RESUMO
AIMS: Piwi-like RNA-mediated gene silencing 4 (PIWIL4) or HIWI2, are seen deregulated in human cancers and possibly play critical roles in tumorigenesis. It is unknown what role HIWI2 plays in the regulation of fibrosarcoma, an early metastatic lethal type of soft tissue sarcoma (STS). The present study aimed to investigate the role of HIWI2 in the tumorigenesis of fibrosarcoma. MAIN METHODS: The expression of HIWI2 in HT1080 fibrosarcoma cells was determined by qRT-PCR and western blotting. The MTT assay, colony formation assay, cell cycle, and PE-AnnexinV/7AAD apoptosis assay using flow cytometry, DNA laddering assay, comet assay, and γH2AX accumulation assay were performed to study the effect of HIWI2 overexpression in HT1080 cells. Further, the effect of silencing of HIWI2 was determined by cell viability assay, transwell migration, and invasion assay. KEY FINDINGS: HIWI2 is under-expressed in STS cell lines and tissues, which is associated with poor disease-free survival, disease-specific survival, and progression-free survival of the patients. Overexpression of HIWI2 in HT1080 cells causes DNA damage by increasing intracellular ROS by inhibiting the expression of antioxidant genes (SOD1, SOD2, GPX1, GPX4, and CAT). Furthermore, an increase in H2AX phosphorylation was observed, which activates p53 that promotes p21 expression and caspase-3 activation, leading to G2/M phase cell cycle arrest and apoptosis. HIWI2 silencing, on the contrary, promotes cell growth, migration, and invasion by activating MMP2 and MMP9. SIGNIFICANCE: These results are the first to show that HIWI2 acts as a tumor suppressor in fibrosarcoma by modulating the ROS/DNA damage/p53 pathway.
Assuntos
Dano ao DNA/fisiologia , Fibrossarcoma/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Proteínas de Ligação a RNA/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Apoptose/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Fibrossarcoma/patologia , Humanos , Proteínas de Ligação a RNA/genética , Proteína Supressora de Tumor p53/antagonistas & inibidoresRESUMO
PURPOSE: Autophagy and cell-cycle checkpoints act in concert to confer cellular radioresistance. We investigated the functional interaction between radiation-induced autophagy and G2 checkpoint activation in highly radioresistant human pancreatic ductal adenocarcinoma (PDAC) cells. METHODS AND MATERIALS: Four human PDAC cell lines (MIA PaCa-2, KP-4, Panc-1, and SUIT-2) were analyzed. These cells were first irradiated using x-rays, and their cell cycle status, autophagy, and cell cycle checkpoint marker expression and ATP production levels were evaluated. Autophagic flux assays and siRNA knockdown were used to evaluate autophagy activity. Double thymidine block experiments were performed to synchronize the cells. Two inhibitors (MK-1775 and SCH 900776) were used to attenuate G2 checkpoint activation. Cell survival assays and animal experiments were performed to evaluate the radiosensitizing effects of the G2 checkpoint inhibitors. RESULTS: Autophagy and G2/M accumulation were synchronously induced in human PDAC cells with an activated G2 checkpoint at 12 hours after x-ray irradiation of 6 Gy. Radiation-induced autophagy produced the ATP levels required for cell survival. Double thymidine block experiments revealed that no autophagy occurred in cells that were solely in G2 phase. MK-1775 or SCH 900776 exposure attenuated not only G2 checkpoint activation but also postirradiation autophagy, indicating the dependence of radiation-induced autophagy on an activated G2 checkpoint. The inhibitors demonstrated a higher radiosensitizing effect in the PDAC cells than the autophagy inhibitor chloroquine. MK-1775 in combination with x-rays significantly suppressed the tumor growth of MIA PaCa-2 xenografts compared with other treatment groups, including radiation or drug exposure alone, to enhance the radiosensitivity of PDAC cells in vivo. CONCLUSIONS: Biological crosstalk exists between the G2 checkpoint activation and radiation-induced autophagy processes that are believed to independently contribute to the radioresistance of human PDAC cells. These findings have important implications for the development of future radiation therapy strategies for PDAC.
Assuntos
Autofagia/efeitos da radiação , Carcinoma Ductal Pancreático/radioterapia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Neoplasias Pancreáticas/radioterapia , Tolerância a Radiação , Trifosfato de Adenosina/biossíntese , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Pirazóis/farmacologia , Pirimidinonas/farmacologia , Quinolinas/farmacologia , Tiazóis/farmacologiaRESUMO
The RIP family plays a key role in mediating cell inflammation, oxidative stress and death. Among them, RIPK1, as an important regulatory factor in the upstream of the NF-κB pathway, is involved in multiple pathways of cell inflammation and death. Epidermal cells constitute the outermost barrier of the human body. Radiation can induce epidermal cell death, inflammation and oxidative stress to cause damage. Therefore, this paper selected HaCaT cell and used CRISPR/Cas technology to construct a cell model of stable knockout of RIPK1 gene, to analyze the effect and regulation of RIPK1 knockout on the function and death of HaCaT cells induced by UVB or TNF-α. The results showed that knockout of RIPK1 had no significant effect on the morphology of HaCaT cells at rest, but it led to slowing cell proliferation and blocking the G2M phase of cell cycle. Compared with HaCaTWT, HaCaTRIP1KO was abnormally sensitive to TNF-α-induced cell death and apoptosis, and may be associated with inhibition of NF-κB pathway. Knocking out RIPK1 led to a more significant inhibition of cell growth by UVB, and up-regulation of the expression of the inflammatory factor IL-1α. P38 MAPK and NF-κB pathways may be involved this process. This study further found that RIPK1 in epidermal cell has a regulatory function on pro-survival signals.
Assuntos
Apoptose/fisiologia , Células Epidérmicas/patologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Raios Ultravioleta/efeitos adversos , Sistemas CRISPR-Cas/genética , Linhagem Celular Transformada , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Técnicas de Inativação de Genes , Células HaCaT , Humanos , Interleucina-1alfa/metabolismo , NF-kappa B/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Transdução de Sinais/fisiologia , Pele/lesões , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Colorectal cancer (CRC) is a leading nonfamilial cause of cancer mortality among men and women. Although various genetic and epigenetic mechanisms have been identified, the full molecular mechanisms deriving CRC tumorigenesis are not fully understood. This study demonstrates that cell adhesion molecule transmembrane and immunoglobulin domain containing 1 (TMIGD1) are highly expressed in mouse and human normal intestinal epithelial cells. TMIGD1 knockout mice were developed, and the loss of TMIGD1 in mice was shown to result in the development of adenomas in small intestine and colon. In addition, the loss of TMIGD1 significantly impaired intestinal epithelium brush border membrane, junctional polarity, and maturation. Mechanistically, TMIGD1 inhibits tumor cell proliferation and cell migration, arrests cell cycle at the G2/M phase, and induces expression of p21CIP1 (cyclin-dependent kinase inhibitor 1), and p27KIP1 (cyclin-dependent kinase inhibitor 1B) expression, key cell cycle inhibitor proteins involved in the regulation of the cell cycle. Moreover, TMIGD1 is shown to be progressively down-regulated in sporadic human CRC, and its downregulation correlates with poor overall survival. The findings herein identify TMIGD1 as a novel tumor suppressor gene and provide new insights into the pathogenesis of colorectal cancer and a novel potential therapeutic target.
Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Neoplasias do Colo/metabolismo , Glicoproteínas de Membrana/metabolismo , Adenoma/genética , Adenoma/metabolismo , Adenoma/patologia , Animais , Movimento Celular/genética , Proliferação de Células/genética , Transformação Celular Neoplásica/metabolismo , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Genes Supressores de Tumor/fisiologia , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: The pathogenesis of psoriasis is still not fully understood. Dynamin-related protein 1 (Drp1) regulates mitochondrial fission and is implicated in various inflammatory conditions, but research regarding Drp1 in the skin is scarce. OBJECTIVES: To examine Drp1 expression in psoriasis vulgaris lesional skin and the effect of Drp1 expression on keratinocyte proliferation and inflammatory mediator release in vitro. MATERIALS AND METHODS: Skin biopsies were collected, and the expression of Drp1 and TNF-α was investigated in the skin. Serum TNF-α level was also examined. The effect of Drp1 on keratinocyte proliferation and inflammatory mediator release was evaluated in HaCaT cells following Drp1 transfection. The effect of TNF-α on Drp1 expression was also studied in HaCaT cells. RESULTS: Drp1 expression was significantly increased and positively correlated with PASI score and TNF-α expression in skin. In HaCaT cells, Drp1 transfection altered cellular ATP and proliferation, induced G2/M arrest, and affected inflammatory mediator (TNF-α, IL-6 and CXCL8) release. Moreover, TNF-α induced Drp1 expression and recovered cellular ATP content and mediator release. CONCLUSION: Drp1 is significantly elevated in psoriasis vulgaris skin and positively correlates with disease severity, moreover, in vitro, Drp1 regulates keratinocyte function.
Assuntos
Proliferação de Células , Dinaminas/metabolismo , Queratinócitos/fisiologia , Psoríase/metabolismo , Psoríase/fisiopatologia , Pele/metabolismo , Trifosfato de Adenosina/metabolismo , Divisão Celular , Dinaminas/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Células HaCaT , Humanos , Mediadores da Inflamação/metabolismo , Dinâmica Mitocondrial , Índice de Gravidade de Doença , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/metabolismo , Regulação para CimaRESUMO
Glutathionylation is an important posttranslational modification that protects proteins from further oxidative damage as well as influencing protein structure and activity. In the present study, we demonstrate that the cysteine-42 residue in protein arginine N-methyltransferase 5 (PRMT5) is glutathionylated in aged mice or in cells that have been exposed to oxidative stress. Deglutathionylation of this protein is catalyzed by glutaredoxin-1 (Grx1). Using mutagenesis and subsequent biochemical analyses, we show that glutathionylation decreased the binding affinity of PRMT5 with methylosome protein-50 (MEP50) and reduced the methyltransferase activity of PRMT5. Furthermore, overexpression of PRMT5-C42A mutant caused a significant increase in histone methylation in HEK293T and A549 cells and promoted cell growth, whereas overexpression of the PRMT5-C42D mutant, a mimic of glutathionylated PRMT5, inhibited cell proliferation. Taken together, our results demonstrate a new mechanism of regulation of PRMT5 methyltransferases activity and suggest that PRMT5 glutathionylation is partly responsible for reactive oxygen species-mediated cell growth inhibition.
Assuntos
Envelhecimento/metabolismo , Proliferação de Células/efeitos dos fármacos , Glutationa/metabolismo , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Bases de Dados de Proteínas , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Glutarredoxinas/metabolismo , Células HEK293 , Histonas/metabolismo , Humanos , Rim/enzimologia , Rim/metabolismo , Metilação , Camundongos , Ligação Proteica , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/genética , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Regulação para CimaRESUMO
Dual-specificity phosphatase 4 (DUSP4), a MAP kinase phosphatase, has been regarded as a tumor suppressor gene in several cancers. However, high-level expression of DUSP4 is occasionally observed in specific cancers and its functional significance in carcinogenesis is not fully understood. In the present study, we showed that downregulation of DUSP4 suppressed the proliferation of cancer cell lines exhibiting high expression of DUSP4 by inducing apoptosis and cell cycle arrest at G2/M phase. Expression microarray analyses and pathway analyses revealed that downregulation of DUSP4 activated the p53 signaling pathway, and might be involved in cell growth suppression. Aberrant accumulation of p53 and induction of p53 downstream target genes were further investigated. Furthermore, cell growth suppression following downregulation of DUSP4 was markedly attenuated in p53-deleted cells established using the CRISPR/Cas9 system. These findings suggest that constitutive expression of DUSP4 in cancer cells contributes to enhanced proliferation through escape from apoptosis and cell cycle arrest. We propose that DUSP4 could be a novel therapeutic target for cancers overexpressing it.
Assuntos
Fosfatases de Especificidade Dupla/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Neoplasias/enzimologia , Neoplasias/patologia , Apoptose/genética , Apoptose/fisiologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/fisiologia , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Regulação para Baixo , Fosfatases de Especificidade Dupla/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Células HCT116 , Humanos , Fosfatases da Proteína Quinase Ativada por Mitógeno/genética , Neoplasias/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
UNC5B is a known tumor suppressor gene in a variety of cancers. As a transmembrane protein, UNC5B also induces apoptosis in a P53-dependent manner. In this study, we demonstrate that UNC5B inhibits proliferation through G2/M phase arrest by mass spectrometry and bioinformatics analysis in bladder cancer cells. By combing with CDC14A and P53, UNC5B dephosphorylated P53 at Ser-315 site. This dephosphorylation facilitated G2/M phase arrest by reducing the expression of cyclin B1 and increasing the expression of p-CDK1, thus inhibiting tumor proliferation. Knockdown of CDC14A suppressed the G2/M phase arrest induced by UNC5B in vitro, and eliminated the inhibitory effect of UNC5B on tumor proliferation in vivo. Our results show that UNC5B-mediated cell cycle arrest may act as a potential treatment for bladder cancer.
Assuntos
Receptores de Netrina/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Humanos , Pessoa de Meia-Idade , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/patologiaRESUMO
Studies have shown that palmatine (PAL) has anti-cancer effects. However, the activity and potential mechanisms of PAL against colorectal cancer remain elusive. The results showed that PAL significantly inhibited the proliferation of colon cancer cells in vitro and in vivo without significant effect on non-tumorigenic colon cells. Target prediction and clinical sample database analysis suggested that PAL may contribute to colon cancer cells phase arrest and apoptosis by targeting aurora kinase A (AURKA). Inhibition and overexpression of AURKA proved that PAL induces G2/M phase arrest and apoptosis in colon cancer cells by targeting AURKA. Moreover, PAL promoted intracellular Reactive oxygen species (ROS) production and decreased mitochondrial membrane potential (ΔΨm). PAL reduced the levels of AURKA, Bcl-xl and Bcl2 proteins, and promoted the expression of pro-apoptotic proteins P53, P73, Caspase3 and Caspase9, as well as the increase of cytochrome c (cyt. c) in cell lysates in vitro and in vivo. Together, our study confirmed that PAL induced G2/M phase arrest and mitochondrial-associated pathway apoptosis in colon cancer cells by targeting AURKA. PAL may provide a novel solution for the treatment of colon cancer by serving as a new AURKA inhibitor.
Assuntos
Aurora Quinase A/antagonistas & inibidores , Alcaloides de Berberina/administração & dosagem , Neoplasias do Colo , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Aurora Quinase A/metabolismo , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos/métodos , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Células HCT116 , Células HT29 , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mitocôndrias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
PURPOSE: Osteosarcoma is one of the rare but fatal malignancies. The high metastatic rate, late diagnosis, emergence of drug resistance against drugs such as doxorubicin, and the lack of therapeutic targets obstructs the treatment of osteosarcoma. This study was undertaken to investigate the role and therapeutic potential of miR-187 in human osteosarcoma cells. METHODS: The WST-1 proliferation assay was used for investigation of cell viability. Transfections were carried out by Lipofectamine 2000 reagent. The qRT-PCR was used for expression analysis. DAPI, acridine orange (AO)/ethidium bromide (EB) and Annexin V/propidium iodide (PI) assay were used for apoptosis. Western blot analysis was used for the determination of protein expression. RESULTS: The expression of miR-187 was significantly downregulated in human osteosarcoma cells. Out of all osteosarcoma cell lines the SAOS-2 showed the lowest expression of miR-187 and therefore this cell line was selected for further studies. Overexpression of miR-187 caused significant inhibition in the proliferation of SAOS-2 osteosarcoma cells. The miR-187-triggered growth inhibition was found to be mainly due to induction of G2/M phase cell cycle arrest of the SAOS-2 cells. The G2/M cell cycle arrest was also accompanied by depletion of Cyclin-B1 expression. Additionally, miR-187 enhanced the chemosensitivity of the osteosarcoma cells to doxorubicin. The wound healing and transwell assay showed that miR-187 overexpression resulted in the suppression of migration and invasion of the SAOS-2 osteosarcoma cells. In silico analysis showed that miR-187 exerts its effects by inhibiting mitogen activated protein kinase 7 (MAPK7). The expression of MAPK7 was found to be significantly upregulated in osteosarcoma cells and overexpression of MAPK7 could nullify the effects of miR-187 on the proliferation of the osteosarcoma cells.
Assuntos
Neoplasias Ósseas/genética , MicroRNAs/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Osteossarcoma/genética , Antibióticos Antineoplásicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/enzimologia , Neoplasias Ósseas/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Regulação para Baixo , Doxorrubicina/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , MicroRNAs/biossíntese , MicroRNAs/genética , Invasividade Neoplásica , Osteossarcoma/tratamento farmacológico , Osteossarcoma/enzimologia , Osteossarcoma/patologiaRESUMO
PURPOSE: Gliomas are aggressive brain tumors accounting for significant mortality across the globe. Biomarkers for early detection and therapeutic targets for efficient treatment are lacking for glioma. This study was undertaken to investigate the role and therapeutic implications of miR-22 in glioma. METHODS: U-87 glioma cell line was used in this study. qRT-PCR was employed for expression analysis. MTT assay was used for determination of cell viability. Lipofectamine 2000 was used for transfection. Flow cytometry was used for cell analysis. Wound healing assay and transwell assay were used for monitoring cell migration and invasion. Western blot analysis was used for estimation of protein expression. RESULTS: The miR-22 expression was found decreased in glioma cells. Overexpression of miR-22 resulted in arrest of the U-87 glioma cells at G2/M checkpoint of the cell cycle. The percentage of apoptotic U-87 cells in G2/M phase were 13.05% in negative control (NC) and 29.06% in miR-22 mimics transfected cells. The cell cycle arrest promoted by miR-22 overexpression was also associated with depletion of cyclin B1 expression in U-87 cells. Furthermore, miR-22 could also significantly increase the sensitivity of glioma U-87 cells to cisplatin. The TargetScan analysis and dual luciferase assay showed SNAIL1 to be the target of miR-22. The expression of SNAIL1 was also enhanced in all the glioma cells and miR-22 overexpression could cause suppression of the SNAIL1 expression in U-87 cells. Furthermore, SNAIL1 silencing could also cause decline in the viability of the U-87 cells. The wound healing assay showed that miR-5 overexpression caused decrease in the migration of U-87 cells, while the transwell assay showed decline in the invasion of miR-22 mimics transfected U-87 cells. CONCLUSION: Taken together, miR-22 may exhibit therapeutic implications in glioma and may prove useful in glioma treatment.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioma/metabolismo , MicroRNAs/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Glioma/genética , Glioma/patologia , Humanos , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , MicroRNAs/genética , Metástase Neoplásica , Fatores de Transcrição da Família Snail/biossíntese , Fatores de Transcrição da Família Snail/genética , TransfecçãoRESUMO
Receptor-interacting protein 1 (RIP1, also known as RIPK1) is not only a tumor-promoting factor in several cancers but also mediates either apoptosis or necroptosis in certain circumstances. In this study we investigated what role RIP1 plays in human ovarian cancer cells. We showed that knockout (KO) of RIP1 substantially suppressed cell proliferation, accompanied by the G2/M checkpoint arrest in two human ovarian cancer cell lines SKOV3 and A2780. On the other hand, RIP1 KO remarkably attenuated cisplatin-induced cytotoxicity, which was associated with reduction of the apoptosis markers PARP cleavage and the necroptosis marker phospho-MLKL. We found that RIP1 KO suppressed cisplatin-induced ROS accumulation in both SKOV3 and A2780 cells. ROS scavenger BHA, apoptosis inhibitor Z-VAD or necroptosis inhibitor NSA could effectively suppress cisplatin's cytotoxicity in the control cells, suggesting that ROS-mediated apoptosis and necroptosis were involved in cisplatin-induced cell death. In addition, blocking necroptosis with MLKL siRNA effectively attenuated cisplatin-induced cytotoxicity. In human ovarian cancer A2780 cell line xenograft nude mice, RIP1 KO not only significantly suppressed the tumor growth but also greatly attenuated cisplatin's anticancer activity. Our results demonstrate a dual role of RIP1 in human ovarian cancer: it acts as either a tumor-promoting factor to promote cancer cell proliferation or a tumor-suppressing factor to facilitate anticancer effects of chemotherapeutics such as cisplatin.
Assuntos
Apoptose/fisiologia , Proliferação de Células/fisiologia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Necroptose/fisiologia , Neoplasias Ovarianas/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Cisplatino/farmacologia , Feminino , Técnicas de Inativação de Genes , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Necroptose/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Paclitaxel/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genéticaRESUMO
Studies have proved the role of GAS5 in the development of different cancers. This study was undertaken to investigate the role and explore therapeutic implications of GAS5 in human cervical cancer. The results showed that GAS5 was significantly (p < 0.05) downregulated in human cervical cancer tissues. The results also showed that cervical cancer progresses with the suppression of GAS5 expression levels. Additionally, the expression of GAS5 was also significantly (p < 0.05) downregulated in human cervical cancer cell lines. Nonetheless, overexpression of GAS5 caused a remarkable decrease in the proliferation of C33A and HeLa cervical cancer cells. The decrease in the proliferation rate was attributed to the induction of apoptosis of C33A and HeLa cells which was accompanied with upregulation of Bax and suppression of Bcl-2. Additionally, GAS5 overexpression also promoted the arrest of C33A and HeLa cells at the G2/M check point of cell cycle via suppression of cyclin B1 and CDK1 expression. The transwell assays showed that GAS5 overexpression significantly (p < 0.05) inhibited the migration and invasion of the C33A and HeLa cervical cancer cells. The bioinformatics analysis as well as the dual luciferase assay showed GAS5 acts as a target of miR-135a. Interestingly, the expression of miR-135a was upregulated in the human cervical cancer cells and its suppression exerted growth inhibitory effects on the C33A and HeLa cells. However, silencing of GAS5 could nullify the effects of miR-135a suppression on the proliferation of C33A and HeLa cells. Taken together, the results of this study point towards the therapeutic implications of GAS5 in the treatment of cervical cancer.
Assuntos
Apoptose/fisiologia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , RNA Longo não Codificante/metabolismo , Neoplasias do Colo do Útero/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , Transdução de Sinais/fisiologia , Regulação para Cima , Neoplasias do Colo do Útero/genéticaRESUMO
Cell-autonomous changes in p53 expression govern the duration and outcome of cell-cycle arrest at the G2 checkpoint for DNA damage. Here, we report that mitogen-activated protein kinase (MAPK) signaling integrates extracellular cues with p53 dynamics to determine cell fate at the G2 checkpoint. Optogenetic tools and quantitative cell biochemistry reveal transient oscillations in MAPK activity dependent on ataxia-telangiectasia-mutated kinase after DNA damage. MAPK inhibition alters p53 dynamics and p53-dependent gene expression after checkpoint enforcement, prolonging G2 arrest. In contrast, sustained MAPK signaling induces the phosphorylation of CDC25C, and consequently, the accumulation of pro-mitotic kinases, thereby relaxing checkpoint stringency and permitting cells to evade prolonged G2 arrest and senescence induction. We propose a model in which this MAPK-mediated mechanism integrates extracellular cues with cell-autonomous p53-mediated signals, to safeguard genomic integrity during tissue proliferation. Early steps in oncogene-driven carcinogenesis may imbalance this tumor-suppressive mechanism to trigger genome instability.
Assuntos
Dano ao DNA , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Expressão Gênica , Humanos , Células MCF-7 , Proteínas Quinases Ativadas por Mitógeno/genética , Fosforilação , Transdução de Sinais , Proteína Supressora de Tumor p53/genéticaRESUMO
Pyroptosis is a form of programmed cell death (PCD) that plays a vital role in immunity and diseases. Although it was recently reported that chemotherapy drugs can induce pyroptosis through caspase-3-dependent cleavage of gasdermin E (GSDME), the role of pyroptosis in osteosarcoma (OS) with dioscin is less understood. In this study, we explored the effects of dioscin on OS in vitro and in vivo and further elucidated the underlying molecular mechanisms and found that dioscin-triggered pyroptosis in GSDME-dependent cell death and that GSDME-N was generated by caspase-3. Furthermore, dioscin inhibited cancer cell growth by inducing G2/M arrest and apoptosis through the JNK/p38 pathway. In vivo, dioscin significantly inhibited OS proliferation. Taken together, our results demonstrate that dioscin can induce apoptosis through the JNK/p38 pathway and GSDME-dependent pyroptosis in OS, identifying it as a potential therapeutic drug for treatment of this disease.
Assuntos
Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Diosgenina/análogos & derivados , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Osteossarcoma/tratamento farmacológico , Apoptose/fisiologia , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/metabolismo , Pontos de Checagem do Ciclo Celular/fisiologia , Morte Celular/fisiologia , Linhagem Celular Tumoral , Diosgenina/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Humanos , Osteossarcoma/metabolismo , Piroptose/fisiologia , Receptores de Estrogênio/efeitos dos fármacos , Receptores de Estrogênio/metabolismoRESUMO
We develop a mathematical model to study the immediate effect of low-dose radiation on the G2 checkpoint and the G2/M transition of the cell cycle via a radiation pathway (the ATM-Chk2 pathway) of an individual mammalian cell. The model consists of a system of nonlinear differential equations describing the dynamics of a network of regulatory proteins that play key roles in the G2/M transition, cell cycle oscillations, and the radiation pathway. We simulate the application of a single pulse of low-dose radiation at different intensities ([Formula: see text] 0-0.4 Gy) and times during the latter part of the G2-phase. We use bifurcation analysis to characterize the effect of radiation on the G2/M transition via the ATM-Chk2 pathway. We show that radiation between 0.1 and 0.3 Gy can delay the G2/M transition, and radiation higher than 0.3 Gy can fully activate the G2 checkpoint. Also, our results show that radiation can be low enough to neither delay the G2/M transition nor activate the G2 checkpoint ([Formula: see text] 0.1 Gy). Our model supports the idea that the cell response to radiation during G2-phase explains hyper-radiosensitivity and increased radioresistance (HRS/IRR) observed at low dose.
Assuntos
Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Modelos Biológicos , Animais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proliferação de Células/fisiologia , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/fisiologia , Sobrevivência Celular/efeitos da radiação , Quinase do Ponto de Checagem 2/metabolismo , Relação Dose-Resposta à Radiação , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Humanos , Conceitos Matemáticos , Dinâmica não Linear , Tolerância a Radiação/fisiologiaRESUMO
Chemokine-like factor 1 (CKLF1) is a cytokine, which has a detrimental effect on the multiple disease progression. Our previous studies reported that arterial injury induced the upregulation of CKLF1 expression in artery at 7-14 days after injury. Here, using a rat carotid balloon injury model, we found that CKLF1 knockdown in the injured site abolished neointimal formation and even decreased medial area; contrarily, CKLF1 overexpression developed a thicker neointima than controls, demonstrating that CKLF1 exerted positive effects on neointimal hyperplasia and the accumulation of vascular smooth muscle cells (VSMC). The mechanism study indicated that CKLF1 reduced susceptibility to the cell cycle G2/M arrest and apoptosis, and thereby speeding up VSMC accumulation. This role of CKLF1 was tightly associated with phosphatidylinositol (PI) 3-kinase signaling pathway. CKLF1 increased the expression of four isoforms of the PI3-kinase catalytic subunits, which in turn activated its downstream targets Akt and an effector NF-κB accepted as critical transcription factors of cell survival and proliferation. Furthermore, RNA-sequencing analysis revealed that CKLF1 had wide-ranging roles in regulating the expression of genes that mainly engaged in cell apoptosis and innate immune response. Collectively, the data allow us to conclude that high level CKLF1 after artery injury switches the balance of VSMC proliferation and apoptosis through PI3K/AKT/NF-κB signaling and consequently leads to neointimal hyperplasia. The findings shed insight into new treatment strategies to limit restenosis based on CKLF1 as a future target.
Assuntos
Apoptose/fisiologia , Quimiocinas/metabolismo , Hiperplasia/metabolismo , Proteínas com Domínio MARVEL/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Neointima/metabolismo , Transdução de Sinais/fisiologia , Animais , Divisão Celular/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Hiperplasia/patologia , Masculino , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , NF-kappa B/metabolismo , Neointima/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Ginsenoside Rg5, a rare saponin belonging to the family of protopanaxadiol ginsenosides, has been demonstrated to have potential anti-tumor effects in various cancers. However, the effect of Rg5 on human gastric cancer and the underlying molecular mechanisms remain to be elucidated. In this study, Rg5 could suppress cell proliferation by causing G2/M phase arrest. Treatment with Rg5 could induce apoptosis through the extrinsic death receptor and intrinsic mitochondrial pathways. Autophagy induction was demonstrated by the formation of autophagosomes and autophagy-related proteins. Rg5-induced cell death was inhibited by the autophagy inhibitor 3-MA and apoptosis inhibitor Z-VAD-FMK. Moreover, the suppression of apoptosis weakened Rg5-induced autophagy, while the inhibition of autophagy attenuated Rg5-induced apoptosis. Further studies revealed that Rg5 induced ROS production and activated MAPK signaling pathways. The ROS scavenger NAC markedly diminished G2/M arrest, apoptosis, autophagy and activation of MAPK pathways induced by Rg5. The p38 inhibitor SB203580 or knockdown of p38 by siRNA clearly reversed Rg5-induced apoptosis and G2/M arrest. The JNK inhibitor SP600125 or knockdown of JNK by siRNA markedly attenuated Rg5-induced G2/M arrest, apoptosis and autophagy. The inhibition of ERK inhibitor U0126 or knockdown of ERK by siRNA clearly restored Rg5-induced apoptosis and autophagy. Finally, Rg5 significantly suppressed the growth of xenograft gastric tumors with fewer side effects. Overall, the evidence suggested that Rg5 is a novel and promising strategy for the treatment of gastric cancer owing to its high efficacy, multiple mechanisms and fewer side effects.
Assuntos
Autofagia/efeitos dos fármacos , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Ginsenosídeos/uso terapêutico , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Gástricas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Autofagia/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Ginsenosídeos/isolamento & purificação , Ginsenosídeos/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Espécies Reativas de Oxigênio/antagonistas & inibidores , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Reports suggest that microRNAs have implications in the development of several diseases including cancer. It is therefore believed that miRs may act as therapeutic targets for cancer treatment. The treatment of ovarian cancer is mainly obstructed by lack of biomarkers and efficient drug targets. Against this backdrop, this study was undertaken to unveil the therapeutic implications of miR-27a in ovarian cancer. The results showed that the expression of miR-27a was significantly elevated in ovarian cancer tissues and cell lines. Inhibition of miR-27a expression resulted in the decrease of proliferation rate and colony formation potential of the SK-OV-3 and OVACAR-3â¯cells via G2/M arrest. The miR-27a inhibition triggered G2/M arrest of SK-OV-3 and OVACAR-3â¯cells was accompanied with depletion of cyclin A and B1 expression levels. TargetScan analysis together with dual reporter assay revealed that miR-27a exerts its effects via modulation of CUL5 expression. The CUL5 was shown to be suppressed in the ovarian cancer tissues and cell lines and suppression of miR-27a expression caused upregulation of CUL5 expression. Overexpression of CUL5 caused inhibition of SK-OV-3 and OVACAR-3â¯cell proliferation via induction of G2/M arrest, similar to that of miR-27a inhibition. Interestingly, CUL5 overexpression reversed the effects of miR-27a inhibition on the viability of SK-OV-3â¯cells. Finally, the suppression of miR-27a could enhance the chemosensitivity of the SK-OV-3â¯cells to cisplatin and docetaxel anticancer drugs and also decreased their migration and invasion. The findings of this study revealed that miR-27a suppression inhibits the growth, chemosensitivity and invasion of ovarian cancer and may prove beneficial in the ovarian cancer management.
Assuntos
Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Proteínas Culina/metabolismo , MicroRNAs/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Docetaxel/farmacologia , Feminino , Pontos de Checagem da Fase G2 do Ciclo Celular/fisiologia , Humanos , Neoplasias Ovarianas/metabolismoRESUMO
The cell cycle is under circadian regulation. Oncogenes can dysregulate circadian-regulated genes to disrupt the cell cycle, promoting tumor cell proliferation. As a regulator of G2/M arrest in response to DNA damage, the circadian gene Timeless Circadian Clock (TIMELESS) coordinates this connection and is a potential locus for oncogenic manipulation. TIMELESS expression was evaluated using RNASeq data from TCGA and by RT-qPCR and western blot analysis in a panel of colon cancer cell lines. TIMELESS expression following ERK inhibition was examined via western blot. Cell metabolic capacity, propidium iodide, and CFSE staining were used to evaluate the effect of TIMELESS depletion on colon cancer cell survival and proliferation. Cell metabolic capacity following TIMELESS depletion in combination with Wee1 or CHK1 inhibition was assessed. TIMELESS is overexpressed in cancer and required for increased cancer cell proliferation. ERK activation promotes TIMELESS expression. TIMELESS depletion increases γH2AX, a marker of DNA damage, and triggers G2/M arrest via increased CHK1 and CDK1 phosphorylation. TIMELESS depletion in combination with Wee1 or CHK1 inhibition causes an additive decrease in cancer cell metabolic capacity with limited effects in non-transformed human colon epithelial cells. The data show that ERK activation contributes to the overexpression of TIMELESS in cancer. Depletion of TIMELESS increases γH2AX and causes G2/M arrest, limiting cell proliferation. These results demonstrate a role for TIMELESS in cancer and encourage further examination of the link between circadian rhythm dysregulation and cancer cell proliferation.