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1.
Trends Immunol ; 45(1): 20-31, 2024 01.
Artículo en Inglés | MEDLINE | ID: mdl-38142147

RESUMEN

Owing to their outstanding performance against COVID-19, mRNA vaccines have brought great hope for combating various incurable diseases, including cancer. Differences in the encoded proteins result in different molecular and cellular mechanisms of mRNA vaccines. With the rapid development of nanotechnology and molecular medicine, personalized antigen-encoding mRNA vaccines that enhance antigen presentation can trigger effective immune responses and prevent off-target toxicities. Herein, we review new insights into the influence of encoded antigens, cytokines, and other functional proteins on the mechanisms of mRNA vaccines. We also highlight the importance of delivery systems and chemical modifications for mRNA translation efficiency, stability, and targeting, and we discuss the potential problems and application prospects of mRNA vaccines as versatile tools for combating cancer.


Asunto(s)
Vacunas contra el Cáncer , Neoplasias , Humanos , Nanomedicina , Vacunas de ARNm , Neoplasias/terapia , Inmunoterapia , Vacunas contra el Cáncer/uso terapéutico
2.
Nature ; 586(7830): 572-577, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32726802

RESUMEN

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a respiratory disease called coronavirus disease 2019 (COVID-19), the spread of which has led to a pandemic. An effective preventive vaccine against this virus is urgently needed. As an essential step during infection, SARS-CoV-2 uses the receptor-binding domain (RBD) of the spike protein to engage with the receptor angiotensin-converting enzyme 2 (ACE2) on host cells1,2. Here we show that a recombinant vaccine that comprises residues 319-545 of the RBD of the spike protein induces a potent functional antibody response in immunized mice, rabbits and non-human primates (Macaca mulatta) as early as 7 or 14 days after the injection of a single vaccine dose. The sera from the immunized animals blocked the binding of the RBD to ACE2, which is expressed on the cell surface, and neutralized infection with a SARS-CoV-2 pseudovirus and live SARS-CoV-2 in vitro. Notably, vaccination also provided protection in non-human primates to an in vivo challenge with SARS-CoV-2. We found increased levels of RBD-specific antibodies in the sera of patients with COVID-19. We show that several immune pathways and CD4 T lymphocytes are involved in the induction of the vaccine antibody response. Our findings highlight the importance of the RBD domain in the design of SARS-CoV-2 vaccines and provide a rationale for the development of a protective vaccine through the induction of antibodies against the RBD domain.


Asunto(s)
Anticuerpos Antivirales/inmunología , Betacoronavirus/inmunología , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Pandemias/prevención & control , Neumonía Viral/inmunología , Neumonía Viral/prevención & control , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , COVID-19 , Vacunas contra la COVID-19 , Humanos , Macaca mulatta/inmunología , Macaca mulatta/virología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos Animales , Modelos Moleculares , Dominios Proteicos , SARS-CoV-2 , Suero/inmunología , Bazo/citología , Bazo/inmunología , Linfocitos T/inmunología , Vacunación
3.
J Am Chem Soc ; 146(10): 6992-7006, 2024 03 13.
Artículo en Inglés | MEDLINE | ID: mdl-38437718

RESUMEN

N6-Methyladenine (6mA) of DNA has emerged as a novel epigenetic mark in eukaryotes, and several 6mA effector proteins have been identified. However, efforts to selectively inhibit the biological functions of these effector proteins with small molecules are unsuccessful to date. Here we report the first potent and selective small molecule inhibitor (13h) of AlkB homologue 1 (ALKBH1), the only validated 6mA demethylase. 13h showed an IC50 of 0.026 ± 0.013 µM and 1.39 ± 0.13 µM in the fluorescence polarization (FP) and enzyme activity assay, respectively, and a KD of 0.112 ± 0.017 µM in the isothermal titration calorimetry (ITC) assay. The potency of 13h was well explained by the cocrystal structure of the 13h-ALKBH1 complex. Furthermore, 13h displayed excellent selectivity for ALKBH1. In cells, compound 13h and its derivative 16 were able to engage ALKBH1 and modulate the 6mA levels. Collectively, our study identified the first potent, isoform selective, and cell-active ALKBH1 inhibitor, providing a tool compound for exploring the biological functions of ALKBH1 and DNA 6mA.


Asunto(s)
ADN , Eucariontes , ADN/metabolismo , Eucariontes/metabolismo , Metilación de ADN
4.
Cancer Immunol Immunother ; 73(9): 173, 2024 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-38953982

RESUMEN

Recent studies have indicated that combining oncolytic viruses with CAR-T cells in therapy has shown superior anti-tumor effects, representing a promising approach. Nonetheless, the localized delivery method of intratumoral injection poses challenges for treating metastatic tumors or distal tumors that are difficult to reach. To address this obstacle, we employed HSV-1-infected CAR-T cells, which systemically delivery HSV into solid tumors. The biological function of CAR-T cells remained intact after loading them with HSV for a period of three days. In both immunocompromised and immunocompetent GBM orthotopic mouse models, B7-H3 CAR-T cells effectively delivered HSV to tumor lesions, resulting in enhanced T-cell infiltration and significantly prolonged survival in mice. We also employed a bilateral subcutaneous tumor model and observed that the group receiving intratumoral virus injection exhibited a significant reduction in tumor volume on the injected side, while the group receiving intravenous infusion of CAR-T cells carrying HSV displayed suppressed tumor growth on both sides. Hence, CAR-THSV cells offer notable advantages in the systemic delivery of HSV to distant tumors. In conclusion, our findings emphasize the potential of CAR-T cells as carriers for HSV, presenting significant advantages for oncolytic virotherapy targeting distant tumors.


Asunto(s)
Inmunoterapia Adoptiva , Viroterapia Oncolítica , Virus Oncolíticos , Receptores Quiméricos de Antígenos , Animales , Ratones , Viroterapia Oncolítica/métodos , Humanos , Virus Oncolíticos/inmunología , Virus Oncolíticos/genética , Inmunoterapia Adoptiva/métodos , Receptores Quiméricos de Antígenos/inmunología , Herpesvirus Humano 1/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto , Línea Celular Tumoral , Linfocitos T/inmunología , Femenino , Glioblastoma/terapia , Glioblastoma/inmunología
5.
Mol Ther ; 31(1): 134-153, 2023 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-36056553

RESUMEN

Glioblastoma (GBM) is the most aggressive primary malignant brain cancer and urgently requires effective treatments. Chimeric antigen receptor T (CAR-T) cell therapy offers a potential treatment method, but it is often hindered by poor infiltration of CAR-T cells in tumors and highly immunosuppressive tumor microenvironment (TME). Here, we armed an oncolytic adenovirus (oAds) with a chemokine CXCL11 to increase the infiltration of CAR-T cells and reprogram the immunosuppressive TME, thus improving its therapeutic efficacy. In both immunodeficient and immunocompetent orthotopic GBM mice models, we showed that B7H3-targeted CAR-T cells alone failed to inhibit GBM growth but, when combined with the intratumoral administration of CXCL11-armed oAd, it achieved a durable antitumor response. Besides, oAd-CXCL11 had a potent antitumor effect and reprogramed the immunosuppressive TME in GL261 GBM models, in which increased infiltration of CD8+ T lymphocytes, natural killer (NK) cells, and M1-polarized macrophages, while decreased proportions of myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and M2-polarized macrophages were observed. Furthermore, the antitumor effect of the oAd-CXCL11 was CD8+ T cell dependent. Our findings thus revealed that CXCL11-armed oAd can improve immune-virotherapy and can be a promising adjuvant of CAR-T therapy for GBM.


Asunto(s)
Neoplasias Encefálicas , Quimiocina CXCL11 , Glioblastoma , Inmunoterapia Adoptiva , Viroterapia Oncolítica , Receptores Quiméricos de Antígenos , Animales , Ratones , Adenoviridae/genética , Línea Celular Tumoral , Quimiocina CXCL11/genética , Glioblastoma/terapia , Receptores Quiméricos de Antígenos/genética , Microambiente Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias Encefálicas/terapia
6.
J Nanobiotechnology ; 22(1): 279, 2024 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-38783333

RESUMEN

BACKGROUND: BCMA-directed autologous chimeric antigen receptor T (CAR-T) cells have shown excellent clinical efficacy in relapsed or refractory multiple myeloma (RRMM), however, the current preparation process for autologous CAR-T cells is complicated and costly. Moreover, the upregulation of CD47 expression has been observed in multiple myeloma, and anti-CD47 antibodies have shown remarkable results in clinical trials. Therefore, we focus on the development of BCMA/CD47-directed universal CAR-T (UCAR-T) cells to improve these limitations. METHODS: In this study, we employed phage display technology to screen nanobodies against BCMA and CD47 protein, and determined the characterization of nanobodies. Furthermore, we simultaneously disrupted the endogenous TRAC and B2M genes of T cells using CRISPR/Cas9 system to generate TCR and HLA double knock-out T cells, and developed BCMA/CD47-directed UCAR-T cells and detected the antitumor activity in vitro and in vivo. RESULTS: We obtained fourteen and one specific nanobodies against BCMA and CD47 protein from the immunized VHH library, respectively. BCMA/CD47-directed UCAR-T cells exhibited superior CAR expression (89.13-98.03%), and effectively killing primary human MM cells and MM cell lines. BCMA/CD47-directed UCAR-T cells demonstrated excellent antitumor activity against MM and prolonged the survival of tumor-engrafted NCG mice in vivo. CONCLUSIONS: This work demonstrated that BCMA/CD47-directed UCAR-T cells exhibited potent antitumor activity against MM in vitro and in vivo, which provides a potential strategy for the development of a novel "off-the-shelf" cellular immunotherapies for the treatment of multiple myeloma.


Asunto(s)
Antígeno de Maduración de Linfocitos B , Antígeno CD47 , Inmunoterapia Adoptiva , Mieloma Múltiple , Receptores Quiméricos de Antígenos , Mieloma Múltiple/terapia , Mieloma Múltiple/inmunología , Humanos , Animales , Antígeno CD47/inmunología , Antígeno de Maduración de Linfocitos B/inmunología , Ratones , Inmunoterapia Adoptiva/métodos , Línea Celular Tumoral , Receptores Quiméricos de Antígenos/inmunología , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/farmacología , Linfocitos T/inmunología , Sistemas CRISPR-Cas , Femenino
7.
Nucleic Acids Res ; 50(18): 10586-10600, 2022 10 14.
Artículo en Inglés | MEDLINE | ID: mdl-36200834

RESUMEN

Type II toxin-antitoxin (TA) systems are widely distributed in bacterial and archaeal genomes and are involved in diverse critical cellular functions such as defense against phages, biofilm formation, persistence, and virulence. GCN5-related N-acetyltransferase (GNAT) toxin, with an acetyltransferase activity-dependent mechanism of translation inhibition, represents a relatively new and expanding family of type II TA toxins. We here describe a group of GNAT-Xre TA modules widely distributed among Pseudomonas species. We investigated PacTA (one of its members encoded by PA3270/PA3269) from Pseudomonas aeruginosa and demonstrated that the PacT toxin positively regulates iron acquisition in P. aeruginosa. Notably, other than arresting translation through acetylating aminoacyl-tRNAs, PacT can directly bind to Fur, a key ferric uptake regulator, to attenuate its DNA-binding affinity and thus permit the expression of downstream iron-acquisition-related genes. We further showed that the expression of the pacTA locus is upregulated in response to iron starvation and the absence of PacT causes biofilm formation defect, thereby attenuating pathogenesis. Overall, these findings reveal a novel regulatory mechanism of GNAT toxin that controls iron-uptake-related genes and contributes to bacterial virulence.


Asunto(s)
Antitoxinas , Toxinas Bacterianas , Acetiltransferasas/metabolismo , Antitoxinas/genética , Antitoxinas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , ADN/metabolismo , Regulación Bacteriana de la Expresión Génica , Homeostasis/genética , Hierro/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo
8.
J Transl Med ; 21(1): 23, 2023 01 13.
Artículo en Inglés | MEDLINE | ID: mdl-36635683

RESUMEN

BACKGROUND: Chimeric antigen receptor (CAR) T cells and immune checkpoint blockades (ICBs) have made remarkable breakthroughs in cancer treatment, but the efficacy is still limited for solid tumors due to tumor antigen heterogeneity and the tumor immune microenvironment. The restrained treatment efficacy prompted us to seek new potential therapeutic methods. METHODS: In this study, we conducted a small molecule compound library screen in a human BC cell line to identify whether certain drugs contribute to CAR T cell killing. Signaling pathways of tumor cells and T cells affected by the screened drugs were predicted via RNA sequencing. Among them, the antitumor activities of JK184 in combination with CAR T cells or ICBs were evaluated in vitro and in vivo. RESULTS: We selected three small molecule drugs from a compound library, among which JK184 directly induces tumor cell apoptosis by inhibiting the Hedgehog signaling pathway, modulates B7-H3 CAR T cells to an effector memory phenotype, and promotes B7-H3 CAR T cells cytokine secretion in vitro. In addition, our data suggested that JK184 exerts antitumor activities and strongly synergizes with B7-H3 CAR T cells or ICBs in vivo. Mechanistically, JK184 enhances B7-H3 CAR T cells infiltrating in xenograft mouse models. Moreover, JK184 combined with ICB markedly reshaped the tumor immune microenvironment by increasing effector T cells infiltration and inflammation cytokine secretion, inhibiting the recruitment of MDSCs and the transition of M2-type macrophages in an immunocompetent mouse model. CONCLUSION: These data show that JK184 may be a potential adjutant in combination with CAR T cells or ICB therapy.


Asunto(s)
Proteínas Hedgehog , Neoplasias , Humanos , Animales , Ratones , Evaluación Preclínica de Medicamentos , Detección Precoz del Cáncer , Inmunoterapia , Citocinas , Inmunoterapia Adoptiva/métodos , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Microambiente Tumoral , Neoplasias/terapia
9.
PLoS Pathog ; 16(10): e1008848, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-33007034

RESUMEN

Colonization factor CFA/I defines the major adhesive fimbriae of enterotoxigenic Escherichia coli and mediates bacterial attachment to host intestinal epithelial cells. The CFA/I fimbria consists of a tip-localized minor adhesive subunit, CfaE, and thousands of copies of the major subunit CfaB polymerized into an ordered helical rod. Biosynthesis of CFA/I fimbriae requires the assistance of the periplasmic chaperone CfaA and outer membrane usher CfaC. Although the CfaE subunit is proposed to initiate the assembly of CFA/I fimbriae, how it performs this function remains elusive. Here, we report the establishment of an in vitro assay for CFA/I fimbria assembly and show that stabilized CfaA-CfaB and CfaA-CfaE binary complexes together with CfaC are sufficient to drive fimbria formation. The presence of both CfaA-CfaE and CfaC accelerates fimbria formation, while the absence of either component leads to linearized CfaB polymers in vitro. We further report the crystal structure of the stabilized CfaA-CfaE complex, revealing features unique for biogenesis of Class 5 fimbriae.


Asunto(s)
Adhesinas Bacterianas/metabolismo , Escherichia coli Enterotoxigénica/fisiología , Proteínas de Escherichia coli/metabolismo , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/fisiología , Chaperonas Moleculares/metabolismo , Secuencia de Aminoácidos , Citoplasma , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas Fimbrias/genética , Chaperonas Moleculares/genética , Conformación Proteica , Homología de Secuencia de Aminoácido
11.
Mol Cancer ; 20(1): 168, 2021 12 18.
Artículo en Inglés | MEDLINE | ID: mdl-34922552

RESUMEN

BACKGROUND: Craniopharyngioma (CP) is rare histologically benign but clinically challenging tumor because of its intimate relationship with the critical structure in the central brain. CP can be divided into two major histologic subtypes: adamantinomatous-type CP (ACP) and papillary-type CP (PCP). Although some genetic aberrations for both categories have been revealed in previous studies, the complete spectrum of genetic changes of this tumor remains unknown. METHODS: In this study, we conducted whole genome sequencing (WGS) on twenty-six CPs including 16 ACPs and 10 PCPs together with their matched blood samples. Somatic variants (SNVs, InDels, SVs and CNVs) were identified and mutational signatures were characterized for each patient. We investigated the impact of a novel CTNNB1 mutant on its protein stability, ubiquitination and Wnt pathway activity. Cell proliferation ability of the CTNNB1 mutant in ACP primary cells was additionally analyzed by CCK8 and colony formation assays. RESULTS: We found that CPs had showed less complexity with fewer somatic mutations compared with malignant tumors. Moreover, mutations in CTNNB1 (68.75% of ACP) and BRAF V600E (70.00% of PCP) are mutually exclusive in ACP and PCP, consolidating that the driving roles of these two genes in ACP and PCP, respectively. A novel mutation in the exon 3 of CTNNB1 which compromised both a transversion and in-frame deletion was identified in ACP. This mutation was experimentally validated to confer ß-catenin increased stability by inhibiting its ubiquitination, thus activating Wnt-signaling pathway and promoting cell proliferation. CONCLUSIONS: Whole genome landscape for CP was revealed by WGS analysis, and a novel mutation in the exon 3 of CTNNB1 was identified. This novel mutation activates Wnt-signaling pathway through increasing the stability of ß-catenin. Our findings provided us with more comprehensive insight into the spectrum of genetic alterations in CP.


Asunto(s)
Craneofaringioma/genética , Mutación , Neoplasias Hipofisarias/genética , beta Catenina/genética , Biomarcadores de Tumor , Biología Computacional/métodos , Craneofaringioma/diagnóstico , Humanos , Mutación INDEL , Neoplasias Hipofisarias/diagnóstico , Polimorfismo de Nucleótido Simple , Pronóstico , Células Tumorales Cultivadas , Secuenciación Completa del Genoma , Vía de Señalización Wnt
12.
Cancer Immunol Immunother ; 70(9): 2453-2465, 2021 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-33543339

RESUMEN

BACKGROUND: T cell with chimeric antigen receptors (CAR-T) has presented remarkable efficacy for blood cancer as an emerging immunotherapy. However, for solid tumors, the therapeutic efficacy is much impaired due to the lack of infiltration and persistence of CAR-T in tumor tissue. Thus, we constructed an interleukin-7-loaded oncolytic adenovirus and combined the use of oncolytic virus and CAR-T to improve the therapeutic outcome. METHODS: We constructed an interleukin-7-loaded oncolytic adenovirus (oAD-IL7) and a B7H3-targeted CAR-T and explored the efficacy of the single use of oAD-IL7, B7H3-CAR-T, or the combined therapy for glioblastoma in vitro and in vivo. The improved CAR-T anti-tumor efficacy was evaluated according to the proliferation, survival, persistence, exhaustion of T cells, and tumor regression. RESULTS: Constructed oAD-IL7 and B7H3-CAR-T presented moderate cytotoxicity during in vitro study, but failed to induce a thorough and persistent anti-tumor therapeutic efficacy in vivo. The combination of oAD-IL7 and B7H3-CAR-T in vitro resulted in enhanced T cell proliferation and reduced T cell apoptosis. The joint efficacy was further confirmed using tumor-bearing xenograft mice. During in vivo study, the mice treated with both oAD-IL7 and B7H3-CAR-T showed prolonged survival and reduced tumor burden. According to the ex vivo study, oAD-IL7 improved the proliferation and persistence of tumor-infiltrating B7H3-CAR-T, but failed to reverse the exhaustion. CONCLUSIONS: Our results indicated that oAD-IL7 is a promising auxiliary therapy to improve the therapeutic efficacy of B7H3-CAR-T in glioblastoma by providing the activating signals for tumor-infiltrating T cells. Our results also lay the basis for the future clinical trials for the combination of IL7-loaded oncolytic adenovirus and CAR-T therapy for glioblastoma.


Asunto(s)
Terapia Genética , Vectores Genéticos/genética , Inmunoterapia Adoptiva , Interleucina-7/genética , Viroterapia Oncolítica , Virus Oncolíticos/genética , Adenoviridae/genética , Animales , Apoptosis/genética , Apoptosis/inmunología , Antígenos B7/antagonistas & inhibidores , Antígenos B7/inmunología , Antígenos B7/metabolismo , Citocinas/metabolismo , Citotoxicidad Inmunológica , Modelos Animales de Enfermedad , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Glioblastoma/etiología , Glioblastoma/metabolismo , Glioblastoma/patología , Glioblastoma/terapia , Humanos , Inmunofenotipificación , Inmunoterapia Adoptiva/métodos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Viroterapia Oncolítica/métodos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
13.
J Nanobiotechnology ; 19(1): 33, 2021 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-33514385

RESUMEN

BACKGROUND: The outbreak and pandemic of coronavirus SARS-CoV-2 caused significant threaten to global public health and economic consequences. It is extremely urgent that global people must take actions to develop safe and effective preventions and therapeutics. Nanobodies, which are derived from single­chain camelid antibodies, had shown antiviral properties in various challenge viruses. In this study, multivalent nanobodies with high affinity blocking SARS-CoV-2 spike interaction with ACE2 protein were developed. RESULTS: Totally, four specific nanobodies against spike protein and its RBD domain were screened from a naïve VHH library. Among them, Nb91-hFc and Nb3-hFc demonstrated antiviral activity by neutralizing spike pseudotyped viruses in vitro. Subsequently, multivalent nanobodies were constructed to improve the neutralizing capacity. As a result, heterodimer nanobody Nb91-Nb3-hFc exhibited the strongest RBD-binding affinity and neutralizing ability against SARS-CoV-2 pseudoviruses with an IC50 value at approximately 1.54 nM. CONCLUSIONS: The present study indicated that naïve VHH library could be used as a potential resource for rapid acquisition and exploitation of antiviral nanobodies. Heterodimer nanobody Nb91-Nb3-hFc may serve as a potential therapeutic agent for the treatment of COVID-19.


Asunto(s)
Anticuerpos de Dominio Único/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Sitios de Unión , Células HEK293 , Humanos , Pruebas de Neutralización , Unión Proteica , Dominios Proteicos , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/antagonistas & inhibidores
14.
Biochem Biophys Res Commun ; 514(2): 379-385, 2019 06 25.
Artículo en Inglés | MEDLINE | ID: mdl-31043272

RESUMEN

Craniopharyngiomas (CPs) are uncommon intracranial benign neoplasms that located in sellar/parasellar region with clinically challenging. B7-H3 is an immune checkpoint molecule highly expressed in many malignant tumors. In this study, we analyzed whether B7-H3 is expressed in 44 CPs samples (adamantinomatous CPs: n = 30 and papillary CPs: n = 14), and whether it could serve as an immunotherapy target in CPs. Immunohistochemical analysis showed that B7-H3 was highly expressed in adamantinomatous CPs (184.3 ±â€¯13.58) and papillary CPs (223.2 ±â€¯11.89), while almost undetectable in normal brain tissue (24 ±â€¯4.9). Besides, B7-H3 expression level was correlated with poor prognosis of patients with CPs. Immunofluorescence and Western blot analysis further suggested that ß-catenin co-localized with B7-H3 and could promote its expression in adaCPs. B7-H3 expression level was positively correlated with staining intensity of IBA1+ cells, but negatively with T cell infiltration in CPs, suggesting that B7-H3 might play a role in the regulation of tumor microenvironment in CPs. Moreover, B7-H3/CD3 bi-specific T cell engager (BiTE) efficiently inhibited the growth of human primary craniopharyngioma cells in a time- and dose-dependent manner. Our results revealed B7-H3 was highly expressed in CPs and targeting B7-H3 might therefore be an effective therapeutic strategy against craniopharyngioma.


Asunto(s)
Antígenos B7/metabolismo , Craneofaringioma/metabolismo , Regulación Neoplásica de la Expresión Génica , Regulación hacia Arriba , Antígenos B7/antagonistas & inhibidores , Complejo CD3/metabolismo , Supervivencia Celular , Craneofaringioma/tratamiento farmacológico , Humanos , Linfocitos Infiltrantes de Tumor/citología , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Pronóstico , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismo , beta Catenina/metabolismo
15.
Biochem Biophys Res Commun ; 516(2): 515-520, 2019 08 20.
Artículo en Inglés | MEDLINE | ID: mdl-31230744

RESUMEN

Glioma is the most common primary brain tumor with high mortality. Given the poor outcomes with standard-of-care treatments, novel treatment strategies are needed. Oncolytic viral therapy for glioma has developed as an exciting therapeutic method in recent years. Zika virus, a member of flavivirus family, has oncolytic activity against glioma cells but the mechanism is unknown. Here, we aimed to determine which viral protein might play a critical role in mitigating glioma cell growth. We examined the tumor suppressor function of four nonstructural proteins NS1, NS3, NS4B and NS5 in human glioma cell line U87. As a result, we found that only NS5 significantly inhibited proliferation, migration and invasion of U87 cells. Moreover, expression of NS5 suppressed tumorigenicity of mouse GL261 glioma cell in vivo. Our findings provide some clues for further exploration of oncolytic Zika virus in the treatment of glioma.


Asunto(s)
Glioma/patología , Proteínas no Estructurales Virales/farmacología , Virus Zika/metabolismo , Animales , Carcinogénesis/efectos de los fármacos , Carcinogénesis/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Invasividad Neoplásica , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/patología
16.
Exp Cell Res ; 366(1): 24-33, 2018 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-29522753

RESUMEN

PTEN deficiency often causes defects in DNA damage repair. Currently, effective therapies for breast cancer are lacking. ATM is an attractive target for cancer treatment. Previous studies suggested a synthetic lethality between PTEN and PARP. However, the synthetically lethal interaction between PTEN and ATM in breast cancer has not been reported. Moreover, the mechanism remains elusive. Here, using KU-60019, an ATM kinase inhibitor, we investigated ATM inhibition as a synthetically lethal strategy to target breast cancer cells with PTEN defects. We found that KU-60019 preferentially sensitizes PTEN-deficient MDA-MB-468 breast cancer cells to cisplatin, though it also slightly enhances sensitivity of PTEN wild-type breast cancer cells. The increased cytotoxic sensitivity is associated with apoptosis, as evidenced by flow cytometry and PARP cleavage. Additionally, the increase of DNA damage accumulation due to the decreased capability of DNA repair, as indicated by γ-H2AX and Rad51 foci, also contributed to this selective cytotoxicity. Mechanistically, compared with PTEN wild-type MDA-MB-231 cells, PTEN-deficient MDA-MB-468 cells have lower level of Rad51, higher ATM kinase activity, and display the elevated level of DNA damage. Moreover, these differences could be further enlarged by cisplatin. Our findings suggest that ATM is a promising target for PTEN-defective breast cancer.


Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Cisplatino/farmacología , Fosfohidrolasa PTEN/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Línea Celular Tumoral , Daño del ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Humanos , Células MCF-7 , Morfolinas/farmacología , Tioxantenos/farmacología
17.
Biochem J ; 475(6): 1107-1119, 2018 03 20.
Artículo en Inglés | MEDLINE | ID: mdl-29382741

RESUMEN

In plants and microorganisms, aspartate kinase (AK) catalyzes an initial commitment step of the aspartate family amino acid biosynthesis. Owing to various structural organizations, AKs from different species show tremendous diversity and complex allosteric controls. We report the crystal structure of AK from Pseudomonas aeruginosa (PaAK), a typical α2ß2 hetero-tetrameric enzyme, in complex with inhibitory effectors. Distinctive features of PaAK are revealed by structural and biochemical analyses. Essentially, the open conformation of Lys-/Thr-bound PaAK structure clarifies the inhibitory mechanism of α2ß2-type AK. Moreover, the various inhibitory effectors of PaAK have been identified and a general amino acid effector motif of AK family is described.


Asunto(s)
Aspartato Quinasa/química , Aspartato Quinasa/metabolismo , Pseudomonas aeruginosa/enzimología , Regulación Alostérica/genética , Sitio Alostérico/genética , Secuencia de Aminoácidos , Aspartato Quinasa/genética , Catálisis , Modelos Moleculares , Organismos Modificados Genéticamente , Dominios y Motivos de Interacción de Proteínas/genética , Pseudomonas aeruginosa/genética , Alineación de Secuencia
18.
J Neurooncol ; 131(1): 21-29, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27640198

RESUMEN

Annexin A2 (AnxA2) is a highly conserved Ca2+-regulated membrane binding protein, which affects cell mobility and tumor progression. Adamantinomatous craniopharyngioma (AdaCP) are a kind of epithelial tumors of the sellar region with high tendency to recur. Robust biomarkers are required to predict tumor behavior and to establish follow-up individualized treatment approaches. In this study, we firstly compared four surgical AdaCP samples with normal brain by two-dimensional gel electrophoresis (2DE) proteomic analysis. Potential prognostic biomarkers were further validated in a large cohort of 65 AdaCPs by immunohistochemistry. The effects of AnxA2 on AdaCP cells proliferation and migration were analyzed in vitro with isolated primary AdaCP cells as well as SV40T-immortalized cells. Finally, the gefitinib sensitivity of AdaCPs with differentially expressed AnxA2 and the potential molecular mechanisms were examined by flow cytometric analysis, Real-time PCR and immunoblot assays. Proteomic analysis indicated that AnxA2 was the protein spot with the most elevated expression in AdaCP samples. Immunohistochemistry assays indicated the expression level of AnxA2 was significantly higher in recurrent AdaCPs compared with primary ones. Moreover, AnxA2+ AdaCP cells exhibited enhanced proliferation and migration ability compared with AnxA2- AdaCP cells in vitro. Further, we show that AnxA2+ AdaCP cells exhibited elevated expression of EGFR and downstream p-AKT (S308) and p-AKT (S473), and were more sensitive to tyrosine kinase inhibitor gefitinib. Our data suggest that AnxA2 may serve as a promising biomarker for AdaCP progression, recurrence and drug susceptibility. Our data support potential clinical implications for the follow-up treatment of AdaCP patients with high AnxA2 expression.


Asunto(s)
Anexina A2/metabolismo , Encéfalo/metabolismo , Craneofaringioma/metabolismo , Neoplasias Hipofisarias/metabolismo , Adolescente , Adulto , Anciano , Anexina A2/genética , Antineoplásicos/farmacología , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Niño , Preescolar , Estudios de Cohortes , Craneofaringioma/patología , Craneofaringioma/fisiopatología , Receptores ErbB/metabolismo , Femenino , Gefitinib , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Masculino , Persona de Mediana Edad , Proteína Oncogénica v-akt/metabolismo , Neoplasias Hipofisarias/patología , Neoplasias Hipofisarias/fisiopatología , Pronóstico , Quinazolinas/farmacología , Transducción de Señal/efectos de los fármacos , Células Tumorales Cultivadas/efectos de los fármacos , Adulto Joven
19.
J Biol Chem ; 290(24): 14927-44, 2015 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-25918161

RESUMEN

PNAS-4, a novel pro-apoptotic gene, was activated during the early response to DNA damage. Our previous study has shown that PNAS-4 induces S phase arrest and apoptosis when overexpressed in A549 lung cancer cells. However, the underlying action mechanism remains far from clear. In this work, we found that PNAS-4 expression in lung tumor tissues is significantly lower than that in adjacent lung tissues; its expression is significantly increased in A549 cells after exposure to cisplatin, methyl methane sulfonate, and mitomycin; and its overexpression induces S phase arrest and apoptosis in A549 (p53 WT), NCI-H460 (p53 WT), H526 (p53 mutation), and Calu-1 (p53(-/-)) lung cancer cells, leading to proliferation inhibition irrespective of their p53 status. The S phase arrest is associated with up-regulation of p21(Waf1/Cip1) and inhibition of the Cdc25A-CDK2-cyclin E/A pathway. Up-regulation of p21(Waf1/Cip1) is p53-independent and correlates with activation of ERK. We further showed that the intra-S phase checkpoint, which occurs via DNA-dependent protein kinase-mediated activation of Chk1 and Chk2, is involved in the S phase arrest and apoptosis. Gene silencing of Chk1/2 rescues, whereas that of ATM or ATR does not affect, S phase arrest and apoptosis. Furthermore, human PNAS-4 induces DNA breaks in comet assays and γ-H2AX staining. Intriguingly, caspase-dependent cleavage of Chk1 has an additional role in enhancing apoptosis. Taken together, our findings suggest a novel mechanism by which elevated PNAS-4 first causes DNA-dependent protein kinase-mediated Chk1/2 activation and then results in inhibition of the Cdc25A-CDK2-cyclin E/A pathway, ultimately causing S phase arrest and apoptosis in lung cancer cells.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/fisiología , Apoptosis/fisiología , Quinasa de Punto de Control 2/metabolismo , Neoplasias Pulmonares/patología , Proteínas Quinasas/metabolismo , Fase S/fisiología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/fisiología , Secuencia de Bases , Liasas de Carbono-Nitrógeno , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Quinasa de Punto de Control 2/genética , Daño del ADN , Cartilla de ADN , Activación Enzimática , Humanos , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/metabolismo , Proteínas Quinasas/genética , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
20.
Biochem Biophys Res Commun ; 457(3): 347-52, 2015 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-25585381

RESUMEN

Antimicrobial peptides (AMPs) are an evolutionarily conserved component of the innate immune response that provides host defence at skin and mucosal surfaces. Here, we report the identification and characterization of a new type human AMPs, termed AP-57 (Antimicrobial Peptide with 57 amino acid residues), which is also known as C10orf99 (chromosome 10 open reading frame 99). AP-57 is a short basic amphiphilic peptide with four cysteines and a net charge +14 (MW = 6.52, PI = 11.28). The highest expression of AP-57 were detected in the mucosa of stomach and colon through immunohistochemical assay. Epithelium of skin and esophagus show obvious positive staining and strong positive staining were also observed in some tumor and/or their adjacent tissues, such as esophagus cancer, hepatocellular carcinoma, squamous cell carcinoma and invasive ductal carcinoma. AP-57 exhibited broad-spectrum antimicrobial activities against Gram-positive Staphylococcus aureus, Actinomyce, and Fungi Aspergillus niger as well as mycoplasma and lentivirus. AP-57 also exhibited DNA binding capacity and specific cytotoxic effects against human B-cell lymphoma Raji. Compared with other human AMPs, AP-57 has its distinct characteristics, including longer sequence length, four cysteines, highly cationic character, cell-specific toxicity, DNA binding and tissue-specific expressing patterns. Together, AP-57 is a new type of multifunctional AMPs worthy further investigation.


Asunto(s)
Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/química , Línea Celular , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Secuencia Conservada , ADN de Neoplasias/metabolismo , Proteínas de Unión al ADN/química , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Distribución Tisular , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo
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