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Proteinoids-simple polymers composed of amino acids-were suggested decades ago by Fox and coworkers to form spontaneously by heat. These special polymers may self-assemble in micrometer structures called proteinoid microspheres, presented as the protocells of life on earth. Interest in proteinoids increased in recent years, in particular for nano-biomedicine. They were produced by stepwise polymerization of 3-4 amino acids. Proteinoids based on the RGD motif were prepared for targeting tumors. Nanocapsules form by heating proteinoids in an aqueous solution and slowly cooling to room temperature. Proteinoid polymers and nanocapsules suit many biomedical applications owing to their non-toxicity, biocompatibility and immune safety. Drugs and/or imaging reagents for cancer diagnostic, therapeutic and theranostic applications were encapsulated by dissolving them in aqueous proteinoid solutions. Here, recent in vitro and in vivo studies are reviewed.
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Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.
Assuntos
Antineoplásicos , Neoplasias , Humanos , Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Esferoides Celulares , Linhagem Celular , Sobrevivência Celular , Linhagem Celular Tumoral , Microambiente TumoralRESUMO
The efficacy/safety of combining palbociclib (a CDK4/6 inhibitor) and sunitinib (a multi-targeted receptor tyrosine kinase inhibitor) was evaluated, using patient-derived xenograft (PDX) models. Twenty-three PDX mice models were developed from patients with various solid tumors. The mice were randomized to 4 groups (5-6 mice in each): control/palbociclib (100 mg/kg)/sunitinib (50 mg/kg)/combination. Drugs were administered orally, 5 days/week. In 17/23 PDX models (74%), the combination demonstrated a synergistic inhibitory effect vs the monotherapies ("responder" models) with no unexpected toxicities. In 13/17 responder models, where standard-of-care (SOC) was an additional comparator, the combination was more effective than SOC in 7 models, as effective in 4, and less effective in 2. The mean ± SEM experiment duration in 15/17 responder models (2/17 were excluded due to technical issues) was 86 ± 12 and 31 ± 5 days for the combination and control groups, respectively (p = 0.0002). The effect of the combination was dose-dependent. Cell-viability experiments in A549/MDA-MB-231/HT-29 cell lines and experiments using tumor-derived primary cell spheroids supported the PDX findings. In conclusion, combination of palbociclib and sunitinib exerts a synergistic anti-tumor effect without adding unexpected toxicity. A clinical trial assessing this combination is underway.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Neoplasias , Animais , Humanos , Camundongos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Quinase 4 Dependente de Ciclina , Modelos Animais de Doenças , Xenoenxertos , Neoplasias/tratamento farmacológico , Piperazinas , Piridinas , Sunitinibe , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Personalized cancer treatment based on specific mutations offers targeted therapy and is preferred over "standard" chemotherapy. Proteinoid polymers produced by thermal step-growth polymerization of amino acids may form nanocapsules (NCs) that encapsulate drugs overcoming miscibility problems and allowing passive targeted delivery with reduced side effects. The arginine-glycine-glutamic acid (RGD) sequence is known for its preferential attraction to αvß3 integrin, which is highly expressed on neovascular endothelial cells that support tumor growth. Here, tumor-targeted RGD-based proteinoid NCs entrapping a synergistic combination of Palbociclib (Pal) and Alpelisib (Alp) were synthesized by self-assembly to induce the reduction of tumor cell growth in different types of cancers. The diameters of the hollow and drug encapsulating poly(RGD) NCs were 34 ± 5 and 22 ± 3 nm, respectively; thereby, their drug targeted efficiency is due to both passive and active targeting. The encapsulation yield of Pal and Alp was 70 and 90%, respectively. In vitro experiments with A549, MCF7 and HCT116 human cancer cells demonstrate a synergistic effect of Pal and Alp, controlled release and dose dependence. Preliminary results in a 3D tumor spheroid model with cells derived from patient-derived xenografts of colon cancer illustrate disassembly of spheroids, indicating that the NCs have therapeutic potential.
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Fluorescent carbon dots (CDs) play a versatile role in materials science. Herein, we have developed alginate-derived nitrogen-doped CDs as a drug carrier and a toughening agent for hydrogels by a microwave-assisted method. In the first phase of work, we carried out covalent conjugation of the drug onto the CD surface for controlled delivery of drug molecules, and in the second phase of work, we demonstrated how CDs could act as a toughening agent as well as a viscosity modifier for poly(acrylic acid-co-methacrylamide) copolymer hydrogels. The hydrogels were evaluated by Fourier transformed infrared spectroscopy, X-ray photoelectron spectroscopy, and solid-state nuclear magnetic resonance. The hybrid hydrogels have been tested to be mechanically robust with extraordinary stretchability (â¼1200% elongation at break), recoverable to the original position (low permanent set), tunable water uptake, and thixotropic character in dynamic stress. The crosslinked structure has been evaluated through void calculation revealing gradual densification of the network with increasing CD content. Exceptional gel strength (ratio of elastic modulus to loss modulus; G'/Gâ³) has been achieved from analogous crosslinking made by CDs. The delayed network rupturing and superstretchability could make this material a good choice for soft biomaterials and soft robotics.
Assuntos
Carbono/química , Hidrogéis/química , Micro-Ondas , Polissacarídeos/química , Pontos Quânticos/química , Doxorrubicina/química , Doxorrubicina/metabolismo , Portadores de Fármacos/química , Módulo de Elasticidade , Ácidos Polimetacrílicos/química , ReologiaRESUMO
Proteinoids are non-toxic biodegradable polymers prepared by thermal step-growth polymerization of amino acids. Here, P(RGD) proteinoids and proteinoid nanocapsules (NCs) based on D-arginine, glycine, and L-aspartic acid were synthesized and characterized for targeted tumor therapy. Doxorubicin (Dox), a chemotherapeutic drug used for treatment of a wide range of cancers, known for its adverse side effects, was encapsulated during self-assembly to form Dox/P(RGD) NCs. In addition, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which can initiate apoptosis in most tumor cells but undergoes fast enzyme degradation, was stabilized by covalent conjugation to hollow P(RGD) NCs. The effect of polyethylene glycol (PEG) conjugation was also studied. Cytotoxicity tests on CAOV-3 ovarian cancer cells demonstrated that Dox/P(RGD) and TRAIL-P(RGD) NCs were as effective as free Dox and TRAIL with cell viability of 2% and 10%, respectively, while PEGylated NCs were less effective. Drug-bearing P(RGD) NCs offer controlled release with reduced side effects for improved therapy.
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The Hippo kinase pathway is emerging as a conserved signaling pathway that is essential for organ growth and tumorigenesis. Recently, we reported that the ubiquitin E3 ligase ITCH negatively regulates LATS1, thereby increasing YAP activity, which leads to increased cell proliferation and decreased apoptosis. Here, we investigated the role of ITCH in breast tumorigenesis. In particular, we show that ITCH enhances epithelial-to-mesenchymal transition (EMT) through boosting YAP oncogenic function. By contrast, a point mutation in the catalytic domain or WW1 domain of ITCH abolished its EMT-mediated effects. Furthermore, while overexpression of ITCH expression in breast cells is associated with increased incidence of mammary tumor formation and progression, its knockdown inhibited breast cancer cell tumorigenicity and metastasis. Importantly, YAP knockdown was able to attenuate ITCH pro-tumorigenic functions. Lastly, we found that ITCH expression is significantly upregulated in invasive and metastatic breast cancer cases and is associated with worse survival. Together, our results reveal that ITCH pro-tumorigenic functions in breast cancer are mediated, at least in part, through inactivation of the Hippo tumor suppressor pathway.
Assuntos
Neoplasias da Mama/patologia , Transformação Celular Neoplásica/patologia , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Animais/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Adesão Celular , Movimento Celular , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Via de Sinalização Hippo , Humanos , Técnicas Imunoenzimáticas , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Neoplasias Mamárias Animais/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Repressoras/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Transcrição , Células Tumorais Cultivadas , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Ubiquitinas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteínas de Sinalização YAPRESUMO
Proper regulation of cell proliferation, cell apoptosis, and cell death are vital for the development and survival of living organisms. Failure or dysfunction of any of these processes can have devastating effects, including cancer. The Hippo pathway, first discovered in Drosophila, has been found to be a major growth-regulatory signaling pathway that controls these crucial processes and has been implicated in cell-progress regulation and organ size determination. Abnormal regulation of this pathway has been found in several cancer types. However, the mechanisms that regulate the pathway and its core members yet have to be elucidated. One of the main core components of this pathway is LATS1, a serine/threonine kinase. Therefore, understanding how LATS1 activity is regulated is expected to shed light on new mechanisms that regulate the Hippo pathway. In the current work, we identified several potential LATS1 regulators and proved that NEDD4 E3 ubiquitin ligase controls LATS1 stability. We demonstrate that NEDD4 directly interacts with LATS1, leading to ubiquitination and decreased levels of LATS1 and, thus, increased YAP localization in the nucleus, which subsequently increases the transcriptional activity of YAP. As such, we show that NEDD4 acts as an additional regulator of the Hippo pathway on the protein level via interactions between WW domain-containing and PPxY motif-containing proteins. These findings might be applied in the development of new therapeutic approaches through the activation of LATS1.