RESUMEN
Background: Immune checkpoint inhibitors (ICI) are routinely used in advanced clear cell renal cell carcinoma (ccRCC). However, a substantial group of patients does not respond to ICI therapy. Radiation is a promising approach to increase ICI response rates since it can generate anti-tumor immunity. Targeted radionuclide therapy (TRT) is a systemic radiation treatment, ideally suited for precision irradiation of metastasized cancer. Therefore, the aim of this study is to explore the potential of combined TRT, targeting carbonic anhydrase IX (CAIX) which is overexpressed in ccRCC, using [177Lu]Lu-DOTA-hG250, and ICI for the treatment of ccRCC. Methods: In this study, we evaluated the therapeutic and immunological action of [177Lu]Lu-DOTA-hG250 combined with aPD-1/a-CTLA-4 ICI. First, the biodistribution of [177Lu]Lu-DOTA-hG250 was investigated in BALB/cAnNRj mice bearing Renca-CAIX or CT26-CAIX tumors. Renca-CAIX and CT26-CAIX tumors are characterized by poor versus extensive T-cell infiltration and homogeneous versus heterogeneous PD-L1 expression, respectively. Tumor-absorbed radiation doses were estimated through dosimetry. Subsequently, [177Lu]Lu-DOTA-hG250 TRT efficacy with and without ICI was evaluated by monitoring tumor growth and survival. Therapy-induced changes in the tumor microenvironment were studied by collection of tumor tissue before and 5 or 8 days after treatment and analyzed by immunohistochemistry, flow cytometry, and RNA profiling. Results: Biodistribution studies showed high tumor uptake of [177Lu]Lu-DOTA-hG250 in both tumor models. Dose escalation therapy studies in Renca-CAIX tumor-bearing mice demonstrated dose-dependent anti-tumor efficacy of [177Lu]Lu-DOTA-hG250 and remarkable therapeutic synergy including complete remissions when a presumed subtherapeutic TRT dose (4 MBq, which had no significant efficacy as monotherapy) was combined with aPD-1+aCTLA-4. Similar results were obtained in the CT26-CAIX model for 4 MBq [177Lu]Lu-DOTA-hG250 + a-PD1. Ex vivo analyses of treated tumors revealed DNA damage, T-cell infiltration, and modulated immune signaling pathways in the TME after combination treatment. Conclusions: Subtherapeutic [177Lu]Lu-DOTA-hG250 combined with ICI showed superior therapeutic outcome and significantly altered the TME. Our results underline the importance of investigating this combination treatment for patients with advanced ccRCC in a clinical setting. Further investigations should focus on how the combination therapy should be optimally applied in the future.
Asunto(s)
Anhidrasa Carbónica IX , Carcinoma de Células Renales , Inhibidores de Puntos de Control Inmunológico , Neoplasias Renales , Animales , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/terapia , Carcinoma de Células Renales/patología , Ratones , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/patología , Neoplasias Renales/terapia , Neoplasias Renales/radioterapia , Anhidrasa Carbónica IX/metabolismo , Anhidrasa Carbónica IX/antagonistas & inhibidores , Humanos , Línea Celular Tumoral , Radioisótopos/uso terapéutico , Radioisótopos/farmacología , Radioisótopos/administración & dosificación , Lutecio/uso terapéutico , Femenino , Antígenos de Neoplasias/metabolismo , Distribución Tisular , Microambiente Tumoral/efectos de los fármacos , Proteína Tumoral Controlada Traslacionalmente 1 , Ensayos Antitumor por Modelo de Xenoinjerto , Terapia Combinada/métodos , Ratones Endogámicos BALB C , Anticuerpos MonoclonalesRESUMEN
A novel class of compounds designed to hit two anti-tumour targets, G-quadruplex structures and human carbonic anhydrases (hCAs) IX and XII is proposed. The induction/stabilisation of G-quadruplex structures by small molecules has emerged as an anticancer strategy, disrupting telomere maintenance and reducing oncogene expression. hCAs IX and XII are well-established anti-tumour targets, upregulated in many hypoxic tumours and contributing to metastasis. The ligands reported feature a berberine G-quadruplex stabiliser scaffold connected to a moiety inhibiting hCAs IX and XII. In vitro experiments showed that our compounds selectively stabilise G-quadruplex structures and inhibit hCAs IX and XII. The crystal structure of a telomeric G-quadruplex in complex with one of these ligands was obtained, shedding light on the ligand/target interaction mode. The most promising ligands showed significant cytotoxicity against CA IX-positive HeLa cancer cells in hypoxia, and the ability to stabilise G-quadruplexes within tumour cells.
Asunto(s)
Antineoplásicos , Anhidrasa Carbónica IX , Inhibidores de Anhidrasa Carbónica , Anhidrasas Carbónicas , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , G-Cuádruplex , Humanos , G-Cuádruplex/efectos de los fármacos , Inhibidores de Anhidrasa Carbónica/farmacología , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Relación Estructura-Actividad , Estructura Molecular , Anhidrasa Carbónica IX/antagonistas & inhibidores , Anhidrasa Carbónica IX/metabolismo , Anhidrasas Carbónicas/metabolismo , Proliferación Celular/efectos de los fármacos , Ligandos , Células HeLa , Antígenos de Neoplasias/metabolismo , Modelos MolecularesRESUMEN
Clear cell renal cell carcinoma (ccRCC) is a lethal malignancy with high metastatic probability. Paired box 2 gene product (PAX2) carbonic anhydrase IX were biomolecules closely linked with ccRCC development and outcomes of multiple malignancies. We aim to explore the role of immunohistochemical staining of PAX2 and CAIX to predict ccRCC prognosis after nephrectomy. Surgical specimens of patients who were pathologically diagnosed as ccRCC were reviewed. Expression levels of PAX2 and CAIX were assessed via immunohistochemical staining. Recurrence-free survival (RFS) and overall survival were compared among different phenotypes. Inverse probability of treatment weighting (IPTW) was used for adjustment of confounding factors. 56 patients were included. Patients with PAX2 and CAIX high-expression (the two-high group, n=8) had significantly longer RFS and OS than those of simultaneously down-expression (the two-low group, n=31). Median RFS was 38.4 (95% CI: 32.3-NA) for the two-high group and 14.8 (95% CI: 13.4-39.0) months for the two-low group (P=0.043). IPTW confirmed PAX2 and CAIX co-expression is associated with less recurrence risk HR: 0.39, 95% CI: 0.17-0.92, P=0.031). Co-expression of PAX2 and CAIX is associated better prognosis of ccRCC. We are looking for validation by large cohort studies.
Asunto(s)
Anhidrasa Carbónica IX , Carcinoma de Células Renales , Inmunohistoquímica , Neoplasias Renales , Nefrectomía , Factor de Transcripción PAX2 , Humanos , Factor de Transcripción PAX2/metabolismo , Factor de Transcripción PAX2/genética , Carcinoma de Células Renales/cirugía , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/mortalidad , Carcinoma de Células Renales/genética , Masculino , Femenino , Anhidrasa Carbónica IX/metabolismo , Anhidrasa Carbónica IX/genética , Nefrectomía/métodos , Persona de Mediana Edad , Estudios Retrospectivos , Neoplasias Renales/cirugía , Neoplasias Renales/patología , Neoplasias Renales/metabolismo , Neoplasias Renales/mortalidad , Neoplasias Renales/genética , Pronóstico , Anciano , Supervivencia sin Enfermedad , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/genética , Adulto , Antígenos de NeoplasiasRESUMEN
Carbonic anhydrase (CA) was among the first proteins whose X-ray crystal structure was solved to atomic resolution. CA proteins have essentially the same fold and similar active centers that differ in only several amino acids. Primary sulfonamides are well defined, strong and specific binders of CA. However, minor variations in chemical structure can significantly alter their binding properties. Over 1000 sulfonamides have been designed, synthesized and evaluated to understand the correlations between the structure and thermodynamics of their binding to the human CA isozyme family. Compound binding was determined by several binding assays: fluorescence-based thermal shift assay, stopped-flow enzyme activity inhibition assay, isothermal titration calorimetry and competition assay for enzyme expressed on cancer cell surfaces. All assays have advantages and limitations but are necessary for deeper characterization of these protein-ligand interactions. Here, the concept and importance of intrinsic binding thermodynamics is emphasized and the role of structure-thermodynamics correlations for the novel inhibitors of CA IX is discussed - an isozyme that is overexpressed in solid hypoxic tumors, and thus these inhibitors may serve as anticancer drugs. The abundant structural and thermodynamic data are assembled into the Protein-Ligand Binding Database to understand general protein-ligand recognition principles that could be used in drug discovery.
Asunto(s)
Anhidrasas Carbónicas , Isoenzimas , Unión Proteica , Sulfonamidas , Termodinámica , Humanos , Cristalografía por Rayos X , Anhidrasas Carbónicas/metabolismo , Anhidrasas Carbónicas/química , Isoenzimas/metabolismo , Isoenzimas/química , Ligandos , Sulfonamidas/química , Sulfonamidas/farmacología , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasa Carbónica IX/metabolismo , Anhidrasa Carbónica IX/química , Modelos MolecularesRESUMEN
BACKGROUND: Advanced clear cell renal cell carcinoma (ccRCC) is a prevalent kidney cancer for which long-term survival rates are abysmal, though immunotherapies are showing potential. Not yet clinically vetted are bispecific T cell engagers (BTEs) that activate T cell-mediated cancer killing through intercellular synapsing. Multiple BTE formats exist, however, with limited cross-characterizations to help optimize new drug design. Here, we developed BTEs to treat ccRCC by targeting carbonic anhydrase 9 (CA9) while characterizing the persistent BTE (PBTE) format and comparing it to a new format, the persistent multivalent T cell engager (PMTE). These antibody therapies against ccRCC are developed as both recombinant and synthetic DNA (synDNA) medicines. METHODS: Antibody formatting effects on binding kinetics were assessed by flow cytometry and intercellular synaptic strength assays while potency was tested using T-cell activation and cytotoxicity assays. Mouse models were used to study antibody plasma and tumor pharmacokinetics, as well as antitumor efficacy as both recombinant and synDNA medicines. Specifically, three models using ccRCC cell line xenografts and human donor T cells in immunodeficient mice were used to support this study. RESULTS: Compared with a first-generation BTE, we show that the PBTE reduced avidity, intercellular synaptic strength, cytotoxic potency by as much as 33-fold, and ultimately efficacy against ccRCC tumors in vivo. However, compared with the PBTE, we demonstrate that the PMTE improved cell avidity, restored intercellular synapses, augmented cytotoxic potency by 40-fold, improved tumor distribution pharmacokinetics by 2-fold, and recovered synDNA efficacy in mouse tumor models by 20-fold. All the while, the PMTE displayed a desirable half-life of 4 days in mice compared with the conventional BTE's 2 hours. CONCLUSIONS: With impressive efficacy, the CA9-targeted PMTE is a promising new therapy for advanced ccRCC, which can be effectively delivered through synDNA. The highly potent PMTE format itself is a promising new tool for future applications in the multispecific antibody space.
Asunto(s)
Anticuerpos Biespecíficos , Carcinoma de Células Renales , Neoplasias Renales , Linfocitos T , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/inmunología , Humanos , Animales , Ratones , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/inmunología , Linfocitos T/inmunología , Anticuerpos Biespecíficos/farmacología , Anticuerpos Biespecíficos/uso terapéutico , Línea Celular Tumoral , Inmunoterapia/métodos , Anhidrasa Carbónica IX/metabolismo , Femenino , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
DNA methylation is a crucial epigenetic modification that regulates gene expression and determines cell fate; however, the triggers that alter DNA methylation levels remain unclear. Recently, we showed that S-nitrosylation of DNA methyltransferase (DNMT) induces DNA hypomethylation and alters gene expression. Furthermore, we identified DBIC, a specific inhibitor of S-nitrosylation of DNMT3B, to suppress nitric oxide (NO)-induced gene alterations. However, it remains unclear how NO-induced DNA hypomethylation regulates gene expression and whether this mechanism is maintained in normal cells and triggers disease-related changes. To address these issues, we focused on carbonic anhydrase 9 (CA9), which is upregulated under nitrosative stress in cancer cells. We pharmacologically evaluated its regulatory mechanisms using human small airway epithelial cells (SAECs) and DBIC. We demonstrated that nitrosative stress promotes the recruitment of hypoxia-inducible factor 1 alpha to the CA9 promoter region and epigenetically induces CA9 expression in SAECs. Our results suggest that nitrosative stress is a key epigenetic regulator that may cause diseases by altering normal cell function.
Asunto(s)
Anhidrasa Carbónica IX , Metilación de ADN , Epigénesis Genética , Células Epiteliales , Óxido Nítrico , Humanos , Óxido Nítrico/metabolismo , Células Epiteliales/metabolismo , Anhidrasa Carbónica IX/metabolismo , Anhidrasa Carbónica IX/genética , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Regiones Promotoras Genéticas , Células CultivadasRESUMEN
New sulfonamide-triazole-glycoside hybrids derivatives were designed, synthesised, and investigated for anticancer efficacy. The target glycosides' cytotoxic activity was studied with a panel of human cancer cell lines. Sulfonamide-based derivatives, 4, 7 and 9 exhibited promising activity against HepG-2 and MCF-7 (IC50 = 8.39-16.90 µM against HepG-2 and 19.57-21.15 µM against MCF-7) comparing with doxorubicin (IC50 = 13.76 ± 0.45, 17.44 ± 0.46 µM against HepG-2 and MCF-7, rescpectively). To detect the probable action mechanism, the inhibitory activity of these targets was studied against VEGFR-2, carbonic anhydrase isoforms hCA IX and hCA XII. Compoumds 7 and 9 gave favorable potency (IC50 = 1.33, 0.38 µM against VEGFR-2, 66, 40 nM against hCA IX and 7.6, 3.2 nM against hCA XII, respectively), comparing with sorafenib and SLC-0111 (IC50 = 0.43 µM, 53 and 4.8 nM, respectively). Moreover, the docking simulation was assessed to supply better rationalization and gain insight into the binding affinity between the promising derivatives and their targeted enzymes that was used for further modification in the anticancer field.
Asunto(s)
Antineoplásicos , Inhibidores de Anhidrasa Carbónica , Glicósidos , Simulación del Acoplamiento Molecular , Sulfonamidas , Triazoles , Receptor 2 de Factores de Crecimiento Endotelial Vascular , Humanos , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/farmacología , Sulfonamidas/química , Sulfonamidas/farmacología , Glicósidos/química , Glicósidos/farmacología , Triazoles/química , Triazoles/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Anhidrasa Carbónica IX/metabolismo , Anhidrasa Carbónica IX/antagonistas & inhibidores , Anhidrasas Carbónicas/metabolismo , Anhidrasas Carbónicas/química , Células MCF-7 , Células Hep G2 , Línea Celular Tumoral , Antígenos de Neoplasias/metabolismo , Relación Estructura-ActividadRESUMEN
A novel series of 1,8-naphthalimide piperazinamide based benzenesulfonamides derivatives were designed and synthesized as carbonic anhydrase IX (CA IX) inhibitors and ferroptosis inducers for the treatment of triple-negative breast cancer (TNBC). The representative compound 9o exhibited more potent inhibitory activity and selective against CA IX over off-target CA II, compared with positive control SLC-0111. Molecular docking study was also performed to gain insights into the binding interactions of 9o in the binding pocket of CAIX. Moreover, compound 9o exhibited superior antitumor activities against breast cancer cells under hypoxia than that of normoxia conditions. Mechanism studies revealed that compound 9o could act as DNA intercalator and effectively suppressed cell migration, arrested the cell cycle at G1/S phase and induced apoptosis in MDA-MB-231 cells, while inducing ferroptosis accompanied by the dissipation of MMP and the elevation intracellular levels of ROS. Notably, in vivo studies demonstrated that 9o effectively inhibited tumor growth and metastasis in a highly metastatic murine breast cancer 4 T1 xenograft model. Taken together, this study suggests that compound 9o represents a potent and selective CA IX inhibitor and ferroptosis inducer for the treatment of TNBC.
Asunto(s)
Antineoplásicos , Bencenosulfonamidas , Anhidrasa Carbónica IX , Inhibidores de Anhidrasa Carbónica , Proliferación Celular , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Ferroptosis , Naftalimidas , Sulfonamidas , Neoplasias de la Mama Triple Negativas , Humanos , Anhidrasa Carbónica IX/antagonistas & inhibidores , Anhidrasa Carbónica IX/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/metabolismo , Inhibidores de Anhidrasa Carbónica/farmacología , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/síntesis química , Ferroptosis/efectos de los fármacos , Sulfonamidas/farmacología , Sulfonamidas/química , Sulfonamidas/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Animales , Estructura Molecular , Proliferación Celular/efectos de los fármacos , Relación Estructura-Actividad , Ratones , Femenino , Naftalimidas/química , Naftalimidas/farmacología , Naftalimidas/síntesis química , Descubrimiento de Drogas , Apoptosis/efectos de los fármacos , Simulación del Acoplamiento Molecular , Piperazinas/farmacología , Piperazinas/química , Piperazinas/síntesis química , Línea Celular Tumoral , Antígenos de NeoplasiasRESUMEN
Carbonic anhydrase IX (CAIX), a zinc metal transmembrane protein, is highly expressed in 95% of clear cell renal cell carcinomas (ccRCCs). A positron emission tomography (PET) probe designed to target CAIX in nuclear medicine imaging technology can achieve precise positioning, is noninvasive, and can be used to monitor CAIX expression in lesions in real time. In this study, we constructed a novel acetazolamide dual-targeted small-molecule probe [68Ga]Ga-LF-4, which targets CAIX by binding to a specific amino acid sequence. After attenuation correction, the radiolabeling yield reached 66.95 ± 0.57% (n = 5) after 15 min of reaction and the radiochemical purity reached 99% (n = 5). [68Ga]Ga-LF-4 has good in vitro and in vivo stability, and in vivo safety and high affinity for CAIX, with a Kd value of 6.62 nM. Moreover, [68Ga]Ga-LF-4 could be quickly cleared from the blood in vivo. The biodistribution study revealed that the [68Ga]Ga-LF-4 signal was concentrated in the heart, lung, and kidney after administration, which was the same as that observed in the micro-PET/CT study. In a ccRCC patient-derived xenograft (PDX) model, the signal significantly accumulated in the tumor after administration, where it was retained for up to 4 h. After competitive blockade with LF-4, uptake at the tumor site was significantly reduced. The SUVmax of the probe [68Ga]Ga-LF-4 at the ccRCC tumor site was three times greater than that in the PC3 group with low CAIX expression at 30 min (ccRCC vs PC3:1.86 ± 0.03 vs 0.62 ± 0.01, t = 48.2, P < 0.0001). These results indicate that [68Ga]Ga-LF-4 is a novel small-molecule probe that targets CAIX and can be used to image localized and metastatic ccRCC lesions.
Asunto(s)
Anhidrasa Carbónica IX , Carcinoma de Células Renales , Radioisótopos de Galio , Neoplasias Renales , Animales , Anhidrasa Carbónica IX/metabolismo , Anhidrasa Carbónica IX/antagonistas & inhibidores , Humanos , Ratones , Carcinoma de Células Renales/diagnóstico por imagen , Carcinoma de Células Renales/metabolismo , Neoplasias Renales/diagnóstico por imagen , Neoplasias Renales/metabolismo , Distribución Tisular , Línea Celular Tumoral , Radiofármacos/farmacocinética , Radiofármacos/química , Ratones Desnudos , Antígenos de Neoplasias/metabolismo , Sondas Moleculares/farmacocinética , Sondas Moleculares/química , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Acetazolamida/farmacocinética , Femenino , Ratones Endogámicos BALB C , Tomografía de Emisión de Positrones/métodos , Masculino , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Lung cancer represents the leading cause of cancer-related deaths. Non-small cell lung cancer (NSCLC), the most common form of lung cancer, is a molecularly heterogeneous disease with intratumoral heterogeneity and a significant mutational burden associated with clinical outcome. Tumor microenvironment (TME) plays a fundamental role in the initiation and progression of primary de novo lung cancer and significantly influences the response of tumor cells to therapy. Hypoxia, an integral part of the tumor microenvironment and a serious clinical phenomenon, is associated with increased genetic instability and a more aggressive phenotype of NSCLC, which correlates with the risk of metastasis. Low oxygen concentration influences all components of TME including the immune microenvironment. Hypoxia-inducible pathway activated in response to low oxygen supply mediates the expression of genes important for the adaptation of tumor cells to microenvironmental changes. A highly active transmembrane hypoxia-induced metalloenzyme - carbonic anhydrase IX (CAIX), as a part of transport metabolon, contributes to the maintenance of intracellular pH within physiological values and to the acidification of the extracellular space. CAIX supports cell migration and invasion and plays an important role in NSCLC tumor tissue and pleural effusion. Due to its high expression, it also represents a potential diagnostic differential biomarker and therapeutic target in NSCLC. To test new potential targeted therapeutic compounds, suitable models are required that more faithfully simulate tumor tissue, TME components, and spatial architecture.
Asunto(s)
Antígenos de Neoplasias , Biomarcadores de Tumor , Anhidrasa Carbónica IX , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Microambiente Tumoral , Humanos , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/patología , Anhidrasa Carbónica IX/metabolismo , Biomarcadores de Tumor/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/metabolismo , Antígenos de Neoplasias/metabolismo , Hipoxia/metabolismoRESUMEN
Carbonic anhydrases (CAs), are metallo-enzymes implicated in several pathophysiological processes where tissue pH regulation is required. CA IX is a tumor-associated CA isoform induced by hypoxia and involved in the adaptation of tumor cells to acidosis. Indeed, several tumor-driving pathways can induce CA IX expression, and this in turn has been associated to cancer cells invasion and metastatic features as well as to induction of stem-like features, drug resistance and recurrence. After its functional and structural characterization CA IX targeting approaches have been developed to inhibit its activity in neoplastic tissues, and to date this field has seen an incredible acceleration in terms of therapeutic options and biological readouts. Small molecules inhibitors, hybrid/dual targeting drugs, targeting antibodies and adoptive (CAR-T based) cell therapy have been developed at preclinical level, whereas a sulfonamide CA IX inhibitor and an antibody entered Phase Ib/II clinical trials for the treatment and imaging of different solid tumors. Here recent advances on CA IX biology and pharmacology in cancer, and its therapeutic targeting will be discussed.
Asunto(s)
Antígenos de Neoplasias , Anhidrasa Carbónica IX , Inhibidores de Anhidrasa Carbónica , Neoplasias , Humanos , Anhidrasa Carbónica IX/antagonistas & inhibidores , Anhidrasa Carbónica IX/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neoplasias/enzimología , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/inmunología , Inhibidores de Anhidrasa Carbónica/uso terapéutico , Inhibidores de Anhidrasa Carbónica/farmacología , Terapia Molecular Dirigida , AnimalesRESUMEN
Obesity is a major public health concern because it increases the risk of several diseases, including cancer. Crosstalk between obesity and cancer seems to be very complex, and the interaction between adipocytes and cancer cells leads to changes in adipocytes' function and their paracrine signaling, promoting a microenvironment that supports tumor growth. Carbonic anhydrase IX (CA IX) is a tumor-associated enzyme that not only participates in pH regulation but also facilitates metabolic reprogramming and supports the migration, invasion, and metastasis of cancer cells. In addition, CA IX expression, predominantly regulated via hypoxia-inducible factor (HIF-1), serves as a surrogate marker of hypoxia. In this study, we investigated the impact of adipocytes and adipocyte-derived factors on the expression of CA IX in colon and breast cancer cells. We observed increased expression of CA9 mRNA as well as CA IX protein in the presence of adipocytes and adipocyte-derived conditioned medium. Moreover, we confirmed that adipocytes affect the hypoxia signaling pathway and that the increased CA IX expression results from adipocyte-mediated induction of HIF-1α. Furthermore, we demonstrated that adipocyte-mediated upregulation of CA IX leads to increased migration and decreased adhesion of colon cancer cells. Finally, we brought experimental evidence that adipocytes, and more specifically leptin, upregulate CA IX expression in cancer cells and consequently promote tumor progression.
Asunto(s)
Adipocitos , Antígenos de Neoplasias , Neoplasias de la Mama , Anhidrasa Carbónica IX , Neoplasias del Colon , Comunicación Paracrina , Animales , Femenino , Humanos , Ratones , Adipocitos/metabolismo , Antígenos de Neoplasias/metabolismo , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Anhidrasa Carbónica IX/metabolismo , Línea Celular Tumoral , Movimiento Celular , Neoplasias del Colon/patología , Neoplasias del Colon/metabolismo , Medios de Cultivo Condicionados/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Leptina/metabolismo , Obesidad/metabolismo , Microambiente TumoralRESUMEN
Microtubule-associated serine-threonine kinase-like (MASTL) has recently been identified as an oncogenic kinase given its overexpression in numerous cancers. Our group has shown that MASTL expression is upregulated in mouse models of sporadic colorectal cancer and colitis-associated cancer (CAC). CAC is one of the most severe complications of chronic inflammatory bowel disease (IBD), but a limited understanding of the mechanisms governing the switch from normal healing to neoplasia in IBD underscores the need for increased research in this area. However, MASTL levels in patients with IBD and its molecular regulation in IBD and CAC have not been studied. This study reveals that MASTL is upregulated by the cytokine interleukin (IL)-22, which promotes proliferation and has important functions in colitis recovery; however, IL-22 can also promote tumorigenesis when chronically elevated. Upon reviewing the publicly available data, we found significantly elevated MASTL and IL-22 levels in the biopsies from patients with late-stage ulcerative colitis compared with controls, and that MASTL upregulation was associated with high IL-22 expression. Our subsequent in vitro studies found that IL-22 increases MASTL expression in intestinal epithelial cell lines, which facilitates IL-22-mediated cell proliferation and downstream survival signaling. Inhibition of AKT activation abrogated IL-22-induced MASTL upregulation. We further found an increased association of carbonic anhydrase IX (CAIX) with MASTL in IL-22-treated cells, which stabilized MASTL expression. Inhibition of CAIX prevented IL-22-induced MASTL expression and cell survival. Overall, we show that IL-22/AKT signaling increases MASTL expression to promote cell survival and proliferation. Furthermore, CAIX associates with and stabilizes MASTL in response to IL-22 stimulation.NEW & NOTEWORTHY MASTL is upregulated in colorectal cancer; however, its role in colitis and colitis-associated cancer is poorly understood. This study is the first to draw a link between MASTL and IL-22, a proinflammatory/intestinal epithelial recovery-promoting cytokine that is also implicated in colon tumorigenesis. We propose that IL-22 increases MASTL protein stability by promoting its association with CAIX potentially via AKT signaling to promote cell survival and proliferation.
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Interleucina-22 , Interleucinas , Mucosa Intestinal , Interleucinas/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/patología , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Animales , Proliferación Celular , Transducción de Señal , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Colitis Ulcerosa/metabolismo , Colitis Ulcerosa/patología , Ratones , Regulación hacia Arriba , Proteínas Proto-Oncogénicas c-akt/metabolismo , Anhidrasa Carbónica IX/metabolismo , Anhidrasa Carbónica IX/genética , Antígenos de NeoplasiasRESUMEN
The efficacy of molecular-targeted photodynamic therapy (MT-PDT) targeting carbonic anhydrase (CA) IX, a cancer-specific molecule, was demonstrated. CA ligand-directed photosensitizers 1-3 were evaluated for their ability to deactivate CAIX protein in cells. Compounds 2 and 3 selectively deactivated CAIX protein under 540 nm light without affecting internal standard proteins. Mechanistic studies revealed that compound 3 not only induced CAIX-selective light inactivation via singlet oxygen but also induced cell membrane damage, resulting in an anti-tumor effect. In vivo studies of CAIX-targeting MT-PDT revealed that treatment with compound 3 followed by light irradiation exhibited remarkable anti-tumor activity, leading to tumor degeneration and necrosis.
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Anhidrasa Carbónica IX , Fotoquimioterapia , Fármacos Fotosensibilizantes , Anhidrasa Carbónica IX/metabolismo , Anhidrasa Carbónica IX/antagonistas & inhibidores , Humanos , Fármacos Fotosensibilizantes/farmacología , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/síntesis química , Animales , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/farmacología , Inhibidores de Anhidrasa Carbónica/síntesis química , Estructura Molecular , Línea Celular Tumoral , Ratones , Relación Estructura-Actividad , Antígenos de Neoplasias/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Relación Dosis-Respuesta a DrogaRESUMEN
Tumor imaging and delivery of therapeutic agents may be achieved by designing high-affinity and high-selectivity compounds recognizing a tumor cell-expressing biomarker, such as carbonic anhydrase IX (CA IX). The CAIX, overexpressed in many hypoxic solid tumors, helps adjust to the energy requirements of the hypoxic environment, reduces intracellular acidification, and participates in the metastatic invasion of adjacent tissues. Here, we designed a series of sulfonamide compounds bearing CAIX-recognizing, high-affinity, and high-selectivity groups conjugated via a PEG linker to near-infrared (NIR) fluorescent probes used in the clinic for optically guided cancer surgery. We determined compound affinities for CAIX and other 11 catalytically active CA isozymes by the thermal shift assay and showed that the affinity Kd value of CAIX was in the subnanomolar range, hundred to thousand-fold higher than those of other CA isozymes. Similar affinities were also observed for CAIX expressed on the cancer cell surface in live HeLa cell cultures, as determined by the competition assay. The NIR-fluorescent compounds showed excellent properties in visualizing CAIX-positive tumors but not CAIX-negative knockout tumors in a nude mice xenograft model. These compounds would therefore be helpful in optically guided cancer surgery and could potentially be developed for anticancer treatment by radiotherapy.
Asunto(s)
Antígenos de Neoplasias , Anhidrasa Carbónica IX , Inhibidores de Anhidrasa Carbónica , Colorantes Fluorescentes , Humanos , Anhidrasa Carbónica IX/metabolismo , Anhidrasa Carbónica IX/antagonistas & inhibidores , Animales , Colorantes Fluorescentes/química , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/farmacología , Inhibidores de Anhidrasa Carbónica/uso terapéutico , Ratones , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/análisis , Células HeLa , Neoplasias/diagnóstico por imagen , Ratones Desnudos , Sulfonamidas/química , Rayos Infrarrojos , Anhidrasas Carbónicas/metabolismo , Imagen Óptica/métodosRESUMEN
Two novel series of hydrazinyl-based benzenesulfonamides 9a-j and 10a-j were designed and synthesized using SLC-0111 as the lead molecule. The newly synthesized compounds were evaluated for their inhibitory activity against four different human carbonic anhydrase (hCA) isoforms I, II, IX, and XII. Both the series reported here were practically inactive against the off-target isozyme hCA I. Notably, derivative 10a exhibited superior potency (Ki of 10.2 nM) than acetazolamide (AAZ) against the cytosolic isoform hCA II. The hCA IX and XII isoforms implicated in tumor progression were effectively inhibited with Kis in the low nanomolar range of 20.5-176.6 nM and 6.0-127.5 nM, respectively. Compound 9g emerged as the most potent and selective hCA IX and XII inhibitor with Ki of 20.5 nM and SI of 200.1, and Ki of 6.0 nM and SI of 683.7, respectively, over hCA I. Furthermore, six compounds (9a, 9h, 10a, 10g, 10i, and 10j) exhibited significant inhibition toward hCA IX (Kis = 27.0, 41.1, 27.4, 25.9, 40.7, and 30.8 nM) relative to AAZ and SLC-0111 (Kis = 25.0 and 45.0 nM, respectively). These findings underscore the potential of these derivatives as potent and selective inhibitors of hCA IX and XII over the off-target hCA I and II.
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Inhibidores de Anhidrasa Carbónica , Anhidrasas Carbónicas , Sulfonamidas , Humanos , Inhibidores de Anhidrasa Carbónica/farmacología , Inhibidores de Anhidrasa Carbónica/síntesis química , Inhibidores de Anhidrasa Carbónica/química , Relación Estructura-Actividad , Anhidrasas Carbónicas/metabolismo , Sulfonamidas/farmacología , Sulfonamidas/química , Sulfonamidas/síntesis química , Estructura Molecular , Diseño de Fármacos , Antígenos de Neoplasias/metabolismo , Isoenzimas/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Anhidrasa Carbónica IX/antagonistas & inhibidores , Anhidrasa Carbónica IX/metabolismo , Anhidrasa Carbónica II/antagonistas & inhibidores , Anhidrasa Carbónica II/metabolismo , Simulación por Computador , Simulación del Acoplamiento Molecular , Compuestos de FenilureaRESUMEN
Ovarian cancer is an aggressive gynecological tumor characterized by a high relapse rate and chemoresistance. Ovarian cancer exhibits the cancer hallmark of elevated glycolysis, yet effective strategies targeting cancer cell metabolic reprogramming to overcome therapeutic resistance in ovarian cancer remain elusive. Here, we revealed that epigenetic silencing of Otubain 2 (OTUB2) is a driving force for mitochondrial metabolic reprogramming in ovarian cancer, which promotes tumorigenesis and chemoresistance. Mechanistically, OTUB2 silencing destabilizes sorting nexin 29 pseudogene 2 (SNX29P2), which subsequently prevents hypoxia-inducible factor-1 alpha (HIF-1α) from von Hippel-Lindau tumor suppressor-mediated degradation. Elevated HIF-1α activates the transcription of carbonic anhydrase 9 (CA9) and drives ovarian cancer progression and chemoresistance by promoting glycolysis. Importantly, pharmacological inhibition of CA9 substantially suppressed tumor growth and synergized with carboplatin in the treatment of OTUB2-silenced ovarian cancer. Thus, our study highlights the pivotal role of OTUB2/SNX29P2 in suppressing ovarian cancer development and proposes that targeting CA9-mediated glycolysis is an encouraging strategy for the treatment of ovarian cancer.
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Anhidrasa Carbónica IX , Silenciador del Gen , Mitocondrias , Neoplasias Ováricas , Tioléster Hidrolasas , Animales , Femenino , Humanos , Ratones , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/genética , Anhidrasa Carbónica IX/metabolismo , Anhidrasa Carbónica IX/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glucólisis/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Reprogramación Metabólica , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Neoplasias Ováricas/tratamiento farmacológico , Tioléster Hidrolasas/genéticaRESUMEN
The aim of this study was to analyze the association between vaginal microbiota, carbonic anhydrase IX (CAIX) and histological findings of cervical intraepithelial neoplasia (CIN). The study included 132 females, among them 66 were diagnosed with high-grade intraepithelial lesion (CIN2, CIN3, and cancer), 14 with low-grade disease, and 52 assigned to the control group. An interview focused on the behavior risk factors, together with vaginal fluid pH measurement, wet mount microscopy, detection of Chlamydia trachomatis, and Trichomonas vaginalis were performed. After colposcopy, high-grade abnormalities were detected via direct biopsies and treated with conization procedure. Conuses were immuno-stained with CAIX antibody. The histological findings were CIN1 (n = 14), and CIN2+ (included CIN2 (n = 10), CIN3 (n = 49), and cancer (n = 7; squamous cell carcinomas)). Prevalence of bacterial vaginosis (BV) was similar between the groups. Moderate or severe aerobic vaginitis (msAV) was diagnosed more often among CIN2+ (53.0%) than CIN1 (21.4%). Moderate or strong immunostaining of CAIX (msCAIX) was not detected among CIN1 cases. Thus, msAV was prevalent in CAIX non-stained group (p = 0.049) among CIN2 patients. Co-location of msAV and msCAIX was found in CIN3. Regression model revealed that msAV associated with high-grade cervical intraepithelial neoplasia independently from smoking and the number of partners.
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Infecciones por Papillomavirus , Displasia del Cuello del Útero , Neoplasias del Cuello Uterino , Vulvovaginitis , Femenino , Humanos , Anhidrasa Carbónica IX , Conización , Papillomaviridae , Infecciones por Papillomavirus/complicaciones , Displasia del Cuello del Útero/patología , Neoplasias del Cuello Uterino/patologíaRESUMEN
A novel series of hydantoins incorporating phthalimides has been synthesised by condensation of activated phthalimides with 1-aminohydantoin and investigated for their inhibitory activity against a panel of human (h) carbonic anhydrase (CA, EC 4.2.1.1): the cytosolic isoforms hCA I, hCA II, and hCA VII, secreted isoform hCA VI, and the transmembrane hCA IX, by a stopped-flow CO2 hydrase assay. Although all newly developed compounds were totally inactive on hCA I and mainly ineffective towards hCA II, they generally exhibited moderate repressing effects on hCA VI, VII, and IX with KIs values in the submicromolar to micromolar ranges. The salts 3a and 3b, followed by derivative 5, displayed the best inhibitory activity of all the evaluated compounds and their binding mode was proposed in silico. These compounds can also be considered interesting starting points for the development of novel pharmacophores for this class of enzyme inhibitors.
Asunto(s)
Anhidrasas Carbónicas , Hidantoínas , Humanos , Anhidrasas Carbónicas/metabolismo , Anhidrasa Carbónica IX , Relación Estructura-Actividad , Anhidrasa Carbónica I , Anhidrasa Carbónica II , Isoformas de Proteínas/metabolismo , Ftalimidas/farmacología , Hidantoínas/farmacología , Inhibidores de Anhidrasa Carbónica/química , Estructura MolecularRESUMEN
Transition metal ions are structural and catalytic cofactors of many proteins including human carbonic anhydrase (CA), a Zn-dependent hydrolase. Sulfonamide inhibitors of CA recognize and form a coordination bond with the Zn ion located in the active site of the enzyme. The Zn ion may be removed or substituted with other metal ions. Such CA protein retains the structure and could serve as a tool to study metal ion role in the recognition and binding affinity of inhibitor molecules. We measured the affinities of selected divalent transition metal ions, including Mn, Fe, Co, Ni, Cu, Cd, Hg, and Zn to metal-free CA isozymes CA I, CA II, and CAIX by fluorescence-based thermal shift assay, prepared metal-substituted CAs, and determined binding of diverse sulfonamide compounds. Sulfonamide inhibitor binding to metal substituted CA followed a U-shape pH dependence. The binding was dissected to contributing binding-linked reactions and the intrinsic binding reaction affinity was calculated. This value is independent of pH and protonation reactions that occur simultaneously upon binding native CA and as demonstrated here, to metal substituted CA. Sulfonamide inhibitor binding to cancer-associated isozyme CAIX diminished in the order: Zn > Co > Hg > Cu > Cd > Mn > Ni. Energetic contribution of the inhibitor-metal coordination bond was determined for all above metals. The understanding of the principles of metal influence on ligand affinity and selectivity should help design new drugs targeting metalloenzymes.