RESUMO
Pancreatic cancer remains one of the deadliest cancers with extremely high mortality rate, and the number of cases is expected to steadily increase with time. Pancreatic cancer is refractory to conventional cancer treatment options, like chemotherapy and radiotherapy, and commercialized immunotherapeutics, owing to its immunosuppressive and desmoplastic phenotype. Due to these reasons, development of an innovative treatment option that can overcome these challenges posed by the pancreatic tumor microenvironment (TME) is in an urgent need. The present review aims to summarize the evolution of oncolytic adenovirus (oAd) engineering and usage as therapeutics (either monotherapy or combination therapy) over the last decade to overcome these hurdles to instigate a potent antitumor effect against desmoplastic and immunosuppressive pancreatic cancer.
Assuntos
Terapia Viral Oncolítica , Vírus Oncolíticos , Neoplasias Pancreáticas , Humanos , Vírus Oncolíticos/genética , Adenoviridae/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/patologia , Linhagem Celular Tumoral , Microambiente TumoralRESUMO
BACKGROUND: While immune checkpoint inhibitors are becoming a standard of care for multiple types of cancer, the majority of patients do not respond to this form of immunotherapy. New approaches are required to overcome resistance to immunotherapies. METHODS: We investigated the effects of adenoviral p53 (Ad-p53) gene therapy in combination with immune checkpoint inhibitors and selective IL2 or IL15 CD122/132 agonists in the aggressive B16F10 tumor model resistant to immunotherapies. To assess potential mechanisms of action, pre- and post- Ad-p53 treatment biopsies were evaluated for changes in gene-expression profiles by Nanostring IO 360 assays. RESULTS: The substantial synergy of "triplet" Ad-p53 + CD122/132 + anti-PD-1 therapy resulted in potential curative effects associated with the complete tumor remissions of both the primary and contralateral tumors. Interestingly, contralateral tumors, which were not injected with Ad-p53 showed robust abscopal effects resulting in statistically significant decreases in tumor size and increased survival (p < 0.001). None of the monotherapies or doublet treatments induced the complete tumor regressions. Ad-p53 treatment increased interferon, CD8+ T cell, immuno-proteosome antigen presentation, and tumor inflammation gene signatures. Ad-p53 treatment also decreased immune-suppressive TGF-beta, beta-catenin, macrophage, and endothelium gene signatures, which may contribute to enhanced immune checkpoint inhibitor (CPI) efficacy. Unexpectedly, a number of previously unidentified, strongly p53 downregulated genes associated with stromal pathways and IL10 expression identified novel anticancer therapeutic applications. CONCLUSIONS: These results imply the ability of Ad-p53 to induce efficacious local and systemic antitumor immune responses with the potential to reverse resistance to immune checkpoint inhibitor therapy when combined with CD122/132 agonists and immune checkpoint blockade. Our findings further imply that Ad-p53 has multiple complementary immune mechanisms of action, which support future clinical evaluation of triplet Ad-p53, CD122/132 agonist, and immune checkpoint inhibitor combination treatment.
Assuntos
Inibidores de Checkpoint Imunológico , Neoplasias , Terapia Genética , Humanos , Imunoterapia/métodos , Neoplasias/tratamento farmacológico , Neoplasias/terapia , Microambiente Tumoral , Proteína Supressora de Tumor p53/genéticaRESUMO
Immunogenic cell death (ICD) is distinguished by the release of tumor-associated antigens (TAAs) and danger-associated molecular patterns (DAMPs). This cell death has been studied in the field of cancer immunotherapy due to the ability of ICD to induce antitumor immunity. Herein, endoplasmic reticulum (ER) stress-mediated ICD inducing fluorinated mitochondria-disrupting helical polypeptides (MDHPs) are reported. The fluorination of the polypeptide provides a high helical structure and potent anticancer ability. This helical polypeptide destabilizes the mitochondrial outer membrane, leading to the overproduction of intracellular reactive oxygen species (ROS) and apoptosis. In addition, this oxidative stress triggers ER stress-mediated ICD. The in vivo results show that cotreatment of fluorinated MDHP and antiprogrammed death-ligand 1 antibodies (αPD-L1) significantly regresses tumor growth and prevents metastasis to the lungs by activating the cytotoxic T cell response and alleviating the immunosuppressive tumor microenvironment. These results indicate that fluorinated MDHP synergizes with the immune checkpoint blockade therapy to eliminate established tumors and to elicit antitumor immune responses.
Assuntos
Adenocarcinoma/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Inibidores de Checkpoint Imunológico/uso terapêutico , Morte Celular Imunogênica/efeitos dos fármacos , Mitocôndrias/metabolismo , Peptídeos/metabolismo , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Terapia Combinada , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Halogenação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T Citotóxicos/efeitos dos fármacosRESUMO
ABSTRACT: Accumulation of excessive extracellular matrix (ECM) and aberrant transforming growth factor ß (TGF-ß) signaling pathway function can be potential therapeutic targets for keloid treatment. In this study, we examined the antifibrotic effect of metformin as a suppressor of TGF-ß signaling pathways in human dermal fibroblasts (HDFs) and keloid spheroids. Human dermal fibroblasts were stimulated with TGF-ß (10 ng/mL) and treated with metformin (10 mM). The mRNA and protein expression of ECM components were evaluated by quantitative polymerase chain reaction, western blot, and immunofluorescence assay. In addition, we immunohistochemically examined the expression levels of ECM proteins in keloid spheroids. After addition of metformin (10 mM), collagen types I and III and elastin mRNA levels were significantly decreased in HDFs, and collagen type I protein level was significantly decreased. In addition, the expression levels of collagen types I and III, fibronectin, and elastin were significantly reduced in keloid spheroids after treatment with metformin (100 mM). Collagen types I and III and p-Smad2/3 complex proteins were decreased in metformin-treated keloid spheroids. These findings indicated that metformin inhibits the expression of ECM components in TGF-ß-stimulated HDFs and keloid spheroids. Therefore, we suggest the potential of metformin as an effective agent for the treatment of keloids.
Assuntos
Queloide , Metformina , Células Cultivadas , Fibroblastos/patologia , Fibrose , Humanos , Queloide/tratamento farmacológico , Queloide/patologia , Metformina/farmacologia , Fator de Crescimento Transformador beta , Fator de Crescimento Transformador beta1RESUMO
BACKGROUND: Currently, several antibody (Ab)-based therapies have shown excellent therapeutic effects in the clinic. Nonetheless, Ab penetration into tumor tissues is limited due to abnormal vasculature, tumor interstitial pressure, and excessive extracellular matrix (ECM) accumulation, thus demanding novel strategies to overcome these barriers. METHODS: The intratumoral distribution of therapeutic Abs were detected by fluorescence microscopy or positron emission tomography in both human gastric xenograft and syngeneic pancreatic hamster tumor models. The antitumor efficacy by combination of oncolytic adenovirus (Ad), which coexpresses relaxin (RLX), interleukin (IL)-12, and granulocyte macrophage colony-stimulating factor (GM-CSF) (oAd/IL12/GM-RLX) and antibody against the programmed cell death protein 1 (αPD-1) was examined in hamster subcutaneous and orthotopic pancreatic tumor models. The immunological aspects of these combination therapy regimen were assessed by flow cytometry or immunohistochemistry in subcutaneous hamster tumor models. RESULTS: Relaxin-expressing oncolytic Ad effectively degraded tumor ECM and enhanced the tumor penetration of trastuzumab in comparison with trastuzumab monotherapy. Based on these results, an oAd/IL12/GM-RLX was used to enhance the potency of immune checkpoint blockade. The combination of the oAd/IL12/GM-RLX and αPD-1 promoted a concomitant degradation of the tumor ECM and amelioration of the immunosuppressive tumor niches, ultimately enhanced intratumoral infiltration of both αPD-1 and activated T cells. Of note, the combination therapy was able to elicit a potent and durable antitumor immune response against cold tumors that were refractory to immune checkpoint inhibitor monotherapy. CONCLUSIONS: Our findings are the first to demonstrate that expression of four genes (IL-12p35, IL-12p40, GM-CSF, and RLX) mediated by a single oncolytic Ad vector can promote remodeling of both physical and immunological aspects of the tumor niches to overcome the major limitations of Ab-based therapies that have emerged in recent clinical trials.
Assuntos
Adenoviridae/genética , Terapia Viral Oncolítica/métodos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Relaxina/uso terapêutico , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Relaxina/farmacologiaRESUMO
An adenoviral vector (Ad) expressing a Wnt decoy receptor (sLRP6E1E2) is known to induce an anti-fibrotic effect by inhibiting Wnt signaling. We evaluated its effects in vivo using pig models and attempted to introduce an alginate gel-matrix system to prolong the effect of the Ad. Transduction efficiency as to the biological activity of Ad in different forms was evaluated. Then, 50 days after the formation of full-thickness skin defects on the backs of Yorkshire pigs, scars were treated with each form of Ad. Therapeutic efficacy and various factors influencing scar formation and collagen rearrangement were analyzed. Inflammatory cell infiltration within the scar tissues was also evaluated. Decoy Wnt receptor (sLRP6E1E2)-expressing adenovirus treatment improved scar quality in a pig model. Loading this construct in alginate gel allows sustained virus release into local tissues and prolongs Ad activity, thus maintaining its therapeutic effect longer in vivo.
Assuntos
Adenoviridae/genética , Alginatos/química , Cicatriz/terapia , Terapia Genética/métodos , Receptores Wnt/genética , Animais , Colágeno/genética , Colágeno/metabolismo , Técnicas de Transferência de Genes , Hidrogéis/química , Receptores Wnt/metabolismo , Pele/metabolismo , Suínos , Via de Sinalização WntRESUMO
Chirality is ubiquitous in nature and hard-wired into every biological system. Despite the prevalence of chirality in biological systems, controlling biomaterial chirality to influence interactions with cells has only recently been explored. Chiral-engineered supraparticles (SPs) that interact differentially with cells and proteins depending on their handedness are presented. SPs coordinated with d-chirality demonstrate greater than threefold enhanced cell membrane penetration in breast, cervical, and multiple myeloma cancer cells. Quartz crystal microbalance with dissipation and isothermal titration calorimetry measurements reveal the mechanism of these chiral-specific interactions. Thermodynamically, d-SPs show more stable adhesion to lipid layers composed of phospholipids and cholesterol compared to l-SPs. In vivo, d-SPs exhibit superior stability and longer biological half-lives likely due to opposite chirality and thus protection from endogenous proteins including proteases. This work shows that incorporating d-chirality into nanosystems enhances uptake by cancer cells and prolonged in vivo stability in circulation, providing support for the importance of chirality in biomaterials. Thus, chiral nanosystems may have the potential to provide a new level of control for drug delivery systems, tumor detection markers, biosensors, and other biomaterial-based devices.
Assuntos
Materiais Biocompatíveis/química , Nanomedicina , Materiais Biocompatíveis/farmacologia , Técnicas Biossensoriais/métodos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cisteína/química , Meia-Vida , Humanos , Bicamadas Lipídicas/metabolismo , Lipídeos/química , Microscopia Confocal , Polietilenoglicóis/química , Técnicas de Microbalança de Cristal de Quartzo , Estereoisomerismo , TermodinâmicaRESUMO
PURPOSE: Relaxin (RLX) is a transforming growth factor-ß1 (TGF-ß1) antagonist that is believed to function as a potent collagen re-arranger and a major suppressor of extracellular matrix components. Adenoviruses (Ads) are accepted vectors for cancer gene therapy. However, repeated treatments of Ad are limited by short-term biological activity in vivo. The efficacy of sustained RLX expression to scar remodeling was assessed using an injectable alginate gel-matrix system. MATERIALS AND METHODS: Pig scar tissue was treated with relaxin-expressing Ad loaded in alginate gel (gel/Ad-RLX). Surface areas, color, and pliability of scars were compared, and various factors influencing scar formation and collagen arrangement were analyzed. RESULTS: Gel/Ad-RLX decreased scar size, color index, and pliability. Immunohistochemistry showed decreased levels of major extracellular matrix proteins in the gel/Ad-RLX-treated group. Furthermore, treatment with gel/Ad-RLX reduced expression of tissue inhibitor of metalloproteinase-1 and alpha-smooth muscle actin and markedly increased expression of matrix metalloproteinase-1 in pig scar tissues. Gel/Ad-RLX also significantly downregulated TGF-ß1 and upregulated TGF-ß3 mRNAs in pig scar tissues. CONCLUSION: These results support a prominent role for RLX in scar remodeling and suggest that gel/Ad-RLX may have therapeutic effects on scar formation.
Assuntos
Adenoviridae/genética , Adenoviridae/metabolismo , Alginatos , Cicatriz/terapia , Colágeno/metabolismo , Relaxina/genética , Relaxina/metabolismo , Animais , Cicatriz/metabolismo , Matriz Extracelular/metabolismo , Metaloproteinase 1 da Matriz , Relaxina/farmacologia , Suínos , Inibidor Tecidual de Metaloproteinase-1 , Fator de Crescimento Transformador beta1RESUMO
High-mobility group box 1 (HMGB1) protein acts as a DNA chaperone for nuclear homeostasis. It translocates into the cytosol and is secreted into extracellular spaces, triggering proinflammatory cytokines and acting as a mediator in fibrosis. We determined whether HMGB1 plays a role in normal dermal fibrosis and keloid, and is involved with transforming growth factor ß. We investigated the translocation and active release of HMGB1 from normal dermal fibroblasts under lipopolysaccharide stimuli, and the redistribution of nuclear HMGB1 into the cytoplasm of keloid fibroblasts. HMGB1 and its effector toll-like receptors and receptors for advanced glycation end product proteins are actively expressed in keloid tissues. Exogenous HMGB1 can induce the proliferation of human dermal fibroblasts, and could act as a profibrogenic molecule to produce collagen, decrease MMP-1, and increase TIMP-1 mRNA expression. Moreover, administration of HMGB1 increased the expression level of TGF-ß1 and internal signaling molecules, such as Smad 2 and 3, phosphorylated Smad 2/3 complex, Erk 1/2, Akt, and NF-κB. Collectively, we demonstrate that HMGB1 treatment increases the expression level of collagen types I and III, elastin, and fibronectin in dermal spheroid cultures, thus making HMGB1 a promising therapeutic target for treatment of profibrogenic diseases.
Assuntos
Fibroblastos/metabolismo , Proteína HMGB1/metabolismo , Queloide/metabolismo , Queloide/patologia , Pele/citologia , Fibroblastos/citologia , Regulação Enzimológica da Expressão Gênica , Humanos , Espaço Intracelular/metabolismo , Metaloproteinase 1 da Matriz/genética , Metaloproteinase 2 da Matriz/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Inibidor Tecidual de Metaloproteinase-1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Inevitable exposure to ionizing radiation from natural and human-made sources has been increasing over time. After nuclear disasters, such as the Fukushima accident, the public concerns on health risk of radiation exposure because of radioactive contamination of the environment have increased. However, it is very difficult to assess the biological effects of exposure caused by environmental radiation. A reliable and rapid bioassay to monitor the physiological effects of radiation exposure is therefore needed. Here, we quantitatively analyzed the changes in cell shape in Drosophila epidermis after irradiation as a model for biomonitoring of radiation. Interestingly, the number of irregularly shaped epithelial cells was increased by irradiation in a dose-dependent manner. A dose-response curve constructed with the obtained data suggests that the measurement of the number of irregular shaped cell in the epidermis is useful for the assessment of radiation dose. In addition, a comparison of the variation in the different samples and the data scored by different observers showed that our evaluation for cellular morphology was highly reliable and accurate and would, therefore, have immense practical application. Overall, our study suggests that detection of morphological changes in the epithelial cells is one of the efficient ways to quantify the levels of exposure to radioactive radiation from the environment.
Assuntos
Forma Celular/efeitos da radiação , Drosophila/efeitos da radiação , Células Epiteliais/efeitos da radiação , Exposição à Radiação/análise , Monitoramento de Radiação/métodos , Animais , Relação Dose-Resposta à Radiação , Drosophila/ultraestrutura , Células Epiteliais/ultraestrutura , Doses de Radiação , Radiação IonizanteRESUMO
Aberrant activation of the canonical Wingless type (Wnt) signaling pathway plays a key role in the development of hypertrophic scars and keloids, and this aberrant activation of Wnt pathway can be a potential target for the development of novel anti-fibrotic agents. In this study, we evaluated the anti-fibrotic potential of a soluble Wnt decoy receptor (sLRP6E1E2)-expressing non-replicating adenovirus (Ad; dE1-k35/sLRP6E1E2) on human dermal fibroblasts (HDFs), keloid fibroblasts (KFs), and keloid tissue explants. Higher Wnt3a and ß-catenin expression was observed in the keloid region compared to the adjacent normal tissues. The activity of ß-catenin and mRNA expression of type-I and -III collagen were significantly decreased following treatment with dE1-k35/sLRP6E1E2 in HDFs and KFs. The expression of LRP6, ß-catenin, phosphorylated glycogen synthase kinase 3 beta, Smad 2/3 complex, and TGF-ß1 were decreased in Wnt3a- or TGF-ß1-activated HDFs, following administration of dE1-k35/sLRP6E1E2. Moreover, dE1-k35/sLRP6E1E2 markedly inhibited nuclear translocation of both ß-catenin and Smad 2/3 complex. The expression levels of type-I and -III collagen, fibronectin, and elastin were also significantly reduced in keloid tissue explants after treatment with dE1-k35/sLRP6E1E2. These results indicate that Wnt decoy receptor-expressing Ad can degrade extracellular matrix in HDFs, KFs, and primary keloid tissue explants, and thus it may be beneficial for treatment of keloids.
Assuntos
Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Queloide/metabolismo , Receptores Wnt/metabolismo , Adenoviridae/genética , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Derme/citologia , Fibronectinas/genética , Fibronectinas/metabolismo , Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Queloide/genética , Queloide/patologia , Receptores Wnt/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genética , Proteína Wnt3A/genética , Proteína Wnt3A/metabolismo , Proteína Wnt3A/farmacologia , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Mortalin (Mot) is a mitochondrial chaperone of the heat shock protein 70 family and it's pro-proliferative and anti-apoptosis functions could be associated with keloid pathogenesis, and blocking of mortalin and its interaction with p53 might be a potential novel target for the treatment of keloid. Therefore, we generated mortalin-specific small hairpin (sh) RNAs (dE1-RGD/GFP/shMot) and introduced into keloid spheroids for examination of its apoptotic and anti-fibrotic effect. On keloid tissues, mortalin expression was higher than adjacent normal tissues and it's protein expressions were activated keloid fibroblasts (KFs). After primary keloid spheroid were transduced with dE1-RGD/GFP/shMot for knockdown of mortalin, expression of type I, III collagen, fibronectin, and elastin was significantly reduced and transforming growth factor-ß1, epidermal growth factor receptor (EGFR), Extracellular Signal-Regulated Kinases 1 and 2 (Erk 1/2), and Smad 2/3 complex protein expression were decreased. In addition, increased TUNEL activities and cytochrome C were observed. Further, for examine of mortalin and p53 interaction, we performed immunofluorescence analysis. Knockdown of mortalin relocated p53 to the cell nucleus in primary keloid spheroids by dE1-RGD/GFP/shMot transduction. These results support the utility of knockdown of mortalin to induce apoptosis and reduce ECMs expression in keloid spheroid, which may be highly beneficial in treating keloids.
Assuntos
Proteínas de Choque Térmico HSP70/deficiência , Queloide/patologia , Esferoides Celulares/patologia , Adenoviridae/metabolismo , Apoptose , Núcleo Celular/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Elastina/metabolismo , Receptores ErbB/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibronectinas/metabolismo , Fibrose , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , RNA Interferente Pequeno/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
BACKGROUND: Relaxin is a transforming growth factor ß1 antagonist. To determine the effects of relaxin on scar reduction, we investigated the scar remodeling process by injecting relaxin-expressing adenoviruses using a pig scar model. METHODS: Scars with full thickness were generated on the backs of Yorkshire pigs. Scars were divided into two groups (relaxin [RLX] and Control). Adenoviruses were injected into the RLX (expressing relaxin) and Control (not expressing relaxin) groups. Changes in the surface areas, color index and pliability of scars were compared. RESULTS: Fifty days after treatment, the surface areas of scars decreased, the color of scars was normalized, and the pliability of scars increased in RLX group. CONCLUSION: Relaxin-expressing adenoviruses improved the surface area, color, and pliability of scars. The mechanism of therapeutic effects on scar formation should be further investigated.
RESUMO
Pancreatic cancer is a highly lethal disease for which limited therapeutic options are available. Pancreatic cancer exhibits a pronounced collagen-rich stromal reaction, which induces chemoresistance by inhibiting drug diffusion into the tumor. Complementary treatment with oncolytic virus such as an oncolytic adenovirus expressing relaxin (YDC002) is an innovative treatment option for combating chemoresistant pancreatic cancer. Here, we examined the ability of combined treatment with gemcitabine and YDC002, which degrades extracellular matrix (ECM), to efficiently treat chemoresistant and desmoplastic pancreatic cancer. Gemcitabine alone exhibited similarly low cytotoxicity toward pancreatic cancer cells throughout the concentration range (1-50 µM) used, whereas the combination of YDC002 and a subtherapeutic dose of gemcitabine (0.01-0.05 µM) resulted in potent anticancer effects through effective induction of apoptosis. Importantly, YDC002 combined with gemcitabine significantly attenuated the expression of major ECM components including collagens, fibronectin, and elastin in tumor spheroids and xenograft tumors compared with gemcitabine alone, resulting in potent induction of apoptosis, gemcitabine-mediated cytotoxicity, and an oncolytic effect through degradation of tumor ECM. Our results demonstrate that YDC002 can selectively degrade aberrant ECM and attenuate the ECM-induced chemoresistance observed in desmoplastic pancreatic tumor, resulting in a potent antitumor effect through effective induction of apoptosis.
Assuntos
Adenoviridae/metabolismo , Desoxicitidina/análogos & derivados , Terapia Viral Oncolítica/métodos , Neoplasias Pancreáticas/terapia , Relaxina/biossíntese , Adenoviridae/genética , Animais , Antimetabólitos Antineoplásicos/farmacologia , Terapia Combinada , Desoxicitidina/farmacologia , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/virologia , Distribuição Aleatória , Relaxina/genética , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Tumor microenvironment is extremely immunosuppressive, preventing efficient induction of antitumor immunity. To overcome tumor-mediated immunosuppression and enhance the potency of immunogene therapy, oncolytic adenovirus (Ad) co-expressing interleukin (IL)-12 and vascular endothelial growth factor (VEGF)-specific short hairpin ribonucleic acid (shVEGF; RdB/IL12/shVEGF) was generated. Intratumoral injection of RdB/IL12/shVEGF induced a strong antitumor effect in an immune competent B16-F10 melanoma model. RdB/IL12/shVEGF restored immune surveillance function in tumor tissues and actively recruited immune cells by elevating the expression levels of IL-12 and interferon-γ. RdB/IL12/shVEGF efficiently suppressed expression of immunosuppressive VEGF, resulting in restoration of the antitumor immune response and prevention of thymic atrophy. In situ delivery of RdB/IL12/shVEGF to tumor tissues resulted in massive infiltration of differentiated CD4+ T cells, CD8+ T cells, natural killer cells, and dendritic cells to tissues surrounding the necrotic region of tumor. Furthermore, RdB/IL12/shVEGF induced a potent tumor-specific T helper type 1 immune response, implying that attenuation of the immunosuppressive environment mediated by downregulation of VEGF can significantly enhance immune stimulatory functions in the tumor milieu. Collectively, these findings indicate the potential of inducing and restoring potent antitumor immunity using intratumorally administered oncolytic Ad co-expressing IL-12 and shVEGF.
Assuntos
Células Dendríticas/fisiologia , Interleucina-12/genética , Linfócitos do Interstício Tumoral/fisiologia , Neoplasias Experimentais/terapia , Terapia Viral Oncolítica , RNA Interferente Pequeno/genética , Células Th1/imunologia , Adenoviridae/genética , Animais , Movimento Celular , Vetores Genéticos , Humanos , Vigilância Imunológica , Interferon gama/metabolismo , Interleucina-12/metabolismo , Ativação Linfocitária , Masculino , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Microambiente Tumoral , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
In order to identify the cellular factors involved in human melanogenesis, we carried out shRNA-mediated loss-of-function screening in conjunction with induction of melanogenesis by 1-oleoyl-2-acetyl-glycerol (OAG) in human melanoma cells using biochemical and visual assays. Gene targets of the shRNAs (that caused loss of OAG-induced melanogenesis) and their pathways, as determined by bioinformatics, revealed involvement of proteins that regulate cell stress response, mitochondrial functions, proliferation, and apoptosis. We demonstrate, for the first time, that the mitochondrial stress chaperone mortalin is crucial for melanogenesis. Upregulation of mortalin was closely associated with melanogenesis in in vitro cell-based assays and clinical samples of keloids with hyperpigmentation. Furthermore, its knockdown resulted in compromised melanogenesis. The data proposed mortalin as an important protein that may be targeted to manipulate pigmentation for cosmetic and related disease therapeutics.
Assuntos
Hiperpigmentação/genética , Queloide/genética , Melaninas/biossíntese , Melaninas/genética , Melanócitos/metabolismo , Pigmentação da Pele/genética , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Diglicerídeos/farmacologia , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Melanoma/patologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética , Pigmentação da Pele/fisiologiaRESUMO
Mortalin/mthsp70 (HSPA9) is a stress chaperone enriched in many cancers that has been implicated in carcinogenesis by promoting cell proliferation and survival. In the present study, we examined the clinical relevance of mortalin upregulation in carcinogenesis. Consistent with high mortalin expression in various human tumors and cell lines, we found that mortalin overexpression increased the migration and invasiveness of breast cancer cells. Expression analyses revealed that proteins involved in focal adhesion, PI3K-Akt and JAK-STAT signaling, all known to play key roles in cell migration and epithelial-to-mesenchymal transition (EMT), were upregulated in mortalin-expressing cancer cells. We further determined that expression levels of the mesenchymal markers vimentin (VIM), fibronectin (FN1), ß-catenin (CTNNB1), CK14 (KRT14) and hnRNP-K were also increased upon mortalin overexpression, whereas the epithelial markers E-cadherin (CDH1), CK8 (KRT8), and CK18 (KRT18) were downregulated. Furthermore, shRNA-mediated and pharmacological inhibition of mortalin suppressed the migration and invasive capacity of cancer cells and was associated with a diminished EMT gene signature. Taken together, these findings support a role for mortalin in the induction of EMT, prompting further investigation of its therapeutic value in metastatic disease models.
RESUMO
CRISPR-Cas9 system is now widely used to edit a target genome in animals and plants. Cas9 protein derived from Streptococcus pyogenes (SpCas9) cleaves double-stranded DNA targeted by a chimeric single-guide RNA (sgRNA). For plant genome editing, Agrobacterium-mediated T-DNA transformation has been broadly used to express Cas9 proteins and sgRNAs under the control of CaMV 35S and U6/U3 promoter, respectively. We here developed a simple and high-throughput binary vector system to clone a 19-20 bp of sgRNA, which binds to the reverse complement of a target locus, in a large T-DNA binary vector containing an SpCas9 expressing cassette. Two-step cloning procedures: (1) annealing two target-specific oligonucleotides with overhangs specific to the AarI restriction enzyme site of the binary vector; and (2) ligating the annealed oligonucleotides into the two AarI sites of the vector, facilitate the high-throughput production of the positive clones. In addition, Cas9-coding sequence and U6/U3 promoter can be easily exchanged via the Gateway(TM) system and unique EcoRI/XhoI sites on the vector, respectively. We examined the mutation ratio and patterns when we transformed these constructs into Arabidopsis thaliana and a wild tobacco, Nicotiana attenuata. Our vector system will be useful to generate targeted large-scale knock-out lines of model as well as non-model plant.
Assuntos
Arabidopsis/genética , Sistemas CRISPR-Cas/genética , Clonagem Molecular/métodos , Edição de Genes/métodos , Genoma de Planta , Sequência de Bases , Genes de Plantas , Vetores Genéticos , Plantas Geneticamente Modificadas , RNA Guia de Cinetoplastídeos/genética , Transformação GenéticaRESUMO
BACKGROUND: Keloids are marked by an overabundance of extracellular matrix. The antifibrotic effect of hepatocyte growth factor (HGF) is achieved by increasing the expression of matrix metalloproteinases (MMPs) that drive extracellular matrix catabolism. As such, we cultivated an RGD-modified HGF-expressing adenovirus (dE1-RGD/lacZ/HGF) for introduction into keloid fibroblasts (KFs), looking at the subsequent impact on MMP-1 expression. METHODS: KFs infected with either test virus as experimental group (dE1-RGD/lacZ/HGF) or its counterpart (dE1-RGD/lacZ) as control group were examined for HGF protein expression using an enzyme-linked immunosorbent assay (ELISA). Collagen (types I and III) and MMP-1 mRNA levels were also determined by reverse transcriptase-polymerase chain reaction, and ELISA was used to monitor MMP-1 protein expression. RESULTS: In KFs harboring the test virus, high levels of HGF were induced at a multiplicity of infection ratio of 50 (3260.6 ± 162.7 pg/ml) after 72 hours of incubation. Furthermore, reverse transcriptase-polymerase chain reaction and ELISA confirmed that MMP-1 mRNA and protein expression rose significantly in KFs after transduction by the test virus (P < 0.05). However, mRNA levels of collagen were unaffected by the experimental group. CONCLUSION: These results suggest that an HGF-expressing adenovirus may be therapeutic for keloids by increasing MMP-1 expression.