RESUMEN
The design of nanomedicine for cancer therapy, especially the treatment of tumor metastasis has received great attention. Proteasome inhibition is accepted as a new strategy for cancer therapy. Despite being a big breakthrough in multiple myeloma therapy, carfilzomib (CFZ), a second-in-class proteasome inhibitor is still unsatisfactory for solid tumor and metastasis therapy. In this study, hollow titanium nitride (TiN) nanoshells are synthesized as a drug carrier of CFZ. The TiN nanoshells have a high loading capacity of CFZ, and their intrinsic inhibitory effect on autophagy synergistically enhances the activity of CFZ. Due to an excellent photothermal conversion efficiency in the second near-infrared (NIR-II) region, TiN nanoshell-based photothermal therapy further induces a synergistic anticancer effect. In vivo study demonstrates that TiN nanoshells readily drain into the lymph nodes, which are responsible for tumor lymphatic metastasis. The CFZ-loaded TiN nanoshell-based chemo-photothermal therapy combined with surgery offers a remarkable therapeutic outcome in greatly inhibiting further metastatic spread of cancer cells. These findings suggest that TiN nanoshells act as an efficient carrier of CFZ for realizing enhanced outcomes for proteasome inhibitor-based cancer therapy, and this work also presents a "combined chemo-phototherapy assisted surgery" strategy, promising for future cancer treatment.
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Nanocáscaras , Neoplasias , Fotoquimioterapia , Humanos , Línea Celular Tumoral , Oro , Metástasis Linfática , Neoplasias/tratamiento farmacológico , Oligopéptidos , Inhibidores de Proteasoma/farmacología , TitanioRESUMEN
Targeted therapies using tyrosine kinase inhibitors (TKIs) against epidermal growth factor receptor (EGFR) have improved the outcomes of patients with non-small cell lung cancer (NSCLC). However, due to genetic mutations of EGFR or activation of other oncogenic pathways, cancer cells can develop resistance to TKIs, resulting in usually temporary and reversible therapeutic effects. Therefore, new anticancer agents are urgently needed to treat drug-resistant NSCLC. In this study, we found that acetyltanshinone IIA (ATA) displayed much stronger potency than erlotinib in inhibiting the growth of drug-resistant NSCLC cells and their-derived xenograft tumors. Our analyses revealed that ATA achieved this effect by the following mechanisms. First, ATA could bind p70S6K at its ATP-binding pocket to prevent phosphorylation, and second by increasing the ubiquitination of p70S6K to cause its degradation. Since phosphorylation of S6 ribosome protein (S6RP) by p70S6K can induce protein synthesis at the ribosome, the dramatic reduction of p70S6K after ATA treatment led to great reductions of new protein synthesis on several cell cycle-related proteins including cyclin D3, aurora kinase A, polo-like kinase, cyclin B1, survivin; and reduced the levels of EGFR and MET. In addition, ATA treatment increased the levels of p53 and p21 proteins, which blocked cell cycle progression in the G1/S phase. Taken together, as ATA can effectively block multiple signaling pathways essential for protein synthesis and cell proliferation, ATA can potentially be developed into a multi-target anti-cancer agent to treat TKI-resistant NSCLC.
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Antineoplásicos , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/patología , Ciclo Celular , Línea Celular Tumoral , Resistencia a Antineoplásicos , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/patología , Mutación , Fenantrenos , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Quinasas S6 Ribosómicas 70-kDaRESUMEN
Acetyltanshinone IIA (ATA), synthesized in our group exhibiting good anti-breast cancer effects, is expected to replace the commonly used anti-ER+ breast cancer (breast cancer cells overexpressing the estrogen receptor) drug tamoxifen. To promote the clinical progress of ATA, polyethylene glycol (PEG)-modified liposomes were used to encapsulate ATA along with improving its bioavailability and in vivo anticancer efficiency. The resulting liposomal ATA exhibited a spherical shape with an average size of 188.5 nm. In vitro evaluations showed that liposomal ATA retained the anti-breast cancer efficacy of ATA while exerting much less cytotoxicity toward noncancerous cells. Significantly, pharmacokinetics analysis showed that the AUC0-24h of liposomal ATA was 59 times higher than that of free ATA, demonstrating increased bioavailability of ATA. Preclinical experiments demonstrated that liposomal ATA reduced the growth of ER-positive human breast tumor xenografts by 73% in nude mice, and the liposomal ATA exhibited a much lower level of toxicity than that of free ATA with respect to zebrafish larval mortality, body formation, and heart function during development. Moreover, 7-day and 21-day tissue toxicity levels were determined in mice by intravenous administration of a maximum dosage of liposomal ATA (120 mg/kg). The results showed no obvious tissue damage in major organs, including the heart, liver, spleen, kidney, and brain. In summary, we have developed a clinical formulation of liposomal ATA with the high bioavailability and potent efficacy for the treatment of ER-positive breast cancer.
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Antineoplásicos/química , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Composición de Medicamentos/métodos , Liposomas/química , Fenantrenos/química , Fenantrenos/uso terapéutico , Animales , Antineoplásicos/farmacocinética , Disponibilidad Biológica , Supervivencia Celular/efectos de los fármacos , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Larva/efectos de los fármacos , Células MCF-7 , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Fenantrenos/farmacocinética , Ratas , Ratas Sprague-Dawley , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Pez Cebra/embriologíaRESUMEN
PURPOSE: Many anti-cancer drugs are used in chemotherapy; however, little is known about their efficacy against circulating tumor cells (CTCs). In this study, we investigated whether the pulsatile fluidic shear stress (SS) in human arteries can affect the efficacy of anti-cancer drugs. METHODS: Cancer cells were circulated in our microfluidic circulatory system, and their responses to drug and SS treatments were determined using various assays. Breast and cervical cancer cells that stably expressed apoptotic sensor proteins were used to determine apoptosis in real-time by fluorescence resonance energy transfer (FRET)-based imaging microscopy. The occurrence of cell death in non-sensor cells were revealed by annexin V and propidium iodide staining. Cell viability was determined by MTT assay. Intracellular reactive oxygen species (ROS) levels were determined by staining cells with two ROS-detecting dyes: 2',7'-dichlorofluorescin diacetate and dihydroethidium. RESULTS: Fluidic SS significantly increased the potency of the ROS-generating drugs doxorubicin (DOX) and cisplatin but had little effect on the non-ROS-generating drugs Taxol and etoposide. Co-treatment with SS and ROS-generating drugs dramatically elevated ROS levels in CTCs, while the addition of antioxidants abolished the pro-apoptotic effects of DOX and cisplatin. More importantly, the synergistic killing effects of SS and DOX or cisplatin were confirmed in circulated lung, breast, and cervical cancer cells, some of which have a strong metastatic ability. CONCLUSIONS: These findings suggest that ROS-generating drugs are more potent than non-ROS-generating drugs for destroying CTCs under pulsatile fluidic conditions present in the bloodstream. This new information is highly valuable for developing novel therapies to eradicate CTCs in the circulation and prevent metastasis.
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Neoplasias de la Mama/tratamiento farmacológico , Células Neoplásicas Circulantes/patología , Estrés Mecánico , Neoplasias del Cuello Uterino/tratamiento farmacológico , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/sangre , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cisplatino/farmacología , Doxorrubicina/farmacología , Etidio/análogos & derivados , Etidio/química , Femenino , Fluoresceínas/química , Transferencia Resonante de Energía de Fluorescencia , Humanos , Dispositivos Laboratorio en un Chip , Especies Reactivas de Oxígeno/metabolismo , Neoplasias del Cuello Uterino/sangre , Neoplasias del Cuello Uterino/patologíaRESUMEN
Treating systemic metastases at the micrometastatic stage is a potential strategy to inhibit cancer metastasis. This study aims to establish an apoptosis sensor-based platform for rapid, effective, and noninvasive identification of drugs that can inhibit the proliferation of micrometastatic cancer cells. We stably transfected the plasmid DNA encoding the fluorescence resonance energy transfer-based caspase-3 sensor into highly metastatic melanoma B16F10 cells. The resulting B16F10-C3 cells were applied for screening of antiproliferative and proapoptotic drugs in two-dimensional (2D) monolayer, three-dimensional (3D) spheroids, and zebrafish xenotransplantation tumors. All studies were conducted in 96-well plates in a high throughput manner. Fourteen compounds including six chemotherapeutic drugs and eight kinase inhibitors were tested. Thirteen compounds failed the tests due to: Drug resistance, low efficacy, poor pharmacokinetic profile, and/or high side effects to zebrafish. The only compound that passed all tests was pan-phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, which inhibited the proliferation of B16F10-C3 cells in both 2D and 3D cultures. More important, it significantly reduced the xenograft tumor size in zebrafish by decreasing the viability of metastatic cancer cells. Our study suggests that the PI3K/AKT pathway is a potential therapeutic target for the reactivation of tumor dormancy and proliferation of micrometastases. Moreover, this integrated approach is effective for rapid identification of systemic antimetastases drugs.
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Antineoplásicos/aislamiento & purificación , Caspasa 3/análisis , Cromonas/aislamiento & purificación , Evaluación Preclínica de Medicamentos/métodos , Morfolinas/aislamiento & purificación , Metástasis de la Neoplasia/tratamiento farmacológico , Inhibidores de las Quinasa Fosfoinosítidos-3 , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacología , Apoptosis , Técnicas Biosensibles/métodos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cromonas/administración & dosificación , Cromonas/farmacología , Modelos Animales de Enfermedad , Transferencia Resonante de Energía de Fluorescencia , Humanos , Melanoma/tratamiento farmacológico , Melanoma/secundario , Modelos Teóricos , Morfolinas/administración & dosificación , Morfolinas/farmacología , Esferoides Celulares , Factores de Tiempo , Trasplante Heterólogo , Células Tumorales Cultivadas , Pez CebraRESUMEN
Angiogenesis marks the transformation of a benign local tumor into a life-threatening disease. Many in vitro assays are available on two-dimensional (2D) platforms, however, limited research has been conducted to investigate the behavior of tumors and endothelial cells (ECs) grown on three-dimensional (3D) platforms. This study provides a 3D co-culture spheroid of tumor cells with ECs to study the interplay between ECs and tumor cells. In a 3D co-culture with HepG2 hepatocellular carcinoma (HCC) cells, ECs differentiate to form tubule networks when in co-culture. Addition of angiogenic factors or angiogenesis inhibitors to the model system enhanced or inhibited endothelial differentiation in the 3D model, enabling investigations of the cellular signaling pathways utilized in HCC development. The 3D model demonstrated similar protein expression levels as a HCC xenograft, as well as exhibited upregulation of essential signaling proteins such as Akt/mTor in the 3D model, which is not reflected in the 2D model. The effects of several anti-angiogenic agents, such as sorafenib, sunitinib, and axitinib were analyzed in the 3D co-culture model by utilizing fluorescent proteins and a fluorescence resonance energy transfer (FRET)-based caspase-3 sensor in the ECs, which can detect apoptosis in real time. The apoptotic capability of a drug to inhibit angiogenesis in the 3D model can be easily distinguished via the FRET sensor, and dual screening of anti-angiogenesis and anti-tumor drugs can be achieved in a single step via the 3D co-culture model. In summary, a 3D co-culture model is constructed, where a HCC tumor microenvironment with a hypoxic core and true gradient penetration of drugs is achieved for drug screening purposes and in vitro studies utilizing a small HCC tumor. Biotechnol. Bioeng. 2017;114: 1865-1877. © 2017 Wiley Periodicals, Inc.
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Proliferación Celular , Técnicas de Cocultivo/métodos , Células Endoteliales/patología , Neoplasias Experimentales/patología , Neovascularización Patológica/patología , Ingeniería de Tejidos/métodos , Diferenciación Celular , Técnicas de Cocultivo/instrumentación , Células Hep G2 , Humanos , Neoplasias Experimentales/fisiopatología , Neovascularización Patológica/fisiopatología , Esferoides Celulares/patología , Ingeniería de Tejidos/instrumentación , Células Tumorales CultivadasRESUMEN
The emerging graphene quantum dots (GQDs) and carbon dots (C-dots) have gained tremendous attention for their enormous potentials for biomedical applications, owing to their unique and tunable photoluminescence properties, exceptional physicochemical properties, high photostability, biocompatibility, and small size. This article aims to update the latest results in this rapidly evolving field and to provide critical insights to inspire more exciting developments. We comparatively review the properties and synthesis methods of these carbon nanodots and place emphasis on their biological (both fundamental and theranostic) applications.
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Carbono/química , Grafito/química , Puntos Cuánticos , Técnicas Biosensibles , Línea Celular , HumanosRESUMEN
Twenty compounds from Garcinia oblongifolia were screened for proapoptotic activity using FRET-based HeLa-C3 sensor cells. Among them, oblongifolins F and G (1 and 2), 1,3,5-trihydroxy-13,13-dimethyl-2H-pyran[7,6-b]xanthone (3), nigrolineaxanthone T (4), and garcicowin B (5) showed significant proapoptotic activity at a concentration of 10 µM. Bioassessments were then performed to evaluate the potential of these compounds for therapeutic application. All five compounds showed significant cytotoxicity and caspase-3-activating ability in cervical cancer HeLa cells, with compounds 1 and 2 having the highest potencies. All five compounds specifically induced caspase-dependent apoptosis, which could be prevented by the pan-caspase inhibitor zVAD-fmk. In particular, 3 induced apoptosis through mitotic arrest. Compounds 1-5 displayed similar IC50 values (3.9-16.5 µM) against the three cancer cell lines HeLa, MDA-MB-435, and HepG2. In addition, compounds 1, 2, and 4 exhibited similar and potent IC50 values (2.4-5.1 µM) against several breast and colon cancer cell lines, including those overexpressing either HER2 or P-glycoprotein. HER2 and P-glycoprotein are known factors that confer resistance to anticancer drugs in cancer cells. This is the first study on the cytotoxicity, caspase-3-activing ability, and specificity of proapoptotic compounds isolated from G. oblongifolia in HeLa cells. The potential application of these compounds against HER2- or P-glycoprotein-overexpressing cancer cells was investigated.
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Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/farmacología , Garcinia/química , Xantonas/aislamiento & purificación , Xantonas/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Antineoplásicos Fitogénicos/química , Caspasa 3/efectos de los fármacos , Caspasa 3/metabolismo , Inhibidores de Caspasas , Femenino , Células HeLa , Células Hep G2 , Humanos , Concentración 50 Inhibidora , Estructura Molecular , Corteza de la Planta/química , Receptor ErbB-2/metabolismo , Xantonas/químicaRESUMEN
Circulating tumor cells (CTCs) shed from primary tumors must overcome the cytotoxicity of immune cells, particularly natural killer (NK) cells, to cause metastasis. The tumor microenvironment (TME) protects tumor cells from the cytotoxicity of immune cells, which is partially executed by cancer-associated mesenchymal stromal cells (MSCs). However, the mechanisms by which MSCs influence the NK resistance of CTCs remain poorly understood. This study demonstrates that MSCs enhance the NK resistance of cancer cells in a gap junction-dependent manner, thereby promoting the survival and metastatic seeding of CTCs in immunocompromised mice. Tumor cells crosstalk with MSCs through an intercellular cGAS-cGAMP-STING signaling loop, leading to increased production of interferon-ß (IFNß) by MSCs. IFNß reversely enhances the type I IFN (IFN-I) signaling in tumor cells and hence the expression of human leukocyte antigen class I (HLA-I) on the cell surface, protecting the tumor cells from NK cytotoxicity. Disruption of this loop reverses NK sensitivity in tumor cells and decreases tumor metastasis. Moreover, there are positive correlations between IFN-I signaling, HLA-I expression, and NK tolerance in human tumor samples. Thus, the NK-resistant signaling loop between tumor cells and MSCs may serve as a novel therapeutic target.
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Interferón beta , Células Asesinas Naturales , Células Madre Mesenquimatosas , Células Neoplásicas Circulantes , Nucleotidiltransferasas , Transducción de Señal , Microambiente Tumoral , Células Madre Mesenquimatosas/inmunología , Células Madre Mesenquimatosas/metabolismo , Animales , Células Asesinas Naturales/inmunología , Ratones , Interferón beta/metabolismo , Interferón beta/inmunología , Nucleotidiltransferasas/metabolismo , Nucleotidiltransferasas/genética , Humanos , Células Neoplásicas Circulantes/inmunología , Células Neoplásicas Circulantes/metabolismo , Microambiente Tumoral/inmunología , Proteínas de la Membrana/metabolismo , Modelos Animales de Enfermedad , Línea Celular TumoralRESUMEN
When circulating tumor cells (CTCs) travel in circulation, they can be killed by detachment-induced anoikis and fluidic shear stress (SS)-mediated apoptosis. Circulatory treatment, which can make CTCs detached but also generate SS, can increase metastasis of cancer cells. To identify SS-specific mechanosensors without detachment impacts, a microfluidic circulatory system is used to generate arteriosus SS and compare transcriptome profiles of circulating lung cancer cells with suspended cells. Half of the cancer cells can survive SS damage and show higher invasion ability. Mesotrypsin (PRSS3), protease-activated receptor 2 (PAR2), and the subunit of activating protein 1, Fos-related antigen 1 (FOSL1), are upregulated by SS, and their high expression is responsible for promoting invasion and metastasis. SS triggers PRSS3 to cleave the N-terminal inhibitory domain of PAR2 within 2 h. As a G protein-coupled receptor, PAR2 further activates the Gαi protein to turn on the Src-ERK/p38/JNK-FRA1/cJUN axis to promote the expression of epithelial-mesenchymal transition markers, and also PRSS3, which facilitates metastasis. Enriched PRSS3, PAR2, and FOSL1 in human tumor samples and their correlations with worse outcomes reveal their clinical significance. PAR2 may serve as an SS-specific mechanosensor cleavable by PRSS3 in circulation, which provides new insights for targeting metastasis-initiating CTCs.
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Neoplasias Pulmonares , Células Neoplásicas Circulantes , Humanos , Línea Celular Tumoral , Neoplasias Pulmonares/patología , Células Neoplásicas Circulantes/patología , Receptor PAR-2 , Receptores Acoplados a Proteínas G , Tripsina/metabolismoRESUMEN
Natural killer (NK) cells kill mutant cells through death receptors and cytotoxic granules, playing an essential role in controlling cancer progression. However, in the tumor microenvironment (TME), NK cells frequently exhibit an exhausted status, which impairs their immunosurveillance function and contributes to tumor immune evasion. Emerging studies are ongoing to reveal the properties and mechanisms of NK cell exhaustion in the TME. In this review, we will briefly introduce the maturation, localization, homeostasis, and cytotoxicity of NK cells. We will then summarize the current understanding of the main mechanisms underlying NK cell exhaustion in the TME in four aspects: dysregulation of inhibitory and activating signaling, tumor cell-derived factors, immunosuppressive cells, and metabolism and exhaustion. We will also discuss the therapeutic approaches currently being developed to reverse NK cell exhaustion and enhance NK cell cytotoxicity in the TME.
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Antineoplásicos , Neoplasias , Humanos , Microambiente Tumoral , Células Asesinas Naturales , Escape del TumorRESUMEN
Triple-negative breast cancer (TNBC) has higher mortality than non-TNBC because of its stronger metastatic capacity. Increasing studies reported that TNBC tumors had more macrophage infiltration than non-TNBC tumors, which promoted the metastasis of TNBC cells. However, how TNBC cells become more malignant after interacting with macrophages is less reported. In this study, it is observed that when TNBC cells are co-cultured with macrophages, they display higher viability and stronger metastatic ability than non-TNBC cells. Mechanistic studies reveal that TNBC cells acquired these abilities via interactions with macrophages in three phases. First, within 12 h of co-culture with macrophages, some TNBC cells have significantly elevated levels of reactive oxygen species (ROS), which upregulate interleukin 1α (IL1α) expression in ERK1/2-c-Jun- and NF-κB-dependent manners at 24-48 h. Second, the secreted IL1α bound to IL1R1 activates the ERK1/2-ZEB1-VIM pathway which increases metastasis. Third, IL1α/IL1R1 facilitates its own synthesis and induces the expression of IL1ß and IL8 at 72-96 h through the MKK4-JNK-c-Jun and NF-κB signaling pathways. Moreover, a higher level of IL1α is positively correlated with more macrophage infiltration and shorter overall survival in breast cancer patients. Thus, reducing ROS elevation or downregulating IL1α expression can serve as new strategies to decrease metastasis of TNBC.
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FN-kappa B , Neoplasias de la Mama Triple Negativas , Humanos , FN-kappa B/metabolismo , Neoplasias de la Mama Triple Negativas/patología , Especies Reactivas de Oxígeno/metabolismo , Interleucina-1alfa/metabolismo , Línea Celular Tumoral , Carcinogénesis/metabolismo , Transformación Celular Neoplásica , Macrófagos/metabolismoRESUMEN
When cancer cells enter the bloodstream, they can interact with platelets to acquire stronger survival and metastatic abilities. To elucidate the underlying mechanisms, we cocultured metastatic melanoma and triple-negative breast cancer cells with species-homologous platelets. We found that cocultured cancer cells displayed higher viabilities in circulation, stronger capacities for cell migration, invasion, and colony formation in vitro, and more tumorigenesis and metastasis in mice. RNA sequencing analysis revealed that the level of serpin family E member 1 (SERPINE1) was significantly upregulated in cocultured cancer cells. Knockdown of SERPINE1 reversed the coculture-elevated survival and metastatic phenotypes of cancer cells. Mechanistic studies indicated that coculture with platelets activated the TGFß/Smad pathway to induce SERPINE1 expression in cancer cells, which encodes plasminogen activator inhibitor 1 (PAI-1). PAI-1 then activated PI3K to increase the phosphorylation of AKTThr308 and Bad to elevate Bcl-2, which enhanced cell survival in circulation. Moreover, higher levels of PAI-1 were detected in metastatic tumors from melanoma and triple-negative breast cancer patients than in normal tissues, and high levels of PAI-1 were associated with a shorter overall survival time and worse disease progression in breast cancer. PAI-1 may act as a potential biomarker for detecting and treating metastatic tumor cells.
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Melanoma , Neoplasias de la Mama Triple Negativas , Animales , Ratones , Humanos , Plaquetas , Inhibidor 1 de Activador Plasminogénico/genética , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt , Técnicas de Cocultivo , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
Chimeric antigen receptor (CAR)-modified natural killer (NK) cells are recognized as promising immunotherapeutic agents for cancer treatment. However, the efficacy and trafficking of CAR-NK cells in solid tumors are hindered by the complex barriers present in the tumor microenvironment (TME). We have developed a novel strategy that utilizes living CAR-NK cells as carriers to deliver anticancer drugs specifically to the tumor site. We also introduce a time-lapse method for evaluating the efficacy and tumor specificity of CAR-NK cells using a two-photon microscope in live mouse models and three-dimensional (3D) tissue slide cultures. Our results demonstrate that CAR-NK cells exhibit enhanced antitumor immunity when combined with photosensitive chemicals in both in vitro and in vivo tumor models. Additionally, we have successfully visualized the trafficking, infiltration, and accumulation of drug-loaded CAR-NK cells in deeply situated TME using non-invasive intravital two-photon microscopy. Our findings highlight that tumor infiltration of CAR-NK cells can be intravitally monitored through the two-photon microscope approach. In conclusion, our study demonstrates the successful integration of CAR-NK cells as drug carriers and paves the way for combined cellular and small-molecule therapies in cancer treatment. Furthermore, our 3D platform offers a valuable tool for assessing the behavior of CAR cells within solid tumors, facilitating the development and optimization of immunotherapeutic strategies with clinical imaging approaches.
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Conventional techniques for in vitro cancer drug screening require labor-intensive formalin fixation, paraffin embedding, and dye staining of tumor tissues at fixed endpoints. This way of assessment discards the valuable pharmacodynamic information in live cells over time. Here, we found endogenous lipofuscin-like autofluorescence acutely accumulated in the cell death process. Its unique red autofluorescence could report the apoptosis without labeling and continuously monitor the treatment responses in 3D tumor-culture models. Lifetime imaging of lipofuscin-like red autofluorescence could further distinguish necrosis from apoptosis of cells. Moreover, this endogenous fluorescent marker could visualize the apoptosis in live zebrafish embryos during development. Overall, this study validates that lipofuscin-like autofluorophore is a generic cell death marker. Its characteristic autofluorescence could label-free predict the efficacy of anti-cancer drugs in organoids or animal models.
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Lipofuscina , Neoplasias , Animales , Lipofuscina/metabolismo , Pez Cebra/metabolismo , Microscopía Fluorescente , Neoplasias/diagnóstico por imagen , Neoplasias/tratamiento farmacológico , Coloración y EtiquetadoRESUMEN
Oblongifolin C (OC) was identified as a potent apoptosis inducer from an herbal plant, Garcinia yunnanensis, during our previous bioassay-guided drug screening. In this study, we investigated the signaling pathways through which OC activated apoptosis in HeLa cells. We also compared the IC(50) values of OC with that of etoposide, paclitaxel and vinblastine in multiple cancer cell lines including HER2 and P-glycoprotein overexpressing cells. In addition, the in vivo antitumor effect of OC was studied in nude mice model. Our results showed that OC induced a caspase-dependent apoptosis by triggering a series of events in HeLa cells including Bax translocation, cytochrome c release, caspase-3 activation, chromosome fragmentation followed by caspase-8 activation, Bid cleavage and eventually cell death. Addition of a pan-caspase inhibitor or overexpression of an anti-apoptotic protein, Bcl-xL, prevented OC-induced cell death. Moreover, OC exhibited a wide anticancer spectrum in multiple cancer cell lines with comparable IC(50) values, regardless of the expression levels of HER2 and P-glycoprotein. In contrast, the IC(50) values of three clinical anticancer drugs, etoposide, paclitaxel and vinblastine were significantly elevated in HER2 and/or P-glycoprotein overexpressing cells. Furthermore, OC showed a similar antitumor effect but lower general toxicity than etoposide against xenografted human tumors in nude mice model. All these data suggested that OC is a promising apoptosis inducer with the potential to be developed into a clinical anticancer drug.
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Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Garcinia/química , Terpenos/farmacología , Proteína X Asociada a bcl-2/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/análisis , Animales , Caspasas/fisiología , Femenino , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Mitocondrias/fisiología , Receptor ErbB-2/análisis , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In this study, we developed a high-throughput endothelial cell apoptosis assay using a fluorescence resonance energy transfer (FRET)-based biosensor. After exposure to apoptotic inducer UV-irradiation or anticancer drugs such as paclitaxel, the fluorescence of the cells changed from green to blue. We developed this method into a high-throughput assay in 96-well plates by measuring the emission ratio of yellow fluorescent protein (YFP) to cyan fluorescent protein (CFP) to monitor the activation of a key protease, caspase-3, during apoptosis. The Z' factor for this assay was above 0.5 which indicates that this assay is suitable for a high-throughput analysis. Finally, we applied this functional high-throughput assay for screening vascular disrupting agents (VDA) which could induce endothelial cell apoptosis from our in-house compounds library and dioscin was identified as a hit. As this assay allows real time and sensitive detection of cell apoptosis, it will be a useful tool for monitoring endothelial cell apoptosis in living cell situation and for identifying new VDA candidates via a high-throughput screening.
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Inhibidores de la Angiogénesis/aislamiento & purificación , Apoptosis/efectos de los fármacos , Descubrimiento de Drogas/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Ensayos Analíticos de Alto Rendimiento , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Inhibidores de la Angiogénesis/farmacología , Técnicas Biosensibles , Caspasa 3/análisis , Caspasa 3/biosíntesis , Línea Celular , Células Endoteliales de la Vena Umbilical Humana/enzimología , Células Endoteliales de la Vena Umbilical Humana/efectos de la radiación , Humanos , Paclitaxel/farmacología , Rayos UltravioletaRESUMEN
A new aggregation-induced emission (AIE) compound 1,2-bis[4-(isothiocyanatomethyl)phenyl]-1,2-diphenylethene (2) was synthesized for use in SDS-PAGE. The molecule is practically nonemissive in solution but becomes highly emissive after reacting with the amine groups of the proteins by either the prestaining or poststaining method. The sensitivity of 2 achieved in the prestaining method is the same as that of Coomassie brilliant blue (CBB), while that observed in the poststaining method is higher than that of CBB. Excellent linear responses with the amount of protein were obtained in both cases. The detection of a mixture of proteins with different molecular weights was successfully achieved.
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Electroforesis en Gel de Poliacrilamida/métodos , Proteínas/aislamiento & purificación , Coloración y Etiquetado/métodos , Estilbenos/química , Aminas/análisis , Colorantes Fluorescentes , Peso Molecular , Colorantes de RosanilinaRESUMEN
Four new compounds, oliganthins A-D (1-4), and one known caged xanthone gaudichaudione H (5) were isolated from the stems of Garcinia oligantha. The structures of the new compounds were elucidated by spectroscopic evidences. All of the five compounds were evaluated for their apoptosis-inducing effects using HeLa-C3 cells which have been genetically engineered to produce a fluorescent biosensor capable of detecting caspase-3 activation. All of them induced cell apoptosis at 10 µM or lower concentrations. The apoptotic activity of oliganthins A, B and gaudichaudione H were further confirmed by detecting the cleavage of PARP, which is the substrate of activated caspase-3, in these compounds-treated cells using the method of Western blot. Moreover, the values of IC(50) were measured for all five compounds on HeLa cells using the MTT assay. Among them, gaudichaudione H had the lowest IC(50) value of 0.90 µM, while the other four new compounds had IC(50) values of 1.58, 1.52, 4.15, and 7.82 µM, respectively. These results show that gaudichaudione H has the strongest apoptosis-inducing effect and cell growth inhibition effect among these xanthones and it may have the potential to be developed into a new anticancer agent.
Asunto(s)
Antineoplásicos Fitogénicos/química , Apoptosis/efectos de los fármacos , Garcinia/química , Xantenos/química , Xantonas/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/farmacología , Western Blotting , Caspasa 3/genética , Caspasa 3/metabolismo , Femenino , Expresión Génica , Genes Reporteros , Células HeLa , Humanos , Espectroscopía de Resonancia Magnética , Extractos Vegetales/química , Tallos de la Planta/química , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/patología , Xantenos/aislamiento & purificación , Xantenos/farmacología , Xantonas/aislamiento & purificación , Xantonas/farmacologíaRESUMEN
The coronavirus disease 19 (COVID-19) has been rampant since 2019, severely affecting global public health, and causing 5.75 million deaths worldwide. So far, many vaccines have been developed to prevent the infection of SARS-CoV-2 virus. However, the emergence of new variants may threat vaccine recipients as they might evade immunological surveillance that depends on the using of anti-SARS-CoV-2 antibody to neutralize the viral particles. Recent studies have found that recipients who received two doses of vaccination plus an additional booster shoot were able to quickly elevate neutralization response and immune response against wild-type SARS-CoV-2 virus and some initially appeared viral variants. In this review, we assessed the real-world effectiveness of different COVID-19 vaccines by population studies and neutralization assays and compared neutralization responses of booster vaccines in vitro. Finally, as the efficacy of COVID-19 vaccine is expected to decline over time, continued vaccination should be considered to achieve a long-term immune protection against coronavirus.