RESUMO
Nowadays, the usage of nanoparticles in various fields such as drug delivery, attracts the attention of many researchers in the treatment of cancers. Graphene oxide (GO) is one of the novel drug delivery systems which is used broadly owing to its unique features. In this survey, doxorubicin (DOX) was accompanied by natural medicine, curcumin (CUR), to diminish its side effects and enhance its efficiency. Cytotoxicity assay in human gastric cancer (AGS), prostate cancer (PC3), and ovarian cancer (A2780), was evaluated. Also, the uptake of DOX and CUR into cells, was assessed using a fluorescence microscope. Moreover, real-time PCR was applied for the evaluation of the expression of RB1 and CDK2 genes, which were involved in the cell cycle. In both separate and simultaneous forms, DOX and CUR were loaded with high efficiency and the release behavior of both drugs was pH-sensitive. The higher release rate was attained at pH 5.5 and 42 °C for DOX (80.23%) and CUR (13.06), respectively. The intensity of fluorescence in the free form of the drugs, was higher than the loaded form. In the same concentration, the free form of CUR and DOX were more toxic than the loaded form in all cell lines. Also, free drugs showed more impact on the expression of RB1 and CDK2 genes. Co-delivery of CUR and DOX into the mentioned cell lines, was more effective than the free form of CUR and DOX due to its lower toxicity to normal cells.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Curcumina/administração & dosagem , Doxorrubicina/administração & dosagem , Portadores de Fármacos , Grafite/química , Neoplasias/tratamento farmacológico , Polímeros Responsivos a Estímulos , Protocolos de Quimioterapia Combinada Antineoplásica/química , Protocolos de Quimioterapia Combinada Antineoplásica/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Curcumina/química , Curcumina/metabolismo , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Doxorrubicina/química , Doxorrubicina/metabolismo , Composição de Medicamentos , Liberação Controlada de Fármacos , Feminino , Humanos , Concentração de Íons de Hidrogênio , Cinética , Masculino , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: Radix Euphorbiae Ebracteolatae (REE) was recently reported to be significantly superior to vitamin A acid ointment in treating multiple plantar warts. However, the effects of REE on HPV18 remain unclear. Therefore, the current study aimed to investigate the effects of REE on the proliferation of HPV18, and explore possible molecular mechanisms underlying the effects. METHODS: HFK and HFK-HPV18 were treated with water-extracted single or compound REE, ethanol-extracted single or compound REE, TNF-α and IFN for 3 days, respectively. In addition, the organotypic rafts containing HFK-HPV18 and HFK were treated with REE, IFN and TNF-α for 7 days, respectively. Cell proliferation rates were measured with Brdu. mRNA expression of E6, L1, p53 and Rb was detected by qPCR. Protein expression of p53, Rb and L1 was detected by Western blot. RESULTS: Compared to HFK group, HFK-HPV18 group had significantly higher expression of E6 and L1. Compared to the control group, HFK-HPV18 treated with REE, TNF-α and IFN displayed significantly lower proliferation rates. The mRNA expression of E6 was markedly lower, and mRNA expression of p53 and Rb was significantly higher after treatment of REE in HFK-HPV18 or in organotypic rafts containing HFK-HPV18. Treatment with REE markedly increased the protein expression of p53 and Rb, and decreased the protein expression of L1 in HFK-HPV18 or in organotypic rafts containing HFK-HPV18. Among all formula of REE, the inhibition of proliferation rates and expression of E6 and L1, and the increase in expression of p53 and Rb in HFK-HPV18 was highest in ethanol-extracted compound REE group. CONCLUSIONS: The proliferation rates are significantly lower in HFK-HPV18 treated with REE. The expression of E6 and L1 is markedly lower, and expression of p53 and Rb is significantly higher after REE treatment in HFK-HPV18 or organotypic rafts containing HFK-HPV18. Among all formula of REE, ethanol-extracted compound REE displays the highest protection against HPV18.
Assuntos
Euphorbia/química , Infecções por Papillomavirus/tratamento farmacológico , Proteínas de Ligação a Retinoblastoma/genética , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genética , Proteínas do Capsídeo/genética , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Prepúcio do Pênis/efeitos dos fármacos , Prepúcio do Pênis/virologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Papillomavirus Humano 18/genética , Papillomavirus Humano 18/patogenicidade , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/virologia , Masculino , Proteínas Oncogênicas Virais/genética , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Extratos Vegetais/química , Extratos Vegetais/farmacologiaRESUMO
Aged skeletal muscle has an attenuated and delayed ability to proliferate satellite cells in response to resistance exercise. The mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway is a focal point for cell growth, however, the effect of postexercise mTORC1 activation on human skeletal muscle satellite cell (SC) proliferation is unknown. To test the proliferative capacity of skeletal muscle SC in aging muscle to a potent mTORC1 activator (i.e., EAA; essential amino acids) we recruited older (~72y) men to conduct leg resistance exercise (8setsx10reps) without (-EAA; n = 8) and with (+EAA: n = 11) ingestion of 10 g of EAA 1 h postexercise. Muscle biopsies were taken before exercise (Pre) and 24 h postexercise (Post) for assessment of expression and fiber type-specific Pax7+ SC, Ki67+Pax7+ SC and MyoD+ SC -EAA did not show an increase in Pax7+ satellite cells at Post(P > 0.82). Although statistical significance for an increase in Pax7 + SC at 24 h post-RE was not observed in +EAA versus -EAA, we observed trends for a treatment difference (P < 0.1). When examining the change from Pre to Post trends were demonstrated (#/myofiber: P = 0.076; and %/myonuclei: P = 0.065) for a greater increase in +EAA versus -EAA Notably, we found an increase SC proliferation in +EAA, but not -EAA with increase in Ki67+ SC and MyoD+ cells (P < 0.05). Ki67+ SC also exhibited a significant group difference Post (P < 0.010). Pax7+ SC in fast twitch myofibers did not change and were not different between groups (P > 0.10). CDK2, MEF2C, RB1 mRNA only increased in +EAA (P < 0.05). Acute muscle satellite cell proliferative capacity may be partially rescued with postexercise EAA ingestion in older men.
Assuntos
Aminoácidos Essenciais/farmacologia , Proliferação de Células , Músculo Esquelético/efeitos dos fármacos , Treinamento Resistido , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Idoso , Aminoácidos Essenciais/administração & dosagem , Estudos de Casos e Controles , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Suplementos Nutricionais , Humanos , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Masculino , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Proteína MyoD/genética , Proteína MyoD/metabolismo , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Proteínas de Ligação a Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/fisiologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Systematic analyses of plants that are used in traditional medicine may lead to the discovery of novel cytotoxic secondary metabolites. Diterpene possesses multiple bioactivities; here, epoxy clerodane diterpene (ECD) was isolated from Tinospora cordifolia (Willd.) stem and shown potential antiproliferative effect in MCF-7 human breast cancer cells. The antiproliferative effect of ECD on MCF-7 cells was systematically analyzed by cell and nuclear morphology, alterations in oxidative stress, and the expression of tumor suppressor and mitochondria-mediated apoptosis-related genes. We found that the IC50 value of ECD was 3.2µM at 24h and 2.4µM at 48h. We observed that the cytotoxicity of ECD was specific to MCF-7 cells, whereas ECD was nontoxic to normal Vero and V79 cells. ECD significantly triggered intracellular ROS generation even from the lower doses of 0.6 and 1.2µM; and it is relative to higher dose of 2.4µM. Further, we used 0.6µM, 1.2µM and 2.4µM as experimental doses to analyze the relative dose-dependent effects. Nuclear staining revealed that cells treated with the 2.4µM dose exhibited characteristic apoptotic morphological changes and that 46% of the cells were apoptotic and 4% were necrotic after 48h. ECD significantly increased the expression of mitochondria-dependent apoptotic pathway-related genes after 48h; we observed significantly (p≤0.05) increased expression of CYP1A, GPX, GSK3ß and TNF-α and downregulated expression of NF-κB. ECD also increased the expression of tumor suppressor genes such as Cdkn2A, Rb1 and p53. In addition, we observed that ECD treatment significantly (p≤0.001) upregulated the expression of apoptotic genes such as Bax, cas-3, cas-8, cas-9 and p21 and downregulated the expression of BCL-2, mdm2 and PCNA. In conclusion, ECD regulates the expression of Cdkn2A, p53 and mdm2 and induces apoptosis via the mitochondrial pathway in MCF-7 human breast cancer cells.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p18/genética , Diterpenos Clerodânicos/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas de Ligação a Retinoblastoma/genética , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Inibidor p16 de Quinase Dependente de Ciclina , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Humanos , Células MCF-7 , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , NF-kappa B/genética , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
BACKGROUND: Retinoblastoma is the most common intraocular malignancy of childhood. There is a paucity of genetic testing and prenatal genetic diagnosis from India, which has the highest incidence worldwide. MATERIALS AND METHODS: RB1 gene screening of an 8-month-old female child with bilateral retinoblastoma was accomplished using next generation sequencing. The results were used for prenatal testing in this family. RESULTS: A heterozygous germline mutation (chr13: 48951119delA; c.1281delA) was detected, which resulted in premature termination of a protein product (p.Glu428Argfs*29). Prenatal testing in maternal DNA revealed carrier status of the mother. Further clinical examination in the family members revealed retinocytomas in both eyes of the mother and maternal grandmother. Prenatal genetic testing of the developing fetus showed positivity for the mutation. As the family preferred to continue the pregnancy, serial 3-D ultrasounds were carried out every 2 weeks in the third trimester. Ten days after delivery, small extrafoveal tumors developed in both eyes, which were then treated successfully with transpupillary thermotherapy. CONCLUSION: We report the significance of genetic testing in the early detection and management of retinoblastoma from India.
Assuntos
Genes do Retinoblastoma , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Diagnóstico Pré-Natal , Neoplasias da Retina/genética , Proteínas de Ligação a Retinoblastoma/genética , Retinoblastoma/genética , Ubiquitina-Proteína Ligases/genética , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carboplatina/uso terapêutico , Análise Mutacional de DNA , Etoposídeo/uso terapêutico , Éxons/genética , Feminino , Testes Genéticos , Humanos , Índia , Lactente , Linhagem , Terapia com Prótons , Neoplasias da Retina/diagnóstico , Retinoblastoma/diagnóstico , Vincristina/uso terapêuticoRESUMO
Merkel cell carcinoma is a rare but highly aggressive cutaneous neuroendocrine carcinoma. Cytokeratin 20 (CK20) is expressed in ~95% of Merkel cell carcinomas and is useful for distinction from morphologically similar entities including metastatic small-cell lung carcinoma. Lack of CK20 expression may make diagnosis of Merkel cell carcinoma more challenging, and has unknown biological significance. Approximately 80% of CK20-positive Merkel cell carcinomas are associated with the oncogenic Merkel cell polyomavirus. Merkel cell carcinomas lacking Merkel cell polyomavirus display distinct genetic changes from Merkel cell polyomavirus-positive Merkel cell carcinoma, including RB1 inactivating mutations. Unlike CK20-positive Merkel cell carcinoma, the majority of CK20-negative Merkel cell carcinomas are Merkel cell polyomavirus-negative, suggesting CK20-negative Merkel cell carcinomas predominantly arise through virus-independent pathway(s) and may harbor additional genetic differences from conventional Merkel cell carcinoma. Hence, we analyzed 15 CK20-negative Merkel cell carcinoma tumors (10 Merkel cell polyomavirus-negative, four Merkel cell polyomavirus-positive, and one undetermined) using the Ion Ampliseq Comprehensive Cancer Panel, which assesses copy number alterations and mutations in 409 cancer-relevant genes. Twelve tumors displayed prioritized high-level chromosomal gains or losses (average 1.9 per tumor). Non-synonymous high-confidence somatic mutations were detected in 14 tumors (average 11.9 per tumor). Assessing all somatic coding mutations, an ultraviolet-signature mutational profile was present, and more prevalent in Merkel cell polyomavirus-negative tumors. Recurrent deleterious tumor suppressor mutations affected TP53 (9/15, 60%), RB1 (3/15, 20%), and BAP1 (2/15, 13%). Oncogenic activating mutations included PIK3CA (3/15, 20%), AKT1 (1/15, 7%) and EZH2 (1/15, 7%). In conclusion, CK20-negative Merkel cell carcinoma display overlapping genetic changes with CK20-positive Merkel cell carcinoma, including RB1 mutations restricted to Merkel cell polyomavirus-negative tumors. However, some CK20-negative Merkel cell carcinomas harbor mutations not previously described in Merkel cell carcinoma. Hence, CK20-negative Merkel cell carcinomas harbor diverse oncogenic drivers which may represent therapeutic targets in individual tumors.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Célula de Merkel/genética , Mutação , Neoplasias Cutâneas/genética , Idoso , Idoso de 80 Anos ou mais , Análise Mutacional de DNA/métodos , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Queratina-20/genética , Masculino , Pessoa de Meia-Idade , Proteínas de Ligação a Retinoblastoma/genética , Proteína Supressora de Tumor p53/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Histone modification is well established as a fundamental mechanism driving the regulation of transcription, replication, and DNA repair through the control of chromatin structure. Likewise, it is apparent that incorrect targeting of histone modifications contributes to misregulated gene expression and hence to developmental disorders and diseases of genomic instability such as cancer. The KMT2 family of SET domain methyltransferases, typified by mixed lineage leukemia protein-1 (MLL1), is responsible for histone H3 lysine 4 methylation, a marker of active genes. To ensure that this modification is correctly targeted, a multiprotein complex associates with the methyltransferase and directs activity. We have identified a novel interaction site on the core complex protein WD repeat protein-5 (WDR5), and we mapped the complementary site on its partner retinoblastoma-binding protein-5 (RbBP5). We have characterized this interaction by x-ray crystallography and show how it is fundamental to the assembly of the complex and to the regulation of methyltransferase activity. We show which region of RbBP5 contributes directly to mixed lineage leukemia activation, and we combine our structural and biochemical data to produce a model to show how WDR5 and RbBP5 act cooperatively to stimulate activity.