RESUMO
Telomere replication is essential for continued proliferation of human cells, such as stem cells and cancer cells. Telomerase lengthens the telomeric G-strand, while C-strand replication is accomplished by CST-polymerase α-primase (CST-PP). Replication of both strands is inhibited by formation of G-quadruplex (GQ) structures in the G-rich single-stranded DNA. TMPyP4 and pyridostatin (PDS), which stabilize GQ structures in both DNA and RNA, inhibit telomerase in vitro, and in human cells they cause telomere shortening that has been attributed to telomerase inhibition. Here, we show that TMPyP4 and PDS also inhibit C-strand synthesis by stabilizing DNA secondary structures and thereby preventing CST-PP from binding to telomeric DNA. We also show that these small molecules inhibit CST-PP binding to a DNA sequence containing no consecutive guanine residues, which is unlikely to form GQs. Thus, while these "telomerase inhibitors" indeed inhibit telomerase, they are also robust inhibitors of telomeric C-strand synthesis. Furthermore, given their binding to GQ RNA and their limited specificity for GQ structures, they may disrupt many other protein-nucleic acid interactions in human cells.
Assuntos
Inibidores Enzimáticos , Quadruplex G , Telomerase , Telômero , Telomerase/antagonistas & inibidores , Telomerase/metabolismo , Telomerase/genética , Humanos , Telômero/metabolismo , Quadruplex G/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Ácidos Picolínicos/farmacologia , Ácidos Picolínicos/química , Replicação do DNA/efeitos dos fármacos , DNA Polimerase I/antagonistas & inibidores , DNA Polimerase I/metabolismo , DNA/metabolismo , Aminoquinolinas , Porfirinas , DNA PrimaseRESUMO
Targeting telomere maintenance has emerged as a promising strategy for hepatocellular carcinoma (HCC) treatment. However, given the duality of the telomere-telomerase axis in telomere maintenance, a comprehensive strategy is urgently needed. Herein, we develop a poly(amino acid) (D-PAAs)-based strategy for spatiotemporal codelivery of telomerase inhibitor, BIBR1523, and AKT inhibitor, isobavachalcone. By leveraging D-PAAs' modifiability, we synthesize polymer-inhibitor conjugates (PB and PI) and a folic acid-decorated tumor-targeting vector (PF). These building blocks undergo micellization to fabricate a codelivery nanomedicine (P-BI@P-FA) by exploiting D-PAAs' noncovalent assembly. P-BI@P-FA improves the pharmacokinetics, tumor selectivity, and bioavailability of small molecule inhibitors and initiates a dual telomere-specific inhibition by combining telomerase deactivation with telomere disruption. Furthermore, a hybrid tumor-targeting magnetic nanosystem is designed using D-PAAs and manganese dioxide to showcase magnetic resonance imaging capacities. Our D-PAAs-based strategy addresses the pressing need for telomere-specific HCC treatment while allowing for diagnostic application, presenting a promising avenue for nanomedicine design.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Imageamento por Ressonância Magnética , Nanomedicina , Telomerase , Telômero , Carcinoma Hepatocelular/diagnóstico por imagem , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Telomerase/antagonistas & inibidores , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/tratamento farmacológico , Humanos , Nanomedicina/métodos , Telômero/metabolismo , Imageamento por Ressonância Magnética/métodos , Animais , Camundongos , Linhagem Celular Tumoral , Aminoácidos/química , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/uso terapêuticoRESUMO
BACKGROUND: Ceranib-2, an acid ceramidase (AC) inhibitor, can inhibit cancer cell proliferation and tumor development. However, poor water solubility and low cellular bioavailability limit its efficacy in cancer treatment. METHODS AND RESULTS: This study aimed to investigate the cell death induced by ceranib-2 and its solid lipid nanoformulation (ceranib-2-SLN) produced by the hot homogenization technique and the synergistic relationship between ceramide and telomerase in vitro and in silico. Furthermore, this study proved the possible mechanism of ceranib-2-induced AC inhibition by in silico studies. The effective cytotoxic concentrations of ceranib-2, telomerase level, and changes in ceramide levels were measured by MTT colorimetric cytotoxicity assay, ELISA, and LC/MS/MS methods, respectively. TEM results showed that ceranib-2-SLN was 13-fold smaller than the size of ceranib-2. Ceranib-2 and ceranib-2-SLN had IC50 concentrations of 31.62 (± 2.1) and 27.69 (± 1.75) µM in A549, and 48.79 (± 1.56) and 67.98 (± 2.33) in Beas-2B cells. These compounds simultaneously increased ceramide levels and decreased telomerase levels in A549 cells. Ceranib-2 increased telomerase levels while decreasing ceramide levels in Beas-2B cells. It was shown how the synergistic impact of ceranib-2-induced ceramide production and ceramide-induced telomerase level reduction on cytotoxicity in A549 cells. CONCLUSIONS: Ceranib-2-SLN was discovered to be more cytotoxic on cancer cells than ceranib-2, suggesting that it could be a promising option for the development of a new anti-cancer agent.
Assuntos
Telomerase , Humanos , Telomerase/metabolismo , Telomerase/antagonistas & inibidores , Linhagem Celular Tumoral , Células A549 , Proliferação de Células/efeitos dos fármacos , Antineoplásicos/farmacologia , Ceramidas/metabolismo , Nanopartículas/química , Sobrevivência Celular/efeitos dos fármacosRESUMO
IMP dehydrogenase (IMPDH) inhibition has emerged as a new target therapy for glioblastoma multiforme (GBM), which remains one of the most refractory tumors to date. TCGA analyses revealed distinct expression profiles of IMPDH isoenzymes in various subtypes of GBM and low-grade glioma (LGG). To dissect the mechanism(s) underlying the anti-tumor effect of IMPDH inhibition in adult GBM, we investigated how mycophenolic acid (MPA, an IMPDH inhibitor) treatment affected key oncogenic drivers in glioblastoma cells. Our results showed that MPA decreased the expression of telomerase reverse transcriptase (TERT) in both U87 and U251 cells, and the expression of O6-methylguanine-DNA methyltransferase (MGMT) in U251 cells. In support, MPA treatment reduced the amount of telomere repeats in U87 and U251 cells. TERT downregulation by MPA was associated with a significant decrease in c-Myc (a TERT transcription activator) in U87 but not U251 cells, and a dose-dependent increase in p53 and CCCTC-binding factor (CTCF) (TERT repressors) in both U87 and U251 cells. In U251 cells, MPA displayed strong cytotoxic synergy with BCNU and moderate synergy with irinotecan, oxaliplatin, paclitaxel, or temozolomide (TMZ). In U87 cells, MPA displayed strong cytotoxic synergy with all except TMZ, acting primarily through the apoptotic pathway. Our work expands the mechanistic potential of IMPDH inhibition to TERT/telomere regulation and reveals a synthetic lethality between MPA and anti-GBM drugs.
Assuntos
Glioblastoma , IMP Desidrogenase , Telomerase , Humanos , Telomerase/metabolismo , Telomerase/antagonistas & inibidores , Telomerase/genética , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioblastoma/genética , Glioblastoma/patologia , Linhagem Celular Tumoral , IMP Desidrogenase/antagonistas & inibidores , IMP Desidrogenase/metabolismo , IMP Desidrogenase/genética , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genética , Apoptose/efeitos dos fármacosRESUMO
Imetelstat is a novel, first-in-class, oligonucleotide telomerase inhibitor in development for the treatment of hematologic malignancies including lower-risk myelodysplastic syndromes and myelofibrosis. A nonlinear mixed-effects model was developed to characterize the population pharmacokinetics of imetelstat and identify and quantify covariates that contribute to its pharmacokinetic variability. The model was developed using plasma concentrations from 7 clinical studies including 424 patients with solid tumors or hematologic malignancies who received single-agent imetelstat via intravenous infusion at various dose levels (0.4-11.7 mg/kg) and schedules (every week to every 4 weeks). Covariate analysis included factors related to demographics, disease, laboratory results, renal and hepatic function, and antidrug antibodies. Imetelstat was described by a two-compartment, nonlinear disposition model with saturable binding/distribution and dose- and time-dependent elimination from the central compartment. Theory-based allometric scaling for body weight was included in disposition parameters. The final covariates included sex, time, malignancy, and dose on clearance; malignancy and sex on volume of the central compartment; and malignancy and spleen volume on concentration of target. Clearance in females was modestly lower, resulting in nonclinically relevant increases in predicted exposure relative to males. No effects on imetelstat pharmacokinetics were identified for mild-to-moderate hepatic or renal impairment, age, race, and antidrug antibody status. All model parameters were estimated with adequate precision (relative standard error < 29%). Visual predictive checks confirmed the capacity of the model to describe the data. The analysis supports the imetelstat body-weight-based dosing approach and lack of need for dose individualizations for imetelstat-treated patients.
Assuntos
Oligonucleotídeos , Telomerase , Humanos , Telomerase/antagonistas & inibidores , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Adulto , Oligonucleotídeos/farmacocinética , Oligonucleotídeos/administração & dosagem , Neoplasias/tratamento farmacológico , Modelos Biológicos , Idoso de 80 Anos ou mais , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/administração & dosagem , Relação Dose-Resposta a Droga , Adulto Jovem , Neoplasias Hematológicas/tratamento farmacológicoRESUMO
Choroidal neovascularization (CNV) is the principal driver of blindness in neovascular age-related macular degeneration (nvAMD). Increased activity of telomerase, has been associated with endothelial cell proliferation, survival, migration, and invasion in the context of tumor angiogenesis. Expanding on this knowledge, we investigated the role of telomerase in the development of CNV in mouse model. We observed increased gene expression and activity of telomerase in mouse CNV. Genetic deficiency of the telomerase components, telomerase reverse transcriptase (Tert) and telomerase RNA component (Terc) suppressed laser-induced CNV in mice. Similarly, a small molecule inhibitor of TERT (BIBR 1532), and antisense oligonucleotides (ASOs) targeting Tert and Terc reduced CNV growth. Bone marrow chimera studies suggested that telomerase activity in non-bone marrow-derived cells is crucial for the development of CNV. Comparison of BIBR 1532 with VEGF neutralizing therapeutic strategy in mouse revealed a comparable level of angiosuppressive activity. However, when BIBR and anti-VEGF antibodies were administered as a combination at sub-therapeutic doses, a statistically significant suppression of CNV was observed. These findings underscore the potential benefits of combining sub-therapeutic doses of BIBR and anti-VEGF antibodies for developing newer therapeutic strategies for NV-AMD. Telomerase inhibition with BIBR 1532 suppressed induction of multiple cytokines and growth factors critical for neovascularization. In conclusion, our study identifies telomerase as a promising therapeutic target for treating neovascular disease of the eye and thus provides a proof of principle for further exploration of telomerase inhibition as a novel treatment strategy for nvAMD.
Assuntos
Neovascularização de Coroide , Modelos Animais de Doenças , Telomerase , Telomerase/antagonistas & inibidores , Telomerase/genética , Telomerase/metabolismo , Animais , Neovascularização de Coroide/patologia , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/tratamento farmacológico , Camundongos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Camundongos Endogâmicos C57BL , Aminobenzoatos/farmacologia , RNA/genética , RNA/metabolismo , Oligonucleotídeos Antissenso/farmacologia , NaftalenosRESUMO
Telomere is a protective structure located at the end of chromosomes of eukaryotes, involved in maintaining the integrity and stability of the genome. Telomeres play an essential role in cancer progression; accordingly, targeting telomere dynamics emerges as an effective approach for the development of cancer therapeutics. Targeting telomere dynamics may work through multifaceted molecular mechanisms; those include the activation of anti-telomerase immune responses, shortening of telomere lengths, induction of telomere dysfunction and constitution of telomerase-responsive drug release systems. In this review, we summarize a wide variety of telomere dynamics-targeted agents in preclinical studies and clinical trials, and reveal their promising therapeutic potential in cancer therapy. As shown, telomere dynamics-active agents are effective as anti-cancer chemotherapeutics and immunotherapeutics. Notably, these agents may display efficacy against cancer stem cells, reducing cancer stem levels. Furthermore, these agents can be integrated with the capability of tumor-specific drug delivery by the constitution of related nanoparticles, antibody drug conjugates and HSA-based drugs.
Assuntos
Antineoplásicos , Neoplasias , Telomerase , Telômero , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/terapia , Telômero/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/química , Telomerase/antagonistas & inibidores , Animais , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , Imunoterapia/métodos , Células-Tronco Neoplásicas/efeitos dos fármacosRESUMO
BACKGROUND: Hotspot mutations in the promoter of telomerase reverse transcriptase (TERT) gene are the most common genetic variants in hepatocellular carcinoma (HCC) and associated with poor prognosis of the disease. However, no drug was currently approved for treating TERT promoter mutation positive HCC patients. Here, we aim to explore the potential therapeutic strategy for targeting TERT promoter mutation in HCC. METHODS: The Liver Cancer Model Repository database was used for screening potential drugs to selectively suppress the growth of TERT promoter mutant HCC cells. RNA-seq, CRISPR-Cas9 technology and siRNA transfection were performed for mechanistic studies. Cell counting kit-8 (CCK8) assay and the xenograft tumour models were used for cell growth detection in vitro and in vivo, respectively. Cell apoptosis and cell cycle arrest were analysed by Annexin V-FITC staining and/or propidium iodide staining. RESULTS: PLK1 inhibitors were remarkably more sensitive to HCC cells harbouring TERT promoter mutation than wild-type cells in vitro and in vivo, which were diminished after TERT promoter mutation was edited to the wild-type nucleotide. Comparing the HCC cells with wild-type promoter of TERT, PLK1 inhibitors specifically downregulated Smad3 to regulate TERT for inducing apoptosis and G2/M arrest in TERT mutant HCC cells. Moreover, knockout of Smad3 counteracted the effects of PLK1 inhibitors in TERT mutant HCC cells. Finally, a cooperative effect of PLK1 and Smad3 inhibition was observed in TERT mutant cells. CONCLUSIONS: PLK1 inhibition selectively suppressed the growth of TERT mutant HCC cells through Smad3, thus contributed to discover a novel therapeutic strategy to treat HCC patients harbouring TERT promoter mutations. KEY POINTS: TERT promoter mutation confers sensitivity to PLK1 inhibitors in HCC. The selective growth inhibition of TERT mutant HCC cells induced by PLK1 inhibitor was mediated by Smad3. Combined inhibition of PLK1 and Smad3 showed a cooperative anti-tumor effect in TERT mutant HCC cells.
Assuntos
Carcinoma Hepatocelular , Proteínas de Ciclo Celular , Neoplasias Hepáticas , Quinase 1 Polo-Like , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas , Telomerase , Telomerase/genética , Telomerase/antagonistas & inibidores , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Regiões Promotoras Genéticas/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Animais , Mutação , Camundongos , Linhagem Celular Tumoral , Apoptose/efeitos dos fármacos , Apoptose/genéticaRESUMO
Human telomerase reverse transcriptase (hTERT) may have noncanonical functions in transcriptional regulation and metabolic reprogramming in cancer cells, but it is a challenging target. We thus developed small-molecule ligands targeting hTERT promoter G-quadruplex DNA structures (hTERT G4) to downregulate hTERT expression. Ligand 5 showed high affinity toward hTERT G4 (Kd = 1.1 µM) and potent activity against triple-negative breast cancer cells (MDA-MB-231, IC50 = 1 µM). In cell-based assays, 5 not only exerts markedly inhibitory activity on classical telomere functions including decreased telomerase activity, shortened telomere length, and cellular senescence but also induces DNA damage, acute cellular senescence, and apoptosis. This study reveals that hTERT G4-targeting ligand may cause mitochondrial dysfunction, disrupt iron metabolism and activate ferroptosis in cancer cells. The in vivo antitumor efficacy of 5 was also evaluated in an MDA-MB-231 xenograft mouse model and approximately 78.7% tumor weight reduction was achieved. No observable toxicity against the major organs was observed.
Assuntos
Antineoplásicos , Regulação para Baixo , Quadruplex G , Regiões Promotoras Genéticas , Telomerase , Neoplasias de Mama Triplo Negativas , Telomerase/antagonistas & inibidores , Telomerase/metabolismo , Humanos , Quadruplex G/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Animais , Ligantes , Feminino , Regulação para Baixo/efeitos dos fármacos , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Apoptose/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Nus , Senescência Celular/efeitos dos fármacos , Camundongos Endogâmicos BALB CRESUMO
BACKGROUND: As one of the main molecules in BCR-ABL signaling, c-Myc acts as a pivotal key in disease progression and disruption of long-term remission in patients with CML. OBJECTIVES: To clarify the effects of c-Myc inhibition in CML, we examined the anti-tumor property of a well-known small molecule inhibitor of c-Myc 10058-F4 on K562 cell line. METHODS: This experimental study was conducted in K562 cell line for evaluation of cytotoxic activity of 10058-F4 using Trypan blue and MTT assays. Flow cytometry and Quantitative RT-PCR analysis were also conducted to determine its mechanism of action. Additionally, Annexin/PI staining was performed for apoptosis assessment. RESULTS: The results of Trypan blue and MTT assay demonstrated that inhibition of c-Myc, as shown by suppression of c-Myc expression and its associated genes PP2A, CIP2A, and hTERT, could decrease viability and metabolic activity of K562 cells, respectively. Moreover, a robust elevation in cell population in G1-phase coupled with up-regulation of p21 and p27 expression shows that 10058-F4 could hamper cell proliferation, at least partly, through induction of G1 arrest. Accordingly, we found that 10058-F4 induced apoptosis via increasing Bax and Bad; In contrast, no significant alterations were observed NF-KB pathway-targeted anti-apoptotic genes in the mRNA levels. Notably, disruption of the NF-κB pathway with bortezomib as a common proteasome inhibitor sensitized K562 cells to the cytotoxic effect of 10058-F4, substantiating the fact that the NF-κB axis functions probably attenuate the K562 cells sensitivity to c-Myc inhibition. CONCLUSIONS: It can be concluded from the results of this study that inhibition of c-Myc induces anti-neoplastic effects on CML-derived K562 cells as well as increases the efficacy of imatinib. For further insight into the safety and effectiveness of 10058-F4 in CML, in vivo studies will be required.
Assuntos
Apoptose , Proliferação de Células , Leucemia Mielogênica Crônica BCR-ABL Positiva , Proteínas Proto-Oncogênicas c-myc , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células K562 , NF-kappa B/metabolismo , NF-kappa B/antagonistas & inibidores , Antineoplásicos/farmacologia , Bortezomib/farmacologia , Células Tumorais Cultivadas , Ácidos Borônicos/farmacologia , RNA Mensageiro/genética , Pirazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Telomerase/antagonistas & inibidoresRESUMO
PURPOSE: To evaluate telomerase activity and proliferation of HS839.T melanoma cells, subjected to the action of AZT. METHODS: Cells were grown in triplicate, AZT at different concentrations: 50, 100 and 200μM, was added and left for 24 and 48 hours, and its effects were compared with the control group. Telomerase activity was detected by PCR and cell proliferation was evaluated by MTT. RESULTS: After 24 hours, there was no inhibition of cell proliferation or telomerase activity when compared to the control group. After 48 hours, there was a momentary decrease, suggesting that the cell lines used in this study are sensitive to AZT, but quickly recover both the enzyme activity and cell proliferation. CONCLUSION: The action of AZT on the melanoma cells studied, at the concentrations and times tested, did not inhibit telomerase activity nor affect cell proliferation.
OBJETIVO: Avaliar a atividade da telomerase e da proliferação de células de melanoma HS839.T submetidas à ação do AZT. MÉTODOS: As células foram cultivadas, em triplicata, com diferentes concentrações de AZT: 50, 100 e 200µM, por 24h e 48h, seus efeitos comparados com o grupo controle. A atividade da telomerase foi detectada por PCR e a proliferação celular avaliada por MTT. RESULTADOS: No tempo de 24 horas, não houve inibição da proliferação celular e da atividade da telomerase em comparação com o grupo controle. No período de 48 horas, houve uma diminuição momentânea, sugerindo que as células das linhagens utilizadas neste estudo são sensíveis ao AZT, mas que recuperam a atividade enzimática e proliferativa. CONCLUSÃO: Nas células de melanoma HS839.T estudadas e nas concentrações e tempos propostos, a ação do AZT não inibiu a atividade da telomerase e não afetou a proliferação celular.