RESUMEN
BACKGROUND: Hepatocellular carcinoma (HCC) cells usually show strong resistance to chemotherapy, which not only reduces the efficacy of chemotherapy but also increases the side effects. Regulation of autophagy plays an important role in tumor treatment. Cell senescence is also an important anti-cancer mechanism, which has become an important target for tumor treatment. Therefore, it is of great clinical significance to find anti-HCC drugs that act through this new mechanism. Platycodin D2 (PD2) is a new saponin compound extracted from the traditional Chinese medicine Platycodon grandiflorum. PURPOSE: Our study aimed to explore the effects of PD2 on HCC and identify the underlying mechanisms. METHODS: First, the CCK8 assay was used to detect the inhibitory effect of PD2 on HCC cells. Then, different pathways of programmed cell death and cell cycle regulators were measured. In addition, we assessed the effects of PD2 on the autophagy and senescence of HCC cells by flow cytometry, immunofluorescence staining, and Western blotting. Finally, we studied the in vivo effect of PD2 on HCC cells by using a mouse tumor-bearing model. RESULTS: Studies have shown that PD2 has a good anti-tumor effect, but the specific molecular mechanism has not been clarified. In this study, we found that PD2 has no obvious toxic effect on normal hepatocytes, but it can significantly inhibit the proliferation of HCC cells, induce mitochondrial dysfunction, enhance autophagy and cell senescence, upregulate NIX and P21, and downregulate CyclinA2. Gene silencing and overexpression indicated that PD2 induced mitophagy in HCC cells through NIX, thereby activating the P21/CyclinA2 pathway and promoting cell senescence. CONCLUSIONS: These results indicate that PD2 induces HCC cell death through autophagy and aging. Our findings provide a new strategy for treating HCC.
RESUMEN
The epidermal growth factor receptor (EGFR) has received significant attention as a potential target for glioblastoma (GBM) therapeutics in the past two decades. However, although cetuximab, an antibody that specifically targets EGFR, exhibits a high affinity for EGFR, it has not yet been applied in the treatment of GBM. Antibody-drug conjugates (ADCs) utilize tumor-targeting antibodies for the selective delivery of cytotoxic drugs, resulting in improved efficacy compared to conventional chemotherapy drugs. However, the effectiveness of cetuximab as a targeted antibody for ADCs in the treatment of GBM remains uncertain. In this study, we synthesized AGCM-22, an EGFR-targeted ADC derived from cetuximab, by conjugating it with the tubulin inhibitor monomethyl auristatin E (MMAE) using our Valine-Alanine Cathepsin B cleavable linker. In vitro experiments demonstrated that AGCM-22 effectively inhibited GBM cell proliferation through increased levels of apoptosis and autophagy-related cell death, whereas cetuximab alone had no anti-GBM effects. Additionally, both mouse and human orthotopic tumor models exhibited the selective tumor-targeting efficacy of AGCM-22, along with favorable metabolic properties and superior anti-GBM activity compared to temozolomide (TMZ). In summary, this study presents a novel ADC for GBM therapy that utilizes cetuximab as the tumor-targeting antibody, resulting in effective delivery of the cytotoxic drug payload.
Asunto(s)
Antineoplásicos , Glioblastoma , Inmunoconjugados , Humanos , Animales , Ratones , Cetuximab/farmacología , Preparaciones Farmacéuticas , Glioblastoma/metabolismo , Anticuerpos , Antineoplásicos/uso terapéutico , Receptores ErbB , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Línea Celular Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Inflammatory response triggered by innate immunity plays a pivotal element in the progress of ischemic stroke. Receptor-interacting kinase 2 (RIP2) is implicated in maintaining immunity homeostasis and regulating inflammatory response. However, the underlying mechanism of RIP2 in ischemic stroke is still not well understood. Hence, the study investigated the role and the ubiquitination regulatory mechanism of RIP2 in ischemic stroke. METHODS: Focal cerebral ischemia was introduced by middle cerebral artery occlusion (MCAO) in wild-type (WT) and OTUD1-deficient (OTUD1-/-) mice, oxygen glucose deprivation and reoxygenation (OGD/R) models in BV2 cells and primary cultured astrocytes were performed for monitoring of experimental stroke. GSK2983559 (GSK559), a RIP2 inhibitor was intraventricularly administered 30 min before MCAO. Mice brain tissues were collected for TTC staining and histopathology. Protein expression of RIP2, OTUD1, p-NF-κB-p65 and IκBα was determined by western blot. Localization of RIP2 and OTUD1 was examined by immunofluorescence. The change of IL-1ß, IL-6 and TNF-α was detected by ELISA assay and quantitative real-time polymerase chain reaction. Immunoprecipitation and confocal microscopy were used to study the interaction of RIP2 and OTUD1. The activity of NF-κB was examined by dual-luciferase assay. RESULTS: Our results showed upregulated protein levels of RIP2 and OTUD1 in microglia and astrocytes in mice subjected to focal cerebral ischemia. Inhibition of RIP2 by GSK559 ameliorated the cerebral ischemic outcome by repressing the NF-κB activity and the inflammatory response. Mechanistically, OTUD1 interacted with RIP2 and sequentially removed the K63-linked polyubiquitin chains of RIP2, thereby inhibiting NF-κB activation. Furthermore, OTUD1 deficiency exacerbated cerebral ischemic injury in response to inflammation induced by RIP2 ubiquitination. CONCLUSIONS: These findings suggested that RIP2 mediated cerebral ischemic lesion via stimulating inflammatory response, and OTUD1 ameliorated brain injury after ischemia through inhibiting RIP2-induced NF-κB activation by specifically cleaving K63-linked ubiquitination of RIP2.
Asunto(s)
Isquemia Encefálica , Accidente Cerebrovascular Isquémico , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor , Proteasas Ubiquitina-Específicas , Animales , Ratones , Isquemia Encefálica/metabolismo , Infarto de la Arteria Cerebral Media/complicaciones , Infarto de la Arteria Cerebral Media/metabolismo , Inflamación/metabolismo , Accidente Cerebrovascular Isquémico/metabolismo , Microglía/metabolismo , FN-kappa B/metabolismo , Daño por Reperfusión/metabolismo , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Proteasas Ubiquitina-Específicas/metabolismoRESUMEN
Coronavirus disease 2019 is a global pandemic caused by SARS-CoV-2. The emergence of its variant strains has posed a considerable challenge to clinical treatment. Therefore, drugs capable of inhibiting SARS-CoV-2 infection, regardless of virus variations, are in urgently need. Our results showed that the endosomal acidification inhibitor, Bafilomycin A1 (Baf-A1), had an inhibitory effect on the viral RNA synthesis of SARS-CoV-2, and its Beta and Delta variants at the concentration of 500 nM. Moreover, the human lung xenograft mouse model was used to investigate the anti-SARS-CoV-2 effect of Baf-A1. It was found that Baf-A1 significantly inhibited SARS-CoV-2 replication in the human lung xenografts by in situ hybridization and RT-PCR assays. Histopathological examination showed that Baf-A1 alleviated SARS-CoV-2-induced diffuse inflammatory infiltration of granulocytes and macrophages and alveolar endothelial cell death in human lung xenografts. In addition, immunohistochemistry analysis indicated that Baf-A1 decreased inflammatory exudation and infiltration in SARS-CoV-2-infected human lung xenografts. Therefore, Baf-A1 may be a candidate drug for SARS-CoV-2 treatment.
Asunto(s)
COVID-19 , Humanos , Animales , Ratones , Xenoinjertos , SARS-CoV-2 , Células Epiteliales Alveolares , Modelos Animales de EnfermedadRESUMEN
OBJECTIVE: Oncolytic adenoviruses are capable of exerting anticancer effects via a variety of mechanisms, including apoptosis and autophagy. In the present study, the dual-specific antitumor oncolytic adenovirus, Ad-Apoptin-hTERT-E1a (ATV), was used to infect cervical cancer cell lines to test its antitumor effects. METHODS: To explore the use of apoptin in tumor gene therapy, a recombinant adenovirus ATV expressing the apoptin protein was assessed to determine its lethal and growth-inhibitory effects on human cervical cancer cell line (HeLa) cells in vitro . Nonapoptotic autophagy of HeLa cells infected with ATV was assessed by examining the cell morphology, development of acidic vesicular organelles and the conversion of microtubule-associated protein 1 light chain 3 (LC3) from its cytoplasmic to autophagosomal membrane form. Using gene silencing (knockdown of LC3 and Belin-1), autophagy-associated molecules (e.g. ATG5, ATG12 and ULK1) were monitored by real-time PCR and western blot. RESULTS: A series of experiments demonstrated that ATV could significantly induce apoptosis and autophagy in cervical cancer cells, and provided evidence that ATV not only induced apoptosis but also autophagy and ATG5, ATG12 and ULK1 related pathways were not entirely dependent on LC3 and Beclin-1. CONCLUSION: These results indicate that ATV may have a potential application in tumor gene therapy.
Asunto(s)
Muerte Celular Autofágica , Viroterapia Oncolítica , Neoplasias del Cuello Uterino , Femenino , Humanos , Adenoviridae/genética , Células HeLa , Línea Celular Tumoral , Apoptosis , Autofagia , Viroterapia Oncolítica/métodosRESUMEN
In this study, we compared the inhibitory effects of recombinant oncolytic adenovirus (Ad-apoptin-hTERTp-E1a, Ad-VT) with that of doxorubicin (DOX), a first-line chemotherapy drug, and tamoxifen (TAM), an endocrine therapy drug, on the proliferation of breast cancer cells. We found that Ad-VT could effectively inhibit the proliferation of breast cancer cells (p < 0.01); the inhibition rate of Ad-VT on normal mammary epithelial MCF-10A cells was less than 20%. DOX can effectively inhibit the proliferation of breast cancer cells and also has a strong inhibitory effect on MCF-10A cells (p < 0.01). TAM also has a strong inhibitory effect on breast cancer cells, among which the oestrogen-dependent MCF-7 cell inhibition was stronger (p < 0.01), At higher concentrations, TAM also had a high rate of inhibition (>70%) on the proliferation of MCF-10A cells. We also found that both recombinant adenovirus and both drugs could successfully induce tumour cell apoptosis. Further Western blot results showed that the recombinant adenovirus killed breast cancer cells through the endogenous apoptotic pathway. Analysis of the nude mouse subcutaneous breast cancer model showed that Ad-VT significantly inhibited tumour growth (the luminescence rate of cancer cells was reduced by more than 90%) and improved the survival rate of tumour-bearing mice (p < 0.01). Compared with DOX and TAM, Ad-VT has a significant inhibitory effect on breast cancer cells, but almost no inhibitory effect on normal breast epithelial cells, and this inhibitory effect is mainly through the endogenous apoptotic pathway. These results indicate that Ad-VT has significant potential as a drug for the treatment of breast cancer.
Asunto(s)
Adenoviridae , Neoplasias , Adenoviridae/genética , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Doxorrubicina/farmacología , Estrógenos/farmacología , Ratones , Tamoxifeno/farmacologíaRESUMEN
Autophagy is thought to be involved in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, how SARS-CoV-2 interferes with the autophagic pathway and whether autophagy contributes to virus infection in vivo is unclear. In this study, we identified SARS-CoV-2-triggered autophagy in animal models, including the long-tailed or crab-eating macaque (Macaca fascicularis), human angiotensin-converting enzyme 2 (hACE2) transgenic mice, and xenografted human lung tissues. In Vero E6 and Huh-7 cells, SARS-CoV-2 induces autophagosome formation, accompanied by consistent autophagic events, including inhibition of the Akt-mTOR pathway and activation of the ULK-1-Atg13 and VPS34-VPS15-Beclin1 complexes, but it blocks autophagosome-lysosome fusion. Modulation of autophagic elements, including the VPS34 complex and Atg14, but not Atg5, inhibits SARS-CoV-2 replication. Moreover, this study represents the first to demonstrate that the mouse bearing xenografted human lung tissue is a suitable model for SARS-CoV-2 infection and that autophagy inhibition suppresses SARS-CoV-2 replication and ameliorates virus-associated pneumonia in human lung tissues. We also observed a critical role of autophagy in SARS-CoV-2 infection in an hACE2 transgenic mouse model. This study, therefore, gives insights into the mechanisms by which SARS-CoV-2 manipulates autophagosome formation, and we suggest that autophagy-inhibiting agents might be useful as therapeutic agents against SARS-CoV-2 infection. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global pandemic with limited therapeutics. Insights into the virus-host interactions contribute substantially to the development of anti-SARS-CoV-2 therapeutics. The novelty of this study is the use of a new animal model: mice xenografted with human lung tissues. Using a combination of in vitro and in vivo studies, we have obtained experimental evidence that induction of autophagy contributes to SARS-CoV-2 infection and improves our understanding of potential therapeutic targets for SARS-CoV-2.
Asunto(s)
Enzima Convertidora de Angiotensina 2/genética , Autofagia , Tratamiento Farmacológico de COVID-19 , COVID-19/virología , Pulmón/virología , SARS-CoV-2 , Replicación Viral , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , Autofagosomas , Línea Celular Tumoral , Chlorocebus aethiops , Humanos , Pulmón/patología , Macaca , Masculino , Ratones , Ratones Transgénicos , Neumonía Viral/tratamiento farmacológico , ARN Interferente Pequeño/metabolismo , Células VeroRESUMEN
Background One of the main challenges in the clinical treatment of lung cancer is resistance to chemotherapeutic drugs. P-glycoprotein (P-gp)-mediated drug resistance is the main obstacle to successfully implementing microtubule-targeted tumor chemotherapy. Purpose In this study, we explored the effect of Ad-hTERTp-E1a-Apoptin (Ad-VT) on drug-resistant cell lines and the molecular mechanism by which Ad-VT combined with chemotherapy affects drug-resistant cells and parental cells. Methods In vitro, cell proliferation, colony formation, resistance index (RI), apoptosis and autophagy assays were performed. Protein expression was analyzed by Western blotting. Finally, a xenograft tumor model in nude mice was used to detect tumor growth and evaluate histological characteristics. Results Our results showed that Ad-VT had an obvious killing effect on A549, A549/GEM and A549/Paclitaxel cancer cells, and the sensitivity of drug-resistant cell lines to Ad-VT was significantly higher than that of parental A549 cells. Compared with A549 cells, A549/GEM and A549/Paclitaxel cells had higher autophagy levels and higher viral replication ability. Ad-VT decreased the levels of p-PI3k, p-Akt and p-mTOR and the expression of P-gp. In vivo, Ad-VT combined with chemotherapy can effectively inhibit the growth of chemotherapy-resistant tumors and prolong the survival of mice. Conclusions Thus, the combination of Ad-VT and chemotherapeutic drugs will be a promising strategy to overcome chemoresistance.
Asunto(s)
Adenocarcinoma del Pulmón , Neoplasias Pulmonares , Adenocarcinoma del Pulmón/tratamiento farmacológico , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Desoxicitidina/análogos & derivados , Resistencia a Antineoplásicos , Humanos , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
BACKGROUND: Apoptin, as a tumor-specific pro-apoptotic protein, plays an important anti-tumoral role, but its mechanism of autophagy activation and the interaction between autophagy and apoptosis have not been accurately elucidated. Here, we studied the mechanism of apoptin-induced apoptosis and autophagy and the interaction between two processes. METHODS: Using crystal violet staining and the CCK-8 assay, we analyzed the effect of apoptin in the inhibition of liver cancer cells in vitro and analyzed the effect of inhibiting liver cancer in vivo by establishing a nude mouse tumor model. Flow cytometry and fluorescence staining were used to analyze the main types of apoptin-induced apoptosis and autophagy. Subsequently, the relationship between the two events was also analyzed. Flow cytometry was used to analyze the effect of ROS on apoptin-mediated apoptosis and autophagy mediated by apoptin. The effect of ROS on two phenomena was analyzed. Finally, the role of key genes involved in autophagy was analyzed using gene silencing. RESULTS: The results showed that apoptin can significantly increase the apoptosis and autophagy of liver cancer cells, and that apoptin can cause mitophagy through the increase in the expression of NIX protein. Apoptin can also significantly increase the level of cellular ROS, involved in apoptin-mediated autophagy and apoptosis of liver cancer cells. The change of ROS may be a key factor causing apoptosis and autophagy. CONCLUSION: The above results indicate that the increase in ROS levels after apoptin treatment of liver cancer cells leads to the loss of mitochondrial transmembrane potential, resulting in endogenous apoptosis and mitophagy through the recruitment of NIX. Therefore, ROS may be a key factor connecting endogenous apoptosis and autophagy induced by apoptin in liver cancer cells. Video abstract.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , Apoptosis , Proteínas de la Cápside , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Ratones , Ratones Desnudos , Mitofagia , Especies Reactivas de OxígenoRESUMEN
Ad-apoptin is a recombinant oncolytic adenovirus constructed by our laboratory that can express apoptin. It can selectively kill tumor cells without damaging normal cells. This study investigated the effects of Ad-apoptin on glycolysis, migration and invasion of non-small cell lung cancer. Cell viability and apoptosis were detected by CCK-8 and flow cytometry, respectively. Glycolysis was investigated by glucose consumption, lactic acid production and glycolytic key enzyme protein levels. Migration and invasion were evaluated via wound healing, transwell assays and epithelial-mesenchymal transition (EMT) protein levels. The interaction between apoptin and AMPK was detected by Co-IP. A nude mice tumor model was established to investigate the anti-cancer role of Ad-apoptin in vivo. The results showed that Ad-apoptin inhibits cell viability and induces apoptosis of A549 and NCI-H23 cells. Ad-apoptin can reduce the glucose uptake and lactic production in lung cancer cells, and reduce the expression of related glycolysis-limiting enzymes. At the same time, Ad-apoptin inhibited the migration and invasion of lung cancer. Immunoprecipitation showed that apoptin and AMPK could interact directly. Moreover, knockdown of AMPK significantly attenuated the inhibitory effect of Ad-apoptin on glycolysis, migration and invasion of A549 and NCI-H23 cells. Ad-apoptin can inhibit the growth of tumors in nude mice. Compared with the control group, Ad-apoptin had a significant inhibitory effect on AMPK knockdown tumors. The immunohistochemical results of tumor tissues were consistent with those in vitro. Collectively, Ad-apoptin targets AMPK and inhibits glycolysis, migration and invasion of lung cancer cells through the AMPK/mTOR signaling pathway. This suggests that Ad-apoptin may have therapeutic potential for lung cancer by targeting AMPK activation.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Adenoviridae/genética , Proteínas de la Cápside/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Glucólisis , Viroterapia Oncolítica/métodos , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/terapia , Ciclo Celular , Movimiento Celular , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Serina-Treonina Quinasas TOR/genética , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Apoptin can specifically kill cancer cells but has no toxicity to normal cells. Human telomerase reverse transcriptase (hTERT) can act as a tumour-specific promoter by triggering the expression of certain genes in tumour cells. This study aims to investigate the inhibitory effects and to explore the inhibitory pathway of a dual cancer-specific recombinant adenovirus (Ad-apoptin-hTERTp-E1a, Ad-VT) on breast cancer stem cells. Breast cancer cell spheres were obtained from MCF-7 cells through serum-free suspension culture. The cell spheres were detected by flow cytometry for CD44+ CD24- cell subsets. The stemness of MCF-7-CSC cells was confirmed by in vivo tumorigenesis experiments. The inhibitory effect of the recombinant adenoviruses on MCF-7-CSC cells was evaluated by CCK-8 assay. In addition, the stemness of adenovirus-infected MCF-7-CSC cells was analysed by testing the presence of CD44+ CD24- cell subsets. The ability of the recombinant adenovirus to induce MCF-7-CSC cell apoptosis was detected by staining JC-1, TMRM and Annexin V. Our results showed that a significantly higher proportion of the CD44+ CD24- cell subsets was present in MCF-7-CSC cells with a significantly increased expression of stem cell marker proteins. The MCF-7-CSC cells, whlist exhibited a strong tumorigenic ability with a certain degree of stemness in mice, were shown to be strongly inhibited by recombinant adenovirus Ad-VT through cell apoptosis. In addition, Ad-VT was shown to exert a killing effect on BCSCs. These results provide a new theoretical basis for the future treatment of breast cancer.
Asunto(s)
Antígeno CD24/metabolismo , Receptores de Hialuranos/metabolismo , Células Madre Neoplásicas/metabolismo , Apoptosis/genética , Apoptosis/fisiología , Western Blotting , Antígeno CD24/genética , Citometría de Flujo , Humanos , Receptores de Hialuranos/genética , Células MCF-7 , Potencial de la Membrana Mitocondrial/genética , Potencial de la Membrana Mitocondrial/fisiologíaRESUMEN
As a potential cancer therapy, we developed a recombinant adenovirus named Ad-VT, which was designed to express the apoptosis-inducing gene (apoptin) and selectively replicate in cancer cells via E1a manipulation. However, how it performs in bladder cancer remains unclear. We examined the antitumor efficacy of Ad-VT in bladder cancers using CCK-8 assays and xenograft models. Autophagy levels were evaluated by western blotting, MDC staining, and RFP-GFP-LC3 aggregates' analyses. Here, we report the selective replication and antitumor efficacy (viability inhibition and apoptosis induction) of Ad-VT in bladder cancer cells. Using xenograft tumor models, we demonstrate that its effects are tumor specific resulting in the inhibition of tumor growth and improvement of the survival of mice models. Most Importantly, Ad-VT induced a complete autophagy flux leading to autophagic cancer cell death through a signaling pathway involving AMPK, raptor and mTOR. Finally, we suggest that treatment combination of Ad-VT and rapamycin results in a synergistic improvement of tumor control and survival compared to monotherapy. This study suggests that Ad-VT can induce selective autophagic antitumor activities in bladder cancer through the AMPK-Raptor-mTOR pathway, which can be further improved by rapamycin.
Asunto(s)
Adenoviridae/genética , Autofagia/genética , Viroterapia Oncolítica/métodos , Neoplasias de la Vejiga Urinaria/terapia , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Proteínas de la Cápside/genética , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteína Reguladora Asociada a mTOR/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Neoplasias de la Vejiga Urinaria/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 and broke out as a global pandemic in late 2019. The acidic pH environment of endosomes is believed to be essential for SARS-CoV-2 to be able to enter cells and begin replication. However, the clinical use of endosomal acidification inhibitors, typically chloroquine, has been controversial with this respect. METHODS: In this study, RT-qPCR method was used to detect the SARS-CoV-2N gene to evaluate viral replication. The CCK-8 assay was also used to evaluate the cytotoxic effect of SARS-CoV-2. In situ hybridization was used to examine the distribution of the SARS-CoV-2 gene in lung tissues. Hematoxylin and eosin staining was also used to evaluate virus-associated pathological changes in lung tissues. RESULTS: In this study, analysis showed that endosomal acidification inhibitors, including chloroquine, bafilomycin A1 and NH4CL, significantly reduced the viral yields of SARS-CoV-2 in Vero E6, Huh-7 and 293T-ACE2 cells. Chloroquine and bafilomycin A1 also improved the viability and proliferation of Vero E6 cells after SARS-CoV-2 infection. Moreover, in the hACE2 transgenic mice model of SARS-CoV-2 infection, chloroquine and bafilomycin A1 reduced viral replication in lung tissues and alleviated viral pneumonia with reduced inflammatory exudation and infiltration in peribronchiolar and perivascular tissues, as well as improved structures of alveolar septum and pulmonary alveoli. CONCLUSIONS: Our research investigated the antiviral effects of endosomal acidification inhibitors against SARS-CoV-2 in several infection models and provides an experimental basis for further mechanistic studies and drug development.
Asunto(s)
Antivirales/farmacología , Tratamiento Farmacológico de COVID-19 , COVID-19/virología , Endosomas/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/fisiología , Replicación Viral/efectos de los fármacos , Cloruro de Amonio/farmacología , Enzima Convertidora de Angiotensina 2/genética , Enzima Convertidora de Angiotensina 2/metabolismo , Animales , COVID-19/metabolismo , COVID-19/patología , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Cloroquina/farmacología , Endosomas/metabolismo , Femenino , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Pulmón/patología , Macrólidos/farmacología , Ratones , Ratones Transgénicos , Distribución Aleatoria , SARS-CoV-2/genética , Células VeroRESUMEN
BACKGROUND: Apoptin can specifically kill cancer cells but has no toxicity to normal cells. Human telomerase reverse transcriptase (hTERT) acts as a tumor-specific promoter, triggering certain genes to replicate or express only in tumor cells, conferring specific replication and killing abilities. This study aimed at investigating the anticancer potential of the recombinant adenovirus Ad-apoptin-hTERTp-E1a (Ad-VT) in ovarian cancer treatment. METHODS: Crystal Violet staining and WST-1 assays were used to analyze the inhibitory effect of Ad-VT on ovarian cancer SKOV3 and OVCAR-3 cells. Ad-VT-induced apoptosis of ovarian cancer cells, was detected using Hoechst, Annexin V-FITC/PI, JC-1 staining. Cell migration and invasion of ovarian cancer cells were detected using cell-scratch and Transwell assays. The pGL4.51 plasmid was used to transfect and to generate SKOV3-LUC cells, that stably express luciferase. The in vivo tumor inhibition effect of Ad-VT was subsequently confirmed using a tumor-bearing nude mouse model. RESULTS: Ad-VT had a strong apoptosis-inducing effect on SKOV3 and OVCAR-3 cells, that was mainly mediated through the mitochondrial apoptotic pathway. The Ad-VT could significantly increase the inhibition of ovarian cancer cell migration and invasion. The Ad-VT also can inhibit tumor growth and reduce toxicity in vivo. CONCLUSIONS: The recombinant adenovirus, comprising the apoptin protein and the hTERTp promoter, was able to inhibit the growth of ovarian cancer cells and promote their apoptosis.
Asunto(s)
Adenoviridae/genética , Carcinoma Epitelial de Ovario/genética , Virus de la Anemia del Pollo/genética , Viroterapia Oncolítica/métodos , Neoplasias Ováricas/genética , Proteínas Virales/genética , Adenoviridae/metabolismo , Animales , Apoptosis/genética , Carcinoma Epitelial de Ovario/metabolismo , Carcinoma Epitelial de Ovario/mortalidad , Carcinoma Epitelial de Ovario/virología , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Virus de la Anemia del Pollo/metabolismo , Femenino , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mitocondrias/genética , Mitocondrias/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/virología , Análisis de Supervivencia , Transgenes , Carga Tumoral , Proteínas Virales/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Oncolytic virus therapy is emerging as important means in cancer treatment. In a previous study, we constructed a dual cancer-specific antitumor recombinant adenovirus, designating it Ad-apoptin-hTERTp-E1a (Ad-VT). This study aimed to investigate the anticancer potential of recombinant adenovirus Ad-apoptin-hTERTp-E1a (Ad-VT) in liver cancer. Crystal Violet staining and CCK-8 assays were used to analyse the inhibitory effect of recombinant adenovirus on human hepatoma cell line QGY-7703 and SMMC-7721. Ad-VT had a significant tumour killing inhibitory effect on QGY-7703 and SMMC-7721 cells that was both dose and a time dependent. Ad-VT-induced apoptosis of QGY-7703 cells was detected using Hoechst, Annexin V, and JC-1 staining, as well as western blotting. Recombinant adenovirus had a strong apoptosis-inducing effect on QGY-7703 cells, and killed QGY-7703 cells mainly through the mitochondrial apoptotic pathway. QGY-7703 cells invasion were detected using cell-scratch and Transwell assays. Recombinant adenovirus could significantly inhibit the invasion of QGY-7703 cells over a short period of time. The pGL4.51 plasmid was used to transfect QGY-7703 cells to construct tumour cells stably expressing luciferase (QGY-7703-LUC). The tumour inhibition effect of Ad-VT in vivo was subsequently confirmed by establishing a tumour-bearing nude mouse model. Ad-VT could effectively inhibit tumour growth and prolong survival of the mice. Recombinant adenovirus Ad-VT has the characteristics of tumour-specific replication and specific tumour killing, and could inhibit the growth of liver cancer QGY-7703 cells and promote their apoptosis.
Asunto(s)
Adenoviridae/genética , Proliferación Celular , Neoplasias Hepáticas/terapia , Viroterapia Oncolítica/métodos , Animales , Apoptosis , Femenino , Humanos , Técnicas In Vitro , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Newcastle disease virus (NDV) can specifically kill cancer cells and has less toxicity to normal cells. The hemagglutinin-neuraminidase (HN) protein is an important structural protein in NDV pathogenesis and has been postulated as a promising candidate for antitumor therapy. The aim of this study was to investigate the anticancer potential of recombinant adenovirus Ad-HN-PEG3p-E1a. An MTS assay was performed to determine viral proliferation after viral infection, the data showed that the proliferation ability of osteosarcoma cells decreased, whereas there was no significant change in normal hepatic cells. DAPI and Annexin V experiments showed that osteosarcoma cells were killed because of apoptosis, active oxygen content, and augmented mitochondrial membrane potential loss. Caspase Activity Assay Kits were used to detect the caspase-3 activities of the treated OS-732 for increased expression. Western blot analysis showed that cytochrome C increased significantly and apoptosis of the virus was confirmed in tumor cells. In-vivo experiments show that NDV has an inhibitory effect on tumor growth. The recombinant adenovirus, which is composed of a HN protein and progressive increment promoter PEG3p, could inhibit the growth of OS-732 and promote the apoptosis of tumor cells. However, there was no clear relationship with normal cell (L02) apoptosis.
Asunto(s)
Neoplasias Óseas/terapia , Hemaglutininas Virales/genética , Neuraminidasa/genética , Virus de la Enfermedad de Newcastle/genética , Viroterapia Oncolítica/métodos , Osteosarcoma/terapia , Animales , Apoptosis/genética , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Neoplasias Óseas/virología , Línea Celular Tumoral , Femenino , Células HEK293 , Hemaglutininas Virales/biosíntesis , Humanos , Ratones , Neuraminidasa/biosíntesis , Virus de la Enfermedad de Newcastle/enzimología , Osteosarcoma/genética , Osteosarcoma/patología , Osteosarcoma/virología , Distribución AleatoriaRESUMEN
Porcine reproductive and respiratory syndrome (PRRS) is almost always caused by the North American strain of PRRS virus (PRRSV) in China; the European genotype of PRRSV has emerged in China. The mixed infection of PRRSV and Porcine circovirus type 2 virus (PCV2) are always found in pigs and PRRSV-augmented PCV2 replication and serious clinical symptoms. Current vaccines cannot protect mixed European PRRSV and PCV2 infections. Therefore, the development of a safe and effective new vaccine to prevent and control the mixed infection of European PRRSV and PCV2 is both urgent and necessary. In this study, we developed a recombinant vaccinia vaccine co-expressing the GP3 and GP5 proteins of European PRRSV and the ORF2 protein of PCV2 and evaluated the immunogenicity and its protective effects and its inactivated vaccine in pigs. The recombinant vaccinia vaccine and its inactivated vaccine both elicited significant humoral and cellular immune responses with a higher level of specific antibody responses and T-lymphocyte proliferation than the control group. Furthermore, the pigs inoculated with the recombinant vaccinia vaccine were completely protected against challenge with 105 TCID50 of European PRRSV strain LV. These data suggest that the recombinant vaccinia vaccine is a potential candidate vaccine against European PRRSV and PCV2.
Asunto(s)
Infecciones por Circoviridae/prevención & control , Proteína Receptora de AMP Cíclico/genética , Inmunogenicidad Vacunal , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , China , Infecciones por Circoviridae/inmunología , Circovirus/genética , Circovirus/inmunología , Europa (Continente) , Vectores Genéticos , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/inmunología , Porcinos , Virus Vaccinia/genética , Proteínas del Envoltorio Viral/genéticaRESUMEN
An HIV candidate vaccine for the Chinese population was designed by constructing a recombinant fowlpox virus expressing HIV-1 gag and HIV gp145 proteins via homologous recombination and plaque screening using enhanced green fluorescent protein (EGFP) as the reporter gene. EGFP in the recombinant was then knocked out with the Cre/Loxp system yielding rFPVHg-Hp, which was identified at the genomic, transcriptional and translational levels. The immunogenicity of rFPVHg-Hp was analyzed by measuring levels of HIV-specific antibodies and IFN-γ-secreting splenocytes by enzyme-linked immunosorbent assay and IFN enzyme-linked immune spot test in the BALB/c mouse model. Results showed that rFPV could not stimulate HIV-1 specific antibodies or IFN-γ-secreting cells by a single immunization. Meanwhile, in the prime-boost strategy, HIV-p24 antibodies (P < 0.01) and IFN-γ-secreting cells (P < 0.05) were induced strongly by the candidate vaccine after the boost immunization. Thus, both humoral and cellular immunity could be elicited by the candidate vaccine in a prime-boost immunization strategy. This study provides a foundation for future preclinical studies on the HIV rFPVHg-Hp candidate vaccine.
RESUMEN
Environmental factors in early life have been demonstrated to increase the risk of neurodevelopmental disorders in offspring, especially the deficiency of the cognitive ability. Leptin has emerged as a key hormone that conveys information on energy stores, but there is growing appreciation that leptin signaling may also play an important role in neurodevelopment. The present study aimed to investigate whether maternal HFD exposure impairs the offspring learning and memory through the programming of central leptin system. We observed that hippocampus-dependent learning and memory were impaired in male but not female offspring from HFD-fed maternal ancestors (C57BL/6 mice), as assessed by novel object recognition and Morris water maze tests. Moreover, the chromatin immunoprecipitation results revealed the maternal HFD consumption led to the increasement in the binding of the histone marker H3K9me3 in male offspring, which mediates gene silencing in the leptin receptor promoter region. Furthermore, there was an increase in the expression of the histone methylase SUV39H1 in male but not female offspring, which regulates H3K9me3. Additionally, it has been observed that IL-6 and IL-1 also could lead to similar alternations when acting on cultured hippocampal neurons in vitro. Taken together, our data suggest that maternal HFD consumption influences male offspring hippocampal cognitive performance in a sex-specific manner, and central leptin signaling may serve as the cross-talk between maternal diet and cognitive impairment in offspring.
Asunto(s)
Dieta Alta en Grasa , Hipocampo , Leptina , Ratones Endogámicos C57BL , Efectos Tardíos de la Exposición Prenatal , Transducción de Señal , Aprendizaje Espacial , Animales , Femenino , Masculino , Hipocampo/metabolismo , Leptina/metabolismo , Dieta Alta en Grasa/efectos adversos , Ratones , Aprendizaje Espacial/fisiología , Efectos Tardíos de la Exposición Prenatal/metabolismo , Embarazo , Transducción de Señal/fisiología , Caracteres Sexuales , Neuronas/metabolismo , Histonas/metabolismo , Receptores de Leptina/metabolismo , Receptores de Leptina/genéticaRESUMEN
OBJECTIVE: The purpose of the current study was to statistically clarify the precise risk age in elderly patients undergoing colorectal surgery and to evaluate the safety and efficacy of laparoscopic colorectal resection in these patients. METHODS: Patients' clinical variables were extracted from the database of the Gastrointestinal Surgery Centre, Third Affiliated Hospital of Sun Yat-sen University, from 2015 to 2019. Logistic regression was conducted to identify independent risk factors of postoperative complications and ORs for each age. Curves of odds ratios (ORs) and CIs for each age were fitted by using a locally weighted scatterplot smoother, and a structural breakpoint was determined by the Chow test to identify a precise cutoff risk age for elderly patients. Comparison and subgroup analysis were conducted between surgical approach groups using the Student t test and χ 2 analysis. RESULTS: Locally weighted scatterplot smoother OR analysis manifested that patients aged 69 years old or older suffered a higher possibility of postoperative complications and should be defined as high-risk age. Comparison according to the high-risk age revealed laparoscopic colorectal surgery is better than laparotomic surgery for elderly individuals in terms of hospital stay (9.46 ± 5.96 vs 15.01 ± 6.34, P < 0.05), the incidence of intensive care unit transfer (4 vs 20, P < 0.05), and incidence of surgical site infection (15 vs 20, P < 0.05). Patients who underwent laparotomic surgery had a greater prevalence of Clavien-Dindo II/III complications ( P < 0.05). These findings remained stable even after propensity matching. Furthermore, such superiority was proved especially significant for patients who underwent left-side colorectal resection. In addition, overall survival was improved in the laparoscopic surgery group, whereas no differences were observed in disease-free survival. CONCLUSION: In our study population, age 69 or older was a cutoff point age suggests a higher possibility of postoperative morbidity after colorectal surgery. Laparoscopic colorectal resection should be regarded as a superior therapeutic choice for these elderly individuals, especially for left-side colorectal surgeries.