RESUMEN
Sigma-2-ligands (S2L) are characterized by high binding affinities to their cognate sigma-2 receptor, overexpressed in rapidly proliferating tumor cells. As such, S2L were developed as imaging probes (ISO1) or as cancer therapeutics, alone (SV119 [C6], SW43 [C10]) and as delivery vehicles for cytotoxic drug cargoes (C6-Erastin, C10-SMAC). However, the exact mechanism of S2L-induced cytotoxicity remains to be fully elucidated. A series of high-affinity S2L were evaluated regarding their cytotoxicity profiles across cancer cell lines. While C6 and C10 displayed distinct cytotoxicities, C0 and ISO1 were essentially non-toxic. Confocal microscopy and lipidomics analysis in cellular and mouse models revealed that C10 induced increases in intralysosomal free cholesterol and in cholesterol esters, suggestive of unaltered intracellular cholesterol trafficking. Cytotoxicity was caused by cholesterol excess, a phenomenon that contrasts the effects of NPC1 inhibition. RNA-sequencing revealed gene clusters involved in cholesterol homeostasis and ER stress response exclusively by cytotoxic S2L. ER stress markers were confirmed by qPCR and their targeted modulation inhibited or enhanced cytotoxicity of C10 in a predicted manner. Moreover, C10 increased sterol regulatory element-binding protein 2 (SREBP2) and low-density lipoprotein receptor (LDLR), both found to be pro-survival factors activated by ER stress. Furthermore, inhibition of downstream processes of the adaptive response to S2L with simvastatin resulted in synergistic treatment outcomes in combination with C10. Of note, the S2L conjugates retained the ER stress response of the parental ligands, indicative of cholesterol homeostasis being involved in the overall cytotoxicity of the drug conjugates. Based on these findings, we conclude that S2L-mediated cell death is due to free cholesterol accumulation that leads to ER stress. Consequently, the cytotoxic profiles of S2L drug conjugates are proposed to be enhanced via concurrent ER stress inducers or simvastatin, strategies that could be instrumental on the path toward tumor eradication.
Asunto(s)
Colesterol , Estrés del Retículo Endoplásmico , Receptores sigma , Colesterol/metabolismo , Receptores sigma/metabolismo , Receptores sigma/genética , Humanos , Animales , Ratones , Estrés del Retículo Endoplásmico/efectos de los fármacos , Ligandos , Línea Celular Tumoral , Muerte Celular/efectos de los fármacos , Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patologíaRESUMEN
Cancer selective apoptosis remains a therapeutic challenge and off-target toxicity has limited enthusiasm for this target clinically. Sigma-2 ligands (S2) have been shown to enhance the cancer selectivity of small molecule drug candidates by improving internalization. Here, we report the synthesis of a novel drug conjugate, which was created by linking a clinically underperforming SMAC mimetic (second mitochondria-derived activator of caspases; LCL161), an inhibitor (antagonist) of inhibitor of apoptosis proteins (IAPinh) with the sigma-2 ligand SW43, resulting in the new chemical entity S2/IAPinh. Drug potency was assessed via cell viability assays across several pancreatic and ovarian cancer cell lines in comparison with the individual components (S2 and IAPinh) as well as their equimolar mixtures (S2 + IAPinh) both in vitro and in preclinical models of pancreatic and ovarian cancer. Mechanistic studies of S2/IAPinh-mediated cell death were investigated in vitro and in vivo using syngeneic and xenograft mouse models of murine pancreatic and human ovarian cancer, respectively. S2/IAPinh demonstrated markedly improved pharmacological activity in cancer cell lines and primary organoid cultures when compared to the controls. In vivo testing demonstrated a marked reduction in tumor growth rates and increased survival rates when compared to the respective control groups. The predicted mechanism of action of S2/IAPinh was confirmed through assessment of apoptosis pathways and demonstrated strong target degradation (cellular inhibitor of apoptosis proteins-1 [cIAP-1]) and activation of caspases 3 and 8. Taken together, S2/IAPinh demonstrated efficacy in models of pancreatic and ovarian cancer, two challenging malignancies in need of novel treatment concepts. Our data support an in-depth investigation into utilizing S2/IAPinh for the treatment of cancer.
Asunto(s)
Antineoplásicos , Neoplasias Ováricas , Humanos , Animales , Ratones , Femenino , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Antineoplásicos/química , Apoptosis , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Caspasas/metabolismo , Línea Celular TumoralRESUMEN
BACKGROUND: Ovarian cancer is initially responsive to frontline chemotherapy. Unfortunately, it often recurs and becomes resistant to available therapies and the survival rate for advanced and recurrent ovarian cancer is unacceptably low. We thus hypothesized that it would be possible to achieve more durable treatment responses by combining cisplatin chemotherapy with SW IV-134, a cancer-targeted peptide mimetic and inducer of cell death. SW IV-134 is a recently developed small molecule conjugate linking a sigma-2 ligand with a peptide analog (mimetic) of the intrinsic death pathway activator SMAC (second-mitochondria activator of caspases). The sigma-2 receptor is overexpressed in ovarian cancer and the sigma-2 ligand portion of the conjugate facilitates cancer selectivity. The effector portion of the conjugate is expected to synergize with cisplatin chemotherapy and the cancer selectivity is expected to reduce putative off-target toxicities. METHODS: Ovarian cancer cell lines were treated with cisplatin alone, SW IV-134 alone and a combination of the two drugs. Treatment efficacy was determined using luminescent cell viability assays. Caspase-3/7, - 8 and - 9 activities were measured as complementary indicators of death pathway activation. Syngeneic mouse models and patient-derived xenograft (PDX) models of human ovarian cancer were studied for response to SW IV-134 and cisplatin monotherapy as well as combination therapy. Efficacy of the therapy was measured by tumor growth rate and survival as the primary readouts. Potential drug related toxicities were assessed at necropsy. RESULTS: The combination treatment was consistently superior in multiple cell lines when compared to the single agents in vitro. The expected mechanism of tumor cell death, such as caspase activation, was confirmed using luminescent and flow cytometry-based assay systems. Combination therapy proved to be superior in both syngeneic and PDX-based murine models of ovarian cancer. Most notably, combination therapy resulted in a complete resolution of established tumors in all study animals in a patient-derived xenograft model of ovarian cancer. CONCLUSIONS: The addition of SW IV-134 in combination with cisplatin chemotherapy represents a promising treatment option that warrants further pre-clinical development and evaluation as a therapy for women with advanced ovarian cancer.
Asunto(s)
Antineoplásicos/uso terapéutico , Compuestos de Azabiciclo/uso terapéutico , Carcinoma Epitelial de Ovario/tratamiento farmacológico , Cisplatino/uso terapéutico , Oligopéptidos/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Resistencia a Antineoplásicos , Femenino , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Pancreatic cancer is a lethal malignancy that frequently acquires resistance to conventional chemotherapies often associated with overexpression of inhibitors of apoptosis proteins (IAPs). We have recently described a novel means to deliver second mitochondria-derived activator of caspases (SMAC) mimetics selectively to cancer cells employing the sigma-2 ligand/receptor interaction. The intrinsic death pathway agonist SMAC offers an excellent opportunity to counteract the anti-apoptotic activity of IAPs. SMAC mimetics have been used to sensitize several cancer types to chemotherapeutic agents but cancer-selective delivery and appropriate cellular localization have not yet been considered. In our current study, we tested the ability of the sigma-2/SMAC drug conjugate SW IV-134 to sensitize pancreatic cancer cells to gemcitabine. METHODS: Using the targeted SMAC mimetic SW IV-134, inhibition of the X-linked inhibitor of apoptosis proteins (XIAP) was induced pharmacologically and its impact on cell viability was studied alone and in combination with gemcitabine. Pathway analyses were performed by assessing caspase activation, PARP cleavage and membrane blebbing (Annexin-V), key components of apoptotic cell death. Single-agent treatment regimens were compared with combination therapy in a preclinical mouse model of pancreatic cancer. RESULTS: The sensitizing effect of XIAP interference toward gemcitabine was confirmed via pharmacological intervention using our recently designed, targeted SMAC mimetic SW IV-134 across a wide range of commonly used pancreatic cancer cell lines at concentrations where the individual drugs showed only minimal activity. On a mechanistic level, we identified involvement of key components of the apoptosis machinery during cell death execution. Furthermore, combination therapy proved superior in decreasing the tumor burden and extending the lives of the animals in a preclinical mouse model of pancreatic cancer. CONCLUSION: We believe that the strong sensitizing capacity of SW IV-134 in combination with clinically relevant doses of gemcitabine represents a promising treatment option that warrants clinical evaluation.
Asunto(s)
Antimetabolitos Antineoplásicos/administración & dosificación , Antineoplásicos/administración & dosificación , Compuestos de Azabiciclo/administración & dosificación , Desoxicitidina/análogos & derivados , Oligopéptidos/administración & dosificación , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Antimetabolitos Antineoplásicos/farmacología , Antineoplásicos/farmacología , Compuestos de Azabiciclo/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Desoxicitidina/administración & dosificación , Desoxicitidina/farmacología , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Oligopéptidos/farmacología , Neoplasias Pancreáticas/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
Cancer-selective drug delivery is an important concept in improving treatment while minimizing off-site toxicities, and sigma-2 receptors, which are overexpressed in solid tumors, represent attractive pharmacologic targets. Select sigma-2 ligands have been shown to be rapidly internalized selectively into cancer cells while retaining the capacity to deliver small molecules as drug cargoes. We utilized the sigma-2-based drug delivery concept to convert Erastin, a clinically underperforming drug, into a potent pancreatic cancer therapeutic. The Erastin derivative des-methyl Erastin (dm-Erastin) was chemically linked to sigma-2 ligand SV119 to create SW V-49. Conjugation increased the killing capacity of dm-Erastin by nearly 35-fold in vitro and reduced the size of established tumors and doubled the median survival in syngeneic and patient-derived xenograft models when compared to non-targeted dm-Erastin. Mechanistic analyses demonstrated that cell death was associated with robust reactive oxygen species production and could be efficiently antagonized with antioxidants. Mass spectrometry was employed to demonstrate selective uptake into pancreatic cancer cells. Thus, targeted delivery of dm-Erastin via conjugation to the sigma-2 ligand SV119 produced efficient tumor control and prolonged animal survival with minimal off-target toxicities, and SW V-49 represents a promising new therapeutic with the potential to advance the fight against pancreatic cancer.
Asunto(s)
Antineoplásicos/farmacocinética , Compuestos de Azabiciclo/farmacología , Carbamatos/farmacología , Sistemas de Liberación de Medicamentos/métodos , Neoplasias Pancreáticas/tratamiento farmacológico , Piperazinas/farmacología , Animales , Antineoplásicos/síntesis química , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Receptores sigma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Because of the complexity and heterogeneity of human neuropsychiatric disorders, it has been difficult to identify animal models that mimic the symptoms of these neuropathologies and can be used to screen for antipsychotic agents. For this study we selected the murine 5HT2A/2C receptor agonist-induced head twitch response (HTR) induced by the administration of 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), which has been proposed as an animal model of symptoms associated with a variety of behavioral and psychiatric conditions. We investigated the DOI-induced HTR in male DBA/2J mice using a panel of D2-like (D2, D3 and D4) and D2 dopamine receptor selective compounds. When DBA/2J mice were administered a daily dose of DOI (5 mg/kg), tolerance to the DOI occurs. However, administrations of the same dose of DOI every other day (48 h) or on a weekly basis did not lead to tolerance and the ability to induce tolerance after daily administration of DOI remains intact after repeated weekly administration of DOI. Subsequently, a panel of D2-like dopamine receptor antagonists was found to effectively inhibit the DOI-induced HTR in DBA/2J mice. However, the benzamide eticlopride, which is a high affinity D2-like antagonist, was a notable exception. SV 293, SV-III-130s and N-methylbenperidol, which exhibit a high affinity for D2 versus the D3 dopamine receptor subtypes (60- to 100-fold binding selectivity), were also found to inhibit the HTR in DBA/2J mice. This observation suggests a functional interaction between dopaminergic and serotonergic systems through D2 dopamine receptors and the 5-HT2A serotonin receptors in vivo.
Asunto(s)
Anfetaminas/toxicidad , Antipsicóticos/farmacología , Antagonistas de los Receptores de Dopamina D2/farmacología , Receptores de Dopamina D3/antagonistas & inhibidores , Agonistas del Receptor de Serotonina 5-HT2/toxicidad , Animales , Conducta Animal/efectos de los fármacos , Antagonistas de los Receptores de Dopamina D2/farmacocinética , Tolerancia a Medicamentos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBARESUMEN
Pancreatic adenocarcinoma is highly resistant to conventional therapeutics and has been shown to evade apoptosis by deregulation of the X-linked and cellular inhibitors of apoptosis proteins (XIAP and cIAP). Second mitochondria-derived activator of caspases (Smac) induces and amplifies cell death by reversing the anti-apoptotic activity of IAPs. Thus, Smac-derived peptide analogues (peptidomimetics) have been developed and shown to represent promising cancer therapeutics. Sigma-2 receptors are overexpressed in many proliferating tumor cells including pancreatic cancer. Selected ligands to this receptor are rapidly internalized by cancer cells. These characteristics have made the sigma-2 receptor an attractive target for drug delivery because selective delivery to cancer cells has the potential to increase therapeutic efficacy while minimizing toxicity to normal tissues. Here, we describe the initial characterization of SW IV-134, a chemically linked drug conjugate between the sigma-2 ligand SW43 and the Smac mimetic SW IV-52 as a novel treatment option for pancreatic adenocarcinoma. The tumor killing characteristics of our dual-domain therapeutic SW IV-134 was far greater than either component in isolation or in an equimolar mix and suggests enhanced cellular delivery when chemically linked to the sigma-2 ligand. One of the key findings was that SW IV-134 retained target selectivity of the Smac cargo with the involvement of the NF-κB/TNFα signaling pathway. Importantly, SW IV-134 slowed tumor growth and improved survival in murine models of pancreatic cancer. Our data support further study of this novel therapeutic and this drug delivery strategy because it may eventually benefit patients with pancreatic cancer.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Compuestos de Azabiciclo/uso terapéutico , Oligopéptidos/uso terapéutico , Páncreas/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Receptores sigma/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Caspasa 3/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Terapia Molecular Dirigida , Páncreas/metabolismo , Páncreas/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologíaRESUMEN
Cancer stem cells (CSCs) are believed to be responsible for the long-term growth of a tumor, as well as its metastasis and recurrence after conventional therapies. Here, it is demonstrated that the sigma-2 receptor is overexpressed on the surface of breast CSCs, and thus could serve as a biomarker for the purpose of targeting. Breast CSCs are targeted with Au nanocages (AuNCs) by functionalizing their surfaces with SV119, a synthetic small molecule capable of binding to the sigma-2 receptor with high specificity. The interiors of the AuNCs could also be loaded with an anticancer drug to be selectively delivered to breast CSCs and released in a controllable fashion. The results demonstrate that the SV119-AuNC conjugate can serve as a new platform to carry out photothermal and chemo therapies simultaneously, eradicating breast CSCs more effectively through a synergetic effect.
Asunto(s)
Antineoplásicos/toxicidad , Compuestos de Azabiciclo/química , Carbamatos/química , Oro/química , Nanoestructuras/química , Células Madre Neoplásicas/efectos de los fármacos , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Doxorrubicina/administración & dosificación , Doxorrubicina/química , Doxorrubicina/toxicidad , Portadores de Fármacos/química , Femenino , Humanos , Rayos Láser , Fototerapia , Unión Proteica , Receptores sigma/química , Receptores sigma/metabolismoRESUMEN
BACKGROUND: Drug resistance is a significant problem in the treatment of ovarian cancer and can be caused by multiple mechanisms. Inhibition of apoptosis by the inhibitor of apoptosis proteins (IAPs) represents one such mechanism, and can be overcome by a mitochondrial protein called second mitochondria-derived activator of caspases (SMAC). We have previously shown that the ligands of sigma-2 receptors effectively induce tumor cell death. Additionally, because sigma-2 receptors are preferentially expressed in tumor cells, their ligands provide an effective mechanism for selective anti-cancer therapy. METHODS: In the current work, we have improved upon the previously described sigma-2 ligand SW43 by conjugating it to a pro-apoptotic small molecule SMAC mimetic SW IV-52, thus generating the novel cancer therapeutic SW IV-134. The new cancer drug was tested for receptor selectivity and tumor cell killing activity in vitro and in vivo. RESULTS: We have shown that SW IV-134 retained adequate sigma-2 receptor binding affinity in the context of the conjugate and potently induced cell death in ovarian cancer cells. The cell death induced by SW IV-134 was significantly greater than that observed with either SW43 or SW IV-52 alone and in combination. Furthermore, the intraperitoneal administration of SW IV-134 significantly reduced tumor burden and improved overall survival in a mouse xenograft model of ovarian cancer without causing significant adverse effects to normal tissues. Mechanistically, SW IV-134 induced degradation of cIAP-1 and cIAP-2 leading to NF-ÒB activation and TNFα-dependent cell death. CONCLUSIONS: Our findings suggest that coupling sigma-2 ligands to SMAC peptidomimetics enhances their effectiveness while maintaining the cancer selectivity. This encouraging proof-of-principle preclinical study supports further development of tumor-targeted small peptide mimetics via ligands to the sigma-2 receptor for future clinical applications.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Compuestos de Azabiciclo/química , Compuestos de Azabiciclo/farmacología , Oligopéptidos/química , Oligopéptidos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Receptores sigma/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Western Blotting , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Femenino , Xenoinjertos , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Ligandos , Ratones , Ratones SCID , Proteínas Mitocondriales/química , Terapia Molecular Dirigida/métodos , Neoplasias Ováricas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The sigma-2 receptor has been identified as a biomarker in proliferating tumors. To date there is no well-established functional assay for defining sigma-2 agonists and antagonists. Many sigma-2 ligands with diverse structures have been shown to induce cell death in a variety of cancer cells by triggering caspase-dependent and independent apoptosis. Therefore, in the current study, we used the cell viability assay and the caspase-3 activity assay to determine sigma-2 agonists and antagonists. Three classes of sigma-2 ligands developed in our laboratory were evaluated for their potency to induce cell death in two tumor cell lines, mouse breast cancer cell line EMT-6 and human melanoma cell line MDA-MB-435. The data showed that the EC50 values of the sigma-2 ligands using the cell viability assay ranged from 11.4µM to >200µM, which were comparable with the EC50 values obtained using the caspase-3 assay. Based on the cytotoxicity of a sigma-2 ligand relative to that of siramesine, a commonly accepted sigma-2 agonist, we have categorized our sigma-2 ligands into agonists, partial agonists, and antagonists. The establishment of functional assays for defining sigma-2 agonists and antagonists will facilitate functional characterization of sigma-2 receptor ligands and sigma-2 receptors.
Asunto(s)
Receptores sigma/agonistas , Receptores sigma/antagonistas & inhibidores , Animales , Compuestos de Azabiciclo/química , Compuestos de Azabiciclo/farmacología , Benzamidas/química , Benzamidas/farmacología , Bioensayo , Caspasa 3/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Indoles/química , Indoles/farmacología , Ligandos , Ratones , Unión Proteica/efectos de los fármacos , Receptores sigma/metabolismo , Compuestos de Espiro/química , Compuestos de Espiro/farmacología , Tropanos/química , Tropanos/farmacologíaRESUMEN
SV 293 [1-([5-methoxy-1H-indol-3-yl]methyl)-4-(4-[methylthio]âphenyl)piperidin-4-ol] binds with 100-fold higher affinity to human D2 receptors compared to the human D3 and D4 dopamine receptor subtypes. We investigated the intrinsic efficacy of this compound at the D2 dopamine receptor subtype using both: (1) a forskolin-dependent adenylyl cyclase inhibition assay and (2) an electrophysiological assay for evaluating coupling to G-protein-coupled inwardly rectifying potassium channels. In both assays SV 293 was found to be a neutral antagonist capable of blocking the effects of the full D2-like receptor agonist quinpirole. Based upon these results we propose that SV 293 is a useful pharmacological tool that can be used for both in vitro and in vivo studies to investigate the role of D2-like dopamine receptor subtypes in neurological, neuropsychiatric and movement disorders where dopaminergic pathways have been implicated.
Asunto(s)
Antagonistas de Dopamina/farmacología , Antagonistas de los Receptores de Dopamina D2 , Indoles/farmacología , Piperidinas/farmacología , Inhibidores de Adenilato Ciclasa , Animales , Línea Celular Tumoral , Colforsina/farmacología , Agonistas de Dopamina/farmacología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/fisiología , Humanos , Ratones , Quinpirol/farmacología , Receptores de Dopamina D2/fisiologíaRESUMEN
A series of microPET imaging studies were conducted in anesthetized rhesus monkeys using the dopamine D2-selective partial agonist, [(11)C]SV-III-130. There was a high uptake in regions of brain known to express a high density of D2 receptors under baseline conditions. Rapid displacement in the caudate and putamen, but not in the cerebellum, was observed after injection of the dopamine D2/3 receptor nonselective ligand S(-)-eticlopride at a low dosage (0.025mg/kg/i.v.); no obvious displacement in the caudate, putamen and cerebellum was observed after the treatment with a dopamine D3 receptor selective ligand WC-34 (0.1mg/kg/i.v.). Pretreatment with lorazepam (1mg/kg, i.v. 30min) to reduce endogenous dopamine prior to tracer injection resulted in unchanged binding potential (BP) values, a measure of D2 receptor binding in vivo, in the caudate and putamen. d-Amphetamine challenge studies indicate that there is a significant displacement of [(11)C]SV-III-130 by d-Amphetamine-induced increases in synaptic dopamine levels.
Asunto(s)
Encéfalo/diagnóstico por imagen , Radioisótopos de Carbono/farmacocinética , Piperazinas/farmacocinética , Tomografía de Emisión de Positrones/métodos , Quinolonas/farmacocinética , Radiofármacos/farmacocinética , Receptores de Dopamina D2/agonistas , Animales , Agonistas de Dopamina/farmacocinética , Macaca mulatta , MasculinoRESUMEN
UNLABELLED: Inhibitor of apoptosis (IAP) proteins play a central role in many types of cancer, and IAP antagonists are in development as anticancer agents. IAP antagonists cause apoptosis in many cells, but they also activate alternative NF-κB signaling through NF-κB-inducing kinase (NIK), which regulates osteoclasts. In bone metastasis, a positive feedback loop between tumors and osteoclasts promotes tumor growth and osteolysis. We therefore tested the effect of IAP antagonists on the bone microenvironment for metastasis. In both drug-sensitive and drug-resistant tumors, growth in bone was favored, as compared with other sites during IAP antagonist treatment. These drugs also caused osteoporosis and increased osteoclastogenesis, mediated by NIK, and enhanced tumor-associated osteolysis. Cotreatment with zoledronic acid, a potent osteoclast inhibitor, reduced IAP antagonist-enhanced tumor growth in bone and osteolysis. Thus, IAP antagonist-based cancer treatment may be compromised by osteoporosis and enhanced skeletal metastasis, which may be prevented by antiresorptive agents. SIGNIFICANCE: Although IAP antagonists are a class of anticancer agents with proven efficacy in multiple cancers, we show that these agents can paradoxically increase tumor growth and metastasis in the bone by stabilizing NIK and activating the alternative NF-κB pathway in osteoclasts. Future clinical trials of IAP antagonist-based therapy may require detailed examination of this potential for enhanced bone metastasis and osteoporosis, as well as possible combination with antiresorptive agents.
Asunto(s)
Conservadores de la Densidad Ósea/farmacología , Neoplasias Óseas/secundario , Proteínas Inhibidoras de la Apoptosis/antagonistas & inhibidores , Osteoclastos/efectos de los fármacos , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Western Blotting , Conservadores de la Densidad Ósea/efectos adversos , Conservadores de la Densidad Ósea/química , Neoplasias Óseas/metabolismo , Huesos/efectos de los fármacos , Huesos/metabolismo , Huesos/patología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Difosfonatos/administración & dosificación , Difosfonatos/farmacología , Humanos , Imidazoles/administración & dosificación , Imidazoles/farmacología , Proteínas Inhibidoras de la Apoptosis/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Estructura Molecular , Oligopéptidos/administración & dosificación , Oligopéptidos/farmacología , Osteoclastos/citología , Osteoclastos/metabolismo , Osteoporosis/inducido químicamente , Osteoporosis/prevención & control , Ensayos Antitumor por Modelo de Xenoinjerto , Ácido ZoledrónicoRESUMEN
We previously reported on the synthesis of substituted phenyl-4-hydroxy-1-piperidyl indole analogues with nanomolar affinity at D2 dopamine receptors, ranging from 10- to 100-fold selective for D2 compared to the D3 dopamine receptor subtype. More recently, we evaluated a panel of aripiprazole analogues, identifying several analogues that also exhibit D2 vs D3 dopamine receptor binding selectivity. These studies further characterize the intrinsic efficacy of the compound with the greatest binding selectivity from each chemical class, 1-((5-methoxy-1H-indol-3-yl)methyl)-4-(4-(methylthio)phenyl)piperidin-4-ol (SV 293) and 7-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butoxy)-3,4-dihydroquinolin-2(1H)-one (SV-III-130s), using an adenylyl cyclase inhibition assay, a G-protein-coupled inward-rectifying potassium (GIRK) channel activation assay, and a cell based phospho-MAPK (pERK1/2) assay. SV 293 was found to be a neutral antagonist at D2 dopamine receptors using all three assays. SV-III-130s is a partial agonist using an adenylyl cyclase inhibition assay but an antagonist in the GIRK and phospho ERK1/2 assays. To define the molecular basis for the binding selectivity, the affinity of these two compounds was evaluated using (a) wild type human D2 and D3 receptors and (b) a panel of chimeric D2/D3 dopamine receptors. Computer-assisted modeling techniques were used to dock these compounds to the human D2 and D3 dopamine receptor subtypes. It is hoped that these studies on D2 receptor selective ligands will be useful in the future design of (a) receptor selective ligands used to define the function of D2-like receptor subtypes, (b) novel pharmacotherapeutic agents, and/or (c) in vitro and in vivo imaging agents.
Asunto(s)
Antagonistas de Dopamina/síntesis química , Antagonistas de los Receptores de Dopamina D2 , Receptores de Dopamina D3/antagonistas & inhibidores , Antagonistas de Dopamina/farmacología , Células HEK293 , Humanos , Modelos Moleculares , Unión Proteica , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , TransfecciónRESUMEN
BACKGROUND: Sigma-2 receptor ligands have been studied for treatment of pancreatic cancer because they are preferentially internalized by proliferating cells and induce apoptosis. This mechanism of apoptosis is poorly understood, with varying reports of caspase-3 dependence. We evaluated multiple sigma-2 receptor ligands in this study, each shown to decrease tumor burden in preclinical models of human pancreatic cancer. RESULTS: Fluorescently labeled sigma-2 receptor ligands of two classes (derivatives of SW43 and PB282) localize to cell membrane components in Bxpc3 and Aspc1 pancreatic cancer cells and accumulate in lysosomes. We found that interactions in the lysosome are critical for cell death following sigma-2 ligand treatment because selective inhibition of a protective lysosomal membrane glycoprotein, LAMP1, with shRNA greatly reduced the viability of cells following treatment. Sigma-2 ligands induced lysosomal membrane permeabilization (LMP) and protease translocation triggering downstream effectors of apoptosis. Subsequently, cellular oxidative stress was greatly increased following treatment with SW43, and the hydrophilic antioxidant N-acetylcysteine (NAC) gave greater protection against this than a lipophilic antioxidant, α-tocopherol (α-toco). Conversely, PB282-mediated cytotoxicity relied less on cellular oxidation, even though α-toco did provide protection from this ligand. In addition, we found that caspase-3 induction was not as significantly inhibited by cathepsin inhibitors as by antioxidants. Both NAC and α-toco protected against caspase-3 induction following PB282 treatment, while only NAC offered protection following SW43 treatment. The caspase-3 inhibitor DEVD-FMK offered significant protection from PB282, but not SW43. CONCLUSIONS: Sigma-2 ligand SW43 commits pancreatic cancer cells to death by a caspase-independent process involving LMP and oxidative stress which is protected from by NAC. PB282 however undergoes a caspase-dependent death following LMP protected by DEVD-FMK and α-toco, which is also known to stabilize the mitochondrial membrane during apoptotic stimuli. These differences in mechanism are likely dependent on the structural class of the compounds versus the inherent sigma-2 binding affinity. As resistance of pancreatic cancers to specific apoptotic stimuli from chemotherapy is better appreciated, and patient-tailored treatments become more available, ligands with high sigma-2 receptor affinity should be chosen based on sensitivities to apoptotic pathways.
Asunto(s)
Antineoplásicos/farmacología , Lisosomas/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Receptores sigma/metabolismo , Animales , Antineoplásicos/farmacocinética , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Femenino , Humanos , Ligandos , Lisosomas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Microscopía Confocal , Neoplasias Pancreáticas/patología , Especies Reactivas de Oxígeno/metabolismo , Receptores sigma/genéticaRESUMEN
We previously reported that the antipsychotic drug haloperidol, a multifunctional D2-like dopamine and sigma receptor subtype antagonist, has neuroprotective properties. In this study we further examined the association between neuroprotection and receptor antagonism by evaluating a panel of novel compounds with varying affinity at sigma and D2-like dopamine receptors. These compounds were evaluated using an in vitro cytotoxicity assay that utilizes a hippocampal-derived cell line, HT-22, in the presence or absence of varying concentrations (5 to 20 mM) of glutamate. While haloperidol was found to be a potent neuroprotective agent in this in vitro cell assay, the prototypic sigma 1 receptor agonist (+)-pentazocine was found not to be neuroprotective. Subsequently, the potency for the neuroprotection of HT-22 cells was evaluated for a) three SV series indoles which have nMolar affinity at D2-like receptors but varying affinity at sigma 1 receptor and b) two benzyl phenylacetamides sigma 1 receptor selective compounds which bind with low affinity at D2-like receptors but have nMolar affinity for the sigma 1 receptor. We observed that cytoprotection correlated with the affinity of the compounds for sigma 1 receptors. Based upon results from the HT-22 cell-based in vitro assay, two phenylacetamides, LS-127 and LS-137, were further evaluated in vivo using a transient middle cerebral artery occlusion (t-MCAO) model of stroke. At a dose of 100 µg/kg, both LS-127 and LS-137 attenuated infarct volume by approximately 50%. These studies provide further evidence that sigma 1 receptor selective compounds can provide neuroprotection in cytotoxic situations. These results also demonstrate that sigma 1 receptor selective benzyl phenylacetamides are candidate pharmacotherapeutic agents that could be used to minimize neuronal death after a stroke or head trauma.
Asunto(s)
Fármacos Neuroprotectores/metabolismo , Receptores sigma/metabolismo , Acetamidas/química , Acetamidas/metabolismo , Acetamidas/uso terapéutico , Animales , Antipsicóticos/química , Antipsicóticos/metabolismo , Antipsicóticos/uso terapéutico , Femenino , Células HEK293 , Humanos , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/prevención & control , Ratones , Fármacos Neuroprotectores/química , Fármacos Neuroprotectores/uso terapéutico , Pentazocina/química , Pentazocina/metabolismo , Pentazocina/uso terapéutico , Unión Proteica/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de Dopamina D2/metabolismo , Receptores sigma/agonistas , Resultado del Tratamiento , Receptor Sigma-1RESUMEN
One major challenge in the development of cancer therapeutics is the selective delivery of the drugs to their cellular targets. In the case of pancreatic cancer, the σ-2 receptor is a unique target that triggers apoptosis upon activation. We have previously developed a series of chemical compounds with high affinity for the σ-2 receptor and showed rapid internalization of the ligands. One particular specific ligand of the σ-2 receptor, SV119, binds to pancreatic cancer cells and induces target cell death in vitro and in vivo. In this study, we characterized the ability of SV119 to selectively deliver other death-inducing cargos to augment the cytotoxic properties of SV119 itself. When conjugated to SV119, small molecules that are known to interfere with intracellular prosurvival pathways retained their ability to induce cell death, the efficiency of which was enhanced by the combinatorial effect of SV119 delivered with its small molecule cargo. Our findings define a simple platform technology to increase the tumor-selective delivery of small molecule therapeutics via σ-2 ligands, permitting chemotherapeutic synergy that can optimize efficacy and patient benefit.
Asunto(s)
Apoptosis , Receptores sigma/metabolismo , Transducción de Señal , Animales , Línea Celular Tumoral , Femenino , Ligandos , Ratones , Ratones Endogámicos C57BL , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologíaRESUMEN
We have functionalized the surface of gold nanocages with SV119, a synthetic small molecule specific to sigma-2 receptors, and then demonstrated the capability of this new class of conjugates for targeting cancer cells.
Asunto(s)
Oro/química , Nanocápsulas/química , Nanomedicina/métodos , Nanoestructuras/química , Neoplasias Experimentales/química , Neoplasias Experimentales/metabolismo , Receptores sigma/metabolismo , Línea Celular Tumoral , Humanos , Ensayo de Materiales , Nanocápsulas/ultraestructura , Nanoestructuras/ultraestructura , Neoplasias Experimentales/patología , Tamaño de la PartículaRESUMEN
We synthesized and characterized two novel fluorescent sigma-2 receptor selective ligands, SW120 and SW116, and evaluated these ligands as potential probes for imaging cell proliferation. Both ligands are highly selective for sigma-2 receptors versus sigma-1 receptors. SW120 and SW116 were internalized into MDA-MB-435 cells, and 50% of the maximum fluorescent intensity was reached in 11 and 24 minutes, respectively. In vitro studies showed that 50% of SW120 or SW116 washed out of cells in 1 hour. The internalization of SW120 was reduced ≈30% by phenylarsine oxide, an inhibitor of endocytosis, suggesting that sigma-2 ligands are internalized, in part, by an endocytotic pathway. Subcellular localization studies using confocal and two-photon microscopy showed that SW120 and SW116 partially colocalized with fluorescent markers of mitochondria, endoplasmic reticulum, lysosomes, and the plasma membrane, suggesting that sigma-2 receptors localized to the cytoplasmic organelles and plasma membrane. SW120 did not colocalize with the nuclear dye 4',6-diamidino-2-phenylindole. In vivo studies showed that the uptake of SW120 in solid tumors and peripheral blood mononuclear cells of mice positively correlated with the expression level of the cell proliferation marker Ki-67, suggesting that sigma-2 fluorescent probes may be used to image cell proliferation in mice.
Asunto(s)
Rastreo Celular/métodos , Colorantes Fluorescentes/química , Técnicas de Sonda Molecular , Receptores sigma/metabolismo , Animales , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Femenino , Colorantes Fluorescentes/farmacocinética , Humanos , Leucocitos Mononucleares/metabolismo , Ligandos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Microscopía de Fluorescencia por Excitación Multifotónica , Neoplasias Experimentales/metabolismoRESUMEN
The sigma-2 receptor, whose gene remains to be cloned, has been validated as a biomarker for tumour cell proliferation. Here we report the use of a novel photoaffinity probe, WC-21, to identify the sigma-2 receptor-binding site. WC-21, a sigma-2 ligand containing both a photoactive azide moiety and a fluorescein isothiocyanate group, irreversibly labels sigma-2 receptors in rat liver; the membrane-bound protein was identified as PGRMC1 (progesterone receptor membrane component 1). Immunocytochemistry reveals that both PGRMC1 and SW120, a fluorescent sigma-2 receptor ligand, colocalize with molecular markers of the endoplasmic reticulum and mitochondria in HeLa cells. Overexpression and knockdown of the PGRMC1 protein results in an increase and a decrease in binding of a sigma-2 selective radioligand, respectively. The identification of the putative sigma-2 receptor-binding site as PGRMC1 should stimulate the development of unique imaging agents and cancer therapeutics that target the sigma-2 receptor/PGRMC1 complex.