RESUMO
Necroptosis is an alternative form of programmed cell death that generally occurs under apoptosis-deficient conditions. Our previous work showed that connexin32 (Cx32) promotes the malignant progress of hepatocellular carcinoma (HCC) by enhancing the ability of resisting apoptosis in vivo and in vitro. Whether triggering necroptosis is a promising strategy to eliminate the apoptosis-resistant HCC cells with high Cx32 expression remains unknown. In this study, we found that Cx32 expression was positively correlated with the expression of necroptosis protein biomarkers in human HCC specimens, cell lines, and a xenograft model. Treatment with shikonin, a well-used necroptosis inducer, markedly caused necroptosis in HCC cells. Interestingly, overexpressed Cx32 exacerbated shikonin-induced necroptosis, but downregulation of Cx32 alleviated necroptosis in vitro and in vivo. Mechanistically, Cx32 was found to bind to Src and promote Src-mediated caspase 8 phosphorylation and inactivation, which ultimately reduced the activated caspase 8-mediated proteolysis of receptor-interacting serine-threonine protein kinase 1/3, the key molecule for necroptosis activation. In conclusion, we showed that Cx32 contributed to the activation of necroptosis in HCC cells through binding to Src and then mediating the inactivation of caspase 8. The present study suggested that necroptosis inducers could be more favorable than apoptosis inducers to eliminate HCC cells with high expression of Cx32.
Assuntos
Carcinoma Hepatocelular/metabolismo , Caspase 8/metabolismo , Conexinas/metabolismo , Neoplasias Hepáticas/metabolismo , Necroptose/genética , Coativador 1 de Receptor Nuclear/metabolismo , Transdução de Sinais/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Conexinas/genética , Técnicas de Silenciamento de Genes , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Naftoquinonas/administração & dosagem , Necroptose/efeitos dos fármacos , Coativador 1 de Receptor Nuclear/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Transdução de Sinais/efeitos dos fármacos , Transfecção , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Proteína beta-1 de Junções ComunicantesRESUMO
MicroRNA is expected to be a novel therapeutic tool for tumors. Gap junctions facilitate the transfer of microRNA, which exerts biological effects on tumor cells. However, the length of microRNA that can pass through certain gap junctions composed of specific connexin remains unknown. To address this question, the present study investigated the permeability of gap junctions composed of various connexins, including connexin 43, connexin 32 or connexin 37, to microRNAs consisting of 18-27 nucleotides in glioma cells and cervical cancer cells. Results indicated that all of the microRNAs were able to be transferred from donor glioma cells to neighboring cells through the connexin 43 composed gap junction, but not the gap junctions composed of connexin 32 or connexin 37, in cervical cancer cells. Downregulation of the function of gap junctions comprising connexin 43 by pharmacological inhibition and shRNA significantly decreased the transfer of these microRNAs. In contrast, gap junction enhancers and overexpression of connexin 43 effectively increased these transfers. In glioma cells, cell proliferation was inhibited by microRNA-34a. Additionally, these effects of microRNA-34a were significantly enhanced by overexpression of connexin 43 in U251 cells, indicating that gap junctions play an important role in the antitumor effect of microRNA by transfer of microRNA to neighboring cells. Our data are the first to clarify the pattern of microRNA transmission through gap junctions and provide novel insights to show that antitumor microRNAs should be combined with connexin 43 or a connexin 43 enhancer, not connexin 32 or connexin 37, in order to improve the therapeutic effect.
Assuntos
Comunicação Celular/genética , Proliferação de Células/genética , Junções Comunicantes/metabolismo , MicroRNAs/genética , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/genética , Técnicas de Cocultura , Conexina 43/genética , Conexinas/genética , Glioma/genética , Glioma/patologia , Células HeLa , Humanos , Interferência de RNA , Proteína beta-1 de Junções Comunicantes , Proteína alfa-4 de Junções ComunicantesRESUMO
Ovarian cancer is the most lethal gynecologic malignancy, and cisplatin is one of the first-line chemotherapeutic agents. However, acquired cisplatin resistance prevents the successful treatment of patients with ovarian cancer. Gap junction (GJ) and connexin (Cx) are closely related to tumor formation, but the relationship between cisplatin resistance and GJ or Cx are undetermined. In this study, we established the cisplatin-resistant human ovarian cancer cell line A2780-CDDP. Here we showed that cisplatin resistance was correlated to the loss of GJ and the upregulation of Cx32 expression. Enhancing GJ in A2780-CDDP cells could increase the apoptotic response to cisplatin treatment. Furthermore, although Cx32 expression was increased in A2780-CDDP cells, it was more localized to the cytoplasm rather than in the membrane, and knockdown of Cx32 in A2780-CDDP cells sensitized them to cisplatin treatment. In summary, Cx32 is involved in cisplatin resistance, and cytoplasmic Cx32 plays an important role in chemoresistance.
Assuntos
Apoptose/efeitos dos fármacos , Cisplatino/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Junções Comunicantes/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Relação Dose-Resposta a Droga , Feminino , Junções Comunicantes/efeitos dos fármacos , Humanos , Neoplasias Ovarianas/patologia , Sistemas de Translocação de Proteínas/efeitos dos fármacos , Sistemas de Translocação de Proteínas/metabolismo , Transporte ProteicoRESUMO
Analgesics and antineoplastic drugs are often used concurrently for cancer patients. Our previous study reported that gap junctions composed of connexin32 (Cx32) was implicated in the effect of analgesics on cisplatin cytotoxicity. However, the effect of analgesic on the most widely expressed connexin (Cx), connexin43 (Cx43), and whether such effect mediates the influence on chemotherapeutic efficiency remain unknown. By manipulation of Cx43 expression or gap junction function, we found that there were gap junction-dependent and independent effect of Cx43 on temozolomide (TMZ) sensitivity in U87 glioblastoma cells. Studies on survival and apoptosis showed widely used analgesic tramadol significantly reduced TMZ-induced cytotoxicity in control and negative control cells but not shCx43-transfected cells. Proliferation assay demonstrated tramadol suppressed TMZ-induced cytotoxicity only on high density (with gap junction formation) but not on low density (without gap junction formation). Tramadol inhibited dye-coupling through gap junctions between U87 cells. Tramadol treatment for 72 h did not alter Cx43 expression, but decreased Cx43 phosphorylation accompanied with reduced p-ERK and p-JNK. Our results indicated that long-term treatment with tramadol reduced TMZ cytotoxicity in U87 cells by suppressing Cx43-composed gap junctions, suggesting identification and usage of antinociceptive drugs which do not downregulate connexin activity should have beneficial therapeutic consequences.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Comunicação Celular/efeitos dos fármacos , Conexina 43/metabolismo , Dacarbazina/análogos & derivados , Junções Comunicantes/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Tramadol/farmacologia , Analgésicos Opioides/farmacologia , Antineoplásicos Alquilantes/farmacologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Comunicação Celular/genética , Linhagem Celular Tumoral , Conexina 43/genética , Dacarbazina/administração & dosagem , Dacarbazina/farmacologia , Interações Medicamentosas , Junções Comunicantes/genética , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Temozolomida , Tramadol/administração & dosagem , TransfecçãoRESUMO
The role of connexin proteins (Cx), which form gap junctions (GJ), in progression and chemotherapeutic sensitivity of cervical cancer (CaCx), is unclear. Using cervix specimens (313 CaCx, 78 controls) and CaCx cell lines, we explored relationships among Cx expression, prognostic variables and mechanisms that may link them. In CaCx specimens, Cx32 was upregulated and cytoplasmically localized, and three other Cx downregulated, relative to controls. Cx32 expression correlated with advanced FIGO staging, differentiation and increased tumor size. In CaCx cell lines, Cx32 expression suppressed streptonigrin/cisplatin-induced apoptosis in the absence of functional GJ. In CaCx specimens and cell lines, expression of Cx32 upregulated epidermal growth factor receptor (EGFR) expression. Inhibition of EGFR signaling abrogated the anti-apoptotic effect of Cx32 expression. In conclusion, upregulated Cx32 in CaCx cells produces anti-apoptotic, pro-tumorigenic effects in vivo and vitro. Abnormal Cx32 expression/localization in CaCx appears to be both a mechanism and biomarker of chemotherapeutic resistance.
Assuntos
Apoptose , Biomarcadores Tumorais/biossíntese , Conexinas/biossíntese , Receptores ErbB/biossíntese , Proteínas de Neoplasias/biossíntese , Neoplasias do Colo do Útero/metabolismo , Conexinas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/genética , Feminino , Células HeLa , Humanos , Proteínas de Neoplasias/genética , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Proteína beta-1 de Junções ComunicantesRESUMO
Tumour necrosis factor α (TNFα) and TNFrelated apoptosis inducing ligand (TRAIL) usually trigger either survival or apoptosis signals in various cell types, and nuclear factor κB (NFκB) is a key factor that regulates their biological effects. Connexin 32 (Cx32) is a gap junction (GJ) protein that plays vital roles in tumourigenesis and tumour progression. Our previous study explored abnormal Cx32 expression in paranuclear areas, exacerbated prognostic parameters and suppressed streptonigrin/cisplatin-induced apoptosis in human cervical cancer (CaCx) cells. In this study, we investigated the role of Cx32 in the extrinsic apoptosis pathway of CaCx cells. In transgenic HeLa cells and C-33A cells, Cx32 expression was manipulated using doxycycline or Cx32 siRNA. GJ inhibitors or low density culturing was used to change the status of gap junction intracellular communication (GJIC). We found that apoptosis induced by TNFα and TRAIL was suppressed by Cx32 expression despite the presence or absense of GJIC. We also found that Cx32 upregulated the expression of nuclear NFκB and its downstream targets c-IAP1, MMP2, and MMP9 in HeLaCx32 and C-33A cells. Following our previous study design, our clinical data showed that NFκB and MMP2 levels increased in human CaCx specimens with high Cx32 expression compared to levels in paracarcinoma of cervical specimens. SC75741 and JSH-23, NFкB signalling pathway inhibitors, inhibited the anti-apoptotic effects of Cx32. In conclusion, Cx32 suppressed TNFα /TRAIL-induced extrinsic apoptosis by upregulating the NFκB signalling pathway. This study demonstrates a novel mechanism for Cx32's anti-apoptotic effect and provides a reasonable explanation for the pro-tumour effect of Cx32 in human CaCx cells.
Assuntos
Conexinas/metabolismo , Doxiciclina/farmacologia , NF-kappa B/metabolismo , RNA Interferente Pequeno/farmacologia , Neoplasias do Colo do Útero/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Conexinas/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Transdução de Sinais/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Neoplasias do Colo do Útero/genética , Proteína beta-1 de Junções ComunicantesRESUMO
Previous work has shown that gap junction intercellular communication (GJIC) enhances cisplatin (Pt) toxicity in testicular tumor cells but decreases it in non-tumor testicular cells. In this study, these different GJIC-propagated effects were demonstrated in tumor versus non-tumor cells from other organ tissues (liver and lung). The downregulation of GJIC by several different manipulations (no cell contact, pharmacological inhibition, and siRNA suppression) decreased Pt toxicity in tumor cells but enhanced it in non-tumor cells. The in vivo results using xenograft tumor models were consistent with those from the above-mentioned cells. To better understand the mechanism(s) involved, we studied the effects of GJIC on Pt accumulation in tumor and non-tumor cells from the liver and lung. The intracellular Pt and DNA-Pt adduct contents clearly increased in non-tumor cells but decreased in tumor cells when GJIC was downregulated. Further analysis indicated that the opposite effects of GJIC on Pt accumulation in normal versus tumor cells from the liver were due to its different effects on copper transporter1 and multidrug resistance-associated protein2, membrane transporters attributed to intracellular Pt transfer. Thus, GJIC protects normal organs from cisplatin toxicity while enhancing it in tumor cells via its different effects on intracellular Pt transfer.