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Assessing programmed death ligand 1 (PD-L1) expression through immunohistochemistry (IHC) is the golden standard in predicting immunotherapy response of non-small cell lung cancer (NSCLC). However, observation of heterogeneous PD-L1 distribution in tumor space is a challenge using IHC only. Meanwhile, immunofluorescence (IF) could support both planar and three-dimensional (3D) histological analyses by combining tissue optical clearing with confocal microscopy. We optimized clinical tissue preparation for the IF assay focusing on staining, imaging, and post-processing to achieve quality identical to traditional IHC assay. To overcome limited dynamic range of the fluorescence microscope's detection system, we incorporated a high dynamic range (HDR) algorithm to restore the post imaging IF expression pattern and further 3D IF images. Following HDR processing, a noticeable improvement in the accuracy of diagnosis (85.7%) was achieved using IF images by pathologists. Moreover, 3D IF images revealed a 25% change in tumor proportion score for PD-L1 expression at various depths within tumors. We have established an optimal and reproducible process for PD-L1 IF images in NSCLC, yielding high quality data comparable to traditional IHC assays. The ability to discern accurate spatial PD-L1 distribution through 3D pathology analysis could provide more precise evaluation and prediction for immunotherapy targeting advanced NSCLC.
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Antígeno B7-H1 , Carcinoma Pulmonar de Células não Pequenas , Imunofluorescência , Imageamento Tridimensional , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Antígeno B7-H1/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/diagnóstico , Imageamento Tridimensional/métodos , Imunofluorescência/métodos , Imuno-Histoquímica/métodos , Microscopia Confocal/métodos , Biomarcadores Tumorais/metabolismoRESUMO
Novel therapeutics have significantly improved the survival and quality of life of patients with malignancies in this century. Versatile precision diagnostic data were used to formulate personalized therapeutic strategies for patients. However, the cost of extensive information depends on the consumption of the specimen, raising the challenges of effective specimen utilization, particularly in small biopsies. In this study, we proposed a tissue-processing cascaded protocol that obtains 3-dimensional (3D) protein expression spatial distribution and mutation analysis from an identical specimen. In order to reuse the thick section tissue evaluated after the 3D pathology technique, we designed a novel high-flatness agarose-embedded method that could improve tissue utilization rate by 1.52 fold, whereas it reduced the tissue-processing time by 80% compared with the traditional paraffin-embedding method. In animal studies, we demonstrated that the protocol would not affect the results of DNA mutation analysis. Furthermore, we explored the utility of this approach in non-small cell lung cancer because it is a compelling application for this innovation. We used 35 cases including 7 cases of biopsy specimens of non-small cell lung cancer to simulate future clinical application. The cascaded protocol consumed 150-µm thickness of formalin-fixed, paraffin-embedded specimens, providing 3D histologic and immunohistochemical information approximately 38 times that of the current paraffin-embedding protocol, and 3 rounds of DNA mutation analysis, offering both essential guidance for routine diagnostic evaluation and advanced information for precision medicine. Our designed integrated workflow provides an alternative way for pathological examination and paves the way for multidimensional tumor tissue assessment.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Animais , Humanos , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Qualidade de Vida , Mutação , DNA , Inclusão em Parafina/métodos , FormaldeídoRESUMO
Background: Perineural invasion (PNI), a form of local invasion defined as the ability of cancer cells to invade in, around, and through nerves, has a negative prognostic impact in oral cavity squamous cell carcinoma (OCSCC). Unfortunately, the diagnosis of PNI suffers from a significant degree of intra- and interobserver variability. The aim of this pilot study was to develop a deep learning-based human-enhanced tool, termed domain knowledge enhanced yield (Domain-KEY) algorithm, for identifying PNI in digital slides. Methods: Hematoxylin and eosin (H&E)-stained whole-slide images (WSIs, n = 85) were obtained from 80 patients with OCSCC. The model structure consisted of two parts to simulate human decision-making skills in diagnostic pathology. To this aim, two semantic segmentation models were constructed (i.e., identification of nerve fibers followed by the diagnosis of PNI). The inferred results were subsequently subjected to post-processing of generated decision rules for diagnostic labeling. Ten H&E-stained WSIs not previously used in the study were read and labeled by the Domain-KEY algorithm. Thereafter, labeling correctness was visually inspected by two independent pathologists. Results: The Domain-KEY algorithm was found to outperform the ResnetV2_50 classifier for the detection of PNI (diagnostic accuracy: 89.01% and 61.94%, respectively). On analyzing WSIs, the algorithm achieved a mean diagnostic accuracy as high as 97.50% versus traditional pathology. The observed accuracy in a validation dataset of 25 WSIs obtained from seven patients with oropharyngeal (cancer of the tongue base, n = 1; tonsil cancer, n = 1; soft palate cancer, n = 1) and hypopharyngeal (cancer of posterior wall, n = 2; pyriform sinus cancer, n = 2) malignancies was 96%. Notably, the algorithm was successfully applied in the analysis of WSIs to shorten the time required to reach a diagnosis. The addition of the hybrid intelligence model decreased the mean time required to reach a diagnosis by 15.0% and 23.7% for the first and second pathologists, respectively. On analyzing digital slides, the tool was effective in supporting human diagnostic thinking. Conclusions: The Domain-KEY algorithm successfully mimicked human decision-making skills and supported expert pathologists in the routine diagnosis of PNI.
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Microscopic examination of biopsied and resected prostatic specimens is the mainstay in the diagnosis of prostate cancer. However, conventional analysis of hematoxylin and eosin (H&E)-stained tissue is time-consuming and offers limited two-dimensional (2D) information. In the current study, we devised a method-termed Prostate Rapid Optical examination for cancer STATus (proSTAT)-for rapid screening of prostate cancer using high-resolution 2D and three-dimensional (3D) confocal images obtained after hydrophilic tissue clearing of 100-µm-thick tissue slices. The results of the proSTAT method were compared with those of traditional H&E stains for the analysis of cores (n=15) obtained from radical prostatectomy specimens (n=5). Gland lumen formation, consistent with Gleason pattern 3, was evident following tracking of multiple optical imaging sections. In addition, 3D rendering allowed visualizing a tubular network of interconnecting branches. Rapid 3D fluorescent labeling of tumor protein p63 accurately distinguished prostate adenocarcinoma from normal tissue and benign lesions. Compared with conventional stains, the 3D spatial and molecular information extracted from proSTAT may significantly increase the amount of available data for pathological assessment of prostate specimens. Our approach is amenable to automation and-subject to independent validation-can find a wide spectrum of clinical and research applications.
Assuntos
Próstata , Neoplasias da Próstata , Corantes , Humanos , Masculino , Microscopia Confocal , Gradação de Tumores , Próstata/diagnóstico por imagem , Próstata/patologia , Prostatectomia/métodos , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/patologiaRESUMO
Nanoparticles (NPs), such as liposomes, effectively evade the severe toxicity of unexpected accumulation and passively shuttle drugs into tumor tissues by enhanced permeability and retention. In the case of non-small cell lung cancer and pancreatic ductal adenocarcinoma, cancer-associated fibroblasts promote the aggregation of a gel-like extracellular matrix that forms a physical barrier in the desmoplastic stroma of the tumor. These stroma are composed of protein networks and glycosaminoglycans (GAGs) that greatly compromise tumor-penetrating performance, leading to insufficient extravasation and tissue penetration of NPs. Moreover, the presence of heparan sulfate (HS) and related proteoglycans on the cell surface and tumor extracellular matrix may serve as molecular targets for NP-mediated drug delivery. Here, a GAG-binding peptide (GBP) with high affinity for HS and high cell-penetrating activity was used to develop an HS-targeting delivery system. Specifically, liposomal doxorubicin (L-DOX) was modified by post-insertion with the GBP. We show that the in vitro uptake of L-DOX in A549 lung adenocarcinoma cells increased by GBP modification. Cellular uptake of GBP-modified L-DOX (L-DOX-GBP) was diminished in the presence of extracellular HS but not in the presence of other GAGs, indicating that the interaction with HS is critical for the cell surface binding of L-DOX-GBP. The cytotoxicity of doxorubicin positively correlated with the molecular composition of GBP. Moreover, GBP modification improved the in vivo distribution and anticancer efficiency of L-DOX, with enhanced desmoplastic targeting and extensive distribution. Taken together, GBP modification may greatly improve the tissue distribution and delivery efficiency of NPs against HS-abundant desmoplastic stroma-associated neoplasm.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Antibióticos Antineoplásicos/administração & dosagem , Antibióticos Antineoplásicos/farmacocinética , Doxorrubicina/análogos & derivados , Heparitina Sulfato/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Células A549 , Adenocarcinoma de Pulmão/metabolismo , Animais , Antibióticos Antineoplásicos/química , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Doxorrubicina/química , Doxorrubicina/farmacocinética , Sistemas de Liberação de Medicamentos , Feminino , Glicosaminoglicanos/metabolismo , Humanos , Lipossomos/administração & dosagem , Lipossomos/síntese química , Lipossomos/química , Lipossomos/farmacocinética , Neoplasias Pulmonares/metabolismo , Camundongos , Células NIH 3T3 , Nanopartículas/administração & dosagem , Nanopartículas/química , Nanopartículas/metabolismo , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Distribuição Tecidual/efeitos dos fármacos , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
More than 80% of infectious bacteria form biofilm, which is a bacterial cell community surrounded by secreted polysaccharides, proteins and glycolipids. Such bacterial superstructure increases resistance to antimicrobials and host defenses. Thus, to control these biofilm-forming pathogenic bacteria requires antimicrobial agents with novel mechanisms or properties. Pseudomonas aeruginosa, a Gram-negative opportunistic nosocomial pathogen, is a model strain to study biofilm development and correlation between biofilm formation and infection. In this study, a recombinant hemolymph plasma lectin (rHPLOE) cloned from Taiwanese Tachypleus tridentatus was expressed in an Escherichia coli system. This rHPLOE was shown to have the following properties: (1) Binding to P. aeruginosa PA14 biofilm through a unique molecular interaction with rhamnose-containing moieties on bacteria, leading to reduction of extracellular di-rhamnolipid (a biofilm regulator); (2) decreasing downstream quorum sensing factors, and inhibiting biofilm formation; (3) dispersing the mature biofilm of P. aeruginosa PA14 to improve the efficacies of antibiotics; (4) reducing P. aeruginosa PA14 cytotoxicity to human lung epithelial cells in vitro and (5) inhibiting P. aeruginosa PA14 infection of zebrafish embryos in vivo. Taken together, rHPLOE serves as an anti-biofilm agent with a novel mechanism of recognizing rhamnose moieties in lipopolysaccharides, di-rhamnolipid and structural polysaccharides (Psl) in biofilms. Thus rHPLOE links glycan-recognition to novel anti-biofilm strategies against pathogenic bacteria.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Ramnose/metabolismo , Células A549 , Animais , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/efeitos dos fármacos , Escherichia coli/metabolismo , Glicolipídeos/metabolismo , Caranguejos Ferradura/metabolismo , Humanos , Lectinas/metabolismo , Polissacarídeos Bacterianos/metabolismo , Percepção de Quorum/efeitos dos fármacos , Peixe-ZebraRESUMO
ST3Gal1 is a key sialyltransferase which adds α2,3-linked sialic acid to substrates and generates core 1 O-glycan structure. Upregulation of ST3Gal1 has been associated with worse prognosis of breast cancer patients. However, the protein substrates of ST3Gal1 implicated in tumor progression remain elusive. In our study, we demonstrated that ST3GAL1-silencing significantly reduced tumor growth along with a notable decrease in vascularity of MCF7 xenograft tumors. We identified vasorin (VASN) which was shown to bind TGF-ß1, as a potential candidate that links ST3Gal1 to angiogenesis. LC-MS/MS analysis of VASN secreted from MCF7, revealed that more than 80% of its O-glycans are sialyl-3T and disialyl-T. ST3GAL1-silencing or desialylation of VASN by neuraminidase enhanced its binding to TGF-ß1 by 2- to 3-fold and thereby dampening TGF-ß1 signaling and angiogenesis, as indicated by impaired tube formation of HUVECs, suppressed angiogenesis gene expression and reduced activation of Smad2 and Smad3 in HUVEC cells. Examination of 114 fresh primary breast cancer and their adjacent normal tissues showed that the expression levels of ST3Gal1 and TGFB1 were high in tumor part and the expression of two genes was positively correlated. Kaplan Meier survival analysis showed a significantly shorter relapse-free survival for those with lower expression VASN, notably, the combination of low VASN with high ST3GAL1 yielded even higher risk of recurrence (p = 0.025, HR = 2.967, 95% CI = 1.14-7.67). Since TGF-ß1 is known to transcriptionally activate ST3Gal1, our findings illustrated a feedback regulatory loop in which TGF-ß1 upregulates ST3Gal1 to circumvent the negative impact of VASN.
Assuntos
Neoplasias da Mama/patologia , Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Recidiva Local de Neoplasia/patologia , Neovascularização Patológica/patologia , Sialiltransferases/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Mama/patologia , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/mortalidade , Progressão da Doença , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Células MCF-7 , Camundongos , Recidiva Local de Neoplasia/epidemiologia , RNA Interferente Pequeno/metabolismo , Ácidos Siálicos/metabolismo , Sialiltransferases/genética , Transdução de Sinais , Análise de Sobrevida , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
Hepatitis B virus (HBV) is an aetiological factor for liver cirrhosis and hepatocellular carcinoma (HCC). Despite current antiviral therapies that successfully reduce the viral load in patients with chronic hepatitis B, persistent hepatitis B surface antigen (HBsAg) remains a risk factor for HCC. To explore whether intrahepatic viral antigens contribute directly to hepatocarcinogenesis, we monitored the mitotic progression of HBV-positive cells. Cytokinesis failure was increased in HBV-positive HepG2.2.15 and 1.3ES2 cells, as well as in HuH-7 cells transfected with a wild-type or X-deficient HBV construct, but not in cells transfected with an HBsAg-deficient construct. We show that expression of viral large surface antigen (LHBS) was sufficient to induce cytokinesis failure of immortalized hepatocytes. Premitotic defects with DNA damage and G2 /M checkpoint attenuation preceded cytokinesis in LHBS-positive cells, and ultimately resulted in hyperploidy. Inhibition of polo-like kinase-1 (Plk1) not only restored the G2 /M checkpoint in these cells, but also suppressed LHBS-mediated in vivo tumourigenesis. Finally, a positive correlation between intrahepatic LHBS expression and hepatocyte hyperploidy was detected in >70% of patients with chronic hepatitis B. We conclude that HBV LHBS provokes hyperploidy by inducing DNA damage and upregulation of Plk1; the former results in atypical chromatin structures, and the latter attenuates the function of the G2 /M DNA damage checkpoint. Our data uncover a mechanism by which genomic integrity of hepatocytes is disrupted by viral LHBS. These findings highlight the role of intrahepatic surface antigen as an oncogenic risk factor in the development of HCC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Assuntos
Carcinoma Hepatocelular/virologia , Citocinese , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/metabolismo , Hepatite B Crônica/virologia , Hepatócitos/virologia , Neoplasias Hepáticas/virologia , Ploidias , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proteínas de Ciclo Celular/metabolismo , Transformação Celular Viral , Dano ao DNA , Modelos Animais de Doenças , Pontos de Checagem da Fase G2 do Ciclo Celular , Células Hep G2 , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B da Marmota/genética , Vírus da Hepatite B da Marmota/metabolismo , Vírus da Hepatite B/genética , Hepatite B Crônica/genética , Hepatite B Crônica/metabolismo , Hepatite B Crônica/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatócitos/transplante , Interações Hospedeiro-Patógeno , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Marmota , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Quinase 1 Polo-LikeRESUMO
Cell penetrating peptide derived from human eosinophil cationic protein (CPPecp) is a 10-amino-acid peptide containing a core heparan sulfate (HS)-binding motif of human eosinophil cationic protein (ECP). It binds and penetrates bronchial epithelial cells without cytotoxic effects. Here we investigated airway-protective effects of CPPecp in BEAS-2B cell line and mite-induced airway allergic inflammation in BALB/c mice. In BEAS-2B cell, CPPecp decreases ECP-induced eotaxin mRNA expression. CPPecp also decreases eotaxin secretion and p-STAT6 activation induced by ECP, as well as by IL-4. In vivo studies showed CPPecp decreased mite-induced airway inflammation in terms of eosinophil and neutrophil count in broncho-alveolar lavage fluid, peri-bronchiolar and alveolar pathology scores, cytokine production in lung protein extract including interleukin (IL)-5, IL-13, IL-17A/F, eotaxin; and pause enhancement from methacholine stimulation. CPPecp treated groups also showed lower serum mite-specific IgE level. In this study, we have demonstrated the in vitro and in vivo anti-asthma effects of CPPecp.
Assuntos
Antiasmáticos/farmacologia , Asma/tratamento farmacológico , Peptídeos Penetradores de Células/farmacologia , Proteína Catiônica de Eosinófilo/química , Mucosa Respiratória/efeitos dos fármacos , Alérgenos/imunologia , Animais , Antiasmáticos/uso terapêutico , Antígenos de Dermatophagoides/imunologia , Asma/imunologia , Asma/patologia , Brônquios/citologia , Brônquios/efeitos dos fármacos , Brônquios/imunologia , Linhagem Celular , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/uso terapêutico , Citocinas/imunologia , Citocinas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Eosinófilos/imunologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Mucosa Respiratória/citologia , Mucosa Respiratória/imunologia , Resultado do TratamentoRESUMO
A 10-residue, glycosaminoglycan-binding peptide, GBPECP, derived from human eosinophil cationic protein has been recently designated as a potent cell-penetrating peptide. A model system containing peptide, glycan, and lipid was monitored by nuclear magnetic resonance (NMR) spectroscopy to determine the cell-penetrating mechanism. Heparin octasaccharide with dodecylphosphocholine (DPC) lipid micelle was titrated into the GBPECP solution. Our data revealed substantial roles for the charged residues Arg5 and Lys7 in recognizing heparin, whereas Arg3 had less effect. The aromatic residue Trp4 acted as an irreplaceable moiety for membrane insertion, as the replacement of Trp4 with Arg4 abolished cell penetration, although it significantly improved the heparin-binding ability. GBPECP bound either heparin or lipid in the presence or absence of the other ligand indicating that the peptide has two alternative binding sites: Trp4 is responsible for lipid insertion, and Arg5 and Lys7 are for GAG binding. We developed a molecular model showing that the two effects synergistically promote the penetration. The loss of either effect would abolish the penetration. GBPECP has been proven to enter cells through macropinocytosis. The GBPECP treatment inhibited A549 lung cancer cell migration and invasion, implying that the cellular microenvironment would be modulated by GBPECP internalization. The intracellular penetration of GBPECP leading to inhibition of epithelial cell migration and invasion depends on the presence of the tryptophan residue in its sequence compared with similar derivative peptides. Therefore, GBPECP shows substantial potential as a novel delivery therapeutic through rapid and effective internalization and interference with cell mobility.
Assuntos
Peptídeos Penetradores de Células/metabolismo , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Triptofano/metabolismo , Animais , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Linhagem Celular Tumoral , Humanos , Microscopia de Fluorescência , Ligação Proteica , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
The oral cancer cell line OC3-I5 with a highly invasive ability was selected and derived from an established OSCC line OC3. In this study, we demonstrated that matrix metalloproteinases protein MMP-13 was up-regulated in OC3-I5 than in OC3 cells. We also observed that expression of epithelial-mesenchymal transition (EMT) markers including Twist, p-Src, Snail1, SIP1, JAM-A, and vinculin were increased in OC3-I5 compared to OC3 cells, whereas E-cadherin expression was decreased in the OC3-I5 cells. Using siMMP-13 knockdown techniques, we showed that siMMP-13 not only reduced the invasion and migration, but also the adhesion abilities of oral cancer cells. In support of the role of MMP-13 in metastasis, we used MMP-13 expressing plasmid-transfected 293T cells to enhance MMP-13 expression in the OC3 cells, transplanting the MMP-13 over expressing OC3 cells into nude mice led to enhanced lung metastasis. In summary, our findings show that MMP-13 promotes invasion and metastasis in oral cancer cells, suggesting altered expression of MMP-13 may be utilized to impede the process of metastasis.
RESUMO
Hepatitis B virus (HBV) is a driver of hepatocellular carcinoma, and two viral products, X and large surface antigen (LHBS), are viral oncoproteins. During chronic viral infection, immune-escape mutants on the preS2 region of LHBS (preS2-LHBS) are gain-of-function mutations that are linked to preneoplastic ground glass hepatocytes (GGHs) and early disease onset of hepatocellular carcinoma. Here, we show that preS2-LHBS provoked calcium release from the endoplasmic reticulum (ER) and triggered stored-operated calcium entry (SOCE). The activation of SOCE increased ER and plasma membrane (PM) connections, which was linked by ER- resident stromal interaction molecule-1 (STIM1) protein and PM-resident calcium release- activated calcium modulator 1 (Orai1). Persistent activation of SOCE induced centrosome overduplication, aberrant multipolar division, chromosome aneuploidy, anchorage-independent growth, and xenograft tumorigenesis in hepatocytes expressing preS2- LHBS. Chemical inhibitions of SOCE machinery and silencing of STIM1 significantly reduced centrosome numbers, multipolar division, and xenograft tumorigenesis induced by preS2-LHBS. These results provide the first mechanistic link between calcium homeostasis and chromosome instability in hepatocytes carrying preS2-LHBS. Therefore, persistent activation of SOCE represents a novel pathological mechanism in HBV-mediated hepatocarcinogenesis.
Assuntos
Canais de Cálcio/metabolismo , Carcinoma Hepatocelular/genética , Instabilidade Cromossômica , Antígenos de Superfície da Hepatite B/metabolismo , Hepatite B/complicações , Neoplasias Hepáticas/genética , Mutação , Precursores de Proteínas/metabolismo , Animais , Cálcio/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/virologia , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Hepatite B/genética , Hepatite B/virologia , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/patogenicidade , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/virologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Precursores de Proteínas/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
As heparan sulfate proteoglycans (HSPGs) are known as co-receptors to interact with numerous growth factors and then modulate downstream biological activities, overexpression of HS/HSPG on cell surface acts as an increasingly reliable prognostic factor in tumor progression. Cell penetrating peptides (CPPs) are short-chain peptides developed as functionalized vectors for delivery approaches of impermeable agents. On cell surface negatively charged HS provides the initial attachment of basic CPPs by electrostatic interaction, leading to multiple cellular effects. Here a functional peptide (CPPecp) has been identified from critical HS binding region in hRNase3, a unique RNase family member with in vitro antitumor activity. In this study we analyze a set of HS-binding CPPs derived from natural proteins including CPPecp. In addition to cellular binding and internalization, CPPecp demonstrated multiple functions including strong binding activity to tumor cell surface with higher HS expression, significant inhibitory effects on cancer cell migration, and suppression of angiogenesis in vitro and in vivo. Moreover, different from conventional highly basic CPPs, CPPecp facilitated magnetic nanoparticle to selectively target tumor site in vivo. Therefore, CPPecp could engage its capacity to be developed as biomaterials for diagnostic imaging agent, therapeutic supplement, or functionalized vector for drug delivery.
Assuntos
Movimento Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/administração & dosagem , Heparitina Sulfato/administração & dosagem , Neoplasias/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Peptídeos Penetradores de Células/química , Proteína Catiônica de Eosinófilo/química , Proteína Catiônica de Eosinófilo/metabolismo , Humanos , Camundongos , Neoplasias/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Horseshoe crab is an ancient marine arthropod that, in the absence of a vertebrate-like immune system, relies solely on innate immune responses by defense molecules found in hemolymph plasma and granular hemocytes for host defense. A plasma lectin isolated from the hemolymph of Taiwanese Tachypleus tridentatus recognizes bacteria and lipopolysaccharides (LPSs), yet its structure and mechanism of action remain unclear, largely because of limited availability of horseshoe crabs and the lack of a heterogeneous expression system. In this study, we have successfully expressed and purified a soluble and functional recombinant horseshoe crab plasma lectin (rHPL) in an Escherichia coli system. Interestingly, rHPL bound not only to bacteria and LPSs like the native HPL but also to selective medically important pathogens isolated from clinical specimens, such as Gram-negative Pseudomonas aeruginosa and Klebsiella pneumoniae and Gram-positive Streptococcus pneumoniae serotypes. The binding was demonstrated to occur through a specific molecular interaction with rhamnose in pathogen-associated molecular patterns (PAMPs) on the bacterial surface. Additionally, rHPL inhibited the growth of P. aeruginosa PAO1 in a concentration-dependent manner. The results suggest that a specific protein-glycan interaction between rHPL and rhamnosyl residue may further facilitate development of novel diagnostic and therapeutic strategies for microbial pathogens.
Assuntos
Proteínas de Artrópodes/metabolismo , Proteínas de Bactérias/metabolismo , Lectinas/metabolismo , Ramnose/metabolismo , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Dicroísmo Circular , Caranguejos Ferradura/metabolismo , Klebsiella pneumoniae/metabolismo , Lectinas/química , Lectinas/genética , Lipopolissacarídeos/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pneumoniae/metabolismoRESUMO
Elevated cathepsin S (Cat S) level is correlated with higher migration ability in tumor cells. This study investigates the inhibitory effect of novel synthetic α-ketoamide compounds on cathepsin activity and cancer cell migration. The effect of several α-ketoamide compounds on the activity of recombinant cathepsins (Cat S, Cat L and Cat K) was examined. Two highly metastatic cancer cell lines were incubated with three Cat S-specific compounds (6n, 6 w and 6r) to analyze their effect on cellular Cat S activity and cell migration. At a 100 nM concentration, compounds 6n, 6r and 6 w effectively inhibited Cat S activity. Cat S activity and cell migration were significantly reduced in CL1-3 cells after treatment with either 6n or 6 w at 5 µM. Similar results were also obtained when A2058 cells were treated with 6n. These results highlight the therapeutic potential of α-ketoamide compounds, especially 6n and 6 w, to prevent or delay cancer metastasis.
Assuntos
Amidas/farmacologia , Antineoplásicos/farmacologia , Catepsinas/antagonistas & inibidores , Movimento Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Amidas/síntese química , Amidas/química , Antineoplásicos/síntese química , Antineoplásicos/química , Catepsinas/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Estrutura Molecular , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Mechanical forces induced by interstitial fluid flow in and surrounding tissues and by blood/lymphatic flow in vessels may modulate cancer cell invasion and metastasis and anticancer drug delivery. Our previous study demonstrated that laminar flow-induced shear stress induces G2/M arrest in tumor cells. However, whether shear stress modulates final cell fate remains unclear. In this study, we investigated the role of flow-induced shear stress in modulating the survival of four human tumor cell lines, i.e., Hep3B hepatocarcinoma cells, MG63 osteosarcoma cells, SCC25 oral squamous carcinoma cells, and A549 carcinomic alveolar basal epithelial cells. Laminar shear stress (LSS) ranging from 0.5 to 12dyn/cm(2) induced death of these four tumor cell lines. In contrast to LSS at 0.5dyn/cm(2), oscillatory shear stress (OSS) at 0.5±4dyn/cm(2) cannot induce cancer cell death. Both LSS and OSS had no effect on human normal hepatocyte, lung epithelial, and endothelial cells. Application of LSS to these four cell lines increased the percentage of cells stained positively for annexin V-FITC, with up-regulations of cleaved caspase-8, -9, and -3, and PARP. In addition, LSS also induced Hep3B cell autophagy, as detected by acidic vesicular organelle formation, LC3B transformation, and p62/SQSTM1 degradation. By transfecting with small interfering RNA, we found that the shear-induced apoptosis and autophagy are mediated by bone morphogenetic protein receptor type (BMPR)-IB, BMPR-specific Smad1 and Smad5, and p38 mitogen-activated protein kinase in Hep3B cells. Our findings provide insights into the molecular mechanisms by which shear stress induces apoptosis and autophagy in tumor cells.
Assuntos
Apoptose , Autofagia , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Neoplasias/patologia , Proteínas Smad/metabolismo , Estresse Mecânico , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Neoplasias/enzimologia , Transdução de Sinais , Proteína Smad1/metabolismo , Proteína Smad5/metabolismoRESUMO
UNLABELLED: Heparan sulfate proteoglycans (HSPGs) are glycoproteins consisting of a core protein to which linear heparan sulfate (HS) side chains are covalently attached. These HS side chains mediate a variety of biologic functions involved in inflammation. Radionuclide imaging of HS side chains in tissues with inflammation may be used for the stratification of patients who would most likely benefit from HSPG-targeting therapy. The goal of this study was to evaluate the feasibility of in vivo radionuclide imaging of HS side chain expression in a mouse model of asthma using the recombinant eosinophil cationic protein (rECP). METHODS: rECP was radioiodinated with (125)I or (123)I using the Chloramine-T method. The 50% inhibitory concentration value for (125)I-labeled rECP was determined in a competitive cell-binding assay using Beas-2B cells. The binding of radiolabeled rECP to HS side chains was evaluated both in vitro and in vivo. The biodistribution of radiolabeled rECP was assessed in asthma mice or in control mice using SPECT imaging, ex vivo biodistribution measurements, and microautoradiography. RESULTS: The 50% inhibitory concentration value for (125)I-rECP was 7.4 ± 0.1 nM. The loss of HS side chains substantially inhibited the cellular and tissue uptake of (125)I- or (123)I-rECP, indicating that HS side chains of HSPGs are required for (125)I- or (123)I-eosinophil cationic protein binding and uptake both in vitro and in vivo. SPECT imaging demonstrated an appreciably higher accumulation of radioactivity in the lungs of asthma mice than in those of control mice. Ex vivo biodistribution studies also confirmed that there was at least a 4-fold increase in the lung-to-muscle ratio of asthma mice, compared with control mice. The accumulation of radiolabeled rECP was linearly correlated with leukocyte infiltration. CONCLUSION: This study illustrates the feasibility of using radiolabeled rECP for the visualization of HS side chains of HSPGs and the evaluation of allergic lung inflammation in living subjects. Our data indicate that radiolabeled rECP is a novel imaging agent for HS side chains of HSPGs in predicting allergic lung inflammation in living mice.
Assuntos
Asma/complicações , Proteína Catiônica de Eosinófilo , Regulação da Expressão Gênica , Heparitina Sulfato/metabolismo , Hipersensibilidade/complicações , Imagem Molecular/métodos , Proteínas Recombinantes , Alérgenos/imunologia , Animais , Asma/diagnóstico por imagem , Asma/imunologia , Transporte Biológico , Linhagem Celular Tumoral , Modelos Animais de Doenças , Estabilidade de Medicamentos , Proteína Catiônica de Eosinófilo/metabolismo , Proteína Catiônica de Eosinófilo/farmacocinética , Feminino , Humanos , Inflamação/diagnóstico por imagem , Inflamação/imunologia , Radioisótopos do Iodo , Marcação por Isótopo , Leucócitos/imunologia , Camundongos , Imagem Multimodal , Tomografia por Emissão de Pósitrons , Radioquímica , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacocinética , Tomografia Computadorizada por Raios XRESUMO
Cell-penetrating peptides (CPPs) are short peptides which can carry various types of molecules into cells; however, although most CPPs rapidly penetrate cells in vitro, their in vivo tissue-targeting specificities are low. Herein, we describe cell-binding, internalization, and targeting characteristics of a newly identified 10-residue CPP, denoted ECP(32-41), derived from the core heparin-binding motif of human eosinophil cationic protein (ECP). Besides traditional emphasis on positively charged residues, the presence of cysteine and tryptophan residues was demonstrated to be essential for internalization. ECP(32-41) entered Beas-2B and wild-type CHO-K1 cells, but not CHO cells lacking of cell-surface glycosaminoglycans (GAGs), indicating that binding of ECP(32-41) to cell-surface GAGs was required for internalization. When cells were cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decreases in ECP(32-41) internalization were observed, suggesting that cell-surface GAGs, especially heparan sulfate proteoglycans were necessary for ECP(32-41) attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major ECP(32-41) internalization routes. ECP(32-41) was demonstrated to transport various cargoes including fluorescent chemical, fluorescent protein, and peptidomimetic drug into cultured Beas-2B cells in vitro, and targeted broncho-epithelial and intestinal villi tissues in vivo. Hence this CPP has the potential to serve as a novel vehicle for intracellular delivery of biomolecules or medicines, especially for the treatment of pulmonary or gastrointestinal diseases.
Assuntos
Peptídeos Penetradores de Células/metabolismo , Proteína Catiônica de Eosinófilo/química , Células Epiteliais/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Microdomínios da Membrana/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Transporte Biológico Ativo , Células CHO , Linhagem Celular , Peptídeos Penetradores de Células/síntese química , Cricetinae , Cricetulus , Cisteína/química , Cisteína/metabolismo , Células Epiteliais/citologia , Proteoglicanas de Heparan Sulfato/química , Humanos , Cinética , Microdomínios da Membrana/química , Dados de Sequência Molecular , Pinocitose , Ligação Proteica , Triptofano/química , Triptofano/metabolismoRESUMO
Doxorubicin is an anticancer drug used in a wide range of cancer therapies; however, doxorubicin-induced drug resistance is one of the most serious obstacles of cancer chemotherapy. Recent studies have indicated that reduced oxidative stress levels in cancer cells induce drug resistance. However, the redox-modifications of resistance - associated cellular targets are largely unknown. Thus, the current study employed cysteine-labeling based two-dimensional differential gel electrophoresis (2D-DIGE) combined with MALDI-TOF mass spectrometry (MALDI-TOF MS) to analyze the effect of doxorubicin resistance on redox regulation in uterine cancer and showed 33 spots that were significantly changed in thiol reactivity. These proteins involve cytoskeleton regulation, signal transduction, redox-regulation, glycolysis, and cell-cycle regulation. The current work shows that the redox 2D-DIGE-based proteomic strategy provides a rapid method to study the molecular mechanisms of doxorubicin-induced drug resistance in uterine cancer. The identified targets may be used to further evaluate their roles in drug-resistance formation and for possible diagnostic or therapeutic applications.