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Mitochondrial optic neuropathy (MON) describes a group of optic neuropathies that exhibit mitochondrial dysfunction in retinal ganglion cells. Pathogenesis of MON includes genetic factors, such as Leber hereditary optic neuropathy and dominant optic atrophy, or acquired factors, such as drug intoxication and nutritional deficiencies, or the combination of both genetic factors and acquired factors. Regardless of different causes, MON shares similar features including bilateral central visual acuity loss, equally normal or slightly sluggish reaction of pupils to light and so on. Many novel therapies, such as pharmacological strategies, genetic therapy and stem cell therapy, are being widely studied in order to limit or reverse the damage of retinal ganglion cells. This article review the pathogenesis, clinical manifestations, ancillary testing, differential diagnosis and treatment progress of MON. (Chin J Ophthalmol, 2021, 57: 386-390).
Assuntos
Atrofia Óptica Autossômica Dominante , Atrofia Óptica Hereditária de Leber , Doenças do Nervo Óptico , DNA Mitocondrial/genética , Humanos , Mitocôndrias , Atrofia Óptica Hereditária de Leber/diagnóstico , Atrofia Óptica Hereditária de Leber/genética , Atrofia Óptica Hereditária de Leber/terapia , Nervo Óptico , Doenças do Nervo Óptico/diagnóstico , Doenças do Nervo Óptico/terapiaRESUMO
Objective: To investigate the effects of miR-191-5p on cell migration, clone formation and proliferation of gastric cancer (GC) cells. Methods: The level of miR-191-5p expression was detected by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in 60 paired GC tissues and their adjacent normal tissues. miR-191-5p overexpression was achieved by transfection of construct pcDNA-miR-191-5p into GC cells. The migration, clone formation and proliferation of GC cells were detected by the scratch wound assay, clone formation assay and cell counting kit-8 (CCK-8), respectively. Low expression of miR-191-5p was achieved with miRNA-191-5p inhibitor. The binding sites of cyclin-dependent kinase 6 (CDK6) and miR-191-5p were analyzed using TargetScan software, and the interaction of CDK6 and miR-191-5p was verified using dual-fluorescence reporter gene expression. Western blot (WB) was used to detect the effect of miR-191-5p on the expression of p21 and CDK6 proteins. Results: miR-191-5p decreased in 53 cases (88%) of GC tissues compared to their controls. Furthermore, overexpression of miR-191-5p effectively inhibited the migration, clone formation and proliferation of GC cells (P<0.05). Dual-fluorescence reporter confirmed that miR-191-5p bound to 3'UTR of CDK6. WB showed that pcDNA-miR-191-5p inhibited the CDK6 expression but promoted the p21. Conclusion: Down-regulation of miR-191-5p has a correlation with the progression of GC. Overexpression of miR-191-5p can decrease the expression of CDK6 and inhibit the growth of GC cells.
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MicroRNAs , Neoplasias Gástricas , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , MicroRNAs/genética , Invasividade Neoplásica/genética , Neoplasias Gástricas/genéticaRESUMO
Objective: To observe the effects of intravenous methylprednisolone pulse (IVMP) therapy on the recovery of visual acuity and its influencing factors in patients with the relapse of aquaporin (AQP) 4 antibody positive neuromyelitis optica related optic neuritis (NMO-ON). Methods: Retrospective case series. Forty-eight eyes of 35 patients diagnosed as NMO-ON in the Neuro-ophthalmology Clinic of Beijing Tongren Hospital from September 2012 to April 2018 were included in this research. All patients were AQP4 antibody seropositive, and had clinical manifestations of acute optic neuritis, with a history of optic neuritis treated with glucocorticoids effectively. They received the treatment of IVMP 500 mg/d or 1 000 mg/d for 3 to 5 days. The post-treatment and pre-treatment visual acuities were compared. Improving four lines or more was considered as markedly effective, improving two or three lines as effective, and improving one line or no change or a decline as no effect. The impacts of age, visual acuity at onset, relapse rate and dosage on the acute exacerbation of NMO-ON were analyzed. Mann-Whitney U test and Kruskal-Wallis test were used for statistical analysis. Results: Among the 35 patients, there were 2 males and 33 females, aged from 15 to 73 years (median, 36 years). In the 48 eyes of recurrence, the treatment was effective 41.7% (20/48), effective 20.8% (10/48), and ineffective 37.5% (18/48). The IVMP therapy was effective in 25 of 34 eyes with one recurrence and 5 of 14 eyes with two or more recurrences, and the difference was statistically significant (Z=2.315, P=0.021). The efficacy in 13 eyes with preoperative visual acuity not lower than 0.05 (10/13) was better than 35 eyes with preoperative visual acuity lower than 0.05 (20/35), and the difference was statistically significant (Z=1.994, P=0.046). Different ages and doses (1 000 mg/d and 500 mg/d) made no significant difference in the efficacy (P=0.273,0.105). Conclusions: The IVMP therapy is effective for the NMO-ON relapse in patients who were AQP4 antibody seropositive. The effect of IVMP treatment at doses of 500 mg/d and 1 000 mg/d is similar. Furthermore, visual acuity less than 0.05 and more relapses reduce the efficacy in relapsed NMO-ON patients. (Chin J Ophthalmol, 2020, 56: 509-513).
Assuntos
Neuromielite Óptica , Neurite Óptica , Adolescente , Adulto , Idoso , Aquaporina 4 , Autoanticorpos , Feminino , Humanos , Masculino , Metilprednisolona/uso terapêutico , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto JovemRESUMO
Resveratrol (RES) is a naturally occurring and effective drug for tumor prevention and treatment. However, its low levels of aqueous solubility, stability, and poor bioavailability limit its application, especially when used as a free drug. In this study, RES was loaded into peptide and sucrose liposomes (PSL) to enhance the physico-chemical properties of RES and exploit RES delivery mediated by liposomes to effectively treat breast cancer. RES loaded PSL (the complex: PSL@RES) were stable, had a good RES encapsulation efficiency, and prolonged RES-release in vitro. PSL@RES was exceptionally efficient for inhibiting the growth of cancer cells, as the IC50 of PSL@RES in MCF-7 cells was found to be only 20.89 µmol L-1. The therapeutic efficacy of PSL@RES was evaluated in mice bearing breast cancer. The results showed that PSL@RES at a dosage of 5 mg kg-1 was more effective than 10 mg kg-1 free RES, and PSL@RES inhibited tumor growth completely at a dosage of 10 mg kg-1. PSL@RES induced apoptosis in breast tumor by upregulation of p53 expression. This then downregulated Bcl-2 and upregulated Bax, thereby inducing Caspase-3 activation. More importantly, encapsulation of RES within peptide liposomes greatly reduced the toxicity of free RES to mice. Overall, the simple formulation of liposomal nanocarriers of RES developed in this study produces satisfactory outcomes to encourage further applications of liposomal carriers for the treatment of breast cancer.
Assuntos
Antineoplásicos , Portadores de Fármacos/química , Lipossomos/química , Neoplasias Mamárias Experimentais/tratamento farmacológico , Resveratrol , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Humanos , Células MCF-7 , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Resveratrol/administração & dosagem , Resveratrol/uso terapêuticoRESUMO
Objective: To observe the effect of intravenous methylprednisolone pulse (IVMP) therapy on the recovery of visual acuity and its influencing factors in patients with the first attack of optic neuritis associated with aquaporin-4(AQP4) antibody seropositive neuromyelitis optica. Methods: Retrospective case series study. A total of 165 eyes of 120 patients diagnosed as optic neuritis related to neuromyelitis optica for the first time in the Neuro-ophthalmology Clinic of Beijing Tongren Hospital from September 2012 to December 2017 were selected in this research. All patients had AQP4 antibody seropositivity and clinical manifestations of acute optic neuritis, excluding other diagnoses. All the patients received the treatment of IVMP 500 mg/d or 1 000 mg/d for 3 days, followed by a slowly tapering course of oral glucocorticoids. The post-treatment and pre-treatment visual acuities were compared. Improving four lines or more was considered as effective markedly, improving two or three lines as effective, and improving one line or no change or a decline as no effect. The onset age, visual acuity before treatment and doses in the acute exacerbation were analyzed. The Mann-Whitney U test and Kruskal-Wallis test were used for statistical analyses. Results: Among the 120 patients, there were 17 males and 103 females, with age ranging from 16 to 80 years (median, 44 years). There were 17.6% (29/165) of the eyes with conspicuous therapy, 33.3% (55/165) of the eyes with effective therapy and 49.1% (81/165) of the eyes with ineffective therapy. The effect of IVMP decreased obviously when the age of onset was over 50 years old [41.1%(23/56) vs. 56.0%(61/109), Z=2.645, P=0.008]. Patients with no light perception and light perception before treatment had better therapeutic effect than those with counting fingers-0.3 before treatment [72.2%(26/36), 72.7%(24/33) vs. 30.1%(25/83), Z=2.726, 2.967; P=0.006, 0.003]. Although the efficacy of patients with visual acuity of onset over 0.3 (9/13) was better than patients with counting fingers-0.3, but the difference was not statistically significant (Z=1.743, P=0.081). Different doses, including IVMP 1 000 mg/d and 500 mg/d, had no significant difference in the effect (Z=1.115, P=0.265). Conclusions: IVMP therapy is only valid for a half of eyes with optic neuritis associated with AQP4 antibody seropositive neuromyelitis optica. The effect of IVMP treatment at doses of 500 mg/d and 1 000 mg/d is similar. Furthermore, the visual acuity from finger counting to 0.3 and age of onset over 50 years old have an influence on the treatment effect. (Chin J Ophthalmol, 2019, 55: 180-185).
Assuntos
Neuromielite Óptica , Neurite Óptica , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Aquaporina 4 , Autoanticorpos , Feminino , Humanos , Masculino , Metilprednisolona , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto JovemRESUMO
Despite advances in early diagnosis and the development of molecularly targeted therapy, curative treatment of colon cancer once it has metastasized is yet to be accomplished. This is closely associated with deregulated CRC cell proliferation and resistance to apoptosis. Here we reveal that upregulation of microRNA-645 (miR-645) through DNA copy number gain is responsible for enhanced proliferation and resistance to apoptosis in colon cancer. MiR-645 was upregulated in most colon cancer tissues related to adjacent normal mucosa. This appeared to be associated with amplification of a section of chromosome 20q13.13, where miR-645 is located. Inhibition of miR-645 reduced proliferation and enhanced sensitivity to apoptosis triggered by the chemotherapeutic drugs 5-fluorouracil and cisplatin in CRC cells, and retarded colon cancer xenograft growth. Conversely, overexpression of miR-645 in normal colon epithelial cells enhanced proliferation and triggered anchorage-independent cell growth. Although SRY-related HMG-box 30 (SOX30) was identified as a miR-645 target, its expression was only partially affected by miR-645, suggesting that miR-645 is a fine-tuning mechanism of SOX30 expression. Moreover, overexpression of SOX30 only moderately inhibited promotion of CRC cell proliferation by miR-645, indicating that miR-645 may have more targets that contribute to its pro-proliferation effect in colon cancer. Together, this study reveals that miR-645 can regulate oncogenesis in colon cancer with SOX30 being one of its targets.
RESUMO
Inositol polyphosphate 4-phosphatase type II (INPP4B) negatively regulates phosphatidylinositol 3-kinase signaling and is a tumor suppressor in some types of cancers. However, we have found that it is frequently upregulated in human colon cancer cells. Here we show that silencing of INPP4B blocks activation of Akt and serum- and glucocorticoid-regulated kinase 3 (SGK3), inhibits colon cancer cell proliferation and retards colon cancer xenograft growth. Conversely, overexpression of INPP4B increases proliferation and triggers anchorage-independent growth of normal colon epithelial cells. Moreover, we demonstrate that the effect of INPP4B on Akt and SGK3 is associated with inactivation of phosphate and tensin homolog through its protein phosphatase activity and that the increase in INPP4B is due to Ets-1-mediated transcriptional upregulation in colon cancer cells. Collectively, these results suggest that INPP4B may function as an oncogenic driver in colon cancer, with potential implications for targeting INPP4B as a novel approach to treat this disease.
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Neoplasias do Colo/genética , Monoéster Fosfórico Hidrolases/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Humanos , Imuno-Histoquímica , Monoéster Fosfórico Hidrolases/metabolismoRESUMO
The three dimensional (3D) Dirac semimetal is a new quantum state of matter that has attracted much attention recently in physics and material science. Here, we report on the growth of large plate-like single crystals of Cd3As2 in two major orientations by a self-selecting vapor growth (SSVG) method, and the optimum growth conditions have been experimentally determined. The crystalline imperfections and electrical properties of the crystals were examined with transmission electron microscopy (TEM), scanning tunneling microscopy (STM), and transport property measurements. This SSVG method makes it possible to control the as-grown crystal compositions with excess Cd or As leading to mobilities near 5-10(5) cm(2)V(-1)s(-1). Zn-doping can effectively reduce the carrier density to reach the maximum residual resistivity ratio (RRRρ300K/ρ5K) of 7.6. A vacuum-cleaved single crystal has been investigated using angle-resolved photoemission spectroscopy (ARPES) to reveal a single Dirac cone near the center of the surface Brillouin zone with a binding energy of approximately 200 meV.
RESUMO
Relatedness between individuals is central to ecological genetics. Multiple methods are available to quantify relatedness from molecular data, including method-of-moment and maximum-likelihood estimators. We describe a maximum-likelihood estimator for autopolyploids, and quantify its statistical performance under a range of biologically relevant conditions. The statistical performances of five additional polyploid estimators of relatedness were also quantified under identical conditions. When comparing truncated estimators, the maximum-likelihood estimator exhibited lower root mean square error under some conditions and was more biased for non-relatives, especially when the number of alleles per loci was low. However, even under these conditions, this bias was reduced to be statistically insignificant with more robust genetic sampling. We also considered ambiguity in polyploid heterozygote genotyping and developed a weighting methodology for candidate genotypes. The statistical performances of three polyploid estimators under both ideal and actual conditions (including inbreeding and double reduction) were compared. The software package POLYRELATEDNESS is available to perform this estimation and supports a maximum ploidy of eight.
Assuntos
Funções Verossimilhança , Modelos Genéticos , Poliploidia , Genótipo , Heterozigoto , Endogamia , Padrões de Herança , Modelos Estatísticos , SoftwareRESUMO
Increased global protein synthesis and selective translation of mRNAs encoding proteins contributing to malignancy is common in cancer cells. This is often associated with elevated expression of eukaryotic translation initiation factor 4 (eIF4E), the rate-limiting factor of cap-dependent translation initiation. We report here that in human melanoma downregulation of miR-768-3p as a result of activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway has an important role in the upregulation of eIF4E and enhancement in protein synthesis. Melanoma cells displayed increased nascent protein production and elevated eIF4E expression, which was associated with the downregulation of miR-768-3p that was predicted to target the 3'-untranslated region of the eIF4E mRNA. Overexpression of miR-768-3p led to the downregulation of the endogenous eIF4E protein, reduction in nascent protein synthesis and inhibition of cell survival and proliferation. These effects were efficiently reversed when eIF4E was co-overexpressed in melanoma cells. On the other hand, introduction of anti-miR-768-3p into melanocytes upregulated endogenous eIF4E protein expression and increased global protein synthesis. Downregulation of miR-768-3p appeared to be mediated by activation of the MEK/ERK pathway, in that treatment of BRAF(V600E) melanoma cells with the mutant BRAF inhibitor PLX4720 or exposure of either BRAF(V600E) or wild-type BRAF melanoma cells to the MEK inhibitor U0126 resulted in the upregulation of miR-768-3p and inhibition of nascent protein synthesis. This inhibition was partially blocked in cells cointroduced with anti-miR-768-3p. Significantly, miR-768-3p was similarly downregulated, which was inversely associated with the expression levels of eIF4E in fresh melanoma isolates. Taken together, these results identify downregulation of miR-768-3p and subsequent upregulation of eIF4E as an important mechanism in addition to phosphorylation of eIF4E responsible for MEK/ERK-mediated enhancement of protein synthesis in melanoma.
Assuntos
Sistema de Sinalização das MAP Quinases , Melanoma/genética , MicroRNAs/genética , Biossíntese de Proteínas , RNA Mensageiro/genética , Transdução de Sinais , Linhagem Celular Tumoral , Regulação para Baixo , Fator de Iniciação 4E em Eucariotos/metabolismo , Humanos , Melanoma/metabolismo , Melanoma/patologia , Regulação para CimaRESUMO
Akt is constitutively activated in up to 70% of human melanomas and has an important role in the pathogenesis of the disease. However, little is known about protein phosphatases that dephosphorylate and thereby inactivate it in melanoma cells. Here we report that suppression of pleckstrin homology domain and leucine-rich repeat Ser/Thr protein phosphatase 1 (PHLPP1) by DNA methylation promotes Akt activation and has an oncogenic role in melanoma. While it is commonly downregulated, overexpression of PHLPP1 reduces Akt activation and inhibits melanoma cell proliferation in vitro, and retards melanoma growth in a xenograft model. In contrast, knockdown of PHLPP1 increases Akt activation, enhances melanoma cell and melanocyte proliferation, and results in anchorage-independent growth of melanocytes. Suppression of PHLPP1 involves blockade of binding of the transcription factor Sp1 to the PHLPP1 promoter. Collectively, these results suggest that suppression of PHLPP1 by DNA methylation contributes to melanoma development and progression.
Assuntos
Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Melanoma/genética , Proteínas Nucleares/genética , Fosfoproteínas Fosfatases/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Metilação de DNA , Regulação para Baixo , Ativação Enzimática , Humanos , Masculino , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Nus , Proteínas Nucleares/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição Sp1/metabolismo , Carga TumoralRESUMO
Epidemiological evidence has linked the development and progression of several cancers including melanoma with obesity. However, whether obesity impinges on responses of cancer cells to treatment remains less understood. Here we report that human adipocytes contribute to resistance of melanoma cells to various therapeutic agents. Exposure to media from adipocyte cultures (adipocyte media) increased cell proliferation and reduced sensitivity of melanoma cells to apoptosis induced by diverse chemotherapeutic drugs, including the DNA-damaging drug cisplatin, the microtubuletargeting agent docetaxel, and the histone deacetylase inhibitor SAHA. This was associated with increased activation of PI3K/Akt and MEK/ERK signaling, and was attenuated by a PI3K or MEK inhibitor. The effect of adipocyte media on melanoma cells was, at least in part, due to the interaction between the adipokine leptin and its long form receptor OB-Rb, in that immunodepletion of leptin in adipocyte media or siRNA knockdown of OB-Rb in melanoma cells reversed the increase in Akt and ERK activation, enhancement in cell proliferation, and importantly, protection of melanoma cells against the drugs. In support, recombinant leptin partially recapitulated the effect of adipocyte media on melanoma cells. Of note, OB-Rb was increased on the surface of melanoma cells compared to melanocytes, whereas leptin short form receptors appeared to be suppressed post-transcriptionally, suggesting that OB-Rb was selectively upregulated in melanoma cells. Collectively, these results indicate that adipocytes contribute to the resistance of melanoma cells to chemotherapeutic drugs and agents targeting the PI3K/Akt and MEK/ERK pathways, and suggest that inhibition of the leptin/ OB-Rb system may be useful to improve the efficacy of multiple therapeutic approaches in the treatment of melanoma.
Assuntos
Adipócitos/metabolismo , Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Queratinócitos/efeitos dos fármacos , Adipócitos/citologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Meios de Cultivo Condicionados/farmacologia , Docetaxel , Inibidores de Histona Desacetilases/farmacologia , Humanos , Queratinócitos/metabolismo , Queratinócitos/patologia , Leptina/deficiência , Leptina/genética , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Terapia de Alvo Molecular , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores para Leptina/antagonistas & inibidores , Receptores para Leptina/genética , Receptores para Leptina/metabolismo , Transdução de Sinais , Taxoides/farmacologiaRESUMO
Past studies have shown that histone deacetylase (HDAC) and mutant BRAF (v-Raf murine sarcoma viral oncogene homolog B1) inhibitors synergistically kill melanoma cells with activating mutations in BRAF. However, the mechanism(s) involved remains less understood. Here, we report that combinations of HDAC and BRAF inhibitors kill BRAF(V600E) melanoma cells by induction of necrosis. Cotreatment with the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) or panobinostat (LBH589) and the BRAF inhibitor PLX4720 activated the caspase cascade, but caspases appeared dispensable for killing, in that inhibition of caspases did not invariably block induction of cell death. The majority of dying cells acquired propidium iodide positivity instantly when they became positive for Annexin V, suggesting induction of necrosis. This was supported by caspase-independent release of high-mobility group protein B1, and further consolidated by rupture of the plasma membrane and loss of nuclear and cytoplasmic contents, as manifested by transmission electron microscopic analysis. Of note, neither the necrosis inhibitor necrostatin-1 nor the small interference RNA (siRNA) knockdown of receptor-interacting protein kinase 3 (RIPK3) inhibited cell death, suggesting that RIPK1 and RIPK3 do not contribute to induction of necrosis by combinations of HDAC and BRAF inhibitors in BRAF(V600E) melanoma cells. Significantly, SAHA and the clinically available BRAF inhibitor vemurafenib cooperatively inhibited BRAF(V600E) melanoma xenograft growth in a mouse model even when caspase-3 was inhibited. Taken together, these results indicate that cotreatment with HDAC and BRAF inhibitors can bypass canonical cell death pathways to kill melanoma cells, which may be of therapeutic advantage in the treatment of melanoma.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Histona Desacetilases/farmacologia , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Técnicas de Silenciamento de Genes , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Masculino , Camundongos , Camundongos Nus , Mutação de Sentido Incorreto , Necrose , Panobinostat , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , RNA Interferente Pequeno/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Sulfonamidas/farmacologia , Vemurafenib , Vorinostat , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Past studies have shown that amplified insulin-like growth factor 1 (IGF1)/IGF1 receptor (IGF1-R) signalling has an important role in colorectal cancer (CRC) development, progression and resistance to treatment. In this report, we demonstrate that downregulation of microRNA-497 (miR-497) as a result of DNA copy number reduction is involved in upregulation of IGF1-R in CRC cells. MiR-497 and miR-195 of the miR-15/16/195/424/497 family that share the same 3' untranslated region (3'UTR) binding seed sequence and are predicted to target IGF1-R were concurrently downregulated in the majority of CRC tissues relative to paired adjacent normal mucosa. However, only overexpression of miR-497 led to suppression of the IGF1-R 3'UTR activity and downregulation of the endogenous IGF1-R protein in CRC cells. This was associated with inhibition of cell survival, proliferation and invasion, and increased sensitivity to apoptosis induced by various stimuli including the chemotherapeutic drugs cisplatin and 5-fluorouracil, and the death ligand tumour necrosis factor-related apoptosis-inducing ligand. The biological effect of miR-497 on CRC cells was largely mediated by inhibition of phosphatidylinositol 3-kinase/Akt signalling, as overexpression of an active form of Akt reversed its impact on cell survival and proliferation, recapitulating the effect of overexpression of IGF1-R. Downregulation of miR-497 and miR-195 appeared to associate with copy number loss of a segment of chromosome 17p13.1, where these miRs are located at proximity. Similarly to miR-195, the members of the same miR family, miR-424 that was upregulated, and miR-15a, miR-15b and miR-16 that were unaltered in expression in CRC tissues compared with paired adjacent normal mucosa, did not appear to have a role in regulating the expression of IGF1-R. Taken together, these results identify downregulation of miR-497 as an important mechanism of upregulation of IGF1-R in CRC cells that contributes to malignancy of CRC.
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Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Variações do Número de Cópias de DNA , MicroRNAs/metabolismo , Receptor IGF Tipo 1/metabolismo , Regiões 3' não Traduzidas , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Cisplatino/farmacologia , Progressão da Doença , Regulação para Baixo , Resistencia a Medicamentos Antineoplásicos/genética , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , MicroRNAs/genética , Invasividade Neoplásica/genética , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genéticaRESUMO
Endoplasmic reticulum (ER) stress triggers apoptosis by activating Bim in diverse types of cells, which involves dephosphorylation of Bim(EL) by protein phosphatase 2A (PP2A). However, melanoma cells are largely resistant to ER stress-induced apoptosis, suggesting that Bim activation is suppressed in melanoma cells undergoing ER stress. We show here that ER stress reduces PP2A activity leading to increased ERK activation and subsequent phosphorylation and proteasomal degradation of Bim(EL). Despite sustained upregulation of Bim at the transcriptional level, the Bim(EL) protein expression was downregulated after an initial increase in melanoma cells subjected to pharmacological ER stress. This was mediated by increased activity of ERK, whereas the phosphatase activity of PP2A was reduced by ER stress in melanoma cells. The increase in ERK activation was, at least in part, due to reduced dephosphorylation by PP2A, which was associated with downregulation of the PP2A catalytic C subunit. Notably, instead of direct dephosphorylation of Bim(EL), PP2A inhibited its phosphorylation indirectly through dephosphorylation of ERK in melanoma cells. Taken together, these results identify downregualtion of PP2A activity as an important protective mechanism of melanoma cells against ER stress-induced apoptosis.
Assuntos
Estresse do Retículo Endoplasmático , Melanoma/metabolismo , Proteína Fosfatase 2/genética , Apoptose , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Melanoma/genética , Melanoma/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosforilação , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Transcrição Gênica , Regulação para CimaRESUMO
Soybean protein hydrolysates (SPHs) bind with calcium, forming soluble SPH-calcium complexes via the carboxyl groups of glutamic and aspartic acid residues. However, their effect on calcium uptake is still unclear. In this study, Caco-2 cells were used to estimate the effect of SPH-calcium complexes with different molecular weights on calcium uptake in vitro. The changes in intracellular calcium ion concentration were measured by Fura-2 loading and expressed in fluorescence intensity. SPH-calcium complexes could promote calcium uptake. Improved fluorescence intensity was significantly different in SPH-calcium complexes (10 to 30 kDa), SPH-calcium complexes (3 to 10 kDa), and SPH-calcium complexes (1 to 3 kDa). The maximum levels of relative fluorescence intensity (18.3) occurred with SPH-calcium complexes (10 to 30 kDa). The effect of SPH-calcium complexes (10 to 30 kDa) on Ca(2+) increase was determined to be concentration dependent in the range of 0.5 to 4 mg/mL. Our results indicate that soybean protein itself might be responsible for promoting calcium absorption.
Assuntos
Cálcio/metabolismo , Hidrolisados de Proteína/metabolismo , Proteínas de Soja/metabolismo , Absorção , Células CACO-2 , Fura-2/metabolismo , Humanos , Peso MolecularRESUMO
The soluble complexes formed between hydrolyzed soybean protein and calcium at pH 7.4 were investigated using dialysis, gel chromatography, and Fourier transform infrared spectrometry (FTIR). The results demonstrate that the amount of calcium bound was significantly different for soybean protein hydrolysates obtained using the proteases neutrase, flavourzyme, protease M, and pepsin. Maximum levels of calcium binding (66.9 mg/g) occurred with hydrolysates produced using protease M. Peptide fragments exhibiting high calcium binding capacity had molecular weights of either 14.4 kDa or 8 to 9 kDa, and the calcium binding capacity was linearly correlated with carboxyl group content (R(2)= 0.8204). FTIR experiments revealed that upon binding calcium, the amide I band underwent a shift to lower wave numbers. A wide, intense Ca-O absorption band also appeared between 400 and 100 cm(-1) in the far-infrared spectrum. The width and intensity of this band increased after treatment of samples with glutaminase. The amount of bound calcium was related to both the molecular weight of the peptides and to the carboxyl group content, and the most likely sites for calcium binding are the carboxyl groups of Asp and Glu.