RESUMO
Microspherule protein 1(MCRS1) is known to be an oncogene in several tumors. However, recent studies have shown that MCRS1 inhibits lymphatic metastasis in gastric cancer (GC) patients by inhibiting telomerase activity. Protein kinase, membrane associated tyrosine/threonine 1(Pkmyt1), a member of the WEE1 family, has been found to interact with MCRS1 by yeast two-hybrid assay; however, how these two proteins interact in GC is still unclear. Hence, this study aimed to investigate the effect of MCRS1 interaction with Pkmyt1 on GC cell proliferation, migration, and invasion. Initially, we observed increased expression of MCRS1 in GC SGC-7901 cells and decreased expression in GC BGC-823 cells. Hence, we down-regulated MCRS1 expression in SGC-7901 cells and up-regulated it in BGC-823 cells. Our results showed that overexpression of MCRS1 inhibits the growth, invasion and migration of GC cells, while downregulation of MCRS1 promotes the growth, invasion and migration of GC cells. When MK1775, an inhibitor of WEE1 kinase, was added after downregulation of MCRS1, phenotypic recovery effects were observed. Overexpression of MCRS1 also inhibited the expression of Pkmyt1 and vice versa. This indicated that there might be a possible interaction between MCRS1 and Pkmyt1. Furthermore, immunoprecipitation assay revealed the interaction between MCRS1 and Pkmyt1 in virto, and immunofluorescence experiments showed that the two proteins were co-localized in the cytoplasm. In conclusion, our study confirmed the specific tumor suppressive activity of MCRS1 in GC proliferation, invasion and migration and suggested that it might inhibit the progression of GC through its interaction with Pkmyt1.
Assuntos
Transição Epitelial-Mesenquimal , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Ligação a RNA/fisiologia , Neoplasias Gástricas/patologia , Linhagem Celular , Movimento Celular , Proliferação de Células , Humanos , Invasividade Neoplásica , Neoplasias Gástricas/metabolismoRESUMO
Apoptosis plays a critical role in normal embryonic development and tissue homeostasis regulation. EndophilinA2 (EndoA2) is widely reported to regulate endocytosis. Additionally, EndoA2 has been demonstrated to be involved in tumor metastasis, neuroregulation and vascular function. In this study, we used siRNA and Ad-EndoA2 transfection strategy to investigate whether EndoA2 provides a protective effect against apoptosis induced by H2O2 in H9C2 cardiomyocytes and the underlying mechanisms. We found that EndoA2 siRNA knockdown promoted H2O2-induced apoptosis in H9C2 cardiomyocytes, evidenced by decreased cell number, increased apoptotic cells, and activation of caspase-3. In contrast, EndoA2 overexpression showed the opposite effects and inhibited H2O2-induced apoptosis in H9C2 cardiomyocytes. Further studies revealed that EndoA2 overexpression strengthened autophagy, evidenced by the increased LC3 II/I ratio and P62 degradation, whereas EndoA2 siRNA knockdown produced the opposite effects. Furthermore, we revealed that there was an interaction between Bif-1 and Beclin-1. Upon H2O2 treatment, the association of Bif-1 and Beclin-1 remarkably increased. EndoA2 overexpression further promoted the binding of Bif-1 with Beclin-1, whereas EndoA2 siRNA knockdown reduced this association. These data strongly suggested that EndoA2 inhibited H2O2-induced apoptosis in H9C2 cardiomyocytes, possibly by promoting Bif-1 to form a complex with Beclin-1 and strengthening autophagy. This study provides a novel target for heart diseases.
Assuntos
Aciltransferases/metabolismo , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cardiotônicos/metabolismo , Peróxido de Hidrogênio/toxicidade , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteína Beclina-1/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , RatosRESUMO
AIM: To investigate the expression of protein kinase CK2α (CK2α) in human thyroid disease and its relationship with thyroid cancer metastasis. MATERIALS AND METHODS: Using immunohistochemistry we measured the expression of CK2α in 76 benign and malignant human thyroid cancer tissues, including 10 pairs of papillary carcinoma tissues with or without lymph node cancerous metastasis and similarly 10 pairs of lymph nodes. RESULTS: The expression of CK2α was found to be higher in thyroid carcinoma cases (papillary carcinoma, follicular carcinoma, anaplastic carcinoma and medullary carcinoma) than in ones such as chronic lymphocytic thyroiditis, nodular goiter and adenoma. These findings were also confirmed by RT-PCR and Western blotting. More strikingly, elevated expression of CK2α in thyroid papillary carcinoma tissues was not only significantly associated with lymph node cancerous metastasis and clinical stage of thyroid cancers; but also correlated with epithelial-mesenchymal transition (EMT) and high tenascin C (TNC) expression. In addition, EMT and high TNC expression in thyroid carcinoma tissues was significantly associated with lymph node cancerous metastasis. CONCLUSIONS: Elevated expression of nuclear CK2α is a poor prognosis indicator in lymph node cancerous metastasis of human thyroid cancers.
Assuntos
Adenocarcinoma Folicular/metabolismo , Carcinoma/metabolismo , Caseína Quinase II/metabolismo , Linfonodos/patologia , Carcinoma Anaplásico da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Adenocarcinoma Folicular/patologia , Adenocarcinoma Folicular/secundário , Adenoma/metabolismo , Adulto , Idoso , Caderinas/metabolismo , Carcinoma/patologia , Carcinoma/secundário , Carcinoma Neuroendócrino , Carcinoma Papilar , Estudos de Casos e Controles , Transição Epitelial-Mesenquimal , Feminino , Bócio Nodular/metabolismo , Humanos , Imuno-Histoquímica , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Prognóstico , Tenascina/metabolismo , Câncer Papilífero da Tireoide , Carcinoma Anaplásico da Tireoide/patologia , Carcinoma Anaplásico da Tireoide/secundário , Neoplasias da Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/secundário , Tireoidite Autoimune/metabolismo , Adulto JovemRESUMO
To further test whether protein kinase A (PKA) can affect the mitotic cell cycle, one-cell stage mouse embryos at S phase (22 h after hCG injection) were incubated in M16 medium containing various concentrations of H-89, a PKA inhibitor. With increasing concentrations of H-89 (0-50 µmol/L), the G(2) phase of eggs was decreased and the cleavage rate was accelerated. A concentration of 40 µmol/L H-89 led to all of the mouse eggs entering the M phase of mitosis. Furthermore, to study the role of PKA in regulating the phosphorylation status of S149 and S321 sites of cell division cycle 25B (CDC25B) on one-cell stage fertilized mouse eggs, pBSK-CDC25B-WT, pBSK-CDC25B-S149A, pBSK-CDC25B-S321A and pBSK-CDC25B-S149A/S321A were transcribed into mRNAs in vitro, then mRNAs were microinjected into S phase of mouse fertilized eggs and cultured in M16 medium pretreated with H-89. Then, the cleavage of fertilized eggs, maturation promoting factor (MPF) activity and phosphorylation status of CDC2-Tyr15 were observed. In the presence of 40 µmol/L H-89, the cleavage rate of fertilized eggs in CDC25B-S/A-mRNAs and CDC25B-WT-mRNA injected groups was significantly higher than that in the control groups, and the peak of MPF activity appeared in the CDC25B-S/A-mRNAs and CDC25B-WT-mRNA injected groups earlier than that in the control groups. CDC2-Tyr15 phosphorylation state was consistent with MPF activity. In conclusion, the present study suggests that PKA regulates the early development of mouse embryos by phosphorylation of S149 and S321 of CDC25B, which plays an important role in the regulation of G(2)/M transition in the mitotic cell cycle of fertilized mouse eggs.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Mitose/efeitos dos fármacos , Zigoto/crescimento & desenvolvimento , Fosfatases cdc25/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/genética , Desenvolvimento Embrionário/fisiologia , Feminino , Masculino , Mesotelina , Camundongos , Microinjeções , Fosforilação , Serina/genética , Serina/metabolismo , Zigoto/citologia , Fosfatases cdc25/genéticaRESUMO
Protein kinase B (PKB, also called Akt) is known as a serine/threonine protein kinase. Some studies indicate that the Akt signalling pathway strongly promotes G2/M transition in mammalian cell cycle progression, but the mechanism remains to be clarified, especially in the fertilized mouse egg. Here, we report that the expression of Akt at both the protein and mRNA level was highest in G2 phase, accompanied by a peak of Akt activity. In addition, the subcellular localization of p21(Cip1/WAF1) has been proposed to be critical in the cell cycle. Hence, we detected the expression and localization of p21(Cip1/WAF1) after injecting fertilized mouse eggs with Akt mRNA. In one-cell stage fertilized embryos microinjected with mRNA coding for a constitutively active myristoylated Akt (myr-Akt), p21(Cip1/WAF1) was retained in the cytoplasm. Microinjection of mRNA of kinase-deficient Akt(Akt-KD) resulted in nuclear localization of p21(Cip1/WAF1) . Meanwhile, microinjection of different types of Akt mRNA affected the phosphorylation status of p21(Cip1/WAF1) . However, there was no obvious difference in the protein expression of p21(Cip1/WAF1) . Therefore, Akt controls the cell cycle by changing the subcellular localization of p21(Cip1/WAF1) , most likely by affecting the phosphorylation status of p21(Cip1/WAF1) .
Assuntos
Divisão Celular , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Desenvolvimento Embrionário , Fase G2 , Zigoto/citologia , Animais , Núcleo Celular/metabolismo , Proliferação de Células , Citoplasma/metabolismo , Feminino , Imunofluorescência , Masculino , Camundongos , Microinjeções , Oócitos/citologia , Oócitos/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Zigoto/metabolismoRESUMO
We investigated the expression of transient receptor potential canonical 6 (TRPC6) protein in benign and malignant human prostate tissues and in prostate cancer cell lines and the association with the stage, grade and androgen responsiveness of the tumors. Immunohistochemical techniques, Western blot and reverse transcription polymerase chain reaction (RT-PCR) were used to investigate TRPC6 expression. TRPC6 protein was detected in 9 of 20 (45.0%) of benign prostatic hyperplasia (BPH) cases, and there was a significant difference compared with prostate cancer (129 of 149 [86.6%])(P < 0.01). TRPC6 expression was associated with the histological grade and extraprostatic extension (P < 0.01). Tumors of higher stage tended to have a higher frequency of TRPC6 protein staining, but the difference was not significant among T2, T3 and T4. TRPC6 expression difference between androgen-independent (AI) tumors and androgen-dependent (AD) tumors was not statistically significant. TRPC6 was also observed in prostate cancer cell lines. In summary, TRPC6 is detected in benign and malignant human prostate tissues and prostate cancer cell lines and is associated with the histological grade, Gleason score and extraprostatic extension of prostate cancer.