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1.
Nat Commun ; 11(1): 4455, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32901005

RESUMO

Dysregulated alternative splicing (AS) driving carcinogenetic mitosis remains poorly understood. Here, we demonstrate that cancer metastasis-associated antigen 1 (MTA1), a well-known oncogenic chromatin modifier, broadly interacts and co-expresses with RBPs across cancers, contributing to cancerous mitosis-related AS. Using developed fCLIP-seq technology, we show that MTA1 binds abundant transcripts, preferentially at splicing-responsible motifs, influencing the abundance and AS pattern of target transcripts. MTA1 regulates the mRNA level and guides the AS of a series of mitosis regulators. MTA1 deletion abrogated the dynamic AS switches of variants for ATRX and MYBL2 at mitotic stage, which are relevant to mitosis-related tumorigenesis. MTA1 dysfunction causes defective mitotic arrest, leads to aberrant chromosome segregation, and results in chromosomal instability (CIN), eventually contributing to tumorigenesis. Currently, little is known about the RNA splicing during mitosis; here, we uncover that MTA1 binds transcripts and orchestrates dynamic splicing of mitosis regulators in tumorigenesis.


Assuntos
Carcinogênese/genética , Carcinogênese/metabolismo , Montagem e Desmontagem da Cromatina/fisiologia , Mitose/fisiologia , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Processamento Alternativo , Animais , Sítios de Ligação/genética , Montagem e Desmontagem da Cromatina/genética , Instabilidade Cromossômica , Feminino , Células HCT116 , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Mitose/genética , Neoplasias/genética , Neoplasias/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Transativadores/antagonistas & inibidores , Transativadores/genética
2.
Chem Biol Interact ; 330: 109236, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32866467

RESUMO

A series of novel pyrrolopyrimidine urea derivatives were synthesized and evaluated for their anticancer activity against colon cancer cell lines. Compounds showed the remarkable cytotoxic activity on HCT-116 wt cell line. The most potent compound 4c (IC50 = 0.14 µM) induced apoptosis in HCT-116 wt and HCT-116 p53-/- cell lines. Otherwise, treatment of HCT-116 BAX-/-BAK-/- cells with compound 4c didn't lead to activation of apoptosis, suggesting that compound 4c induces apoptotic cell death by activating BAX/BAK-dependent pathway. Moreover, while the compound 4c increase the activation of caspase-3 and caspase-9 levels in HCT-116 wt and HCT-116 p53-/- cells, caspase-3 or caspase-9 activation was not observed in HCT-116 BAX-/-BAK-/- cells. In addition, compound 4c induced mitochondrial apoptosis in cells grown as oncospheroids, which better mimic the in vivo milieu of tumors. 4c treatment also activated JNK along with inhibition of prosurvival kinases such as Akt and ERK 1/2 in HCT-116 wt and HCT-116 p53 -/- cells as well as in HCT-116 BAX-/-BAK-/- cells. Notably, our results indicated that compound 4c induced mitochondrial apoptosis through activation p53-independent apoptotic signaling pathways.


Assuntos
Apoptose/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Pirimidinas/farmacologia , Pirróis/farmacologia , Proteína Supressora de Tumor p53/fisiologia , Caspase 3/genética , Caspase 9/genética , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Técnicas de Inativação de Genes , Células HCT116 , Humanos , Mitocôndrias/metabolismo , Pirimidinas/síntese química , Pirimidinas/química , Pirróis/síntese química , Pirróis/química , Proteína Supressora de Tumor p53/genética , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genética
3.
PLoS Biol ; 18(9): e3000825, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32886690

RESUMO

Microbial dysbiosis in the upper digestive tract is linked to an increased risk of esophageal squamous cell carcinoma (ESCC). Overabundance of Porphyromonas gingivalis is associated with shorter survival of ESCC patients. We investigated the molecular mechanisms driving aggressive progression of ESCC by P. gingivalis. Intracellular invasion of P. gingivalis potentiated proliferation, migration, invasion, and metastasis abilities of ESCC cells via transforming growth factor-ß (TGFß)-dependent Drosophila mothers against decapentaplegic homologs (Smads)/Yes-associated protein (YAP)/Transcriptional coactivator with PDZ-binding motif (TAZ) activation. Smads/YAP/TAZ/TEA domain transcription factor1 (TEAD1) complex formation was essential to initiate downstream target gene expression, inducing an epithelial-mesenchymal transition (EMT) and stemness features. Furthermore, P. gingivalis augmented secretion and bioactivity of TGFß through glycoprotein A repetitions predominant (GARP) up-regulation. Accordingly, disruption of either the GARP/TGFß axis or its activated Smads/YAP/TAZ complex abrogated the tumor-promoting role of P. gingivalis. P. gingivalis signature genes based on its activated effector molecules can efficiently distinguish ESCC patients into low- and high-risk groups. Targeting P. gingivalis or its activated effectors may provide novel insights into clinical management of ESCC.


Assuntos
Infecções por Bacteroidaceae/complicações , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Porphyromonas gingivalis/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adulto , Idoso , Animais , Infecções por Bacteroidaceae/metabolismo , Infecções por Bacteroidaceae/mortalidade , Infecções por Bacteroidaceae/patologia , Células Cultivadas , Progressão da Doença , Drosophila , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/microbiologia , Neoplasias Esofágicas/mortalidade , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/microbiologia , Carcinoma de Células Escamosas do Esôfago/mortalidade , Feminino , Seguimentos , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Transdução de Sinais/fisiologia , Proteínas Smad/metabolismo , Análise de Sobrevida , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo
4.
J Environ Pathol Toxicol Oncol ; 39(2): 101-111, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32749120

RESUMO

Long noncoding RNAs (lncRNAs) have been reported to be involved in cancer initiation and evolution, including colorectal cancer (CRC). Nuclear-enriched abundant transcript 1 (NEAT1) exerts important functions in multiple cancers; however, the specific modulatory mechanism in CRC demands in-depth exploration. The expression levels of NEAT1, microRNA-195-5p (miR-195-5p), and centrosomal protein 55 (CEP55) were examined using quantitative real-time polymerase chain reaction (qRT-PCR), and protein expression of CEP55 was detected by Western blot assay. Cell proliferation and apoptosis were measured by 3-(4,5-dimethylthiazole-2-y1)-2,5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry. Transwell migration and invasion assays were applied to evaluate cell metastasis ability. Dual-luciferase reporter assay was used to analyze the correlation among NEAT1, miR-195-5p and CEP55. The expression of NEAT1 was up-regulated in CRC tissues and cells, and overall survival was lower with high expression of NEAT1. Knockdown of NEAT1 repressed cell proliferation, migration, and invasion, while inducing apoptosis in CRC cells. NEAT1 targeted miR-195-5p and inhibited the expression of miR-195-5p. Silence of NEAT1 inhibited CRC cell proliferation, migration, and invasion, and promoted apoptosis by up-regulating miR-195-5p. MiR-195-5p targeted and suppressed CEP55 expression, and CEP55 reverted the effects induced by miR-195-5p. NEAT1 regulated the expression of CEP55 through miR-195-5p. NEAT1 promotes colorectal cancer cellular processes by regulating CEP55 expression via the sponging of miR-195-5p. Therefore, NEAT1 might play a crucial role in CRC treatments.


Assuntos
Neoplasias Colorretais/genética , RNA Longo não Codificante/genética , Apoptose/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Movimento Celular/genética , Proliferação de Células/genética , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Mucosa Gástrica/citologia , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Estimativa de Kaplan-Meier , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo
5.
Nat Commun ; 11(1): 4124, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807787

RESUMO

In response to DNA damage, a synthetic lethal relationship exists between the cell cycle checkpoint kinase MK2 and the tumor suppressor p53. Here, we describe the concept of augmented synthetic lethality (ASL): depletion of a third gene product enhances a pre-existing synthetic lethal combination. We show that loss of the DNA repair protein XPA markedly augments the synthetic lethality between MK2 and p53, enhancing anti-tumor responses alone and in combination with cisplatin chemotherapy. Delivery of siRNA-peptide nanoplexes co-targeting MK2 and XPA to pre-existing p53-deficient tumors in a highly aggressive, immunocompetent mouse model of lung adenocarcinoma improves long-term survival and cisplatin response beyond those of the synthetic lethal p53 mutant/MK2 combination alone. These findings establish a mechanism for co-targeting DNA damage-induced cell cycle checkpoints in combination with repair of cisplatin-DNA lesions in vivo using RNAi nanocarriers, and motivate further exploration of ASL as a generalized strategy to improve cancer treatment.


Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Reparo do DNA/fisiologia , Animais , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Células HCT116 , Humanos , Immunoblotting , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Nanomedicina/métodos , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
6.
Nat Commun ; 11(1): 4338, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859893

RESUMO

Reversible phosphorylation of Pol II and accessory factors helps order the transcription cycle. Here, we define two kinase-phosphatase switches that operate at different points in human transcription. Cdk9/cyclin T1 (P-TEFb) catalyzes inhibitory phosphorylation of PP1 and PP4 complexes that localize to 3' and 5' ends of genes, respectively, and have overlapping but distinct specificities for Cdk9-dependent phosphorylations of Spt5, a factor instrumental in promoter-proximal pausing and elongation-rate control. PP1 dephosphorylates an Spt5 carboxy-terminal repeat (CTR), but not Spt5-Ser666, a site between Kyrpides-Ouzounis-Woese (KOW) motifs 4 and 5, whereas PP4 can target both sites. In vivo, Spt5-CTR phosphorylation decreases as transcription complexes pass the cleavage and polyadenylation signal (CPS) and increases upon PP1 depletion, consistent with a PP1 function in termination first uncovered in yeast. Depletion of PP4-complex subunits increases phosphorylation of both Ser666 and the CTR, and promotes redistribution of promoter-proximally paused Pol II into gene bodies. These results suggest that switches comprising Cdk9 and either PP4 or PP1 govern pause release and the elongation-termination transition, respectively.


Assuntos
Quinase 9 Dependente de Ciclina/metabolismo , RNA Polimerase II/metabolismo , Transcrição Genética/fisiologia , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Células HCT116 , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Fator B de Elongação Transcricional Positiva/metabolismo , Interferência de RNA , RNA Polimerase II/genética , Receptores de Neuropeptídeo Y/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo
7.
Int J Nanomedicine ; 15: 5417-5432, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32801697

RESUMO

Introduction: Green-based materials have been increasingly studied to circumvent off-target cytotoxicity and other side-effects from conventional chemotherapy. Materials and Methods: Here, cellulose fibers (CF) were isolated from rice straw (RS) waste by using an eco-friendly alkali treatment. The CF network served as an anticancer drug carrier for 5-fluorouracil (5-FU). The physicochemical and thermal properties of CF, pure 5-FU drug, and the 5-FU-loaded CF (CF/5-FU) samples were evaluated. The samples were assessed for in vitro cytotoxicity assays using human colorectal cancer (HCT116) and normal (CCD112) cell lines, along with human nasopharyngeal cancer (HONE-1) and normal (NP 460) cell lines after 72-hours of treatment. Results: XRD and FTIR revealed the successful alkali treatment of RS to isolate CF with high purity and crystallinity. Compared to RS, the alkali-treated CF showed an almost fourfold increase in surface area and zeta potential of up to -33.61 mV. SEM images illustrated the CF network with a rod-shaped structure and comprised of ordered aggregated cellulose. TGA results proved that the thermal stability of 5-FU increased within the drug carrier. Based on UV-spectroscopy measurements for 5-FU loading into CF, drug loading encapsulation efficiency was estimated to be 83 ±0.8%. The release media at pH 7.4 and pH 1.2 showed a maximum drug release of 79% and 46%, respectively, over 24 hours. In cytotoxicity assays, CF showed almost no damage, while pure 5-FU killed most of the both normal and cancer cells. Impressively, the drug-loaded sample of CF/5-FU at a 250 µg/mL concentration demonstrated a 58% inhibition against colorectal cancer cells, but only a 23% inhibition against normal colorectal cells. Further, a 62.50 µg/mL concentration of CF/5FU eliminated 71% and 39% of nasopharyngeal carcinoma and normal nasopharyngeal cells, respectively. Discussion: This study, therefore, showed the strong potential anticancer activity of the novel CF/5-FU formulations, warranting their further investigation.


Assuntos
Celulose/química , Portadores de Fármacos/química , Fluoruracila/administração & dosagem , Fluoruracila/farmacologia , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/farmacologia , Linhagem Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/farmacocinética , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Fluoruracila/farmacocinética , Células HCT116 , Humanos , Neoplasias Nasofaríngeas/tratamento farmacológico , Oryza/química , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Termogravimetria , Difração de Raios X
8.
Life Sci ; 258: 118226, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32771555

RESUMO

AIM: Colorectal carcinoma (CRC) is one of the most prevalent cancers throughout the world. Circulating serum-derived microRNAs (miRNAs, miRs) can be used as non-invasive biomarkers for CRC diagnosis. This study aimed to identify a panel of six serum exosomal miRNAs as novel diagnostic biomarkers for CRC. MAIN METHODS: Exosomes were isolated and characterized from the conditioned media of the human colon adenocarcinoma cells (HCT-116 and Caco2). Sera were isolated from peripheral blood of 45 CRC and also 45 healthy individuals. The expression levels and diagnostic value of candidate circulating miRNAs (miR-19a, miR-20a, miR-150, miR-143, miR-145, and let-7a) were measured through quantitative real-time PCR. Receiver operating characteristic (ROC) curves were applied to evaluate the diagnostic accuracy of selected miRNAs. The association of candidate miRNAs and clinicopathological characteristics e.g. tumor node metastasis (TNM) staging and lymph node metastasis (LNM) were further evaluated. KEY FINDINGS: Circulating serum miR-19a, miR-20a, miR-150, and let-7a were significantly up-regulated in CRC patients, while miR-143 and miR-145 showed a significant down-regulation. The higher levels of miR-143 and miR-145 in patients with TNM stage I-II were detected, whereas miR-19a, miR-20a, miR-150, and let-7a were highly expressed in TNM stage III. The expression levels of miR-19a, miR-20a, and miR-150 were positively correlated with LNM status, while the expression levels of miR-143 and miR-145 were lower in patients with LNM. Area under the ROC curves of miR-19a, miR-20a, miR-150, miR-143, miR-145, and let-7a were 0.87, 0.83, 0.75, 0.76, 0.78 and 0.71, respectively. SIGNIFICANCE: We established a panel of six-circulating miRNA signature (i.e. miR-19a, miR-20a, miR-143, miR-145, miR-150, and let-7a) in serum as a non-invasive biomarker for CRC diagnosis. These findings confirm that serum-derived miRNAs have a strong potential to be a diagnostic biomarker for patients with CRC.


Assuntos
Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Neoplasias Colorretais/sangue , Neoplasias Colorretais/genética , MicroRNAs/sangue , MicroRNAs/genética , Adulto , Idoso , Células CACO-2 , Exossomos/genética , Exossomos/metabolismo , Feminino , Redes Reguladoras de Genes/genética , Células HCT116 , Humanos , Masculino , Pessoa de Meia-Idade
9.
Nat Genet ; 52(7): 709-718, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32601473

RESUMO

Propagation of clonal regulatory programs contributes to cancer development. It is poorly understood how epigenetic mechanisms interact with genetic drivers to shape this process. Here, we combine single-cell analysis of transcription and DNA methylation with a Luria-Delbrück experimental design to demonstrate the existence of clonally stable epigenetic memory in multiple types of cancer cells. Longitudinal transcriptional and genetic analysis of clonal colon cancer cell populations reveals a slowly drifting spectrum of epithelial-to-mesenchymal transcriptional identities that is seemingly independent of genetic variation. DNA methylation landscapes correlate with these identities but also reflect an independent clock-like methylation loss process. Methylation variation can be explained as an effect of global trans-acting factors in most cases. However, for a specific class of promoters-in particular, cancer-testis antigens-de-repression is correlated with and probably driven by loss of methylation in cis. This study indicates how genetic sub-clonal structure in cancer cells can be diversified by epigenetic memory.


Assuntos
Evolução Clonal , Epigênese Genética , Neoplasias/genética , Neoplasias/patologia , Transcrição Genética , Linhagem Celular , Proliferação de Células/genética , Metilação de DNA , DNA de Neoplasias/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , RNA Neoplásico/metabolismo , Análise de Célula Única
10.
Ecotoxicol Environ Saf ; 203: 110930, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32684523

RESUMO

Benzo[a]pyrene(BaP), a polycyclic aromatic hydrocarbons (PAH) of environmental pollutants, is one of the main ingredients in cigarettes and an agonist of the aryl hydrocarbon receptor (AhR). Mesenchymal stem cells (MSCs) including C3H10T1/2 and MEF cells, adult multipotent stem cells, can be differentiated toward osteoblasts during the induction of osteogenic induction factor-bone morphogenetic protein 2(BMP2). Accumulating evidence suggests that BaP decreases bone development in mammals, but the further mechanisms of BaP on BMP2-induced bone formation involved are unknown. Here, we researched the role of BaP on BMP2-induced osteoblast differentiation and bone formation. We showed that BaP significantly suppressed early and late osteogenic differentiation, and downregulated the runt-related transcription factor 2(Runx2), osteocalcin(OCN) and osteopontin (OPN) during the induction of BMP2 in MSCs. Consistent with in vitro results, administration of BaP inhibited BMP2-induced subcutaneous ectopic osteogenesis in vivo. Interestingly, blocking AhR reversed the inhibition of BaP on BMP2-induced osteogenic differentiation, which suggested that AhR played an important role in this process. Moreover, BaP significantly decreased BMP2-induced Smad1/5/8 phosphorylation. Furthermore, BaP significantly reduced bone morphogenetic protein receptor 2(BMPRII) expression and excessively activated Hey1. Thus, our data demonstrate the role of BaP in BMP2-induced bone formation and suggest that impaired BMP/Smad pathways through AhR regulating BMPRII and Hey1 may be an underlying mechanism for BaP inhibiting BMP2-induced osteogenic differentiation.


Assuntos
Benzo(a)pireno/toxicidade , Proteína Morfogenética Óssea 2/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Benzo(a)pireno/metabolismo , Linhagem Celular , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Células HCT116 , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Camundongos , Camundongos Nus , Osteoblastos/metabolismo
11.
Nat Commun ; 11(1): 3289, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620890

RESUMO

The spatial organization of chromosomes has key functional roles, yet how chromosomes fold remains poorly understood at the single-molecule level. Here, we employ models of polymer physics to investigate DNA loci in human HCT116 and IMR90 wild-type and cohesin depleted cells. Model predictions on single-molecule structures are validated against single-cell imaging data, providing evidence that chromosomal architecture is controlled by a thermodynamics mechanism of polymer phase separation whereby chromatin self-assembles in segregated globules by combinatorial interactions of chromatin factors that include CTCF and cohesin. The thermodynamics degeneracy of single-molecule conformations results in broad structural and temporal variability of TAD-like contact patterns. Globules establish stable environments where specific contacts are highly favored over stochastic encounters. Cohesin depletion reverses phase separation into randomly folded states, erasing average interaction patterns. Overall, globule phase separation appears to be a robust yet reversible mechanism of chromatin organization where stochasticity and specificity coexist.


Assuntos
Cromatina/química , Conformação Molecular , Fenômenos Físicos , Polímeros/química , Análise de Célula Única/métodos , Fator de Ligação a CCCTC/química , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Células HCT116 , Humanos , Ligação Proteica , Processos Estocásticos , Termodinâmica
12.
Anticancer Res ; 40(8): 4687-4694, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727793

RESUMO

BACKGROUND/AIM: The Japanese apricot "Prunus mume" is a traditional Japanese medicine. MK615, a compound extract from Prunus mume has been reported to have anti-tumor effects. Herein, we used 3D floating (3DF) culture to evaluate the anticancer effects of MK615 against human colorectal cancer (CRC) cells that contain mutant (mt) KRAS. MATERIALS AND METHODS: HKe3 cells exogenously expressing mtKRAS (HKe3-mtKRAS) were treated with MK615 in 3DF cultures. The protein levels of hypoxia-inducible factor 1 (HIF-1) and E-cadherin were quantified by western blotting. RESULTS: MtKRAS enhanced hypoxia tolerance via up-regulation of HIF-1. The expression of HIF-1 protein was suppressed by constitutive overexpression of E-cadherin in CRC HCT116 spheroids. MK615 increased the expression of E-cadherin and decreased the expression of HIF-1 in HKe3-mtKRAS. These results suggest that MK615 suppresses hypoxia tolerance by up-regulation of E-cadherin in CRC cells with mtKRAS. CONCLUSION: MK615 exhibits properties useful for the potential treatment of CRC patients with mtKRAS.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Hipóxia Celular/fisiologia , Neoplasias do Colo/metabolismo , Neoplasias Colorretais/metabolismo , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Regulação para Cima/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Células HCT116 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Prunus/química , Ativação Transcricional/efeitos dos fármacos
13.
Anticancer Res ; 40(8): 4505-4511, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727780

RESUMO

BACKGROUND/AIM: The expression of corticotropin-releasing hormone (CRH)-related peptides involved in stress response in colorectal cancer has been reported. We examined the involvement of CRH-related peptides in cellular stress caused by anticancer drugs in colorectal cancer. MATERIALS AND METHODS: Changes in the expression levels of CRH-related peptides and their receptors in HCT116, DLD-1, and SW480 cell lines after fluorouracil (5-FU) loading were evaluated. Effects of the receptor antagonist against DNA synthesis disorder caused by 5-FU and SN-38 were evaluated using the 3H-labeled deoxyribonucleoside incorporation assay. RESULTS: No changes in the mRNA expression levels of CRH-related peptides (UCN and UCN2) -and their receptors (CRHR1 and CRHR2) were observed. Addition of antagonists to cells with DNA synthesis disorder caused by 5-FU and SN-38 showed no differences in the incorporation of 3H-labeled deoxyribonucleoside. CONCLUSION: CRH-related peptides showed no effect on the stress response to anticancer drugs nor on DNA synthesis disorder in colorectal cancer.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/genética , Hormônio Liberador da Corticotropina/genética , Receptores de Hormônio Liberador da Corticotropina/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Irinotecano/farmacologia
14.
Proc Natl Acad Sci U S A ; 117(30): 17535-17542, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32661159

RESUMO

Mismatch repair (MMR) deficiencies are a hallmark of various cancers causing accumulation of DNA mutations and mismatches, which often results in chemotherapy resistance. Metalloinsertor complexes, including [Rh(chrysi)(phen)(PPO)]Cl2 (Rh-PPO), specifically target DNA mismatches and selectively induce cytotoxicity within MMR-deficient cells. Here, we present an in vivo analysis of Rh-PPO, our most potent metalloinsertor. Studies with HCT116 xenograft tumors revealed a 25% reduction in tumor volume and 12% increase in survival with metalloinsertor treatment (1 mg/kg; nine intraperitoneal doses over 20 d). When compared to oxaliplatin, Rh-PPO displays ninefold higher potency at tumor sites. Pharmacokinetic studies revealed rapid absorption of Rh-PPO in plasma with notable accumulation in the liver compared to tumors. Additionally, intratumoral metalloinsertor administration resulted in enhanced anticancer effects, pointing to a need for more selective delivery methods. Overall, these data show that Rh-PPO inhibits xenograft tumor growth, supporting the strategy of using Rh-PPO as a chemotherapeutic targeted to MMR-deficient cancers.


Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Ródio , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacocinética , Pareamento Incorreto de Bases/efeitos dos fármacos , Complexos de Coordenação/administração & dosagem , Complexos de Coordenação/química , Complexos de Coordenação/farmacocinética , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Modelos Animais de Doenças , Células HCT116 , Humanos , Camundongos , Estrutura Molecular , Ródio/química , Distribuição Tecidual , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Life Sci ; 257: 118122, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32702446

RESUMO

AIMS: Berberine is an isoquinoline alkaloid extracted from the root, rhizome and stem bark of Coptidis Rhizoma. Previous studies have revealed the anti-tumor potential of berberine against various types of cancer cells. However, the underlying mechanisms are not yet fully understood. In this study, we focused on the effects of berberine on fatty acid synthesis and extracellular vesicles formation in cancer cells, and revealed the internal mechanism of berberine inhibition on cancer cell proliferation. MATERIALS AND METHODS: Anti-proliferative activity of berberine was determined by cell counting and microscope observation and cell cycle analysis. Activities of AMPK and ACC, expression of extracellular vesicles markers were detected by western blotting. 13C labeling metabolic flux analysis was used for determination of de novo synthesis of fatty acids. The excreted extracellular vesicles in culture mediums were separated by both polyethylene glycol enrichment of extracellular vesicles and differential centrifugation separation. KEY FINDINGS: Among our early experiments, 5-10 µmol/L berberine exhibited the substantial anti-proliferative effect against human colon cancer cell line HCT116, cervical cancer cell line HeLa and other cancer cells. It was also revealed that, through activating AMPK, berberine inhibited ACC activity then suppressed intracellular fatty acid synthesis, finally decreased the biogenesis of extracellular vesicles. Moreover, supplement with citrate acid, palmitic acid, as well as exogenous extracellular vesicles, could rescue the inhibitory effect of berberine on cell proliferation, suggesting that inhibited ACC activity, suppressed fatty acid synthesis and decreased extracellular vesicles production were important mechanisms account for berberine inhibiting cancer cell proliferation. SIGNIFICANCE: Our study indicates that berberine suppresses cancer cell proliferation through inhibiting the synthesis of fatty acids and decreasing biogenesis and secretion of extracellular vesicles, suggests that berberine is a promising candidate for the development of new therapies for cancer.


Assuntos
Antineoplásicos/farmacologia , Berberina/farmacologia , Vesículas Extracelulares/metabolismo , Ácidos Graxos/metabolismo , Neoplasias/tratamento farmacológico , Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/metabolismo , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ácido Cítrico/farmacologia , Vesículas Extracelulares/efeitos dos fármacos , Células HCT116/efeitos dos fármacos , Células HeLa/efeitos dos fármacos , Humanos
16.
Life Sci ; 257: 118126, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32707053

RESUMO

AIMS: Rab31, a Rab5 subfamily member, has emerged as a modulator of membrane trafficking. Our study serves to clarify the role and mechanism of Rab31 in colorectal carcinoma (CRC) pathogenesis. MATERIALS AND METHODS: The differential expression of Rab31 was examined in paired normal and cancerous colonic tissues by quantitative PCR, western blot and immunochemistry. The prognostic significance of Rab31 was analysed by univariate and multivariate survival analyses. We also investigated the effects of Rab31 on tumour growth in vitro. KEY FINDINGS: We observed that Rab31, which is related to histological differentiation in CRC, was markedly overexpressed in CRC cells. Moreover, patients who showed higher Rab31 levels had a shortened survival period relative to those with low Rab31 levels. Rab31 knockdown significantly downregulated cyclin D1, p-mTOR, and p-p70S6K expression. Moreover, the expression of Rab31-induced p-p70S6K was almost inhibited by rapamycin, a well-established inhibitor of mTOR. Similarly, rapamycin also significantly decreased the stimulatory effect of Rab31 on the expression of cyclin D1. SIGNIFICANCE: These findings suggested that Rab31 enhanced proliferation, promoted cell cycle progression, and inhibited apoptosis of colorectal carcinoma cells through the mTOR pathway.


Assuntos
Neoplasias Colorretais/metabolismo , Ciclina D1/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Idoso , Apoptose , Western Blotting , Células CACO-2 , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Células HCT116 , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sobrevida , Regulação para Cima
17.
Mol Cell ; 79(3): 406-415.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32692975

RESUMO

Protein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation channel formed by the Sec61 complex. Unrelated small-molecule natural products and synthetic compounds inhibit Sec61 with differential effects for different substrates or for Sec61 from different organisms, making this a promising target for therapeutic intervention. To understand the mode of inhibition and provide insight into the molecular mechanism of this dynamic translocon, we determined the structure of mammalian Sec61 inhibited by the Mycobacterium ulcerans exotoxin mycolactone via electron cryo-microscopy. Unexpectedly, the conformation of inhibited Sec61 is optimal for substrate engagement, with mycolactone wedging open the cytosolic side of the lateral gate. The inability of mycolactone-inhibited Sec61 to effectively transport substrate proteins implies that signal peptides and transmembrane domains pass through the site occupied by mycolactone. This provides a foundation for understanding the molecular mechanism of Sec61 inhibitors and reveals novel features of translocon function and dynamics.


Assuntos
Macrolídeos/farmacologia , Microssomos/química , Ribossomos/química , Canais de Translocação SEC/química , Animais , Sítios de Ligação , Sistema Livre de Células/metabolismo , Cães , Expressão Gênica , Células HCT116 , Células HEK293 , Humanos , Macrolídeos/química , Macrolídeos/isolamento & purificação , Microssomos/metabolismo , Simulação de Dinâmica Molecular , Mutação , Mycobacterium ulcerans/química , Mycobacterium ulcerans/patogenicidade , Pâncreas/química , Pâncreas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Ribossomos/metabolismo , Canais de Translocação SEC/antagonistas & inibidores , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo , Homologia Estrutural de Proteína , Especificidade por Substrato
18.
Proc Natl Acad Sci U S A ; 117(29): 16938-16948, 2020 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-32616570

RESUMO

Despite nearly four decades of effort, broad inhibition of oncogenic RAS using small-molecule approaches has proven to be a major challenge. Here we describe the development of a pan-RAS biologic inhibitor composed of the RAS-RAP1-specific endopeptidase fused to the protein delivery machinery of diphtheria toxin. We show that this engineered chimeric toxin irreversibly cleaves and inactivates intracellular RAS at low picomolar concentrations terminating downstream signaling in receptor-bearing cells. Furthermore, we demonstrate in vivo target engagement and reduction of tumor burden in three mouse xenograft models driven by either wild-type or mutant RAS Intracellular delivery of a potent anti-RAS biologic through a receptor-mediated mechanism represents a promising approach to developing RAS therapeutics against a broad array of cancers.


Assuntos
Toxina Diftérica/metabolismo , Endopeptidases/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Proteólise , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Animais , Antineoplásicos/uso terapêutico , Células Cultivadas , Toxina Diftérica/química , Toxina Diftérica/genética , Endopeptidases/química , Endopeptidases/genética , Feminino , Células HCT116 , Humanos , Masculino , Camundongos , Camundongos Nus , Mutação , Sinais Direcionadores de Proteínas , Proteínas Recombinantes/uso terapêutico , Proteínas ras/genética
19.
Nat Commun ; 11(1): 3387, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636417

RESUMO

Biosynthesis of glycosylphosphatidylinositol (GPI) is required for anchoring proteins to the plasma membrane, and is essential for the integrity of the fungal cell wall. Here, we use a reporter gene-based screen in Saccharomyces cerevisiae for the discovery of antifungal inhibitors of GPI-anchoring of proteins, and identify the oligocyclopropyl-containing natural product jawsamycin (FR-900848) as a potent hit. The compound targets the catalytic subunit Spt14 (also referred to as Gpi3) of the fungal UDP-glycosyltransferase, the first step in GPI biosynthesis, with good selectivity over the human functional homolog PIG-A. Jawsamycin displays antifungal activity in vitro against several pathogenic fungi including Mucorales, and in vivo in a mouse model of invasive pulmonary mucormycosis due to Rhyzopus delemar infection. Our results provide a starting point for the development of Spt14 inhibitors for treatment of invasive fungal infections.


Assuntos
Antifúngicos/farmacologia , Glicosiltransferases/antagonistas & inibidores , Policetídeos/farmacologia , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Animais , Proliferação de Células , Modelos Animais de Doenças , Fermentação , Genes Reporter , Glicosilfosfatidilinositóis/biossíntese , Células HCT116 , Células Hep G2 , Humanos , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Células K562 , Pulmão/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Mucorales , Família Multigênica , Rhizopus , Saccharomyces cerevisiae
20.
PLoS One ; 15(7): e0236403, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32716961

RESUMO

Autophagy, a self-degradative physiological process, is critical for homeostasis maintenance and energy source balancing in response to various stresses, including nutrient deprivation. It is a highly conserved catabolic process in eukaryotes and is indispensable for cell survival as it involves degradation of unessential or excessive components and their subsequent recycling as building blocks for the synthesis of necessary molecules. Although the dysregulation of autophagy has been reported to broadly contribute to various diseases, including cancers and neurodegenerative diseases, the molecular mechanisms underlying the epigenetic regulation of autophagy are poorly elucidated. Here, we report that the level of lysine demethylase 3B (KDM3B) increases in nutrient-deprived HCT116 cells, a colorectal carcinoma cell line, resulting in transcriptional activation of the autophagy-inducing genes. KDM3B was found to enhance the transcription by demethylating H3K9me2 on the promoter of these genes. Furthermore, we observed that the depletion of KDM3B inhibited the autophagic flux in HCT116 cells. Collectively, these data suggested the critical role of KDM3B in the regulation of autophagy-related genes via H3K9me2 demethylation and induction of autophagy in nutrient-starved HCT116 cells.


Assuntos
Autofagia/genética , Histona Desmetilases com o Domínio Jumonji/metabolismo , Ativação Transcricional/genética , Aminoácidos/deficiência , Autofagia/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Células HCT116 , Células HEK293 , Histonas/metabolismo , Humanos , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Sirolimo/farmacologia , Ativação Transcricional/efeitos dos fármacos , Proteína com Valosina/metabolismo
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