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PURPOSE: Guanfacine is an α2A-adrenergic receptor agonist, FDA-approved to treat attention-deficit hyperactivity disorder and high blood pressure, typically as an extended-release formulation up to 7 mg/day. In our dysautonomia clinic, we observed that off-label use of short-acting guanfacine at 1 mg/day facilitated symptom relief in two families with multiple members presenting with severe generalized anxiety. We also noted anecdotal improvements in associated dysautonomia symptoms such as hyperhidrosis, cognitive impairment, and palpitations. We postulated that a genetic deficit existed in these patients that might augment guanfacine susceptibility. METHODS: We used whole-exome sequencing to identify mutations in patients with shared generalized anxiety and dysautonomia symptoms. Guanfacine-induced changes in the function of voltage-gated Na+ channels were investigated using voltage-clamp electrophysiology. RESULTS: Whole-exome sequencing uncovered the p.I739V mutation in SCN9A in the proband of two nonrelated families. Moreover, guanfacine inhibited ionic currents evoked by wild-type and mutant NaV1.7 encoded by SCN9A, as well as other NaV channel subtypes to a varying degree. CONCLUSION: Our study provides further evidence for a possible pathophysiological role of NaV1.7 in anxiety and dysautonomia. Combined with off-target effects on NaV channel function, daily administration of 1 mg short-acting guanfacine may be sufficient to normalize NaV channel mutation-induced changes in sympathetic activity, perhaps aided by partial inhibition of NaV1.7 or other channel subtypes. In a broader context, expanding genetic and functional data about ion channel aberrations may enable the prospect of stratifying patients in which mutation-induced increased sympathetic tone normalization by guanfacine can support treatment strategies for anxiety and dysautonomia symptoms.
Assuntos
Doenças do Sistema Nervoso Autônomo , Guanfacina , Humanos , Guanfacina/uso terapêutico , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Mutação , Ansiedade/tratamento farmacológico , Ansiedade/genética , Agonistas alfa-AdrenérgicosRESUMO
Small non-coding RNAs (sncRNAs) are defined by being less than 200 nucleotides (nt) in length, and consequently, have been divided into many different subclasses including mature microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), protein functional effector sncRNA (pfeRNA), precursor miRNA (pre-miRNA), small nucleolar RNA (snoRNA), 5S ribosome RNA (5SrRNA), 5.8SrRNA, and small nuclear RNA (snRNA). Except for the class of pfeRNAs, the discovery, identification, biogenesis, characterization, and function of other sncRNAs have been well documented. Herein, we provide a review, written especially for clinicians, of the least understood class of functional sncRNAs, the pfeRNAs, focusing on their initial discovery, identification, unique features, function, as well as their exciting clinical translational potential.
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MicroRNAs , Pequeno RNA não Traduzido , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , MicroRNAs/genética , RNA Interferente Pequeno/genética , RNA de Interação com PiwiRESUMO
Protein functional effector sncRNAs (pfeRNAs) are approximately 30-60 nucleotides (nt), of which the extraction method from plasma has not yet been reported. Silver staining in a high-resolution polyacrylamide gel suggested that the majority of plasma sncRNAs extracted by some broadly used commercial kits were sncRNAs from 100 nt upwards. Additionally, TRIzol's protocol is for long RNA but not sncRNA recovery. Here, we report a TRIzol-based frozen precipitation method (TFP method), which shows rigor and reproducibility in high yield and quality for plasma sncRNAs approximately 30-60 nt. In contrast to the yields by the commercial kit, plasma sncRNAs extracted by the TFP method enriched more sncRNAs. We used four different pfeRNAs of 34 nt, 45 nt, 53 nt, and 58 nt to represent typical sizes of sncRNAs from 30 to 60 nt and compared their levels in the recovered sncRNAs by the TFP method and by the commercial kit. The TFP method showed lower cycle threshold (CT) values by 2.01-9.17 cycles in 38 plasma samples from 38 patients, including Caucasian, Asian, African American, Latin, Mexican, and those who were a mix of more than one race. In addition, pfeRNAs extracted by two organic-based extraction methods and four commercial kits were undetermined in 22 of 38 samples. Thus, the quick and unbiased TFP method enriches plasma sncRNA ranging from 30 to 60 nt.
Assuntos
Pequeno RNA não Traduzido , Guanidinas , Humanos , Nucleotídeos , Fenóis , Pequeno RNA não Traduzido/genética , Reprodutibilidade dos TestesRESUMO
The ability to differentiate between benign, suspicious, and malignant pulmonary nodules is imperative for definitive intervention in patients with early stage lung cancers. Here, we report that plasma protein functional effector sncRNAs (pfeRNAs) serve as non-invasive biomarkers for determining both the existence and the nature of pulmonary nodules in a three-stage study that included the healthy group, patients with benign pulmonary nodules, patients with suspicious nodules, and patients with malignant nodules. Following the standards required for a clinical laboratory improvement amendments (CLIA)-compliant laboratory-developed test (LDT), we identified a pfeRNA classifier containing 8 pfeRNAs in 108 biospecimens from 60 patients by sncRNA deep sequencing, deduced prediction rules using a separate training cohort of 198 plasma specimens, and then applied the prediction rules to another 230 plasma specimens in an independent validation cohort. The pfeRNA classifier could (1) differentiate patients with or without pulmonary nodules with an average sensitivity and specificity of 96.2% and 97.35% and (2) differentiate malignant versus benign pulmonary nodules with an average sensitivity and specificity of 77.1% and 74.25%. Our biomarkers are cost-effective, non-invasive, sensitive, and specific, and the qPCR-based method provides the possibility for automatic testing of robotic applications.
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Cancer recurrence after surgery remains an unresolved clinical problem1-3. Myeloid cells derived from bone marrow contribute to the formation of the premetastatic microenvironment, which is required for disseminating tumour cells to engraft distant sites4-6. There are currently no effective interventions that prevent the formation of the premetastatic microenvironment6,7. Here we show that, after surgical removal of primary lung, breast and oesophageal cancers, low-dose adjuvant epigenetic therapy disrupts the premetastatic microenvironment and inhibits both the formation and growth of lung metastases through its selective effect on myeloid-derived suppressor cells (MDSCs). In mouse models of pulmonary metastases, MDSCs are key factors in the formation of the premetastatic microenvironment after resection of primary tumours. Adjuvant epigenetic therapy that uses low-dose DNA methyltransferase and histone deacetylase inhibitors, 5-azacytidine and entinostat, disrupts the premetastatic niche by inhibiting the trafficking of MDSCs through the downregulation of CCR2 and CXCR2, and by promoting MDSC differentiation into a more-interstitial macrophage-like phenotype. A decreased accumulation of MDSCs in the premetastatic lung produces longer periods of disease-free survival and increased overall survival, compared with chemotherapy. Our data demonstrate that, even after removal of the primary tumour, MDSCs contribute to the development of premetastatic niches and settlement of residual tumour cells. A combination of low-dose adjuvant epigenetic modifiers that disrupts this premetastatic microenvironment and inhibits metastases may permit an adjuvant approach to cancer therapy.
Assuntos
Epigênese Genética , Terapia Genética , Células Supressoras Mieloides/fisiologia , Neoplasias/terapia , Microambiente Tumoral , Animais , Azacitidina/farmacologia , Benzamidas/farmacologia , Diferenciação Celular , Movimento Celular/efeitos dos fármacos , Quimioterapia Adjuvante , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Camundongos , Células Supressoras Mieloides/citologia , Metástase Neoplásica/terapia , Neoplasias/cirurgia , Piridinas/farmacologia , Receptores CCR2/genética , Receptores de Interleucina-8B/genética , Microambiente Tumoral/efeitos dos fármacosRESUMO
PIWI-interacting RNA Likes (piR-Ls) were recently reported to regulate functions of their target phospho-Proteins (p-Proteins) in somatic lung cells. However, the mechanism underlying this functionality remains unclear. piR-Ls interact with their targets through direct binding but do not follow base-pairing rules, known to have important roles at levels of transcription, RNA processing and translation for small non-coding RNA (sncRNA). These observations imply a fundamentally different type of sncRNA with behavior that causes a molecular response in their target p-Proteins. Furthermore, the interaction of piR-Ls with their targets regulates the functional efficacy of target p-Proteins. In addition, except for writers (kinase) and erasers (phosphatase), the functional efficacy of p-Proteins on their readers still remains unknown. It is reasonable to consider the existence of protein functional effector sncRNAs (pfeRNAs), which were identified by deep sequencing the immunoprecipitation products of antibodies targeting phosphorylated residues in proteins, as well as by functional analysis. pfeRNAs harbor unique features in size distribution, 3' terminal modification, shared core sequences, and functional manner, and could be new players in lung physiological and pathological conditions.
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Biomarcadores Tumorais/genética , Neoplasias Pulmonares/genética , Fosfoproteínas/genética , Pequeno RNA não Traduzido/genética , Animais , Biomarcadores Tumorais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Fosfoproteínas/metabolismo , Fosforilação , Pequeno RNA não Traduzido/metabolismo , Transcrição GênicaRESUMO
We recently reported that PIWI-interacting RNAs likes (piR-Ls) could regulate functions of the interacting phosphorylated proteins (p-Proteins). In addition, except for writers and erasers, functional efficacy of p-Proteins on their readers still remains unknown. We, therefore, reasoned there was a type of sncRNAs which could regulate functional efficacy of p-Proteins. Here, we profiled sncRNAs interacting with phosphorylated -Ser, -Thr and -Tyr residues in 3 HBE and 4 lung SCC cell lines, investigated effects and mechanisms of phosphorylated-residue-interacting sncRNAs. Our results demonstrated sncRNAs regulating functional efficacy of p-Proteins and we thus referred them as Protein Functional Effector sncRNAs (pfeRNAs). pfeRNAs were distributed among 26 to 50 nucleotides, shared some core sequences and showed distinctive expression patterns between HBE and SCC cells. Core sequences 417 (CS417), showing consistent upregulation in all 4 SCC cells, bound directly to p-Nucleolin (NCL), which was dependent on the key elements CGCG of CS417 and p-Ser619 of NCL. The CS417/p-NCL interaction was critical for functional efficacy of p-NCL in basic activities of lung normal and cancer cells. Thus, we revealed a novel type of pfeRNAs controlling functional efficacy of p-Proteins in lung somatic cells.
Assuntos
Carcinoma de Células Escamosas/genética , Neoplasias Pulmonares/genética , Pulmão/fisiologia , Pequeno RNA não Traduzido/genética , Humanos , Células Híbridas/citologia , Células Híbridas/metabolismo , Células Híbridas/fisiologia , Pulmão/citologia , Pulmão/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Fosforilação , Pequeno RNA não Traduzido/metabolismo , TransfecçãoRESUMO
Lung cancer is the leading cause of cancer-related death worldwide. Although advanced drugs have benefitted patients, therapeutic success has largely been hampered because of rapid development of resistance. Here we report that PIWI-interacting RNA likes (piR-Ls), a novel type of functional sncRNAs, play key roles in chemoresistance to cisplatin (CDDP)-based chemotherapy in lung squamous cell carcinoma (LSCC). piR-L-138 was upregulated upon CDDP-based chemotherapy both in LSCC cells and in patient-derived xenograft (PDX) LSCC models. Further, targeting upregulated piR-L-138 led to increased apoptosis in CDDP-treated LSCC cells and LSCC xenograft mice treated with CDDP. In addition, piR-L-138 directly interacted with p60-MDM2 and inhibited CDDP-activated apoptosis in p53-mutated LSCC. We identified the upregulated piR-L-138 upon CDDP-based chemotherapy, confirmed the enhanced sensitivity of LSCC to agents by targeting the upregulated piR-L-138 both in vitro and in vivo, and revealed mechanisms underlying piR-L-138 in chemoresistance, bolstering a new emerging clinical modality where novel functional piR-Ls provide potential strategies to overcome chemoresistance for patients with LSCC.
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Protein glycosylation plays a fundamental role in a multitude of biological processes, and the associated aberrant expression of glycoproteins in cancer has made them attractive biomarkers and therapeutic targets. In this study, we examined differentially expressed glycoproteins in cell lines derived from three different states of lung tumorigenesis: an immortalized bronchial epithelial cell (HBE) line, a non-small cell lung cancer (NSCLC) cell line harboring a Kirsten rat sarcoma viral oncogene homolog (KRAS) activation mutation and a NSCLC cell line harboring an epidermal growth factor receptor (EGFR) activation deletion. Using a Triple SILAC proteomic quantification strategy paired with hydrazide chemistry N-linked glycopeptide enrichment, we quantified 118 glycopeptides in the three cell lines derived from 82 glycoproteins. Proteomic profiling revealed 27 glycopeptides overexpressed in both NSCLC cell lines, 6 glycopeptides overexpressed only in the EGFR mutant cells and 19 glycopeptides overexpressed only in the KRAS mutant cells. Further investigation of a panel of NSCLC cell lines found that Cellular repressor of E1A-stimulated genes (CREG1) overexpression was closely correlated with KRAS mutation status in NSCLC cells and could be down-regulated by inhibition of KRAS expression. Our results indicate that CREG1 is a down-stream effector of KRAS in a sub-type of NSCLC cells and a novel candidate biomarker or therapeutic target for KRAS mutant NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Glicoproteínas/análise , Proteoma/análise , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Repressoras/metabolismo , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , HumanosRESUMO
PIWI-interacting RNAs (piRNAs) are thought to silence transposon and gene expression during development. However, the roles of piRNAs in somatic tissues are largely unknown. Here we report the identification of 555 piRNAs in human lung bronchial epithelial (HBE) and non-small cell lung cancer (NSCLC) cell lines, including 295 that do not exist in databases termed as piRNA-like sncRNAs or piRNA-Ls. Distinctive piRNA/piRNA-L expression patterns are observed between HBE and NSCLC cells. piRNA-like-163 (piR-L-163), the top downregulated piRNA-L in NSCLC cells, binds directly to phosphorylated ERM proteins (p-ERM), which is dependent on the central part of UUNNUUUNNUU motif in piR-L-163 and the RRRKPDT element in ERM. The piR-L-163/p-ERM interaction is critical for p-ERM's binding capability to filamentous actin (F-actin) and ERM-binding phosphoprotein 50 (EBP50). Thus, piRNA/piRNA-L may play a regulatory role through direct interaction with proteins in physiological and pathophysiological conditions.
Assuntos
Actinas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Proteínas do Citoesqueleto/metabolismo , Neoplasias Pulmonares/genética , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , RNA Interferente Pequeno/metabolismo , Mucosa Respiratória/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Northern Blotting , Brônquios/citologia , Brônquios/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Humanos , Imunoprecipitação , Neoplasias Pulmonares/metabolismo , Espectrometria de Massas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Piwi-interacting RNAs (piRNAs), a newly identified class of small non-coding RNAs, direct the Piwi-dependent transposon silencing, heterochromatin modification and germ cell maintenance. Owing to our limited knowledge regarding their biogenesis, piRNAs are considered as the most mysterious class of small regulatory RNAs, particularly in pathogenesis such as tumorigenesis. Recently, several lines of evidence have emerged to suggest that piRNAs may be dis-regulated and play crucial roles in tumorigenesis in previously unsuspected ways. In this prospective piece, we will discuss the emerging insights into the potential novel roles of piRNAs in carcinogenesis and highlight their potential implications in cancer detection, classification and therapy.
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Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Neoplasias/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Apoptose , Biomarcadores Tumorais , Epigênese Genética , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Prognóstico , RNA Interferente Pequeno/genética , Transcrição GênicaRESUMO
OBJECTIVE: Lung cancer remains number one cause of cancer related deaths worldwide. Cell cycle deregulation plays a major role in the pathogenesis of Non-Small Cell Lung Cancer (NSCLC). CDC25A represents a critical cell cycle regulator that enhances cell cycle progression. In this study we aimed to investigate the role of a novel CDC25A transcriptional variant, CDC25A(Q110del), on the regulation of the CDC25A protein, and its impact on prognosis of NSCLC patients. METHODOLOGY/PRINCIPAL FINDINGS: Here we report a novel CDC25A transcript variant with codon 110 (Glutamine) deletion, that we termed CDC25A(Q110del) in NSCLC cells. In 9 (75%) of the 12 NSCLC cell lines, CDC25A(Q110del) expression accounted for more than 20% of the CDC25A transcripts. Biological effects of CDC25A(Q110del) were investigated in H1299 and HEK-293F cells using UV radiation, flowcytometry, cyclohexamide treatment, and confocal microscopy. Compared to CDC25A(wt), CDC25A(Q110del) protein had longer half-life; cells expressing CDC25A(Q110del) were more resistant to UV irradiation and showed more mitotic activity. Taqman-PCR was used to quantify CDC25A(Q110del) expression levels in 88 primary NSCLC tumor/normal tissue pairs. In patients with NSCLC, Kaplan Meier curves showed tumors expressing higher levels of CDC25A(Q110del) relative to the adjacent lung tissues to have significantly inferior overall survival (P = .0018). SIGNIFICANCE: Here we identified CDC25A(Q110del) as a novel transcriptional variant of CDC25A in NSCLC. The sequence-specific nature of the abnormality could be a prognostic indicator in NSCLC patients as well as a candidate target for future therapeutic strategies.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Isoformas de Proteínas/genética , Fosfatases cdc25/genética , Sequência de Aminoácidos , Sequência de Bases , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Primers do DNA , Citometria de Fluxo , Humanos , Neoplasias Pulmonares/patologia , Microscopia Confocal , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Fosfatases cdc25/químicaRESUMO
Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related death. Developing minimally invasive techniques that can diagnose NSCLC, particularly at an early stage, may improve its outcome. Using microarray platforms, we previously identified 12 microRNAs (miRNAs) the aberrant expressions of which in primary lung tumors are associated with early-stage NSCLC. Here, we extend our previous research by investigating whether the miRNAs could be used as potential plasma biomarkers for NSCLC. We initially validated expressions of the miRNAs in paired lung tumor tissues and plasma specimens from 28 stage I NSCLC patients by real-time quantitative reverse transcription PCR, and then evaluated diagnostic value of the plasma miRNAs in a cohort of 58 NSCLC patients and 29 healthy individuals. The altered miRNA expressions were reproducibly confirmed in the tumor tissues. The miRNAs were stably present and reliably measurable in plasma. Of the 12 miRNAs, five displayed significant concordance of the expression levels in plasma and the corresponding tumor tissues (all r>0.850, all P<0.05). A logistic regression model with the best prediction was defined on the basis of the four genes (miRNA-21, -126, -210, and 486-5p), yielding 86.22% sensitivity and 96.55% specificity in distinguishing NSCLC patients from the healthy controls. Furthermore, the panel of miRNAs produced 73.33% sensitivity and 96.55% specificity in identifying stage I NSCLC patients. In addition, the genes have higher sensitivity (91.67%) in diagnosis of lung adenocarcinomas compared with squamous cell carcinomas (82.35%) (P<0.05). Altered expressions of the miRNAs in plasma would provide potential blood-based biomarkers for NSCLC.
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Biomarcadores Tumorais/sangue , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/diagnóstico , MicroRNAs/sangue , Adenocarcinoma/diagnóstico , Adenocarcinoma de Pulmão , Idoso , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/diagnóstico , Feminino , Humanos , Modelos Logísticos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Non-small-cell lung cancer (NSCLC) is the leading cause of cancer death. Early detection of NSCLC will improve its outcome. The current techniques for NSCLC early detection are either invasive or have low accuracy. Molecular analyses of clinical specimens present promising diagnostic approaches. Non-coding RNAs (ncRNAs) play an important role in tumorigenesis and could be developed as biomarkers for cancer. Here we aimed to develop small nucleolar RNAs (snoRNAs), a common class of ncRNAs, as biomarkers for NSCLC early detection. The study comprised three phases: (1) profiling snoRNA signatures in 22 NSCLC tissues and matched noncancerous lung tissues by GeneChip Array, (2) validating expressions of the signatures by RT-qPCR in the tissues, and (3) evaluating plasma expressions of the snoRNAs in 37 NSCLC patients, 26 patients with chronic obstructive pulmonary disease (COPD), and 22 healthy subjects. RESULTS: In the surgical tissues, six snoRNAs were identified, which were overexpressed in all tumour tissues compared with their normal counterparts. The overexpressions of the genes in tumors were confirmed by RT-qPCR. The snoRNAs were stably present and reliably detectable in plasma. Of the six genes, three (SNORD33, SNORD66 and SNORD76) displayed higher plasma expressions in NSCLC patients compared with the cancer-free individuals (All < 0.01). The use of the three genes produced 81.1% sensitivity and 95.8% specificity in distinguishing NSCLC patients from both normal and COPD subjects. The plasma snoRNA expressions were not associated with stages and histological types of NSCLC (All > 0.05). CONCLUSIONS: The identified snoRNAs provide potential markers for NSCLC early detection.
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Biomarcadores Tumorais/análise , Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , RNA Nuclear Pequeno/genética , Estudos de Casos e Controles , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In this study, fluorescent metal nanoshells were synthesized as a molecular imaging agent to detect single microRNA (miRNA) molecules in the cells positive to lung cancer. These metal nanoshells were composed of silica spheres with encapsulated Ru(bpy)(3)(2+) complexes as cores and thin silver layers as shells. Compared with the silica spheres in the absence of metal, the metal nanoshells displayed an enhanced emission intensity, shortened lifetime, and extended photostability. The single-stranded probe oligonucleotides were covalently bound on the metal nanoshells to hybridize with the target miRNA-486 molecules in the cells. It was shown that with stronger emission intensity and longer lifetime, the conjugated metal nanoshells were isolated distinctly from the cellular autofluorescence on the cell images. These emission spots on the cell images were counted accurately and analyzed with a pool of cells representing the miRNA-486 expression levels in the cells. The results may reflect a genomic signal change and provide a reference to lung cancer early diagnosis as well as other diseases.
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Corantes Fluorescentes/química , Neoplasias Pulmonares/patologia , MicroRNAs/análise , Nanoconchas/química , Sequência de Bases , Linhagem Celular Tumoral , Humanos , MicroRNAs/química , Imagem Molecular , Hibridização de Ácido Nucleico , Sondas de Ácido Nucleico/química , Sondas de Ácido Nucleico/genética , Compostos Organometálicos/química , Prata/química , Espectrometria de FluorescênciaRESUMO
Human cytomegalovirus (HCMV) infection is usually implicated in thrombocytopenia occurring in newborns and immunocompromised patients. However, the underlying mechanisms remain elusive. This study was conducted to investigate the effects of HCMV infection on the viability of megakaryocytic CHRF-288-11 cells and the underlying mechanisms involved. RT-PCR for determining mRNA expression of HCMV immediate early gene 1 and Western blot for measuring protein expression of late HCMV gene pp65 showed that CHRF-288-11 cells were susceptible to HCMV infection. HCMV infection reduced the viability of CHRF-288-11 cells via apoptosis in a dose- and time-dependent manner. Both caspase 3 and c-Jun terminal kinase (JNK) signaling pathway were activated in the HCMV-treated CHRF-288-11 cells. z-DEVD-fmk (a caspase inhibitor) and SP600125 (a JNK inhibitor) significantly prevented the death of CHRF-288-11 cells induced by HCMV, respectively. Furthermore, inhibition of JNK activity could reduce the formation of active caspase 3 induced by HCMV. Interestingly, the co-application of antivirus drug ganciclovir and SP600125 synergistically prevented the death of CHRF-288-11 cells induced by HCMV. Collectively, these findings suggest that HCMV infection may induce the caspase-dependent apoptosis of megakaryocytic CHRF-288-11 cells by the activation of JNK signaling pathway.
Assuntos
Apoptose/fisiologia , Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/patologia , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Megacariócitos , Antracenos/farmacologia , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Inibidores de Cisteína Proteinase/farmacologia , Infecções por Citomegalovirus/tratamento farmacológico , Ganciclovir/farmacologia , Humanos , Megacariócitos/metabolismo , Megacariócitos/patologia , Megacariócitos/virologia , Oligopeptídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Trombocitopenia/metabolismo , Trombocitopenia/patologia , Trombocitopenia/virologiaRESUMO
Prostate cancer (PCa) contains a small population of cancer stem cells (CSCs) that contribute to its initiation and progression. The development of specific markers for identification of the CSCs may lead to new diagnostic strategies of PCa. Increased aldehyde dehydrogenase 1A1 (ALDH1A1) activity has been found in the stem cell populations of leukemia and some solid tumors. The aim of the study was to investigate the stem-cell-related function and clinical significance of the ALDH1A1 in human PCa. ALDEFLUOR assay was used to isolate ALDH1A1(+) cells from PCa cell lines. Stem cell characteristics of the ALDH1A1(+) cells were then investigated by in vitro and in vivo approaches. The ALDH1A1 expression was also analyzed by immunohistochemistry in 18 normal prostate and 163 PCa tissues. The ALDH1A1(+) PCa cells showed high clonogenic and tumorigenic capacities, and serially reinitiated transplantable tumors that resembled histopathologic characteristics and heterogeneity of the parental PCa cells in mice. Immunohistochemical analysis of human prostate tissues showed that ALDH1A1(+) cells were sparse and limited to the basal component in normal prostates. However, in tumor specimens, increased ALDH1A1 immunopositivity was found not only in secretory type cancer epithelial cells but also in neuroendocrine tumor populations. Furthermore, the high ALDH1A1 expression in PCa was positively correlated with Gleason score (P=0.01) and pathologic stage (P=0.01), and inversely associated with overall survival and cancer-specific survival of the patients (P=0.00093 and 0.00017, respectively). ALDH1A1 could be a prostate CSC-related marker. Measuring its expression might provide a potential approach to study tumorigenesis of PCa and predict outcome of the disease.
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Aldeído Desidrogenase/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Células-Tronco , Idoso , Família Aldeído Desidrogenase 1 , Animais , Testes de Carcinogenicidade , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Transplante de Neoplasias , Prognóstico , Retinal Desidrogenase , Transplante HeterólogoRESUMO
Cerebellar granule neurons (CGNs) undergo apoptosis when deprived of depolarizing concentration of potassium. A key regulator of cell cycle, E2F1, was believed to play a role in CGN apoptosis induced by potassium deprivation. However, here we demonstrated that although E2F1 was upregulated in wild type CGNs following potassium deprivation, CGNs that derived from E2F1 knockout mice underwent apoptosis at a similar rate as the wild type. Analysis of the apoptotic neurons revealed no difference in the activation of caspase-3 in E2F1 null and wild type CGNs. Furthermore, knockdown of E2F1 expression by RNA interference failed to attenuate the apoptosis of CGNs induced by potassium deprivation. Taken together, our results suggested that E2F1 is not essential for apoptosis induced by potassium deprivation in CGNs.
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Apoptose/efeitos dos fármacos , Cerebelo/citologia , Fator de Transcrição E2F1/fisiologia , Neurônios/efeitos dos fármacos , Potássio/farmacologia , Animais , Animais Recém-Nascidos , Apoptose/genética , Apoptose/fisiologia , Contagem de Células/métodos , Células Cultivadas , Relação Dose-Resposta a Droga , Fator de Transcrição E2F1/deficiência , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Camundongos , Camundongos Knockout , Neurônios/fisiologia , Interferência de RNA/fisiologia , Fatores de TempoRESUMO
The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is closely associated with nasopharyngeal carcinoma (NPC). In this study, we investigated that the effect of silencing LMP1 on cell cycle distribution and chemosensitivity in EBV-positive naso-pharyngeal carcinoma C666-1 cells. Silencing of LMP1 by specific siRNA induced G1 arrest in C666-1 cells. The protein expression of CDK4 and cyclin D1 decreased and P27 was upregulated following LMP1 knockdown. Phosphorylation of AKT and its downstream targets IkappaB, FKHR was inhibited by LMP1 siRNA. The chemosensitivity of C666-1 cells to bleomycin and cisplatin was enhanced by siRNA targeting LMP1. The cells treated with LMP1 siRNA showed enhanced cleavage of the effector caspase3 and PARP, and Bax had the tendency to exhibit higher expression. Also, cotransfection of constitutive active AKT plasmid with LMP-1 siRNA plasmid abrogates sensitivity of C666-1 to bleomycin and cisplatin. It is reported for the first time that AKT signaling pathway was directly involved in the effects induced by siRNA targeting LMP1. Our findings confirm LMP1 as a rational therapeutic target in NPC.
Assuntos
Antineoplásicos/farmacologia , Carcinoma/patologia , Ciclo Celular/fisiologia , Herpesvirus Humano 4/fisiologia , Neoplasias Nasofaríngeas/patologia , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Proteínas da Matriz Viral/antagonistas & inibidores , Bleomicina/farmacologia , Carcinoma/metabolismo , Carcinoma/virologia , Caspase 3/metabolismo , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral/citologia , Linhagem Celular Tumoral/efeitos dos fármacos , Transformação Celular Viral , Cisplatino/farmacologia , Sistemas de Liberação de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/patologia , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/metabolismo , Fase G1 , Genes bcl-2 , Humanos , Proteínas I-kappa B/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/virologia , Proteínas de Neoplasias/genética , Fosforilação , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Transdução de Sinais , Transfecção , Proteínas da Matriz Viral/fisiologia , Proteína X Associada a bcl-2/biossíntese , Proteína X Associada a bcl-2/genéticaRESUMO
Epstein-Barr Virus (EBV) is closely associated with B cell malignancies. However, whether EBV appears to be absolutely required for cell proliferation and survival in lymphoma cells is still unknown. In this study, small interfering RNA (siRNA) targeting LMP1 was employed to investigate the effect of LMP1 on cell proliferation in EBV-positive lymphoblastoid B-cell line. A plasmid stable encoding 21-nt small RNA specifically and efficiently interfering LMP1 was constructed, resulting in a substantial loss of LMP1 mRNA and a significantly decreased LMP1 protein expression. Our data demonstrated that cell proliferation was completely inhibited and apoptosis was induced after knockdown of LMP1 gene in lymphoblastoid B-cell line. Also, we found that suppression of LMP1 caused downregulation of telomerase protein expression and decreased telomerase activity in lymphoma cells. In EBV-negative NPC cell line, transfection of plasmid expressing LMP1 greatly enhanced telomerase protein expression. Our results suggested that siRNA targeting LMP1 can induce apoptosis in EBV-positive lymphoma cells and is associated with inhibition of telomerase activity and expression. siRNA-directed LMP1 silencing may be of the therapeutic value for preventing and treating those EBV-associated tumors.