the thiol-sensing 1,2-dithiolan-3-one 1-oxide unit of the natural product.

Reaction of cellular thiols with the 1,2-dithiolan-3-one 1-oxide moiety of leinamycin triggers the generation of DNA-damaging reactive intermediates. Studies with small, synthetic analogues of leinamycin reveal that the macrocyclic portion of the natural product imparts remarkable hydrolytic stability to the 1,2-dithiolan-3-one 1-oxide heterocycle without substantially compromising its thiol-sensing property.

Heterogeneous nuclear ribonucleoprotein K and nucleolin as transcriptional activators of the vascular endothelial growth factor promoter through interaction with secondary DNA structures.

The human vascular endothelial growth factor (VEGF) promoter contains a polypurine/polypyrimidine (pPu/pPy) tract that is known to play a critical role in its transcriptional regulation. This pPu/pPy tract undergoes a conformational transition between B-DNA, single-stranded DNA, and atypical secondary DNA structures such as G-quadruplexes and i-motifs. We studied the interaction of the cytosine-rich (C-rich) and guanine-rich (G-rich) strands of this tract with transcription factors heterogeneous nuclear ribonucleoprotein (hnRNP) K and nucleolin, respectively, both in vitro and in vivo and their potential role in the transcriptional control of VEGF. Using chromatin immunoprecipitation (ChIP) assay for our in vivo studies and electrophoretic mobility shift assay (EMSA) for our in vitro studies, we demonstrated that both nucleolin and hnRNP K bind selectively to the G- and C-rich sequences, respectively, in the pPu/pPy tract of the VEGF promoter. The small interfering RNA (siRNA)-mediated silencing of either nucleolin or hnRNP K resulted in the down-regulation of basal VEGF gene, suggesting that they act as activators of VEGF transcription. Taken together, the identification of transcription factors that can recognize and bind to atypical DNA structures within the pPu/pPy tract will provide new insight into mechanisms of transcriptional regulation of the VEGF gene.

Evidence of the formation of G-quadruplex structures in the promoter region of the human vascular endothelial growth factor gene.

The polypurine/polypyrimidine (pPu/pPy) tract of the human vascular endothelial growth factor (VEGF) gene is proposed to be structurally dynamic and to have potential to adopt non-B DNA structures. In the present study, we further provide evidence for the existence of the G-quadruplex structure within this tract both in vitro and in vivo using the dimethyl sulfate (DMS) footprinting technique and nucleolin as a structural probe specifically recognizing G-quadruplex structures. We observed that the overall reactivity of the guanine residues within this tract toward DMS was significantly reduced compared with other guanine residues of the flanking regions in both in vitro and in vivo footprinting experiments. We also demonstrated that nucleolin, which is known to bind to G-quadruplex structures, is able to bind specifically to the G-rich sequence of this region in negatively supercoiled DNA. Our chromatin immunoprecipitation analysis further revealed binding of nucleolin to the promoter region of the VEGF gene in vivo. Taken together, our results are in agreement with our hypothesis that secondary DNA structures, such as G-quadruplexes, can be formed in supercoiled duplex DNA and DNA in chromatin in vivo under physiological conditions similar to those formed in single-stranded DNA templates.

Thiol-dependent DNA cleavage by aminomethylated Beaucage's reagent.

Aminomethylated Beaucage's reagent 1 was found to be more potent than 3H-1,2-benzodithiol-3-one 1,1-dioxide (Beaucage's reagent) in causing DNA cleavage. The current study demonstrated the importance of the amino functionality in enhancing DNA-cleaving activities, and such findings may facilitate development of novel sulfur-containing DNA-cleaving molecules in cancer therapy.

Synthesis and antiproliferating activity of iron chelators of hydroxyamino-1,3,5-triazine family.

We synthesized and evaluated new specific tridentate iron(III) chelators of 2,6-bis[hydroxyamino]-1,3,5-triazine (BHT) family for use in iron deprivation cancer therapy. Physical properties of BHT chelators are easily customizable allowing easy penetration through cellular membranes. Antiproliferative activity of new BHT chelators was studied on MDA-MB-231 and MiaPaCa cells and compared to a clinically available new oral iron chelator, deferasirox (DFX). The antiproliferative activity of new chelators was found to correlate with iron(III) chelation ability and some of analogs showed substantially higher antiproliferative activity than DFX.

In vitro footprinting of promoter regions within supercoiled plasmid DNA.

Polypurine/polypyrimidine (pPu/pPy) tracts, which exist in the promoter regions of many growth-related genes, have been proposed to be very dynamic in their conformation. In this chapter, we describe a detailed protocol for DNase I and S1 nuclease footprinting experiments with supercoiled plasmid DNA containing the promoter regions to probe whether there are conformational transitions to B-type DNA, melted DNA, and G-quadruplex structures within this tract. This is demonstrated with the proximal promoter region of the human vascular endothelial growth factor (VEGF) gene, which also contains multiple binding sites for Sp1 and Egr-1 transcription factors.

Biochemical techniques for the characterization of G-quadruplex structures: EMSA, DMS footprinting, and DNA polymerase stop assay.

The proximal promoter region of many human growth-related genes contains a polypurine/polypyrimidine tract that serves as multiple binding sites for Sp1 or other transcription factors. These tracts often contain a guanine-rich sequence consisting of four runs of three or more contiguous guanines separated by one or more bases, corresponding to a general motif known for the formation of an intramolecular G-quadruplex. Recent results provide strong evidence that specific G-quadruplex structures form naturally within these polypurine/polypyrimidine tracts in many human promoter regions, raising the possibility that the transcriptional control of these genes can be modulated by G-quadruplex-interactive agents. In this chapter, we describe three general biochemical methodologies, electrophoretic mobility shift assay (EMSA), dimethylsulfate (DMS) footprinting, and the DNA polymerase stop assay, which can be useful for initial characterization of G-quadruplex structures formed by G-rich sequences.

Characterization of DNA damage induced by a natural product antitumor antibiotic leinamycin in human cancer cells.

Leinamycin is a structurally novel Streptomyces-derived natural product that displays very potent activity against various human cancer cell lines (IC(50) values in the low nanomolar range). Previous in vitro biochemical studies have revealed that leinamycin alkylates DNA, generates apurinic (AP) sites and reactive oxygen species (ROS), and causes DNA strand breaks. However, it is not clear whether these events occur inside cells. In the present study, we have determined the endogenous amount of AP sites and DNA strand breaks in genomic DNA and the amount of oxidative stress in a human pancreatic carcinoma cell line, MiaPaCa, treated with leinamycin by utilizing the aldehyde-reactive probe assay, the comet assay, and fluorescent probes, respectively. We demonstrated that AP sites are formed rapidly following exposure to leinamycin, and the number of AP sites was increased up to seven-fold in a dose-dependent manner. However, only 25-50% of these sites remain 2 h after media containing drug molecules were aspirated and replaced with fresh media. We also observed leinamycin-induced ROS generation and a concomitant increase in apoptosis of MiaPaCa cells. Because both AP sites and ROS have the potential to generate strand breaks in cellular DNA, the comet assay was utilized to detect damage to nuclear DNA in leinamycin-treated MiaPaCa cell cultures. Both alkaline and neutral electrophoretic analysis revealed that leinamycin produces both single- and double-stranded DNA damage in drug-treated cells in a dose-dependent manner. Taken together, the results suggest that rapid conversion of leinamycin-guanine (N7) adducts into AP sites to produce DNA strand breaks, in synergy with leinamycin-derived ROS, accounts for the exceedingly potent biological activity of this natural product.

Identification and characterization of nucleolin as a c-myc G-quadruplex-binding protein.

myc is a proto-oncogene that plays an important role in the promotion of cellular growth and proliferation. Understanding the regulation of c-myc is important in cancer biology, as it is overexpressed in a wide variety of human cancers, including most gynecological, breast, and colon cancers. We previously demonstrated that a guanine-rich region upstream of the P1 promoter of c-myc that controls 85-90% of the transcriptional activation of this gene can form an intramolecular G-quadruplex (G4) that functions as a transcriptional repressor element. In this study, we used an affinity column to purify proteins that selectively bind to the human c-myc G-quadruplex. We found that nucleolin, a multifunctional phosphoprotein, binds in vitro to the c-myc G-quadruplex structure with high affinity and selectivity when compared with other known quadruplex structures. In addition, we demonstrate that upon binding, nucleolin facilitates the formation and increases the stability of the c-myc G-quadruplex structure. Furthermore, we provide evidence that nucleolin overexpression reduces the activity of a c-myc promoter in plasmid presumably by inducing and stabilizing the formation of the c-myc G-quadruplex. Finally, we show that nucleolin binds to the c-myc promoter in HeLa cells, which indicates that this interaction occurs in vivo. In summary, nucleolin may induce c-myc G4 formation in vivo.

NM23-H2 may play an indirect role in transcriptional activation of c-myc gene expression but does not cleave the nuclease hypersensitive element III(1).

The formation of G-quadruplex structures within the nuclease hypersensitive element (NHE) III(1) region of the c-myc promoter and the ability of these structures to repress c-myc transcription have been well established. However, just how these extremely stable DNA secondary structures are transformed to activate c-myc transcription is still unknown. NM23-H2/nucleoside diphosphate kinase B has been recognized as an activator of c-myc transcription via interactions with the NHE III(1) region of the c-myc gene promoter. Through the use of RNA interference, we confirmed the transcriptional regulatory role of NM23-H2. In addition, we find that further purification of NM23-H2 results in loss of the previously identified DNA strand cleavage activity, but retention of its DNA binding activity. NM23-H2 binds to both single-stranded guanine- and cytosine-rich strands of the c-myc NHE III(1) and, to a lesser extent, to a random single-stranded DNA template. However, it does not bind to or cleave the NHE III(1) in duplex form. Significantly, potassium ions and compounds that stabilize the G-quadruplex and i-motif structures have an inhibitory effect on NM23-H2 DNA-binding activity. Mutation of Arg(88) to Ala(88) (R88A) reduced both DNA and nucleotide binding but had minimal effect on the NM23-H2 crystal structure. On the basis of these data and molecular modeling studies, we have proposed a stepwise trapping-out of the NHE III(1) region in a single-stranded form, thus allowing single-stranded transcription factors to bind and activate c-myc transcription. Furthermore, this model provides a rationale for how the stabilization of the G-quadruplex or i-motif structures formed within the c-myc gene promoter region can inhibit NM23-H2 from activating c-myc gene expression.