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Chimeric antigen receptor T cell (CAR-T) therapy has been applied in the treatment of B-cell lymphoma; however, CAR-T manufacturing requires virus- or non-virus-based genetic modification, which causes high manufacturing costs and potential safety concerns. Antibody-cell conjugation (ACC) technology, which originated from bio-orthogonal click chemistry, provides an efficient approach for arming immune cells with cancer-targeting antibodies without genetic modification. Here, we applied ACC technology in Vγ9Vδ2 T (γδ2 T) cells to generate a novel off-the-shelf CD20-targeting cell therapy ACE1831 (rituximab-conjugated γδ2 T cells) against relapsed/refractory B-cell lymphoma. ACE1831 exhibited superior cytotoxicity against B-cell lymphoma cells and rituximab-resistant cells compared to γδ2 T cells without rituximab conjugation. The in vivo xenograft study demonstrated that ACE1831 treatment strongly suppressed the aggressive proliferation of B-cell lymphoma and prolonged the survival of tumor-bearing mice with no observed toxicity. Mass spectrometry analysis indicated that cell activation receptors including the TCR complex, integrins and cytokine receptors were conjugated with rituximab. Intriguingly, the antigen recognition of the ACC-linked antibody/receptor complex stimulated NFAT activation and contributed to ACE1831-mediated cytotoxicity against CD20-expressing cancer cells. This study elucidates the role of the ACC-linked antibody/receptor complex in cytotoxicity and supports the potential of ACE1831 as an off-the-shelf γδ2 cell therapy against relapsed/refractory B-cell lymphoma.
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Natural killer (NK) cells harbor efficient cytotoxicity against tumor cells without causing life-threatening cytokine release syndrome (CRS) or graft-versus-host disease (GvHD). When compared to chimeric antigen receptor (CAR) technology, Antibody-Cell Conjugation (ACC) technology has been developed to provide an efficient platform to arm immune cells with cancer-targeting antibodies to recognize and attack cancer cells. Recently, we established an endogenous CD16-expressing oNK cell line (oNK) with a favorable expression pattern of NK activation/inhibitory receptors. In this study, we applied ACC platform to conjugate oNK with trastuzumab and an anti-human epidermal growth factor receptor 2 (HER2) antibody. Trastuzumab-conjugated oNK, ACE-oNK-HER2, executed in vitro and in vivo cytotoxicity against HER2-expressing cancer cells and showed enhanced T cell-recruiting capability and secretion of IFNγ. The irradiated and cryopreserved ACE-oNK-HER2, designated as ACE1702, retained superior HER2-specific in vitro and in vivo potency with no tumorigenic potential. In conclusion, this study provides the evidence to support the potential clinical application of ACE1702 as a novel off-the-shelf NK cell therapy against HER2-expressing solid tumors.
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Natural killer (NK) cells, as a potential source for off-the-shelf cell therapy, attack tumor cells with low risk of severe cytokine release syndrome (CRS) or graft-versus-host disease (GvHD). Fcγ receptor IIIA, also known as CD16, further confers NK cells with antibody-dependent cell-mediated cytotoxicity (ADCC), one mechanism of action of antibody-based immunotherapy. Here, we establish a novel human NK cell line, oNK-1, endogenously expressing CD16 along with high levels of NK activation markers and low levels of NK inhibitory markers. The long-term expansion and CD16 expression of oNK-1 cells were demonstrated. Furthermore, oNK-1 cells elicit superior cytotoxicity against cancer cells than primary NK cells. In conclusion, this study suggests that endogenous CD16-expressing oNK-1 has the potential to develop an effective NK-based therapy.
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A promising approach in cancer therapy is the inhibition of cell proliferation using small molecules. In this study, we report the synthesis of suramin derivatives and their applications. We used NMR spectroscopy and docking simulations to confirm binding sites and three-dimensional models of the ligand-protein complex. The WST-1 assay was used to assess cell viability and cell proliferation in vitro to evaluate the inhibition of protein-protein interactions and to investigate the anti-proliferative activities in a breast cancer cell line. All the suramin derivatives showed anti-proliferative activity by blocking FGF1 binding to its receptor FGFRD2. The dissociation constant was measured by fluorescence spectroscopy. The suramin compound derivatives synthesized herein show potential as novel therapeutic agents for their anti-proliferative activity via the inhibition of protein-protein interactions. The cytotoxicity of these suramin derivatives was lower than that of the parent suramin compound, which may be considered a significant advancement in this field. Thus, these novel suramin derivatives may be considered superior anti-metastasis molecules than those of suramin.
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The inhibition of protein function by small compounds plays a critical role in controlling cell proliferation. We report on a new class of small molecule (NCTU-Alan-2026) inhibitors for cell proliferation. NCTU-Alan-2026 blocks the interaction between FGF1 and its receptor FGF1R2D2. Extensive NMR studies combined with fluorescence experiments provided the specific mechanism of how NCTU-Alan-2026 could inhibit cell proliferation. We describe an innovative therapeutic approach for anti-proliferation and demonstrate an example of inhibition of small molecules by blocking the protein-protein interaction. We found that the compound NCTU-Alan-2026 blocked the interaction between the two proteins FGF1 and FGF1R2D2 and inhibited cell proliferation. The toxicity of NCTU-Alan-2026 is lower than that of suramin. Thus, NCTU-Alan-2026 could be a better drug than suramin in the treatment of cancer.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Fator 1 de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Suramina/química , Suramina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Ligação Proteica/efeitos dos fármacosRESUMO
Terpenoids are a large group of important secondary metabolites that are involved in a variety of physiological mechanisms, and many are used commercially in the cosmetics and pharmaceutical industries. During the past decade, the topic of seasonal variation in terpenoid biosynthesis has garnered increasing attention. Formosan sweet gum ( Liquidambar formosana Hance) is a deciduous tree species. The expression of terpene synthase and accumulation of terpenoids in leaves may vary in different seasons. Here, four sesquiterpene synthases (i.e., LfTPS01, LfTPS02, LfTPS03, and LfTPS04) and a bifunctional mono/sesquiterpene synthase ( LfTPS05) were identified from Formosan sweet gum. The gene expression of LfTPS01, LfTPS02, and LfTPS03 showed seasonal diversification, and, in addition, expression of LfTPS04 and LfTPS05 was induced by methyl jasmonate treatment. The major products LfTPS01, LfTPS02, LfTPS04, and LfTPS05 are hedycaryol, α-selinene, trans-ß-caryophyllene, α-copaene/δ-cadinene, and nerolidol/linalool, respectively. The data indicated that the sesquiterpenoid content in the essential oil of Formosan sweet gum leaves shows seasonal differences that were correlated to the sesquiterpene synthase gene expression.
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Alquil e Aril Transferases/genética , Expressão Gênica/genética , Liquidambar/genética , Proteínas de Plantas/genética , Sesquiterpenos/metabolismo , Monoterpenos Acíclicos , Monoterpenos/metabolismo , Folhas de Planta/genética , Sesquiterpenos Policíclicos , Estações do AnoRESUMO
Antodia cinnamomea, a precious brown-rot fungus endemic to Taiwan, has pharmaceutical applications due to its diverse array of metabolites. The terpenoids found in A. cinnamomea contribute to its most important bioactivities. We identified several terpenoid compounds in A. cinnamomea and revealed that their content in mycelium and fruiting body were significantly different. Using next-generation sequencing and an in-house transcriptome database, we identified several terpene synthase (TPS) candidates. After sequence analysis and functional characterization, 10 out of 12 candidates were found to have single or multiple terpene synthesis functions. Most of the terpenoid compounds were found to confer important bioactivities. RT-PCR results showed a positive correlation between terpene synthase expression pattern and terpenoid content. In addition, we identified several modification enzyme candidates that may be involved in the postmodification of terpenoid compounds with a genomic DNA scaffold, and a putative genetic network.
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Antrodia/metabolismo , Carpóforos/metabolismo , Redes Reguladoras de Genes , Micélio/genética , Terpenos/metabolismo , Antrodia/genética , Antrodia/crescimento & desenvolvimento , Carpóforos/genética , Carpóforos/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Micélio/crescimento & desenvolvimento , Micélio/metabolismo , TranscriptomaRESUMO
BACKGROUND: Recent advances in sequencing technology have opened a new era in RNA studies. Novel types of RNAs such as long non-coding RNAs (lncRNAs) have been discovered by transcriptomic sequencing and some lncRNAs have been found to play essential roles in biological processes. However, only limited information is available for lncRNAs in Drosophila melanogaster, an important model organism. Therefore, the characterization of lncRNAs and identification of new lncRNAs in D. melanogaster is an important area of research. Moreover, there is an increasing interest in the use of ChIP-seq data (H3K4me3, H3K36me3 and Pol II) to detect signatures of active transcription for reported lncRNAs. RESULTS: We have developed a computational approach to identify new lncRNAs from two tissue-specific RNA-seq datasets using the poly(A)-enriched and the ribo-zero method, respectively. In our results, we identified 462 novel lncRNA transcripts, which we combined with 4137 previously published lncRNA transcripts into a curated dataset. We then utilized 61 RNA-seq and 32 ChIP-seq datasets to improve the annotation of the curated lncRNAs with regards to transcriptional direction, exon regions, classification, expression in the brain, possession of a poly(A) tail, and presence of conventional chromatin signatures. Furthermore, we used 30 time-course RNA-seq datasets and 32 ChIP-seq datasets to investigate whether the lncRNAs reported by RNA-seq have active transcription signatures. The results showed that more than half of the reported lncRNAs did not have chromatin signatures related to active transcription. To clarify this issue, we conducted RT-qPCR experiments and found that ~95.24% of the selected lncRNAs were truly transcribed, regardless of whether they were associated with active chromatin signatures or not. CONCLUSIONS: In this study, we discovered a large number of novel lncRNAs, which suggests that many remain to be identified in D. melanogaster. For the lncRNAs that are known, we improved their characterization by integrating a large number of sequencing datasets (93 sets in total) from multiple sources (lncRNAs, RNA-seq and ChIP-seq). The RT-qPCR experiments demonstrated that RNA-seq is a reliable platform to discover lncRNAs. This set of curated lncRNAs with improved annotations can serve as an important resource for investigating the function of lncRNAs in D. melanogaster.
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Drosophila melanogaster/genética , RNA Longo não Codificante/genética , Animais , Cromatina/genética , Imunoprecipitação da Cromatina , Anotação de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNARESUMO
Antrodia cinnamomea is a scarce, epiphyte, host-specific, brown-rot fungus that produces diverse bioactive compounds with potent biological activity. Natural wild-type fruiting bodies of A. cinnamomea are rare and highly valued, but their artificial culture poses challenges. Triterpenoids are a group of secondary metabolites that contribute to the bioactivities of A. cinnamomea. 2,3-Oxidosqualene cyclase (OSC) is a key enzyme in triterpenoid biosynthesis, which converts 2,3-oxidosqualene (OS) into polycyclic triterpenoids. In this study, we isolated a 2,3-oxidosqualene cyclase gene from A. cinnamomea with degenerate primers and designated it as AcOSC. The full length AcOSC cDNA was subcloned into a yeast expression vector, and AcOSC activity was confirmed. RT-PCR results showed that AcOSC expression was highest in the wild-type fruiting body and correlated with a higher concentration of triterpenoids. Agrobacterium-mediated gene transformation was conducted to enhance the triterpenoid synthesis capacity of the cultured mycelium. Metabolite profiling was conducted by LC-MS/MS and principal component analysis (PCA). The compositions and contents of metabolites in the AcOSC transgenic lines were different from those in the wild-type mycelium and vector control. The levels of two important triterpenoids, dehydrosulphurenic acid (DSA) and dehydroeburicoic acid (DEA), were increased in A. cinnamomea oxidosqualene cyclase overexpression strains compared to controls. In summary an Agrobacterium-mediated gene transformation procedure was established that successfully increased the level of transgene expression and enhanced the triterpenoid content in cultured A. cinnamomea.
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Antrodia/genética , Transferases Intramoleculares/isolamento & purificação , Triterpenos/metabolismo , Antrodia/química , Carpóforos/química , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Estrutura Molecular , Micélio , Esqualeno/análogos & derivados , Esqualeno/química , TaiwanRESUMO
Antrodia cinnamomea, a precious, host-specific brown-rot fungus that has been used as a folk medicine in Taiwan for centuries is known to have diverse bioactive compounds with potent pharmaceutical activity. In this study, different fermentation states of A. cinnamomea (wild-type fruiting bodies and liquid cultured mycelium) were sequenced using the next-generation sequencing (NGS) technique. A 45.58 Mb genome encoding 6,522 predicted genes was obtained. High quality reads were assembled into a total of 13,109 unigenes. Using a previously constructed pipeline to search for microRNAs (miRNAs), we then identified 4 predicted conserved miRNA and 63 novel predicted miRNA-like small RNA (milRNA) candidates. Target prediction revealed several interesting proteins involved in tri-terpenoid synthesis, mating type recognition, chemical or physical sensory protein and transporters predicted to be regulated by the miRNAs and milRNAs.
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Antrodia/crescimento & desenvolvimento , Antrodia/genética , MicroRNAs/genética , RNA Fúngico/genética , Antrodia/metabolismo , Sequência de Bases , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação Fúngica da Expressão Gênica , Ontologia Genética , Genoma Fúngico , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/química , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Fúngico/químicaRESUMO
OBJECTIVES: A recent genome-wide association study or GWAS identified that anthrax roxin receptor 2 (ANTXR2) was one of the risk loci for ankylosing spondylitis (AS). Previous study also showed that ANTXR2 could potentially affect new bone formation. This study aimed to investigate the possible mechanisms of ANTXR2 involved in AS pathogenesis. METHODS: The expression level of ANTXR2 and miR-124 in peripheral blood was detected by quantitative real-time polymerase chain reaction or qRT-PCR. ANTXR2 was predicted to be a target gene of miR-124 by TargetScan, which was confirmed by luciferase reporter assays. Western blot analysis was used to further investigate the effect of miR-124 on c-Jun N-terminal kinase (JNK) activation and evaluate the activated status of autophagy. RESULTS: We evidenced that ANTXR2 was downregulated and miR-124 was upregulated in peripheral blood from AS patients. Intriguingly, miR-124 targeted ANTXR2 and overexpression of miR-124 in Jurkat cells notably inhibited ANTXR2 expression. ANTXR2 inhibition by miR-124 promoted JNK activation and induced autophagy. CONCLUSIONS: Our results suggested that miR-124 might induce autophagy to participate in AS by targeting ANTXR2, which might be implicated in pathological process of AS.
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DNA/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Receptores de Peptídeos/genética , Espondilite Anquilosante/genética , Adulto , Western Blotting , Células Cultivadas , Citometria de Fluxo , Estudo de Associação Genômica Ampla , Humanos , Masculino , MicroRNAs/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Peptídeos/biossíntese , Espondilite Anquilosante/diagnóstico , Espondilite Anquilosante/metabolismo , Tomografia Computadorizada por Raios XRESUMO
Antrodia cinnamomea, a polyporus mushroom of Taiwan, has long been used as a remedy for cancer, hypertension, and hangover, with an annual market of over $100 million (US) in Taiwan. We obtained a 32.15-Mb genome draft containing 9,254 genes. Genome ontology enrichment and pathway analyses shed light on sexual development and the biosynthesis of sesquiterpenoids, triterpenoids, ergostanes, antroquinonol, and antrocamphin. We identified genes differentially expressed between mycelium and fruiting body and 242 proteins in the mevalonate pathway, terpenoid pathways, cytochrome P450s, and polyketide synthases, which may contribute to the production of medicinal secondary metabolites. Genes of secondary metabolite biosynthetic pathways showed expression enrichment for tissue-specific compounds, including 14-α-demethylase (CYP51F1) in fruiting body for converting lanostane to ergostane triterpenoids, coenzymes Q (COQ) for antroquinonol biosynthesis in mycelium, and polyketide synthase for antrocamphin biosynthesis in fruiting body. Our data will be useful for developing a strategy to increase the production of useful metabolites.
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Antrodia/metabolismo , Carpóforos/metabolismo , Proteínas Fúngicas/metabolismo , Micélio/metabolismo , Esterol 14-Desmetilase/metabolismo , Transcriptoma/fisiologia , Antrodia/genética , Carpóforos/genética , Proteínas Fúngicas/genética , Perfilação da Expressão Gênica , Genômica , Humanos , Micélio/genética , Esterol 14-Desmetilase/genética , TaiwanRESUMO
BACKGROUND: Mutations or single nucleotide polymorphism (SNP) of relevant genes may affect the risk and prognosis of malignancies. The purpose of this study was to analyze whether the KRAS polymorphisms and mutations can be useful prognostic or risk markers in oral squamous cell carcinoma (OSCC). METHODS: DNA was extracted from tumor tissues of 47 patients with OSCC and blood cells of 84 normal controls and subjected to sequencing for the KRAS. RESULTS: No mutation in the KRAS was found in 47 OSCC samples. However, 2 polymorphisms (rs1137282 and rs712) were detected. Individuals with KRAS SNP rs712 genotypes of G/T or T/T have a reduced risk for OSCC than those with genotype G/G (hazard ratio [HR], 0.26; 95% confidence interval [CI], 0.10-0.60; p = .004). The overall survival between different SNPs were not statistically significant (p = .147 for rs1137282 and p = .202 for rs712). CONCLUSION: These data demonstrate a role for rs712 polymorphism of the KRAS in susceptibility of OSCC.
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Carcinoma de Células Escamosas/genética , Neoplasias Bucais/genética , Mutação , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas/genética , Proteínas ras/genética , Carcinoma de Células Escamosas/mortalidade , Estudos de Casos e Controles , Primers do DNA , Éxons , Feminino , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/mortalidade , Reação em Cadeia da Polimerase , Prognóstico , Proteínas Proto-Oncogênicas p21(ras)RESUMO
Medicinal mushroom Antrodia cinnamomea is a higher Basidiomycetes endemic to Taiwan, where it is commonly used as a traditional folk medicine. It is well known for its multiple biologic activities and its potential for commercial development. Here, ten full lengths of cytochrome P450 (CYP) genes (ac-1 to ac-10) from A. cinnamomea were cloned and identified. With the exception of ac-3 and ac-8, which will probably be assigned as new CYP families, these genes had more than 40% amino acid identity and close evolutionary relationships to known CYPs. Among the ten genes, only Ac-7 did not possess a transmembrane domain but had an N-terminal signal peptide, so it was considered a novel extracellular CYP. The ten A. cinnamomea CYPs had different expression profiles in different growth conditions. In general, they were strongly expressed during the formation of fruiting bodies, especially in natural basidiomycetes. The expression of six CYPs of A. cinnamomea (ac-1 to ac-3 and ac-5 to ac-7) were strictly inhibited in the mycelia cell type. It was therefore concluded that these CYPs are most active in the fruiting bodies of A. cinnamomea.
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Antrodia/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Carpóforos/crescimento & desenvolvimento , Carpóforos/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Sequência de Aminoácidos , Clonagem Molecular , DNA Complementar/genética , DNA Fúngico/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Modelos Moleculares , Anotação de Sequência Molecular , Filogeografia , Conformação Proteica , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The EGFR is an oncogene known to be involved in the development and progression of many cancers. It has been reported that the expression of EGFR and EGFR CA simple sequence repeat 1 (CA SSR1) repeat numbers in tumors can be useful prognostic factors in several cancer types. The objective of the present study was to analyze whether the EGFR polymorphism can be a useful prognostic factor in OSCC in the Taiwanese population. OSCC tissues were collected from 47 patients by surgical excision. The genotyping of EGFR were performed with the ABI Prism 3100 Genetic Analyzer. OSCC patients had a tendency toward an allelic imbalance of CA SSR1. The results also suggested that OSCC patients who were homozygous for CA SSR1 had a poorer prognosis than those who were heterozygous (P<0.001). Besides, patients with an allelic imbalance of CA SSR1 had significantly lower overall and disease free survival rates than those without, using the Kaplan-Meier method (P<0.001). This suggests that the status of CA SSR1 has the potential to be a useful prognosis factor in OSCC.
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Carcinoma de Células Escamosas/genética , Genes erbB-1/genética , Neoplasias Bucais/genética , Polimorfismo Genético , Adulto , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Bucais/mortalidade , Neoplasias Bucais/patologia , Prognóstico , TaiwanRESUMO
Antrodia cinnamomea is peculiar to Taiwan. It only grows on one host and is highly valued as an important component of several traditional Chinese medicines. In this study, the different protein expression profiles of artificially cultivated vegetative mycelium and wild-type basidiomatal fruiting bodies were compared and unique protein spots from wild-type basidiomatal fruiting body were investigated using 2D polyacrylamide gel electrophoresis and LC-MS/MS protein identification. Most of the wild-type proteins not seen in the artificially cultivated mycelium were associated to function in metabolism, cell stress, ROS scavenging, and cell growth. Several proteins from wild-type basidiomes, such as catalase, aryl-alcohol dehydrogenase, S-adenosyl-L-homocysteine hydrolase, intradiol dioxygenase, haloacid dyhydrogenase, alpha- and beta-form tubulin, prohibitin, septin, chaperone, and HSP90 ATPase, showed higher expression than those from artificially cultured mycelium at the mRNA level.