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1.
J Immunother Cancer ; 12(4)2024 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-38658031

RESUMO

BACKGROUND: Tigilanol tiglate (TT) is a protein kinase C (PKC)/C1 domain activator currently being developed as an intralesional agent for the treatment of various (sub)cutaneous malignancies. Previous work has shown that intratumoral (I.T.) injection of TT causes vascular disruption with concomitant tumor ablation in several preclinical models of cancer, in addition to various (sub)cutaneous tumors presenting in the veterinary clinic. TT has completed Phase I dose escalation trials, with some patients showing signs of abscopal effects. However, the exact molecular details underpinning its mechanism of action (MoA), together with its immunotherapeutic potential in oncology remain unclear. METHODS: A combination of microscopy, luciferase assays, immunofluorescence, immunoblotting, subcellular fractionation, intracellular ATP assays, phagocytosis assays and mixed lymphocyte reactions were used to probe the MoA of TT in vitro. In vivo studies with TT used MM649 xenograft, CT-26 and immune checkpoint inhibitor refractory B16-F10-OVA tumor bearing mice, the latter with or without anti-programmed cell death 1 (PD-1)/anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) mAb treatment. The effect of TT at injected and non-injected tumors was also assessed. RESULTS: Here, we show that TT induces the death of endothelial and cancer cells at therapeutically relevant concentrations via a caspase/gasdermin E-dependent pyroptopic pathway. At therapeutic doses, our data demonstrate that TT acts as a lipotoxin, binding to and promoting mitochondrial/endoplasmic reticulum (ER) dysfunction (leading to unfolded protein responsemt/ER upregulation) with subsequent ATP depletion, organelle swelling, caspase activation, gasdermin E cleavage and induction of terminal necrosis. Consistent with binding to ER membranes, we found that TT treatment promoted activation of the integrated stress response together with the release/externalization of damage-associated molecular patterns (HMGB1, ATP, calreticulin) from cancer cells in vitro and in vivo, characteristics indicative of immunogenic cell death (ICD). Confirmation of ICD in vivo was obtained through vaccination and rechallenge experiments using CT-26 colon carcinoma tumor bearing mice. Furthermore, TT also reduced tumor volume, induced immune cell infiltration, as well as improved survival in B16-F10-OVA tumor bearing mice when combined with immune checkpoint blockade. CONCLUSIONS: These data demonstrate that TT is an oncolytic small molecule with multiple targets and confirms that cell death induced by this compound has the potential to augment antitumor responses to immunotherapy.


Assuntos
Inibidores de Checkpoint Imunológico , Morte Celular Imunogênica , Animais , Camundongos , Morte Celular Imunogênica/efeitos dos fármacos , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Linhagem Celular Tumoral , Feminino , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/terapia
2.
Molecules ; 27(13)2022 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-35807225

RESUMO

Tigilanol tiglate (EBC-46) is a small-molecule natural product under development for the treatment of cancers in humans and companion animals. The drug is currently produced by purification from the Australian rainforest tree Fontainea picrosperma (Euphorbiaceae). As part of a selective-breeding program to increase EBC-46 yield from F. picrosperma plantations, we investigated potential gene biomarkers associated with biosynthesis of EBC-46. Initially, we identified individual plants that were either high (>0.039%) or low EBC-46 (<0.008%) producers, then assessed their differentially expressed genes within the leaves and roots of these two groups by quantitative RNA sequencing. Compared to low EBC-46 producers, high-EBC-46-producing plants were found to have 145 upregulated genes and 101 downregulated genes in leaves and 53 upregulated genes and 82 downregulated genes in roots. Most of these genes were functionally associated with defence, transport, and biosynthesis. Genes identified as expressed exclusively in either the high or low EBC-46-producing plants were further validated by quantitative PCR, showing that cytochrome P450 94C1 in leaves and early response dehydration 7.1 and 2-alkenal reductase in roots were consistently and significantly upregulated in high-EBC-46 producers. In summary, this study has identified biomarker genes that may be used in the selective breeding of F. picrosperma.


Assuntos
Diterpenos , Euphorbiaceae , Marcadores Genéticos , Diterpenos/química , Ésteres/química , Euphorbiaceae/química , Euphorbiaceae/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Melhoramento Vegetal , Folhas de Planta/química , Folhas de Planta/genética , Raízes de Plantas/química , Raízes de Plantas/genética
3.
Mol Ecol Resour ; 15(4): 953-66, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25494938

RESUMO

A key strategy to reduce coral loss is the development of effective control method for the corallivorous crown-of-thorns sea star (Acanthaster planci), an omnipresent scourge and threat to the biodiversity of reefs in the Indo-Pacific region. Limited genetic resources are available for this highly fecund species. In this study, we explored one aspect at the heart of A. planci outbreaks, the male reproductive system. Using high-throughput sequencing technology, we report for first time the production of a comprehensive transcriptomic data set for the testes of A. placni that can aid in understanding the molecular mechanisms involved in A. planci spermatogenesis and fertilization. Through de novo transcriptome sequencing, we produced 52 965 998 raw reads corresponding to 4.76 Gb clean read data. From this, 243 870 contigs were assembled with Trinity and used to construct 92 792 unigenes. Distinct genes were then annotated with blastx yielding 30 810 unigenes above the cut-off E-value set at 10(-5) , with ESTScan database query analyses yielding up to 5366 unigenes to known hits. The identification of genes directly involved in sperm development (DEAD-box family proteins), motility, fertilization and signalling (Bindin/Speract receptor) are also discussed.


Assuntos
Espermatogênese , Estrelas-do-Mar/genética , Estrelas-do-Mar/fisiologia , Transcriptoma , Animais , Masculino , Anotação de Sequência Molecular , Dados de Sequência Molecular , Análise de Sequência de DNA , Testículo
4.
BMC Complement Altern Med ; 13: 60, 2013 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-23497063

RESUMO

BACKGROUND: Phytochemical products have a critical role in the drug discovery process. This promising possibility, however, necessitates the need to confirm their scientific verification before use. Hence, this study aims to evaluate (1) the antioxidant activity, (2) cytotoxicity potential, and (3) the effect on ultrastructural alteration in gastric cancer cell lines through exposure to fractions of three local Northeastern Thai edible plants. METHODS: Plants, Syzygium gratum, Justicia gangetica and Limnocharis flava were extracted with ethyl acetate, and each crude extract analysed for their total phenolics content by Folin-Ciocalteu method. Their antioxidant activity was assessed using the ABTS system. The extracts were then assayed for cytotoxicity on two gastric cancer cell lines Kato-III and NUGC-4, and compared with Hs27 fibroblasts as a control using the MTT assay. The cell viability (%), IC50 values, as well as the ultrastructural alterations were evaluated after treatment with one way analysis of variance (ANOVA). RESULTS: The total phenolic values of the ethyl acetate extracts were well correlated with the antioxidant capacity, with extracted product of S. gratum displaying the highest level of antioxidant activity (a 10-fold greater response) over J. gangetica and L. flava respectively. Exposure of S. gratum and J. gangetica extracts to normal cell lines (Hs27) resulted in marginal cytotoxicity effects. However, through a dose-dependent assay S. gratum and J. gangetica extracts produced cytotoxicological effects in just over 75 percent of Kato-III and NUGC-4 cell lines. In addition, apoptotic characteristic was shown under TEM in both cancer cell lines with these two extracts, whereas characteristics of autophagy was found in cell lines after post exposure to extracts from L. flava. CONCLUSIONS: From these three plants, S. gratum had the highest contents of phenolic compounds and antioxidant capacity. All of them found to contain compound(s) with cytotoxicity in vitro on cancer cells but not on normal cell lines as resolved in tissue culture and ultrastructural analysis. This is the first report to show the effect on cellular alteration as apoptosis of an ethyl acetate extract of S. gratum and J. gangetica. Further studies are now focused on individual isolates and their function, prioritizing on S. gratum and J. gangetica for the development of novel therapeutics and combatants against cancer.


Assuntos
Acanthaceae/química , Alismataceae/química , Antioxidantes/farmacologia , Fenóis/farmacologia , Extratos Vegetais/farmacologia , Plantas Comestíveis/química , Neoplasias Gástricas/ultraestrutura , Syzygium/química , Proliferação de Células/efeitos dos fármacos , Humanos , Microscopia Eletrônica de Transmissão , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/fisiopatologia , Tailândia
5.
Artigo em Inglês | MEDLINE | ID: mdl-23103673

RESUMO

The crustacean X-organ-sinus gland (XO-SG) complex controls molt-inhibiting hormone (MIH) production, although extra expression sites for MIH have been postulated. Therefore, to explore the expression of MIH and distinguish between the crustacean hyperglycemic hormone (CHH) superfamily, and MIH immunoreactive sites (ir) in the central nervous system (CNS), we cloned a CHH gene sequence for the crab Portunus pelagicus (Ppel-CHH), and compared it with crab CHH-type I and II peptides. Employing multiple sequence alignments and phylogenic analysis, the mature Ppel-CHH peptide exhibited residues common to both CHH-type I and II peptides, and a high degree of identity to the type-I group, but little homology between Ppel-CHH and Ppel-MIH (a type II peptide). This sequence identification then allowed for the use of MIH antisera to further confirm the identity and existence of a MIH-ir 9kDa protein in all neural organs tested by Western blotting, and through immunohistochemistry, MIH-ir in the XO, optic nerve, neuronal cluster 17 of the supraesophageal ganglion, the ventral nerve cord, and cell cluster 22 of the thoracic ganglion. The presence of MIH protein within such a diversity of sites in the CNS, and external to the XO-SG, raises new questions concerning the established mode of MIH action.


Assuntos
Proteínas de Artrópodes , Braquiúros , Sistema Nervoso Central/metabolismo , Hormônios de Invertebrado , Proteínas do Tecido Nervoso , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Sequência de Bases , Braquiúros/genética , Braquiúros/metabolismo , Clonagem Molecular , Crustáceos , Hormônios de Invertebrado/genética , Hormônios de Invertebrado/metabolismo , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Peptídeos/genética , Filogenia , Alinhamento de Sequência
6.
Microsc Res Tech ; 75(10): 1402-15, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22648947

RESUMO

The ultrastructure of oogenesis in Macrobrachium rosenbergii, with reference to vitellogenesis, has not been reported. We used light and electron microscopy, as well as vitellin (Vn) purification and antibody production, to study the temporal and spatial production of Vn in the ovary by immunofluorescence. Histologically, the ovary is subdivided into cone-shaped ovarian pouches with a central core containing layers of oogonia. These divide to produce oocytes that migrate outwardly and differentiate into mature oocytes. During the course of differentiation, oocytes undergo modifications, including the rearrangement of nuclear chromatin, the accumulation of ribosomes, rough endoplasmic reticulum (RER), and lipid, and the formation of secretory and yolk granules, resulting in four stages. Ultrastructurally, early previtellogenic oocytes (Oc(1)) are characterized by the accumulation of new ribosomal aggregates, translocated from the nucleus. Late previtellogenic oocytes (Oc(2)) show nuclear heterochromatin with a "clock face" pattern, the presence of RER, and three types of secretory granules. Follicular cells occupy the intercellular spaces and surround the Oc(2). Early vitellogenic oocytes (Oc(3)) are larger, with nuclei containing predominantly decondensed euchromatin, and cytoplasm with yolk and secretory granules, and few lipid droplets. Late vitellogenic oocytes (Oc(4)) are characterized by completely euchromatic nuclei, an indistinct plasma membrane, yolk platelets and secretory granules, and abundant lipid. Vitellogenin (Vg) in ovaries of M. rosenbergii consist of two main bands at MW 90 and 102 kDa. Our data indicates that Vn is present, and probably synthesized in Oc(3) and Oc(4), but there may be some undetected exogenous Vg production.


Assuntos
Palaemonidae/ultraestrutura , Animais , Microscopia/métodos , Oócitos/metabolismo , Oócitos/ultraestrutura , Vitelinas/análise , Vitelinas/isolamento & purificação , Vitelogênese
7.
Tissue Cell ; 42(3): 137-50, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20413138

RESUMO

Spermatogenesis in the blue swimming crab, Portunus pelagicus, is described by light and electron microscopy. The testis is composed of anterior (AT) and posterior (PT) lobes, that are partitioned into lobules by connective tissue trabecula, and further divided into zones (germinal, transformation and evacuation), each with various stages of cellular differentiation. The vas deferens is classified into three distinct regions: anterior (AVD), median (MVD), and posterior (PVD), on the presence of spermatophores and two secretions, termed substance I and II. Based on the degree and patterns of heterochromatin, spermatogenesis is classified into 13 stages: two spermatogonia (SgA and SgB), six primary spermatocytes (leptotene, zygotene, pachytene, diplotene, diakinesis, and metaphase), a secondary spermatocyte (SSc), three spermatids (St 1-3), and a mature spermatozoon. Spermatid stages are differentiated by chromatin decondensation and the formation of an acrosomal complex, which is unique to brachyurans. Mature spermatozoa are aflagellated, and have a nuclear projection and a spherical acrosome. AUT-PAGE and Western blots show that, during chromatin decondensation, there is a reduction of most histones, with only small amounts of H2B and H3 remaining in mature spermatozoa.


Assuntos
Braquiúros/citologia , Braquiúros/metabolismo , Núcleo Celular/metabolismo , Histonas/metabolismo , Maturação do Esperma/fisiologia , Espermatozoides/metabolismo , Natação/fisiologia , Acrossomo/metabolismo , Animais , Western Blotting , Núcleo Celular/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Técnica Indireta de Fluorescência para Anticorpo , Masculino , Espermatozoides/citologia , Espermatozoides/ultraestrutura , Ducto Deferente/citologia , Ducto Deferente/metabolismo , Ducto Deferente/ultraestrutura
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