RESUMEN
In eukaryotes, the localization of small ribosomal subunits to mRNA transcripts requires the translation of Kozak elements at the starting site. The sequence of Kozak elements affects the translation efficiency of protein synthesis. However, whether the upstream nucleotide of Kozak sequence affects the expression of recombinant proteins in Chinese hamster ovary (CHO) cells remains unclear. In order to find the optimal sequence to enhance recombinant proteins expression in CHO cells, -10 to +4 sequences around ATG in 100 CHO genes were compared, and the extended Kozak elements with different translation intensities were constructed. Using the classic Kozak element as control, the effects of optimized extended Kozak elements on the secreted alkaline phosphatase (SEAP) and human serum albumin (HSA) gene were studied. The results showed that the optimized extended Kozak sequence can enhance the stable expression level of recombinant proteins in CHO cells. Furthermore, it was found that the increased expression level of the recombinant protein was not related with higher transcription level. In summary, optimizing extended Kozak elements can enhance the expression of recombinant proteins in CHO cells, which contributes to the construction of an efficient expression system for CHO cells.
Asunto(s)
Fosfatasa Alcalina , Cricetulus , Proteínas Recombinantes , Células CHO , Animales , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fosfatasa Alcalina/genética , Fosfatasa Alcalina/metabolismo , Cricetinae , HumanosRESUMEN
Acevaltrate is a natural product isolated from the roots of Valeriana glechomifolia F.G.Mey. (Valerianaceae) and has been shown to exhibit anti-cancer activity. However, the mechanism by which acevaltrate inhibits tumor growth is not fully understood. We here demonstrated the effect of acevaltrate on hypoxia-inducible factor-1α (HIF-1α) expression. Acevaltrate showed a potent inhibitory activity against HIF-1α induced by hypoxia in various cancer cells. This compound markedly decreased the hypoxia-induced accumulation of HIF-1α protein dose-dependently. Further analysis revealed that acevaltrate inhibited HIF-1α protein synthesis and promoted degradation of HIF-1α protein, without affecting the expression level of HIF-1α mRNA. Moreover, the phosphorylation levels of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6K), and eIF4E binding protein-1 (4E-BP1) were significantly suppressed by acevaltrate. In addition, acevaltrate promoted apoptosis and inhibited proliferation, which was potentially mediated by suppression of HIF-1α. We also found that acevaltrate administration inhibited tumor growth in mouse xenograft model. Taken together, these results suggested that acevaltrate was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of acevaltrate against cancers.
Asunto(s)
Apoptosis , Subunidad alfa del Factor 1 Inducible por Hipoxia , Neoplasias , Animales , Humanos , Ratones , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Apoptosis/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Valeriana/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mammalian cell lines are frequently used as the preferred host cells for producing recombinant therapeutic proteins (RTPs) having post-translational modified modification similar to those observed in proteins produced by human cells. Nowadays, most RTPs approved for marketing are produced in Chinese hamster ovary (CHO) cells. Recombinant therapeutic antibodies are among the most important and promising RTPs for biomedical applications. One of the issues that occurs during development of RTPs is their degradation, which caused by a variety of factors and reducing quality of RTPs. RTP degradation is especially concerning as they could result in reduced biological functions (antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity) and generate potentially immunogenic species. Therefore, the mechanisms underlying RTP degradation and strategies for avoiding degradation have regained an interest from academia and industry. In this review, we outline recent progress in this field, with a focus on factors that cause degradation during RTP production and the development of strategies for overcoming RTP degradation. KEY POINTS: ⢠The recombinant therapeutic protein degradation in CHO cell systems is reviewed. ⢠Enzymatic factors and non-enzymatic methods influence recombinant therapeutic protein degradation. ⢠Reducing the degradation can improve the quality of recombinant therapeutic proteins.
Asunto(s)
Apoptosis , Industrias , Animales , Cricetinae , Humanos , Células CHO , Cricetulus , ProteolisisRESUMEN
We have previously developed a non-viral episomal vector based on matrix attachment region (MAR) that can facilitate plasmid replication episomally in mammal cells. In this study, we have focused on the development of an alternative tissue specific episomal vector by incorporating into cis-acting elements. We found that AAT promoter demonstrated the highest eGFP expression level in HepG2, Huh-7 and HL-7702 hepatic cells. Furthermore, hCMV enhancer when combined with AAT promoter significantly improved the eGFP expression level in the transfected HepG2 cells. The mean fluorescence intensity of eGFP in hCMV2 group was 1.33 fold, which was higher than that of the control (p < 0.01), followed by the hCMV1 group (1.21 fold). In addition, the percentages of eGFP-expressing cells in hCMV1 and hCMV2 groups were observed to be 49.3% and 57.2%, which were significantly higher than that of the enhancer-devoid control vector (44.3%) (p < 0.05). Moreover, the eGFP protein were up to 3.5 fold and 5.1 fold (p < 0.05), respectively. This observation could be related with the activities of some specific transcription factors (TFs) during the transcriptional process, such as SRF, REL and CREB1. The composite CMV/AAT promoter can be thus used for efficient transgene expression of MAR-based episomal vector in liver cells and as a potential gene transfer tools for the management of liver diseases. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03774-x.
RESUMEN
Chinese hamster ovary (CHO) cells can produce proteins with complex structures and post-translational modifications which are similar to human-derived cells, and they have been the ideal host cells for the production of recombinant therapy proteins (RTPs). Nearly 70% of approved RTPs are produced by CHO cells. In recent years, a series of measures have been developed to increase the expression of RTPs to achieve the lower production cost during the process of large-scale industrial production of recombinant protein in CHO cells. Among of them, the addition of small molecule additives in the culture medium can improve the expression and production efficiency of recombinant proteins, and has become an effective and simple method. In this paper, the characteristics of CHO cells, the effect and mechanism of small molecule additives are reviewed. KEY POINTS: ⢠Small molecular additives on the expression of RTPs in CHO cells are reviewed ⢠Small molecular additives improve the yield of RTPs ⢠Small molecular additives provide methods for the optimization of serum-free medium.
Asunto(s)
Procesamiento Proteico-Postraduccional , Cricetinae , Animales , Humanos , Cricetulus , Células CHO , Proteínas Recombinantes/metabolismoRESUMEN
Nearly 80% of the approved human therapeutic antibodies are produced by Chinese Hamster Ovary (CHO) cells. To achieve better cell growth and high-yield recombinant protein, fed-batch culture is typically used for recombinant protein production in CHO cells. According to the demand of nutrients consumption, feed medium containing multiple components in cell culture can affect the characteristics of cell growth and improve the yield and quality of recombinant protein. Fed-batch optimization should have a connection with comprehensive factors such as culture environmental parameters, feed composition, and feeding strategy. At present, process intensification (PI) is explored to maintain production flexible and meet forthcoming demands of biotherapeutics process. Here, CHO cell culture, feed composition in fed-batch culture, fed-batch culture environmental parameters, feeding strategies, metabolic byproducts in fed-batch culture, chemostat cultivation, and the intensified fed-batch are reviewed. KEY POINTS: ⢠Fed-batch culture in CHO cells is reviewed. ⢠Fed-batch has become a common technology for recombinant protein production. ⢠Fed batch culture promotes recombinant protein production in CHO cells.
Asunto(s)
Técnicas de Cultivo Celular por Lotes , Reactores Biológicos , Cricetinae , Animales , Humanos , Cricetulus , Células CHO , Proteínas Recombinantes/metabolismo , InmunoglobulinasRESUMEN
Chinese hamster ovary (CHO) cells are currently the most widely used host cells for recombinant therapeutic protein (RTP) production. Currently, the RTP yields need to increase further to meet the market needs and reduce costs. In this study, three stabilizing and anti-repressor (SAR) elements from the human genome were selected, including human SAR7, SAR40, and SAR44 elements. SAR elements were cloned upstream of the promoter in the eukaryotic vector, followed by transfection into CHO cells, and were screened under G418 pressure. Flow cytometry was used to detect enhanced green fluorescent protein (eGFP) expression levels. The gene copy numbers and mRNA expression levels were determined through quantitative real-time PCR. Furthermore, the effect of the stronger SAR elements on adalimumab was investigated. The results showed that transgene expression levels in the SAR-containing vectors were higher than that of the control vector, and SAR7 and SAR40 significantly increased and maintained the long-term expression of the transgene in CHO cells. In addition, the transgene expression level increase was related with gene copy numbers and mRNA expression levels. Collectively, SAR elements can enhance the transgene expression and maintain the long-term expression of a transgene in transfected CHO cells, which may be used to increase recombinant protein production in CHO cells.
RESUMEN
In recent years, mammalian cells have become the primary host cells for the production of recombinant therapeutic proteins (RTPs). Despite that the expression of RTPs in mammalian cells can be improved by directly optimizing or engineering the expression vectors, it is still influenced by the low stability and efficiency of gene integration. Transposons are mobile genetic elements that can be inserted and cleaved within the genome and can change their inserting position. The transposon vector system can be applied to establish a stable pool of cells with high efficiency in RTPs production through facilitating the integration of gene of interest into transcriptionally active sites under screening pressure. Here, the structure and optimization of transposon vector system and its application in expressing RTPs at high level in mammalian cells are reviewed.
RESUMEN
Colon cancer patients with mutant KRAS are resistant to cetuximab, an antibody directed against the epidermal growth factor receptor. New treatment options are needed to improve survival in patients with KRAS mutated colorectal cancer. Digitoxin is a cardiotonic drug, which has been demonstrated to exhibit anticancer effects in a number of cancers. However, the anticancer mechanisms of digitoxin in KRAS mutant human colon cancer cells remain elusive. Our result demonstrated that digitoxin but not cetuximab markedly decreased the expression of hypoxia-inducible factor-1α (HIF-1α), signal transducer and activator of transcription 3 (STAT3) and p-STAT3 protein in KRAS mutant colon cancer cells. Further analysis revealed that digitoxin inhibited HIF-1α protein synthesis, without affecting the expression level of HIF-1α mRNA or degradation of HIF-1α protein. The phosphorylation levels of ribosomal protein S6 kinase (p70S6K) and eIF4E binding protein-1 (4E-BP1) were significantly suppressed by digitoxin. Digitoxin inhibited the expression and activation of STAT3 through upregulation of phosphatase and tensin homolog deleted on chromosome ten (PTEN), SHP1 and protein inhibitors of activated STAT3 (PIAS3) and direct binding to STAT3. Meanwhile, digitoxin inhibited HIF-1α in STAT3-independent manner in KRAS mutant colon cancer cells. Moreover, digitoxin promoted apoptosis and inhibited proliferation and migration, which was potentially mediated by suppression of HIF-1α and STAT3. We also found that digitoxin administration inhibited tumor growth in a mouse xenograft model. Taken together, our findings highlight the therapeutic potential of digitoxin for the treatment of cetuximab-resistant human colon cancer.
Asunto(s)
Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Digitoxina/uso terapéutico , Neoplasias/tratamiento farmacológico , Animales , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones , Mutación , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Gene delivery systems play a vital role in gene therapy and recombinant protein production. The advantages of using gene delivery reagents for non-viral vector include the capacity to accommodate a large packaging load and their low or absent immunogenicity. Furthermore, they are easy to produce at a large scale and preserve. Gene delivery reagents for non-viral vector are commonly used for transfecting a variety of cells and tissues. It is mainly composed of liposomes and non-liposome cationic polymers. According to the different head structures used, the non-viral cationic transfection reagents include a quaternary ammonium salt, amine, amino acid or polypeptide, guanidine salt, and a heterocyclic ring. This article summarizes these approaches and developments of types and components of transfection reagents and optimization of gene delivery. The optimization of mammalian cell transient recombinant protein expression system and cationic reagents for clinical or clinical trials are also discussed.
Asunto(s)
Técnicas de Transferencia de Gen , Terapia Genética , Animales , Cationes , Indicadores y Reactivos , TransfecciónRESUMEN
Multicistronic vectors can increase transgene expression and decrease the imbalance of gene expression in the Chinese hamster ovary (CHO) cell expression system. Small, self-cleaving 2A peptides have a high cleavage efficiency and are essential for constructing high-expression multicistronic vectors. In this study, we investigated the effects of two different 2A peptides on transgene expression in CHO cells via their mediating action on tricistronic vectors. The enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP) genes were linked by the porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) peptides in a multicistronic vector. We transfected CHO cells with these vectors and screened for the presence of blasticidin-resistant colonies. Flow cytometry and real-time quantitative PCR (qPCR) were used to detect the expression levels of eGFP and RFP and the copy numbers of stably transfected cells. The results showed that P2A could enhance eGFP and RFP expression by 1.48- and 1.47-fold, respectively, compared to T2A. The expression levels of the genes were not proportional to their copy numbers. In conclusion, we found that P2A can effectively drive transgene expression in CHO cells and a potent 2A peptide can be used for recombinant protein production in the CHO cell system.
Asunto(s)
Vectores Genéticos/genética , Péptidos/genética , Proteínas Recombinantes/genética , Transgenes/genética , Animales , Células CHO , Cricetinae , Cricetulus , Dosificación de Gen , Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Péptidos/química , Péptidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transfección , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Dictamnine (DTM) is a natural alkaloid isolated from the root of Dictamnus dasycarpus Turcz and has been shown to exhibit multiple biological functions, including anti-inflammatory, antifungal, anti-angiogenic and anticancer activity. However, the mechanisms by which dictamnine inhibits tumor growth are not fully understood. In this study, we investigated the effectiveness of dictamnine as a treatment for cancer and to identify the underlying mechanisms of its anticancer activity. Here, dictamnine showed the potent inhibitory activity against HIF-1α and Slug activation induced by hypoxia in various human cancer cell lines. This compound markedly decreased the hypoxia-induced accumulation of HIF-1α and Slug protein in a dose-dependent manner. Further analysis revealed that dictamnine inhibited HIF-1α protein synthesis, without affecting its degradation. Our results demonstrated that dictamnine reduced HIF-1α protein synthesis by downregulating the mTOR/p70S6K/eIF4E and MAPK pathways, and reduced the expression of Slug by inhibiting the GSK-3ß/Slug signaling pathway. Moreover, epithelial-mesenchymal transition (EMT) was inhibited in dictamnine-treated tumors by downregulation of HIF-1α and Slug, as reflected by the upregulation of E-cadherin and Occludin, and the downregulation of N-cadherin and Vimentin. Phenomenological experiments showed that dictamnine reduced migration and invasion, inhibited HCT116â¯cell proliferation and promoted HCT116â¯cell apoptosis by downregulating HIF-1α and Slug. In vivo studies further confirmed that dictamnine treatment caused significant inhibition of tumor growth in a xenograft tumor model. These findings suggest that dictamnine is a potent cancer inhibitor, providing a rationale for anticancer pathway-targeted therapy.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Quinolinas/farmacología , Factores de Transcripción de la Familia Snail/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Proliferación Celular/efectos de los fármacos , Dictamnus/química , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Neoplasias/diagnóstico por imagen , Neoplasias/metabolismo , Neoplasias Experimentales/diagnóstico por imagen , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Raíces de Plantas/química , Quinolinas/química , Quinolinas/aislamiento & purificación , Transducción de Señal/efectos de los fármacos , Factores de Transcripción de la Familia Snail/metabolismo , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
BACKGROUND AND PURPOSE: IL-1ß is a cytokine of critical importance in inflammatory, infectious and autoimmune diseases. Zinc finger protein 91 (ZFP91) has been reported to be involved in multiple biological processes. Here, we identified a previously unknown role for ZFP91 in the production of biologically active IL-1ß and investigated the underlying mechanisms of its effects. EXPERIMENTAL APPROACH: In vitro, the underlying mechanisms of ZFP91 at inhibiting the expression of IL-1ß were investigated by ELISA, RT-PCR, Western blotting, immunoprecipitation and immunofluorescence assays. In vivo, colitis was induced by giving 4% dextran sulfate sodium (DSS) p.o. in drinking water for 5 days. Peritonitis was induced by injecting 700 µg alum i.p. for 12 h. KEY RESULTS: ZFP91 activated the non-canonical caspase-8 inflammasome, which resulted in robust IL-1ß secretion. Using an immunoprecipitation assay and immunofluorescence assay, we found that ZFP91 promoted the assembly of the non-canonical caspase-8 inflammasome complex. Moreover, ZFP91 enhanced the activation of ERK, p38 MAPK and JNK in macrophages. In addition, our data demonstrate that the synthesis of pro-IL-1ß is dependent on activation of these MAPK signalling pathways. In vivo experiments, the symptoms and colonic inflammation associated with DSS-induced colitis were ameliorated in mice deficient in ZFP91. Furthermore, the inflammation in alum-induced peritonitis was also attenuated in mice deficient in ZFP91. CONCLUSIONS AND IMPLICATIONS: Our research describes a mechanism by which ZFP91 promotes production of IL-1ß under physiological conditions and suggests that ZFP91 may be a promising therapeutic target for intervention in inflammatory, infectious and autoimmune-related diseases.
Asunto(s)
Caspasa 8/metabolismo , Inflamasomas/metabolismo , Interleucina-1beta/biosíntesis , Macrófagos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Administración Oral , Animales , Células Cultivadas , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Colitis/metabolismo , Sulfato de Dextran/administración & dosificación , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células THP-1 , Ubiquitina-Proteína Ligasas/deficienciaRESUMEN
Dictamnus dasycarpus is a traditional Chinese medicine thathas been commonly used in the treatment of cancer. Fraxinellone is a natural product isolated from the D. dasycarpus plant, which has been shown to exhibit neuroprotective and anti-inflammatory activities. However, whether fraxinellone exerts anticancer effects and the mechanisms by which it may inhibit tumor growth remain unknown. Here, we found that fraxinellone, in a dose-dependented manner, inhibited the expression of programmed cell death ligand-1 (PD-L1), which plays a pivotal role in tumorigenesis. It was subsequently shown that fraxinellone reduced HIF-1α protein synthesis via the mTOR/p70S6K/eIF4E and MAPK pathways. It also inhibited activation of STAT3 via the JAK1, JAK2, and Src pathways. Immunoprecipitation and western blotting assays showed that fraxinellone inhibited PD-L1 expression by reducing STAT3 and HIF-1α cooperatively. Flow cytometry, colony formation, and EdU incorporation assays demonstrated that fraxinellone inhibited cell proliferation through suppression of PD-L1. Tube formation, migration, and invasion assays showed that fraxinellone inhibits angiogenesis by suppressing PD-L1. In vivo studies further supported anticancer role for fraxinellone, demonstrating that fraxinellone treatment inhibited the growth of tumor xenografts. We concluded that fraxinellone inhibits PD-L1 expression by downregulating the STAT3 and HIF-1α signaling pathways, subsequently inhibiting proliferation and angiogenesis in cancer cells. These studies reveal previously unknown characteristics of fraxinellone and provide new perspectives into the mechanism of cancer inhibition of the compound.
Asunto(s)
Antineoplásicos/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Benzofuranos/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Factor de Transcripción STAT3/antagonistas & inhibidores , Animales , Antineoplásicos/uso terapéutico , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Benzofuranos/uso terapéutico , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Femenino , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/fisiología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ratones Endogámicos BALB C , Ratones Desnudos , Simulación del Acoplamiento Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a complex that regulates several hundreds of genes, including those involved in immunity and inflammation, survival, proliferation, and the negative feedback of NF-κB signaling. Chelidonine, a major bioactive, isoquinoline alkaloid ingredient in Chelidonium majus, exhibits antiinflammatory pharmacological properties. However, its antiinflammatory molecular mechanisms remain unclear. In this work, we explored the effect of chelidonine on TNF-induced NF-κB activation in HCT116 cells. We found chelidonine inhibited the phosphorylation and degradation of the inhibitor of NF-κB alpha and nuclear translocation of RELA. Furthermore, by inhibiting the activation of NF-κB, chelidonine downregulated target genes involved in inflammation, proliferation, and apoptosis. Chelidonine also inhibited mitogen-activated protein kinase pathway activation by blocking c-Jun N-terminal kinase and p38 phosphorylation. These results suggest that chelidonine may be a potential therapeutic agent against inflammatory diseases in which inhibition of NF-κB activity plays an important role.
Asunto(s)
Benzofenantridinas/uso terapéutico , Alcaloides de Berberina/uso terapéutico , Células HCT116/metabolismo , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Apoptosis , Benzofenantridinas/administración & dosificación , Benzofenantridinas/farmacología , Alcaloides de Berberina/administración & dosificación , Alcaloides de Berberina/farmacología , Humanos , Transducción de Señal , TransfecciónRESUMEN
CONTEXT: Amorfrutin A is a natural product isolated from the fruits of Amorpha fruticosa L. and has been shown to exhibit multiple bioeffector functions. In the present study, we investigated whether amorfrutin A exerts anticancer effects by inhibiting STAT3 activation in cervical cancer cells. OBJECTIVE: To investigate the effectiveness of amorfrutin A as a treatment of cancer, and determine the underlying pharmacological mechanism of action. MATERIALS AND METHODS: HeLa, SK-Hep1, MDA-MB-231 and HCT116 cells were used in this study. Major assays were luciferase reporter assay, MTT, Western blot analysis, immunofluorescence assay, reverse transcription-PCR (RT-PCR), flow cytometric analysis, EdU labeling and immunofluorescence, xenografted assay. RESULTS: Amorfrutin A significantly inhibited tumor necrosis factor-α (TNF-α)-induced phosphorylation and nuclear translocation of STAT3 in human cervical carcinoma cells. Amorfrutin A also inhibited activation of the upstream kinases Janus-activated kinase 1 (JAK1), JAK2 and Src signaling pathways. Furthermore, amorfrutin A increased the expression of p53, p21, p27, induced cell cycle arrest in the G1 phase as well as decreased levels of various oncogene protein products. In vivo studies further confirmed the inhibitory effect of amorfrutin A on the expression of STAT3 proteins, leading to a decrease growth of HeLa cells in a xenograft tumor model. DISCUSSION AND CONCLUSIONS: The results indicated that amorfrutin A is a potent inhibitor of STAT3 and provide new perspectives into the mechanism of its anticancer activity.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Janus Quinasa 1/metabolismo , Janus Quinasa 2/metabolismo , Factor de Transcripción STAT3/metabolismo , Salicilatos/farmacología , Estilbenos/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Femenino , Puntos de Control de la Fase G1 del Ciclo Celular/efectos de los fármacos , Células HCT116 , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacosRESUMEN
The NF-κB signaling pathway plays a pivotal role in regulating the immune response and inflammation. However, it has been shown that NF-κB also has a major role in oncogenesis. Therefore, NF-κB inhibitors have been considered as potential drugs against cancer. Herein, we searched for NF-κB inhibitors from natural sources and identified mollugin from the roots of Rubia cordifolia L. as an inhibitor of NF-κB activation. We found that mollugin significantly inhibited the expression of an NF-κB reporter gene induced by tumor necrosis factor (TNF)-α in a dose-dependent manner. Moreover, mollugin inhibited TNF-α-induced phosphorylation and nuclear translocation of p65, phosphorylation and degradation of inhibitor of κB (IκBα), and IκB kinase (IKK) phosphorylation. Furthermore, we discovered that pretreatment of cells with mollugin prevented the TNF-α-induced expression of NF-κB target genes, such as genes related to proliferation (COX-2, Cyclin D1 and c-Myc), anti-apoptosis (Bcl-2, cIAP-1 and survivin), invasion (MMP-9 and ICAM-1), and angiogenesis (VEGF). We also demonstrated that mollugin potentiated TNF-α-induced apoptosis and inhibited proliferation of HeLa cells. We further demonstrated in vivo that mollugin suppressed the growth of tumor xenografts derived from HeLa cells. Taken together, mollugin may be a valuable candidate for cancer treatment by targeting NF-κB.
Asunto(s)
FN-kappa B/genética , Neoplasias/tratamiento farmacológico , Piranos/administración & dosificación , Factor de Necrosis Tumoral alfa/genética , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Proteínas I-kappa B/genética , Ratones , Inhibidor NF-kappaB alfa/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/patología , Piranos/química , Factor de Transcripción ReIA/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Hypoxia enhances the development of solid tumors. Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that is dominantly expressed under hypoxia in solid tumor cells and is a key factor of tumor regulation. HIF-1α regulates several target genes involved in many aspects of cancer progression, including angiogenesis, metastasis, and cell proliferation, as well as imparting resistance to cancer treatment. In this study, we assessed shikonin, which derives from the traditional medical herb Lithospermum erythrorhizon, for its anti-cancer effects in hypoxia-induced human colon cancer cell lines. Shikonin showed potent inhibitory activity against hypoxia-induced HIF-1α activation in various human cancer cell lines and efficient scavenging activity of hypoxia-induced reactive oxygen species in tumor cells. Further analysis revealed that shikonin inhibited HIF-1α protein synthesis without affecting the expression of HIF-1α mRNA or degrading HIF-1α protein. It was subsequently shown to attenuate the activation of downstream mTOR/p70S6K/4E-BP1/eIF4E kinase. Shikonin also dose-dependently caused the cell cycle arrest of activated HCT116 cells and inhibited the proliferation of HCT116 and SW620 cells. Moreover, it significantly inhibited tumor growth in a xenograft modal. These findings suggest that shikonin could be considered for use as a potential drug in human colon cancer therapy.
Asunto(s)
Antineoplásicos Fitogénicos/toxicidad , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Naftoquinonas/toxicidad , Transducción de Señal/efectos de los fármacos , Animales , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células HCT116 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Lithospermum/química , Lithospermum/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Naftoquinonas/química , Naftoquinonas/aislamiento & purificación , Naftoquinonas/uso terapéutico , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Trasplante HeterólogoRESUMEN
Inflammation contributes to development and progression in a variety of cancers, including cervical cancer, which is the second leading cause of cancer deaths in women worldwide. In this study, we examined the anti-inflammatory effects of imperatorin, a psoralen-type furanocoumarin from the fruits of Angelica dahurica, in tumor necrosis factor-α (TNF-α)-stimulated HeLa cells by investigating its impact on the production and expression of cytokines and the major signal-transduction pathways. We found this compound significantly inhibited the TNF-α-induced expression of NF-κB target genes, such as COX-2, cyclin D1, MMP-9, VEGF, IL-6 and Bcl-xL in a concentration-dependent manner. Further analysis revealed that imperatorin was a potent inhibitor of NF-κB activation by the suppression of TNF-α-induced IKKα/ß phosphorylation, IκB phosphorylation and degradation, and NF-κB p65 nuclear translocation. We also demonstrated that imperatorin downregulated TNF-α-induced activation of PI3K/Akt. Furthermore, our findings show that imperatorin inhibits TNF-α-induced ROS generation. Taken together, imperatorin can blunt inflammation by inhibiting the ROS-mediated activation of the PI3K/Akt/NF-κB pathway.
Asunto(s)
Furocumarinas/administración & dosificación , Inflamación/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/genética , Neoplasias del Cuello Uterino/tratamiento farmacológico , Angelica/química , Femenino , Frutas/química , Furocumarinas/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Inflamación/genética , Inflamación/patología , FN-kappa B/genética , Proteínas de Neoplasias/genética , Fosfatidilinositol 3-Quinasas/genética , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/patologíaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Angelica dahurica is a commonly used traditional Chinese medicine to treat migraine headache, toothache and cancer. Imperatorin is an active natural furocoumarin component originating from Angelica dahurica and has been shown to exhibit multiple bioeffector functions, including anti-cancer activity. However, the mechanism by which imperatorin inhibits tumor growth is not fully understood. AIM OF THE STUDY: The aim of this study was to investigate the effectiveness of imperatorin as a treatment of cancer and to identify the underlying mechanisms of its anticancer activity. MATERIALS AND METHODS: HCT116, HeLa, and Hep3B cells were used in this study. Major assays were promoter-reporter gene assay, MTT, western blot analysis, immunofluorescence assay, reverse transcription-PCR (RT-PCR), flow cytometric analysis, clonogenic assay, EdU labeling and immunofluorescence, xenografted assay, and VEGF ELISA. RESULTS: We here demonstrated the effect of imperatorin on hypoxia-inducible factor-1 (HIF-1) activation. Imperatorin showed a potent inhibitory activity against HIF-1 activation induced by hypoxia in various human cancer cell lines. This compound markedly decreased the hypoxia-induced accumulation of HIF-1α protein dose-dependently, whereas it did not affect the expressions of HIF-1ß and topoisomerase-I (Topo-I). Further analysis revealed that imperatorin inhibited HIF-1α protein synthesis, without affecting the expression level of HIF-1α mRNA or degradation of HIF-1α protein. Moreover, the phosphorylation levels of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6K), eIF4E binding protein-1 (4E-BP1), eukaryotic initiation factor 4E (eIF4E), extracellular signal-regulated kinase-1/2 (ERK1/2), SAPK/JNK and p38 were significantly suppressed by imperatorin. Furthermore, imperatorin prevented hypoxia-induced expression of HIF-1 target genes and flow cytometric analysis indicated that imperatorin induced G1 phase arrest in human colon cancer cell (HCT116). We found that imperatorin administration inhibits tumor growth and blocks tumor angiogenesis in a xenograft tumor model. CONCLUSIONS: These results show that imperatorin inhibited HIF-1α protein synthesis by downregulating the mTOR/p70S6K/4E-BP1 and MAPK pathways. These conclusions suggest that imperatorin is an effective inhibitor of HIF-1 and provide new perspectives into the mechanism of its anticancer activity.