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1.
Theranostics ; 11(19): 9415-9430, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34646378

RESUMO

The feasibility of personalized medicine for cancer treatment is largely hampered by costly, labor-intensive and time-consuming models for drug discovery. Herein, establishing new pre-clinical models to tackle these issues for personalized medicine is urgently demanded. Methods: We established a three-dimensional tumor slice culture (3D-TSC) platform incorporating label-free techniques for time-course experiments to predict anti-cancer drug efficacy and validated the 3D-TSC model by multiphoton fluorescence microscopy, RNA sequence analysis, histochemical and histological analysis. Results: Using time-lapse imaging of the apoptotic reporter sensor C3 (C3), we performed cell-based high-throughput drug screening and shortlisted high-efficacy drugs to screen murine and human 3D-TSCs, which validate effective candidates within 7 days of surgery. Histological and RNA sequence analyses demonstrated that 3D-TSCs accurately preserved immune components of the original tumor, which enables the successful achievement of immune checkpoint blockade assays with antibodies against PD-1 and/or PD-L1. Label-free multiphoton fluorescence imaging revealed that 3D-TSCs exhibit lipofuscin autofluorescence features in the time-course monitoring of drug response and efficacy. Conclusion: This technology accelerates precision anti-cancer therapy by providing a cheap, fast, and easy platform for anti-cancer drug discovery.

2.
Int J Biol Sci ; 17(6): 1521-1529, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33907515

RESUMO

The COVID-19 pandemic has been raging worldwide for more than a year. Many efforts have been made to create vaccines and develop new antiviral drugs to cope with the disease. Here, we propose the application of short interfering RNAs (siRNAs) to degrade the viral genome, thus reducing viral infection. By introducing the concept of the probability of binding efficiency (PBE) and combining the secondary structures of RNA molecules, we designed 11 siRNAs that target the consensus regions of three key viral genes: the spike (S), nucleocapsid (N) and membrane (M) genes of SARS-CoV-2. The silencing efficiencies of the siRNAs were determined in human lung and endothelial cells overexpressing these viral genes. The results suggested that most of the siRNAs could significantly reduce the expression of the viral genes with inhibition rates above 50% in 24 hours. This work not only provides a strategy for designing potentially effective siRNAs against target genes but also validates several potent siRNAs that can be used in the clinical development of preventative medication for COVID-19 in the future.


Assuntos
COVID-19/virologia , Regulação Viral da Expressão Gênica/fisiologia , Genes Virais , RNA Interferente Pequeno/fisiologia , SARS-CoV-2/genética , Células A549 , Células Endoteliais da Veia Umbilical Humana , Humanos , Mutação , Probabilidade , Glicoproteína da Espícula de Coronavírus/genética
3.
Oncogene ; 40(12): 2165-2181, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33627781

RESUMO

Cellular heterogeneity and the lack of metastatic biomarkers limit the diagnosis of and development of therapies for metastatic triple-negative breast cancer (TNBC). Thus, development of new clinically relevant markers is urgently needed. By using RNA-seq analysis, we found that nerve growth factor receptor (NGFR) was highly expressed in metastatic lung clones of MDA-MB-231 cells. This high level of NGFR expression was necessary for TNBC cells to grow into tumor spheres under nonadhesive conditions, resist anoikis, promote primary tumor growth and increase metastasis in mice. NGFR was also expressed at a high level in a greater number of TNBC patients (45%) than non-TNBC patients (23%), enriched in higher grade tumors, and negatively correlated with the overall survival of TNBC patients. Mechanistic analysis indicated that NGFR exerted its prometastatic effects by binding with neurotrophic receptor tyrosine kinase 3 (TrkC) mainly through a ligand-independent manner, which activated the MEK-ERK1-ZEB1 and PI3K-AKT signaling pathways, increased the level of fibronectin, and decreased the expression of PUMA. Notably, we observed that NGFR expression in TrkC-positive metastatic clones reduced cellular sensitivity to anti-Trk therapy. Moreover, WNT family member 5a (WNT5A) and TrkC activated NGFR transcription in a ZEB1-dependent manner. Taken together, this study identified NGFR as a novel driver for transforming TNBC into higher grade metastatic tumors. Our findings provide the basis for the future development of NGFR as a diagnostic and prognostic marker for determining the metastatic potential of TNBC and as a therapeutic target for treating TNBC patients.


Assuntos
Neoplasias Pulmonares/genética , Proteínas do Tecido Nervoso/genética , Receptor trkC/genética , Receptores de Fator de Crescimento Neural/genética , Neoplasias de Mama Triplo Negativas/genética , Proteína Wnt-5a/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética , Animais , Proliferação de Células/genética , Evolução Clonal/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Camundongos , Proteína Quinase 3 Ativada por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Metástase Neoplásica , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt/genética , RNA-Seq , Transdução de Sinais , Neoplasias de Mama Triplo Negativas/patologia
4.
J Hazard Mater ; 408: 124826, 2021 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-33421851

RESUMO

Zebrafish are widely used for detecting toxic agents because of their unique advantages. The conventional zebrafish-based tests use lethal rates and morphological changes as criteria to evaluate the toxicity. To increase the sensitivity of using zebrafish to detect toxic agents, a fluorescence resonance energy transfer-based apoptotic biosensor was introduced into zebrafish genome to generate transgenic sensor zebrafish. Seven chemicals including heavy metals, nanomaterials and DNA-damaging agents were used to treat the sensor zebrafish to determine the sensitivity of the sensor zebrafish. The results showed that sensor zebrafish can detect the toxicity of the tested agents with single-cell sensitivity. Using the sensor zebrafish, we found that, at 100 nM, heavy metal cadmium (Cd) induced apoptosis of zebrafish cells, while no obvious morphological or behavioral changes were observed from the sensor zebrafish. Even at 44.5 nM (the maximum allowable concentration in drinking water), Cd induced a significant increase of apoptosis in sensor zebrafish. ZnO nanoparticles caused apoptosis in sensor zebrafish at a very low concentration of 100 ng/mL. DNA-damaging agents induced the apoptosis of many cells in sensor zebrafish. The sensor zebrafish are much more sensitive than the conventional zebrafish-based tests and can serve as a powerful tool for detecting toxic agents.


Assuntos
Técnicas Biossensoriais , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Cádmio/toxicidade , Transferência Ressonante de Energia de Fluorescência
5.
J Hematol Oncol ; 14(1): 21, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514401

RESUMO

BACKGROUND: B7-H3, an immune-checkpoint molecule and a transmembrane protein, is overexpressed in non-small cell lung cancer (NSCLC), making it an attractive therapeutic target. Here, we aimed to systematically evaluate the value of B7-H3 as a target in NSCLC via T cells expressing B7-H3-specific chimeric antigen receptors (CARs) and bispecific killer cell engager (BiKE)-redirected natural killer (NK) cells. METHODS: We generated B7-H3 CAR and B7-H3/CD16 BiKE derived from an anti-B7-H3 antibody omburtamab that has been shown to preferentially bind tumor tissues and has been safely used in humans in early-phase clinical trials. Antitumor efficacy and induced-immune response of CAR and BiKE were evaluated in vitro and in vivo. The effects of B7-H3 on aerobic glycolysis in NSCLC cells were further investigated. RESULTS: B7-H3 CAR-T cells effectively inhibited NSCLC tumorigenesis in vitro and in vivo. B7-H3 redirection promoted highly specific T-cell infiltration into tumors. Additionally, NK cell activity could be specially triggered by B7-H3/CD16 BiKE through direct CD16 signaling, resulting in significant increase in NK cell activation and target cell death. BiKE improved antitumor efficacy mediated by NK cells in vitro and in vivo, regardless of the cell surface target antigen density on tumor tissues. Furthermore, we found that anti-B7-H3 blockade might alter tumor glucose metabolism via the reactive oxygen species-mediated pathway. CONCLUSIONS: Together, our results suggest that B7-H3 may serve as a target for NSCLC therapy and support the further development of two therapeutic agents in the preclinical and clinical studies.


Assuntos
Anticorpos Biespecíficos/uso terapêutico , Antígeno B7-H1/imunologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Imunoterapia Adotiva/métodos , Neoplasias Pulmonares/terapia , Receptores de Antígenos Quiméricos/uso terapêutico , Animais , Anticorpos Biespecíficos/imunologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Linhagem Celular Tumoral , Feminino , Humanos , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/imunologia , Ativação Linfocitária , Camundongos Endogâmicos NOD , Camundongos SCID , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Linfócitos T/transplante
6.
Cancer Lett ; 490: 1-11, 2020 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-32585412

RESUMO

High expression of human epidermal factor receptor 2 (HER2) is directly related to tumor progression, malignancy and drug resistance in HER2-positive breast cancer (HER2-PBC). The major limitation of current anti-HER2 therapies is that they cannot reduce the levels of HER2 protein. Here, we investigated the effect of acetyltanshinone IIA (ATA) in lapatinib-resistant HER2-PBC cells. Our data showed that ATA exhibited more potent effects than lapatinib against drug-resistant HER2-PBC cells in terms of (1) inhibiting cell growth, (2) reducing phosphorylated and total HER2 levels, (3) inhibiting tumor xenograft growth in nude mice, and (4) reducing HER2 protein levels in tumor xenografts. A mechanistic study revealed that ATA promoted HER2 degradation via increasing c-Cbl and CHIP-mediated HER2 ubiquitination and subsequent HER2 degradation by the proteasome or lysosome. ATA also reduced the levels of other tyrosine kinase receptors (TKRs), such as HER3, IGF-1R and MET, in lapatinib-resistant cells. Our findings suggest that direct degradation of HER2 and other TKRs can be an effective strategy for combatting drug resistance. They also indicate the potential utilization of ATA in treating breast cancer that is resistant or nonresponsive to current HER2-targeted therapies.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fenantrenos/farmacologia , Receptor ErbB-2/metabolismo , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Lapatinib/farmacologia , Camundongos , Camundongos Nus , Receptor ErbB-2/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
7.
ACS Sens ; 5(3): 823-830, 2020 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-32090557

RESUMO

Apoptosis plays crucial roles during development and in disease conditions. While there are some methods to detect apoptosis in vitro, most of them are end-point assays that cannot be used to detect apoptosis in the physiological context of live animals. In this study, transgenic sensor zebrafish were generated that specifically produce a fluorescence resonance energy transfer (FRET)-based biosensor in the zebrafish skin. Under normal conditions, the skin cells of the sensor zebrafish emit green fluorescence; when caspase-3 is activated during apoptosis, the skin cells of the sensor zebrafish switch to emitting blue fluorescence. Through time-lapse FRET imaging with the sensor zebrafish, we observed that caspase-3 can be activated within 5 min and apoptosis can be completed in around 30 min in live zebrafish, no matter the apoptosis occurs several hours after UV irradiation or during the normal development. Using the sensor zebrafish, we found that apoptosis can occur in different parts of the zebrafish skin including the skin covering the trunk, eye, yolk sac, and head during development. Interestingly, we observed that the yolk sac diameter of the zebrafish reduced from 723.8 ± 25.1 µm at 24 h postfertilization (hpf) to 346.1 ± 24.6 µm at 120 hpf. To accommodate this dramatic reduction of the yolk sac size, we found that some excess skin cells on the surface of the yolk sac were removed by apoptosis during this process. The sensor zebrafish provide a powerful and convenient tool for the noninvasive and real-time detection of apoptosis at the single-cell resolution in live zebrafish.


Assuntos
Apoptose , Técnicas Biossensoriais , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Embrião não Mamífero , Transferência Ressonante de Energia de Fluorescência , Larva , Microscopia Confocal , Análise de Célula Única , Pele/efeitos da radiação , Raios Ultravioleta , Peixe-Zebra/genética
8.
EBioMedicine ; 49: 157-171, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31678002

RESUMO

BACKGROUND: Liver is one of the most preferred destinations of distant metastasis in gastric cancer (GC). As effective treatment is still limited, the prognosis of GC patients bearing liver metastasis is poor. We filter out lysyl oxidase (LOX) to study its function in the tumor microenvironment (TME) and seek for potential therapeutic targets. METHODS: Transcription analysis on 6 cases of liver metastasis of GC patients with respective paired primary tumors and adjacent normal livers was performed. The filtration out of LOX was done using 5 datasets. 69 GC liver metastasis tissues were utilized to perform immunohistochemistry (IHC) and analyze prognosis. Computed Tomography (CT) combined 3D organ reconstruction bioluminescence imaging was performed to precisely evaluate the metastatic tumor burden on liver of intrasplenic injection mouse model. Human and mouse cancer associated fibroblasts (CAFs) in liver metastasis were separated to culture to study the interaction of LOX and TGF-ß1. Patients-derived xenograft (PDX) model was established using liver metastasis of patients to evaluate the therapeutic value of LOX inhibitor ß-aminopropionitrile (BAPN). RESULTS: CAFs-derived LOX at liver metastatic niche of GC promotes niche formation and outgrowth thus predicts poor prognosis. Meanwhile tumor cells in niche secrete TGF-ß1 to nourish CAFs and stimulate them to produce more LOX in turn. The mechanism involved in LOX-mediated proliferation facilitation is enhancement of Warburg effect. The inhibitor of LOX, BPAN could hamper the effect brought by LOX in vivo and in vitro. INTERPRETATION: Our study has unveiled a positive feedback loop between CAFs and tumor cells in liver metastasis niche of GC. The core molecule is LOX which facilitates Warburg effect. Targeting LOX with its inhibitor BAPN might serve as a potential therapeutic strategy. FUND: This research was supported by the National Natural Science Foundation of China (31872740), the 100-member plan of the Shanghai Municipal Commission of Health and Family Planning (2017BR043), Shanghai Science and Technology Commission Project(17ZR1416800), Renji Hospital Training Fund (PYMDT-003, PYIII-17-015), National Natural Science Foundation of China (81672358), the Shanghai Municipal Education Commission-Gao feng Clinical MedicineGrant Support (20181708), Program of Shanghai Academic/Technology Research Leader(19XD1403400), Science and Technology Commission of Shanghai Municipality (18410721000), Shanghai Municipal Health Bureau (2018BR32), China Postdoctoral Science Foundation (2018M640403), National Natural Science Foundation of China (81701945) and Youth project of Shanghai Municipal Health Commission(20164Y0045).


Assuntos
Fibroblastos Associados a Câncer/enzimologia , Fibroblastos Associados a Câncer/patologia , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Proteína-Lisina 6-Oxidase/metabolismo , Neoplasias Gástricas/patologia , Aminopropionitrilo/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Glicólise , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Prognóstico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Células Estromais/patologia , Fator de Crescimento Transformador beta1/metabolismo , Regulação para Cima
9.
Mol Pharm ; 16(9): 3873-3886, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31389706

RESUMO

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.


Assuntos
Antineoplásicos/química , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Composição de Medicamentos/métodos , Lipossomos/química , Fenantrenos/química , Fenantrenos/uso terapêutico , Animais , Antineoplásicos/farmacocinética , Disponibilidade Biológica , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Larva/efeitos dos fármacos , Células MCF-7 , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fenantrenos/farmacocinética , Ratos , Ratos Sprague-Dawley , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-Zebra/embriologia
10.
Nanomicro Lett ; 11(1): 93, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-34138046

RESUMO

Photothermal agents with strong light absorption in the second near-infrared (NIR-II) region (1000-1350 nm) are strongly desired for successful photothermal therapy (PTT). In this work, titania-coated Au nanobipyramids (NBP@TiO2) with a strong plasmon resonance in the NIR-II window were synthesized. The NBP@TiO2 nanostructures have a high photothermal conversion efficiency of (93.3 ± 5.2)% under 1064-nm laser irradiation. They are also capable for loading an anticancer drug combretastatin A-4 phosphate (CA4P). In vitro PTT studies reveal that 1064-nm laser irradiation can efficiently ablate human lung cancer A549 cells and enhance the anticancer effect of CA4P. Moreover, the CA4P-loaded NBP@TiO2 nanostructures combined with PTT induce a synergistic antiangiogenesis effect. In vivo studies show that such CA4P-loaded NBP@TiO2 nanostructures under mild 1064-nm laser irradiation at an optical power density of 0.4 W cm-2, which is lower than the skin tolerance threshold value, exhibit a superior antitumor effect. This work presents not only the development of the NBP@TiO2 nanostructures as a novel photothermal agent responsive in the NIR-II window but also a unique combined chemo-photothermal therapy strategy for cancer therapy.

11.
Free Radic Biol Med ; 129: 46-58, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30193891

RESUMO

Circulation of cancer cells in the bloodstream is a vital step for distant metastasis, during which cancer cells are exposed to hemodynamic shear stress (SS). The actions of SS on tumor cells are complicated and not fully understood. We previously reported that fluidic SS was able to promote migration of breast cancer cells by elevating the cellular ROS level. In this study, we further investigated the mechanisms regulating SS-promoted cell migration and identified the role of MnSOD in the related pathway. We found that SS could enhance tumor cell adhesion to extracellular matrix and endothelial monolayer, and MnSOD also regulated this process. Briefly, SS stimulates the generation of mitochondrial superoxide in tumor cells. MnSOD then converts superoxide into hydrogen peroxide, which activates ERK1/2 to promote tumor cell migration and activates FAK to promote tumor cell adhesion. Combining our previous and present studies, we present experimental evidence on the pro-metastatic effects of hemodynamic SS and reveal the underlying mechanism. Our findings provide new insights into the nature of cancer metastasis and the understanding of tumor cell responses to external stresses and have valuable implications for cancer therapy development.


Assuntos
Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Mitocôndrias/metabolismo , Superóxido Dismutase/genética , Superóxidos/metabolismo , Fenômenos Biomecânicos , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Células Epiteliais/patologia , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Células Alimentadoras , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Estresse Mecânico , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/metabolismo
12.
Breast Cancer Res Treat ; 172(2): 297-312, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30117065

RESUMO

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.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Células Neoplásicas Circulantes/patologia , Estresse Mecânico , Neoplasias do Colo do Útero/tratamento farmacológico , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Doxorrubicina/farmacologia , Etídio/análogos & derivados , Etídio/química , Feminino , Fluoresceínas/química , Transferência Ressonante de Energia de Fluorescência , Humanos , Dispositivos Lab-On-A-Chip , Espécies Reativas de Oxigênio/metabolismo , Neoplasias do Colo do Útero/sangue , Neoplasias do Colo do Útero/patologia
13.
Biotechnol Bioeng ; 115(11): 2828-2843, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30102771

RESUMO

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.


Assuntos
Antineoplásicos/isolamento & purificação , Caspase 3/análise , Cromonas/isolamento & purificação , Avaliação Pré-Clínica de Medicamentos/métodos , Morfolinas/isolamento & purificação , Metástase Neoplásica/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Apoptose , Técnicas Biossensoriais/métodos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromonas/administração & dosagem , Cromonas/farmacologia , Modelos Animais de Doenças , Transferência Ressonante de Energia de Fluorescência , Humanos , Melanoma/tratamento farmacológico , Melanoma/secundário , Modelos Teóricos , Morfolinas/administração & dosagem , Morfolinas/farmacologia , Esferoides Celulares , Fatores de Tempo , Transplante Heterólogo , Células Tumorais Cultivadas , Peixe-Zebra
14.
Diab Vasc Dis Res ; 14(6): 534-539, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28830235

RESUMO

OBJECTIVE: The haptoglobin 2-2 genotype is associated with lower haptoglobin concentrations and atherosclerosis in diabetes. Endothelial cell apoptosis contributes significantly to atherosclerosis. We studied endothelial cell apoptosis in diabetes patients with haptoglobin 2-2 and non-haptoglobin 2-2 genotype. Approach and results: We pooled plasma from 10 patients with haptoglobin 2-2 and non-haptoglobin 2-2 genotype and quantified endothelial cell apoptosis using a hemodynamic lab-on-chip system. Then, we conducted similar experiments on individual diabetes plasma samples with the haptoglobin 2-2 ( n = 20) and non-haptoglobin 2-2 genotype ( n = 20). Haptoglobin beta concentrations were measured by Western blot analysis. We looked for association with demographic, metabolic variables, inflammation and oxidative stress. In pooled plasma, endothelial cell apoptosis was higher in haptoglobin 2-2 group (haptoglobin 2-2: 23.18% vs non-haptoglobin 2-2:15.32%). In individual samples, univariate analysis showed that endothelial cell apoptosis correlated with haptoglobin beta concentration [ ß = -10.29 (95% confidence interval: -13.44, -7.14), p < 0.001] and total haptoglobin concentration [ ß = -0.03 (95% confidence interval: -0.05, -0.002), p = 0.03]. After multivariable analysis, only haptoglobin beta concentrations remained significant [ ß = -9.24 (95% confidence interval: -13.10, -5.37), p < 0.001]. The interaction term between haptoglobin genotypes and haptoglobin beta was not significant ( p > 0.05). CONCLUSION: These results show that regardless of the haptoglobin genotype, haptoglobin is associated with prevention of endothelial cell apoptosis in diabetes.


Assuntos
Apoptose , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/patologia , Haptoglobinas/análise , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Biomarcadores/sangue , Estudos de Casos e Controles , Células Cultivadas , Distribuição de Qui-Quadrado , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/genética , Genótipo , Haptoglobinas/genética , Humanos , Dispositivos Lab-On-A-Chip , Procedimentos Analíticos em Microchip , Análise Multivariada , Fenótipo , Projetos Piloto
15.
Biotechnol Bioeng ; 114(8): 1865-1877, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28369747

RESUMO

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.


Assuntos
Proliferação de Células , Técnicas de Cocultura/métodos , Células Endoteliais/patologia , Neoplasias Experimentais/patologia , Neovascularização Patológica/patologia , Engenharia Tecidual/métodos , Diferenciação Celular , Técnicas de Cocultura/instrumentação , Células Hep G2 , Humanos , Neoplasias Experimentais/fisiopatologia , Neovascularização Patológica/fisiopatologia , Esferoides Celulares/patologia , Engenharia Tecidual/instrumentação , Células Tumorais Cultivadas
16.
Sci Rep ; 7: 39975, 2017 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-28054593

RESUMO

Circulating tumor cells (CTCs) are the primary targets of cancer treatment as they cause distal metastasis. However, how CTCs response to exercise-induced high shear stress is largely unknown. To study the effects of hemodynamic microenvironment on CTCs, we designed a microfluidic circulatory system that produces exercise relevant shear stresses. We explore the effects of shear stresses on breast cancer cells with different metastatic abilities, cancer cells of ovarian, lung and leukemic origin. Three major findings were obtained. 1) High shear stress of 60 dynes/cm2 achievable during intensive exercise killed more CTCs than low shear stress of 15 dynes/cm2 present in human arteries at the resting state. 2) High shear stress caused necrosis in over 90% of CTCs within the first 4 h of circulation. More importantly, the CTCs that survived the first 4 h-circulation, underwent apoptosis during 16-24 h of post-circulation incubation. 3) Prolonged high shear stress treatment effectively reduced the viability of highly metastatic and drug resistant breast cancer cells. As high shear stress had much less damaging effects on leukemic cells mimicking the white blood cells, we propose that intensive exercise may be a good strategy for generating high shear stress that can destroy CTCs and prevent cancer metastasis.


Assuntos
Células Neoplásicas Circulantes , Estresse Mecânico , Linhagem Celular Tumoral , Sobrevivência Celular , Exercício Físico , Humanos , Microfluídica , Modelos Biológicos
17.
Oncotarget ; 8(7): 12013-12030, 2017 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-28061455

RESUMO

The Poly (ethylene glycol) methyl ether-block-poly (lactide-co-glycolide) (mPEG-PLGA) nanoparticles carrying acetyltanshinone IIA (ATA), a novel anti-breast cancer agent, were prepared by ultrasonic emulsion method to enhance the bioavailability and reduce the toxicity. Systematic optimization of encapsulation process was achieved using an orthogonal design. Drug efficacy analysis showed that ATA nanoparticles were as effective as free ATA against estrogen receptor positive breast cancer cells, but much less toxic towards human endothelial cells. Furthermore, in zebrafish, ATA nanoparticles displayed much lower toxicity than free ATA. More importantly, the blood concentration of ATA nanoparticles indicated by 24 hour-area under the curve (AUC0-24h) was 10 times higher than free ATA. These results indicated the potential of ATA-loaded mPEG-PLGA nanoparticles for the delivery of ATA in a clinical formulation, and their potential for use in tumor therapy in the future.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Nanopartículas/metabolismo , Fenantrenos/farmacocinética , Animais , Disponibilidade Biológica , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Química Farmacêutica , Emulsões , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Células MCF-7 , Masculino , Nanopartículas/administração & dosagem , Nanopartículas/química , Fenantrenos/administração & dosagem , Fenantrenos/química , Poliésteres , Polietilenoglicóis , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Ultrassom/métodos , Peixe-Zebra
18.
J Med Chem ; 60(1): 504-510, 2017 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-27977181

RESUMO

An orally active and metabolically stable peptide TIBA was successfully engineered as a chimera by fusing an analgesic bradykinin receptor antagonist peptide and the trypsin inhibitory loop of sunflower trypsin inhibitor-1. As a fusion cyclic peptide, the metabolically labile analgesic peptide is protected from degradation by exopeptidases as well as the endopeptidases, and its serum half-life extended from <5 min to >6 h as a chimera. Moreover, the chimera TIBA was also found to be orally active in an animal pain model using a hot plate assay.


Assuntos
Antagonistas de Receptor B1 da Bradicinina/farmacologia , Helianthus/química , Inibidores da Tripsina/farmacologia , Administração Oral , Antagonistas de Receptor B1 da Bradicinina/administração & dosagem , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Inibidores da Tripsina/administração & dosagem
19.
Cancer Lett ; 388: 239-248, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-27965040

RESUMO

Cancer cells are shed into the blood stream and are exposed to hemodynamic shear stress during metastasis. It has been shown that shear stress can destroy circulating tumor cells (CTCs) both in vitro and in vivo. However, it remains unclear whether shear stress can modulate the properties and functions of tumor cells in a manner that might help CTCs to exit circulation. In this study, we established a microfluidic circulatory system to apply physiological fluid shear stress on breast cancer cells and demonstrated that an arterial level of shear stress significantly enhanced tumor cell migration in transwell and wound healing assays, and enhanced extravasation in a transendothelial assay. Circulatory treatment elevated the intracellular levels of reactive oxygen species (ROS), which is an early and indispensable event for activating the extracellular signal-regulated kinases (ERK1/2). Subsequently, ERK1/2 activation promoted the migration of tumor cells and enhanced their extravasation. Finally, reducing cellular ROS production suppressed tumor cell extravasation in both a transendothelial assay and a zebrafish model. This new understanding of how fluid shear stress promotes tumor cell migration has important implications in cancer treatment and can help us to identify potential therapeutic targets for inhibiting tumor progression.


Assuntos
Hemodinâmica/genética , Células Neoplásicas Circulantes/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Movimento Celular , Humanos , Células Neoplásicas Circulantes/patologia , Estresse Mecânico
20.
Oncotarget ; 7(31): 50239-50257, 2016 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-27384484

RESUMO

Understanding the survival mechanism of metastatic cancer cells in circulation will provide new perspectives on metastasis prevention and also shed new light on metastasis-derived drug resistance. In this study, we made it feasible to detect apoptosis of circulating tumor cells (CTCs) in real-time by integrating a fluorescence resonance energy transfer (FRET)-based caspase sensor into one in vitro microfluidic circulatory system, and two in vivo models: zebrafish circulation and mouse lung metastatic model. Our study demonstrated that fluid shear stresses triggered apoptosis of breast cancer cells in circulation by elevating the mitochondrial production of the primary free radical, superoxide anion. Cancer cells with high levels of manganese superoxide dismutase (MnSOD) exhibited stronger resistance to shear force-induced apoptosis and formed more lung metastases in mice. These metastasized cells further displayed higher resistance to chemotherapeutic agent doxorubicin, which also generates superoxide in mitochondria. Specific siRNA-mediated MnSOD knockdown reversed all three phenotypes. Our findings therefore suggest that MnSOD plays an important integrative role in supporting cancer cell survival in circulation, metastasis, and doxorubicin resistance. MnSOD can serve as a new biomarker for identifying metastatic CTCs and a novel therapeutic target for inhibiting metastasis and destroying doxorubicin-resistant breast cancer cells.


Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/metabolismo , Células Neoplásicas Circulantes/metabolismo , Superóxido Dismutase/metabolismo , Animais , Apoptose , Biomarcadores Tumorais , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Radicais Livres , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/patologia , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Microfluídica , Metástase Neoplásica , Transplante de Neoplasias , Fenótipo , Resistência ao Cisalhamento , Peixe-Zebra
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