RESUMO
Lymphoid leukosis is a poultry neoplastic disease caused by avian leukosis virus (ALV) and is characterized by high morbidity and variable mortality rates in chicks. Currently, no effective treatment and vaccination is the only means to control it. This study exploited the immunoinformatics approaches to construct multi-epitope vaccine against ALV. ABCpred and IEDB servers were used to predict B and T lymphocytes epitopes from the viral proteins, respectively. Antigenicity, allergenicity and toxicity of the epitopes were assessed and used to construct the vaccine with suitable adjuvant and linkers. Secondary and tertiary structures of the vaccine were predicted, refined and validated. Structural errors, solubility, stability, immune simulation, dynamic simulation, docking and in silico cloning were also evaluated.The constructed vaccine was hydrophilic, antigenic and non-allergenic. Ramchandran plot showed most of the residues in the favored and additional allowed regions. ProsA server showed no errors in the vaccine structure. Immune simulation showed significant immunoglobulins and cytokines levels. Stability was enhanced by disulfide engineering and molecular dynamic simulation. Docking of the vaccine with chicken's TLR7 revealed competent binding energies.The vaccine was cloned in pET-30a(+) vector and efficiently expressed in Escherichia coli. This study provided a potent peptide vaccine that could assist in tailoring a rapid and cost-effective vaccine that helps to combat ALV. However, experimental validation is required to assess the vaccine efficiency.
Assuntos
Vírus da Leucose Aviária , Animais , Simulação de Acoplamento Molecular , Vacinas de Subunidades Proteicas , Imunoinformática , Galinhas , Epitopos de Linfócito T , Simulação de Dinâmica Molecular , Epitopos de Linfócito B , Vacinas de Subunidades Antigênicas , Biologia ComputacionalRESUMO
RNA-sequencing has been proposed as a valuable technique to develop individualized therapy concepts for cancer patients based on their tumor-specific mutational profiles. Here, we aimed to identify drugs and inhibitors in an individualized therapy-based drug repurposing approach focusing on missense mutations for 35 biopsies of cancer patients. The missense mutations belonged to 9 categories (ABC transporter, apoptosis, angiogenesis, cell cycle, DNA damage, kinase, protease, transcription factor, tumor suppressor). The highest percentages of missense mutations were observed in transcription factor genes. The mutational profiles of all 35 tumors were subjected to hierarchical heatmap clustering. All 7 leukemia biopsies clustered together and were separated from solid tumors. Based on these individual mutation profiles, two strategies for the identification of possible drug candidates were applied: Firstly, virtual screening of FDA-approved drugs based on the protein structures carrying particular missense mutations. Secondly, we mined the Drug Gene Interaction (DGI) database (https://www.dgidb.org/) to identify approved or experimental inhibitors for missense mutated proteins in our dataset of 35 tumors. In conclusion, our approach based on virtual drug screening of FDA-approved drugs and DGI-based inhibitor selection may provide new, individual treatment options for patients with otherwise refractory tumors that do not respond anymore to standard chemotherapy.
Assuntos
Neoplasias , Transcriptoma , Humanos , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Detecção Precoce de Câncer , Neoplasias/tratamento farmacológico , Neoplasias/genética , Fatores de Transcrição/genéticaRESUMO
BACKGROUND: Sheep pulmonary adenocarcinoma (OPA) is a contagious lung cancer of sheep caused by the Jaagsiekte retrovirus (JSRV). OPA typically has a serious economic impact worldwide. A vaccine has yet to be developed, even though the disease has been globally spread, along with its complications. This study aimed to construct an effective multi-epitopes vaccine against JSRV eliciting B and T lymphocytes using immunoinformatics tools. RESULTS: The designed vaccine was composed of 499 amino acids. Before the vaccine was computationally validated, all critical parameters were taken into consideration; including antigenicity, allergenicity, toxicity, and stability. The physiochemical properties of the vaccine displayed an isoelectric point of 9.88. According to the Instability Index (II), the vaccine was stable at 28.28. The vaccine scored 56.51 on the aliphatic index and -0.731 on the GRAVY, indicating that the vaccine was hydrophilic. The RaptorX server was used to predict the vaccine's tertiary structure, the GalaxyWEB server refined the structure, and the Ramachandran plot and the ProSA-web server validated the vaccine's tertiary structure. Protein-sol and the SOLPro servers showed the solubility of the vaccine. Moreover, the high mobile regions in the vaccine's structure were reduced and the vaccine's stability was improved by disulfide engineering. Also, the vaccine construct was docked with an ovine MHC-1 allele and showed efficient binding energy. Immune simulation remarkably showed high levels of immunoglobulins, T lymphocytes, and INF-γ secretions. The molecular dynamic simulation provided the stability of the constructed vaccine. Finally, the vaccine was back-transcribed into a DNA sequence and cloned into a pET-30a ( +) vector to affirm the potency of translation and microbial expression. CONCLUSION: A novel multi-epitopes vaccine construct against JSRV, was formed from B and T lymphocytes epitopes, and was produced with potential protection. This study might help in controlling and eradicating OPA.
Assuntos
Adenocarcinoma de Pulmão , Retrovirus Jaagsiekte de Ovinos , Neoplasias Pulmonares , Doenças dos Ovinos , Vacinas , Adenocarcinoma de Pulmão/veterinária , Animais , Epitopos , Neoplasias Pulmonares/veterinária , Ovinos , Doenças dos Ovinos/prevenção & controleRESUMO
BACKGROUND: Prostate cancer (PCa) is the most prominent malignancy among men worldwide. PCa cells have a high tendency to metastasize to various distant organs, and this activity is the main cause of PCa mortality. Nimbolide is a promising phytochemical constituent of neem Azadirachta indica (Meliaceae). Previous studies showed that nimbolide exhibited potent anticancer activity however, its role against PCa tumorigenesis has not been fully elucidated. PURPOSE: Our work aims to explore the role of nimbolide in regulating the essential tumor-associated processes involved in the metastatic cascade in PCa cells. STUDY DESIGN: Cytotoxicity assay, wound healing and spheroid invasion assays, western blotting, immunofluorescence, tube-formation assay, in vivo and immunohistochemistry. METHODS: The cytotoxicity of nimbolide towards PCa cell lines was assessed by resazurin assays. The cell mobility and migration of nimbolide-treated DU145 cells were determined by wound healing and spheroid invasion assays. Tubulin network was visualized using U2OS cells and DU145 cells. The effect of nimbolide on E-cadherin, ß-catenin, acetylated α-tubulin and HDAC6 protein expressions levels were measured by Western blot. The potentiality of nimbolide to inhibit angiogenesis was revealed by HUVEC tube-formation assay. Nimbolide antitumor effect was studied in a syngeneic model of murine prostate cancer. RESULTS: The current study indicated that nimbolide negatively affected the migratory and invasive capacity of DU145 prostate cancer cells in 2D and three-dimensional (3D) spheroid cultures. Interestingly, nimbolide induced downregulation of E-cadherin without any influence on the expression level of ß-catenin. Additionally, we demonstrated that nimbolide influenced the microtubule network which was supported by the upregulation of acetylated α-tubulin and the reduction in HDAC6 protein. Moreover, the inhibitory effect of nimbolide on angiogenesis was clearly observed in HUVEC tube formation assay. In vivo experiments revealed the significant suppression of PCa growth and targeting of the B-RAF/p.ERK signaling pathway by nimbolide. CONCLUSION: Our results showed that nimbolide inhibited 2D and 3D prostate cancer cells migration and downregulated E-cadherin protein expression, a marker for metastatic chemoresistance and tumor recurrence. Nimbolide stabilized the microtubules, combated angiogenesis and suppressed B.RAF/ERK-mediated in vivo tumor growth. Nimbolide may be considered as potential therapeutic agent for metastatic and advanced PCa patients and merits further investigations.
Assuntos
Neoplasias da Próstata , Animais , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Limoninas , Masculino , Camundongos , Microtúbulos , Neoplasias da Próstata/tratamento farmacológicoRESUMO
AIMS: Epidermal growth factor receptor (EGFR) is not only involved in carcinogenesis, but also in chemoresistance. We characterized U87.MGΔEGFR glioblastoma cells with constitutively active EGFR due to deletion at the ligand binding domain in terms of gene expression profiling and chromosomal aberrations. Wild-type U87.MG cells served as control. MATERIALS AND METHODS: RNA sequencing and network analyses (Ingenuity Pathway Analysis) were performed to identify novel drug resistance mechanisms related to expression of mutation activated EGFR. Chromosomal aberrations were characterized by multicolor fluorescence in situ hybridization (mFISH) and array comparative genomic hybridization (aCGH). KEY FINDINGS: U87.MGΔEGFR cells presented much more chromosomal aberrations, amplifications and deletions than wild-type U87.MG cells. Both cell lines were near-triploid. Numerous genes were overexpressed in U87.MGΔEGFR cells, some of which have been already linked to drug resistance. PXDN, which is associated with epithelial mesenchymal transition, was the most upregulated gene (901.8-fold). TENM1 was 331.6-fold upregulated, and it was previously reported to modulate neural development. EGFR-AS1 (161.2-fold upregulated) has been reported to increase the EGFR mRNA stability and its expression - in accordance with that of EGFR - was upregulated (85.5-fold). In addition to well-known resistance genes, numerous novel genes and genomic aberrations were identified. ANGPT2 upregulation and CPM downregulation were validated by Western blotting. SIGNIFICANCE: Transcriptomics and genomics analyses in U87.MGΔEGFR cells unraveled a range of novel drug resistance mechanisms including apoptosis, DNA repair, ferroptosis, glutathione related gene activities, heat shock, oxidative stress, transcription factor activities, which may have important implications for future treatment strategies.
Assuntos
Neoplasias Encefálicas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Genômica , Glioblastoma/genética , Mutação/genética , Transcriptoma/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Aberrações Cromossômicas , Regulação para Baixo/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Glioblastoma/patologia , Humanos , Metáfase , Mapas de Interação de Proteínas/genética , Transdução de Sinais/genética , Regulação para Cima/genéticaRESUMO
TP53 (p53) is a pivotal player in tumor suppression with fifty percent of all invasive tumors displaying mutations in the TP53 gene. In the present study, we characterized colon cancer cells (HCT116 p53 -/-) with TP53 deletion, a sub-line derived from HCT116-p53 +/+ cells. RNA sequencing and network analyses were performed to identify novel drug resistance mechanisms. Chromosomal aberrations were identified by multicolor fluorescence in situ hybridization (mFISH) and array comparative genomic hybridization (aCGH). Numerous genes were overexpressed in HCT116 p53 -/- cells: RND3/RhoE (235.6-fold up-regulated), DCLK1 (60.2-fold up-regulated), LBH (31.9-fold up-regulated), MYB (28.9-fold up-regulated), TACSTD2 (110.1-fold down-regulated), NRIP1 (81.5-fold down-regulated) and HLA-DMB (69.7-fold down-regulated) are among the identified genes with potential influence on multidrug resistance (MDR) and they are associated with cancer progression and tumorigenesis, according to previously published studies. Probably due to TP53 deletion, disturbances in DNA repair and apoptosis are leading to aberrancies in cellular and organismal organization, ultimately increasing tumorigenesis and cancer progression potential. With NFκB, PI3K and HSP70, being at the center of merged protein network, and TH1-2 pathways, being among the influenced pathways, it can be speculated that the inflammatory pathway contributes to a resistance phenotype together with cell cycle regulation and heat-shock response. HCT116-p53 -/- cells have more chromosomal aberrations, gains and losses in copy numbers than HCT116-p53 +/+ cells. In conclusion, numerous genomic aberrations, which might be associated with yet unknown drug resistance mechanisms, were identified. This may have important implications for future treatment strategies.
Assuntos
Aberrações Cromossômicas , Neoplasias do Colo/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Proteína Supressora de Tumor p53/genética , Antineoplásicos/farmacologia , Apoptose/genética , Neoplasias do Colo/genética , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA/genética , Reparo do DNA/genética , Progressão da Doença , Resistência a Múltiplos Medicamentos/genética , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Hibridização in Situ Fluorescente , Mutação , Análise de Sequência de RNARESUMO
BACKGROUND: Apigenin is one of the most abundant dietary flavonoids that possesses multiple bio-functions. PURPOSE: This study was designed to determine the influence of apigenin on gene expressions, cancer cells, as well as STAT1/COX-2/iNOS pathway mediated inflammation and tumorigenesis in HEK293-STAT1 cells. Furthermore, the cytotoxic activity toward multiple myeloma (MM) cell lines was investigated. METHODS: Bioinformatic analyses were used to predict the sensitivity and resistance of tumor cells toward apigenin and to determine cellular pathways influenced by this compound. The cytotoxic and ferroptotic activity of apigenin was examined by the resazurin reduction assay. Additionally, we evaluated apoptosis, and cell cycle distribution, induction of reactive oxygen species (ROS) and loss of integrity of mitochondrial membrane (MMP) by using the flow cytometry analysis. DAPI staining was used to detect characteristic apoptotic features. Furthermore, we verified its anti-inflammatory and additional mechanism of cell death by western blotting. RESULTS: COMPARE and hierarchical cluster analyses exhibited that 29 of 55 tumor cell lines were sensitive against apigenin (p < 0.001). The Ingenuity Pathway Analysis data showed that important bio-functions affected by apigenin were: gene expression, cancer, hematological system development and function, inflammatory response, and cell cycle. The STAT1 transcription factor was chosen as target protein on the basis of gene promoter binding motif analyses. Apigenin blocked cell proliferation of wild-type HEK293 and STAT1 reporter cells (HEK293-STAT1), promoted STAT1 suppression and subsequent COX-2 and iNOS inhibition. Apigenin also exhibited synergistic activity in combination with doxorubicin toward HEK293-STAT1 cells. Apigenin exerted excellent growth-inhibitory activity against MM cells in a concentration-dependent manner with the greatest activity toward NCI-H929 (IC50 value: 10.73 ± 3.21 µM). Apigenin induced apoptosis, cell cycle arrest, ferroptosis and autophagy in NCI-H929 cells. CONCLUSION: Apigenin may be a suitable candidate for MM treatment. The inhibition of the STAT1/COX-2/iNOS signaling pathway by apigenin is an important mechanism not only in the suppression of inflammation but also in induction of apoptosis.
Assuntos
Apigenina/farmacologia , Ciclo-Oxigenase 2/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Fator de Transcrição STAT1/genética , Antineoplásicos Fitogênicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apigenina/administração & dosagem , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Biologia Computacional/métodos , Relação Dose-Resposta a Droga , Doxorrubicina/administração & dosagem , Células HEK293 , Humanos , Mieloma Múltiplo/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT1/metabolismoRESUMO
BACKGROUND: Sesquiterpene lactones having α-methylene-γ-lactone moiety are promising natural metabolites showing various biological activity. One of the major metabolites isolated from Pulicaria undulata, 2α-hydroxyalantolactone (PU-1), has not been investigated in detail yet. Multidrug resistance (MDR) represents a major obstacle for cancer chemotherapy and the capability of novel natural products to overcoming MDR is of great interest. PURPOSE: Exploring the molecular modes of action for potent natural product metabolites. METHODS: The resazurin reduction assay was employed to evaluate the cytotoxicity of PU-1 on sensitive and their corresponding drug-resistant cell lines (overexpressing P-glycoprotein, BCRP, ABCB5, ΔEGFR, or TP53 knockout). Gene expression profiling was performed by transcriptome-wide mRNA microarray in the human CCRF-CEM leukemic cells after treatment with PU-1. The top significantly up- or down-regulated genes were identified by Chipster program and analyzed using Ingenuity Pathway Analysis (IPA) software. Finally, flow cytometry and Western blotting were performed for cell cycle analyses and apoptosis detection. RESULTS: The sesquiterpene lactone, PU-1, showed potent cytotoxicity towards the drug-sensitive and -resistant cell lines. Transcriptome-wide mRNA expression profiling and pathway analysis pointed to genes involved in DNA damage response and G2/M cell cycle arrest. G2/M arrest was verified by flow cytometry and further confirmed by the upregulation of p21 and downregulation of p-CDC25C expression in Western blotting. Moreover, the suggested DNA damage checkpoint regulation was confirmed by immunofluorescence and Western blotting by upregulation of pS345 Chk1, p-H3 and γ-H2AX. Furthermore, PU-1 inhibited PI3K/AKT pathway, which is involved in signaling DNA damage and G2/M arrest. Cells ultimately induced apoptosis upon PU-1 treatment. CONCLUSIONS: PU-1 is a potent natural product inhibiting otherwise drug-resistant human tumor cell growth through DNA damage, G2/M cell cycle arrest and apoptosis.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Leucemia/tratamento farmacológico , Pulicaria/química , Sesquiterpenos/farmacologia , Antineoplásicos Fitogênicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Sesquiterpenos/químicaRESUMO
AIMS: We systematically characterized metastatic murine B16-F10 melanoma, a sub-line derived from murine melanoma B16-F1 cells. MATERIALS AND METHODS: RNA-sequencing and network analyses (Ingenuity Pathway Analysis) were performed to identify novel potential metastasis mechanisms. Chromosomal aberrations were identified by multicolor fluorescence in situ hybridization (mFISH) using all 21 murine whole chromosome painting probes. KEY FINDINGS: Numerous genes were overexpressed in B16-F10 cells, some of which have been already described as being metastasis-linked. Nr5a1/sf1, a known prognostic marker for adrenal tumors, was 177-fold upregulated in B16-F10 cells compared to B16-F1 cells. Hoxb8 was 75-fold upregulated, which was previously associated with gastric cancer progression and metastasis. Ptk7, which is linked with tumorigenesis and metastasis of esophageal squamous carcinoma, was 67-fold upregulated. B16-F10 cells acquired additional chromosomal aberrations compared to B16-F1 cells, including dic(4)(pter->qter:qter->pter), +dic(6;15), +der(10)t(10;?1;16). SIGNIFICANCE: In addition to well-known metastatic genes, numerous novel genes and genomic aberrations were identified, which may serve as targets for treatment in the future. Transcriptomic and genetic analyses in B16-F10 cells unraveled a range of novel metastasis mechanisms, which may also have important implications for future treatment strategies.
Assuntos
Melanoma Experimental/genética , Metástase Neoplásica/genética , Animais , Linhagem Celular Tumoral , Genoma/genética , Genômica/métodos , Hibridização in Situ Fluorescente/métodos , Melanoma/genética , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência de RNA/métodos , Transcriptoma/genéticaRESUMO
BACKGROUND: Carnosic acid (CA) is one of the main constituents in rosemary extract. It possesses valuable pharmacological properties, including anti-oxidant, anti-inflammatory, anti-microbial and anti-cancer activities. Numerous in vitro and in vivo studies investigated the anticancer profile of CA and emphasized its potentiality for cancer treatment. Nevertheless, the role of multidrug-resistance (MDR) related mechanisms for CA's anticancer effect is not yet known. PURPOSE: We investigated the cytotoxicity of CA against known mechanisms of anticancer drug resistance (P-gp, ABCB5, BCRP, EGFR and p53) and determined novel putative molecular factors associated with cellular response towards CA. STUDY DESIGN: Cytotoxicity assays, bioinformatic analysis, flow cytometry and western blotting were performed to identify the mode of action of CA towards cancer cells. METHODS: The cytotoxicity to CA was assessed using the resazurin assays in cell lines expressing the mentioned resistance mechanisms. A pharmacogenomic characterization of the NCI 60 cell line panel was applied via COMPARE, hierarchical cluster and network analyses. Flow cytometry was used to detect cellular mode of death and ROS generation. Changes in proteins-related to apoptosis were determined by Western blotting. RESULTS: Cell lines expressing ABC transporters (P-gp, BCRP or ABCB5), mutant EGFR or p53 were not cross-resistant to CA compared to their parental counterparts. By pharmacogenomic approaches, we identified genes that belong to different functional groups (e.g. signal transduction, regulation of cytoskeleton and developmental regulatory system). These genes were predicted as molecular determinants that mediate CA tumor cellular responses. The top affected biofunctions included cellular development, cellular proliferation and cellular death and survival. The effect of CA-mediated apoptosis in leukemia cells, which were recognized as the most sensitive tumor type, was confirmed via flow cytometry and western blot analysis. CONCLUSION: CA may provide a novel treatment option to target refractory tumors and to effectively cooperate with established chemotherapy. Using pharmacogenomic approaches and network pharmacology, the relationship between cancer complexity and multi-target potentials of CA was analyzed and many putative molecular determinants were identified. They could serve as novel targets for CA and further studies are needed to translate the possible implications to clinical cancer treatment.
Assuntos
Abietanos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Linhagem Celular Tumoral , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Proteínas de Neoplasias/metabolismo , Farmacogenética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Nimbolide is considered a promising natural product in cancer prevention and treatment. However, it is not known yet, whether the different mechanisms of multidrug resistance (MDR) influence its anticancer activity. In this study, well-known MDR mechanisms (ABCB1, ABCG2, ABCB5, TP53, EGFR) were evaluated against nimbolide. The P-glycoprotein (ABCB1/MDR1)-overexpressing CEM/ADR5000 cell line displayed remarkable hypersensitivity to nimbolide, which was mediated through upregulation of the tumor suppressor, PTEN, and its downstream components resulted in significant downregulation in ABCB1/MDR1 mRNA and P-glycoprotein. In addition, nimbolide targeted essential cellular metabolic-regulating elements including HIF1α, FoxO1, MYC and reactive oxygen species. The expression of breast cancer resistance protein (BCRP) as well as epidermal growth factor receptor (EGFR) and mutant tumor suppressor TP53 did not correlate to nimbolide's activity. Furthermore, this paper looked for other molecular determinants that might determine tumor cellular response towards nimbolide. COMPARE and hierarchical cluster analyses of transcriptome-wide microarray-based mRNA expressions of the NCI 60 cell line panel were performed, and a set of 40 genes from different functional groups was identified. The data suggested NF-κB as master regulator of nimbolide's activity. Interestingly, HIF1α was determined by COMPARE analysis to mediate sensitivity to nimbolide, which would be of great benefit in targeted therapy.
RESUMO
Betulinic acid (BetA) is a naturally occurring pentacyclic triterpene isolated from the outer bark of white-barked birch trees and many other medicinal plants. Here, we studied betulinic acid's cytotoxic activity against drug-resistant tumor cell lines. P-glycoprotein (MDR1/ABCB1) and BCRP (ABCG2) are known ATP-binding cassette (ABC) drug transporters that mediating MDR. ABCB5 is a close relative to ABCB1, which also mediates MDR. Constitutive activation of the EGF receptor is tightly linked to the development of chemotherapeutic resistance. BetA inhibited P-gp, BCRP, ABCB5 and mutation activated EGFR overexpressing cells with similar efficacy as their drug-sensitive parental counterparts. Furthermore, the mRNA expressions of ABCB1, BCRP, ABCB5 and EGFR were not related to the 50% inhibition concentrations (IC50) for BetA in a panel of 60 cell lines of the National Cancer Institute (NCI), USA. In addition to well-established MDR mechanisms, we attempted to identify other molecular mechanisms that play a role in mediating BetA's cytotoxic activity. For this reason, we performed COMPARE and hierarchical cluster analyses of the transcriptome-wide microarray-based mRNA expression of the NCI cell lines panel. Various genes significantly correlating to BetA's activity were involved in different biological processes, e.g., cell cycle regulation, microtubule formation, signal transduction, transcriptional regulation, chromatin remodeling, cell adhesion, tumor suppression, ubiquitination and proteasome degradation. Immunoblotting and in silico analyses revealed that the inhibition of AMFR activity might be one of the mechanisms for BetA to overcome MDR phenotypes. In conclusion, BetA may have therapeutic potential for the treatment of refractory tumors.
RESUMO
For decades, natural products represented a significant source of diverse and unique bioactive lead compounds in drug discovery field. In Clinical oncology, complete tumors remission is hampered by the development of drug-resistance. Therefore, development of cytotoxic agents that may overcome drug resistance is urgently needed. Here, the natural benzophenanthridine alkaloid sanguinarine has been studied for its cytotoxic activity against multidrug resistance (MDR) cancer cells. We investigated the role of the ATP-binding cassette (ABC) transporters BCRP/ABCG2, P-glycoprotein/ABCB1 and its close relative ABCB5 in drug resistance. Further drug resistance mechanisms analyzed in this study were the tumor suppressor TP53 and the epidermal growth factor receptor (EGFR). Multidrug resistant cells overexpressing BCRP, ABCB5 and mutated ΔEGFR were not cross-resistant toward sanguinarine. Interestingly, P-gp overexpressing cells were hypersensitive to sanguinarine. Doxorubicin uptake assay carried by flow cytometry revealed that sanguinarine is a potent inhibitor of the P-gp transporter. Moreover, immunoblotting analysis proved that P-gp was downregulated in a dose dependent manner after treating P-gp overexpressing cells with sanguinarine. It was surmised that The inhibition of NFκB activity might explain the collateral sensitivity in CEM/ADR5000 cells. The COMPARE and hierarchical cluster analyses of transcriptome-wide expression profiles of tumor cell lines of the National Cancer Institute identified genes involved in various cellular processes (immune response, inflammation signaling, cell migration and microtubule formation) significantly correlated with log10IC50 values for sanguinarine. In conclusion, sanguinarine may have therapeutic potential for treating multidrug resistant tumors.
RESUMO
An outbreak of Ebola virus disease (EVD) in Liberia began in March 2014 and ended in January 2016. Epidemiological information on the EVD cases was collected and managed nationally; however, collection and management of the data were challenging at the time because surveillance and reporting systems malfunctioned during the outbreak. EVD diagnostic laboratories, however, were able to register basic demographic and clinical information of patients more systematically. Here we present data on 16,370 laboratory samples that were tested between April 4, 2014 and March 29, 2015. A total of 10,536 traceable individuals were identified, of whom 3,897 were confirmed cases (positive for Ebola virus RNA). There were significant differences in sex, age, and place of residence between confirmed and suspected cases that tested negative for Ebola virus RNA. Age (young children and the elderly) and place of residence (rural areas) were the risk factors for death due to the disease. The case fatality rate of confirmed cases decreased from 80% to 63% during the study period. These findings may help support future investigations and lead to a fuller understanding of the outbreak in Liberia.
Assuntos
Surtos de Doenças/história , Ebolavirus/isolamento & purificação , Doença pelo Vírus Ebola/diagnóstico , Doença pelo Vírus Ebola/mortalidade , Adolescente , Adulto , Distribuição por Idade , Criança , Pré-Escolar , Demografia , Feminino , História do Século XXI , Humanos , Laboratórios , Libéria/epidemiologia , Modelos Logísticos , Masculino , Programas de Rastreamento/métodos , Pessoa de Meia-Idade , Fatores de Risco , Distribuição por Sexo , Adulto JovemRESUMO
Ebola virus is known to persist in semen of male survivors of Ebola virus disease (EVD). However, maximum duration of, or risk factors for, virus persistence are unknown. We report an EVD survivor with preexisting HIV infection, whose semen was positive for Ebola virus RNA 565 days after recovery from EVD.
Assuntos
Ebolavirus/isolamento & purificação , Infecções por HIV/complicações , Doença pelo Vírus Ebola/virologia , RNA Viral/isolamento & purificação , Sêmen/virologia , Humanos , Masculino , Pessoa de Meia-Idade , RNA Viral/química , Fatores de Tempo , Proteínas do Core Viral/química , Proteínas da Matriz Viral/químicaRESUMO
According to World Health Organization (WHO) data, the Ebola virus disease (Ebola) outbreak that began in West Africa in 2014 has resulted in 28,603 cases and 11,301 deaths (1). In March 2015, epidemiologic investigation and genetic sequencing in Liberia implicated sexual transmission from a male Ebola survivor, with Ebola virus detected by reverse transcription-polymerase chain reaction (RT-PCR) 199 days after symptom onset (2,3), far exceeding the 101 days reported from an earlier Ebola outbreak (4). In response, WHO released interim guidelines recommending that all male survivors, in addition to receiving condoms and sexual risk reduction counseling at discharge from an Ebola treatment unit (ETU), be offered semen testing for Ebola virus RNA by RT-PCR 3 months after disease onset, and every month thereafter until two consecutive semen specimens collected at least 1 week apart test negative for Ebola virus RNA (5). Male Ebola survivors should also receive counseling to promote safe sexual practices until their semen twice tests negative. When these recommendations were released, testing of semen was not widely available in Liberia. Challenges in establishing and operating the first nationwide semen testing and counseling program for male Ebola survivors included securing sufficient resources for the program, managing a public health semen testing program in the context of ongoing research studies that were also collecting and screening semen, identification of adequate numbers of trained counselors and appropriate health communication messages for the program, overcoming Ebola survivor-associated stigma, identification and recruitment of male Ebola survivors, and operation of mobile teams.