This study was designed to evaluate MEK5 and ERK5 expression in colon cancer progression and to a... more This study was designed to evaluate MEK5 and ERK5 expression in colon cancer progression and to ascertain the relevance of MEK5/ERK5 signalling in colon cancer. Expression of MEK5 and ERK5 was evaluated in 323 human colon cancer samples. To evaluate the role of MEK5/ERK5 signalling in colon cancer, we developed a stable cell line model with differential MEK5/ERK5 activation. Impact of differential MEK5/ERK5 signalling was evaluated on cell cycle progression by flow cytometry and cell migration was evaluated by wound healing and transwell migration assays. Finally, we used an orthotopic xenograft mouse model of colon cancer to assess tumour growth and progression. Our results demonstrated that MEK5 and ERK5 are overexpressed in human adenomas (Po0.01) and adenocarcinomas (Po0.05), where increased ERK5 expression correlated with the acquisition of more invasive and metastatic potential (Po0.05). Interestingly, we observed a significant correlation between ERK5 expression and NF-κB activation in human adenocarcinomas (Po0.001). We also showed that ERK5 overactivation significantly accelerated cell cycle progression (Po0.05) and increased cell migration (Po0.01). Furthermore, cells with overactivated ERK5 displayed increased NF-κB nuclear translocation and transcriptional activity (Po0.05), together with increased expression of the mesenchymal marker vimentin (Po0.05). We further demonstrated that increased NF-κB activation was associated with increased IκB phosphorylation and degradation (Po0.05). Finally, in the mouse model, lymph node metastasis was exclusively seen in orthotopically implanted tumours with overactivated MEK5/ERK5, and not in tumours with inhibited MEK5/ERK5. Our results suggested that MEK5/ERK5/NF-κB signalling pathway is important for tumour onset, progression and metastasis, possibly representing a novel relevant therapeutic target in colon cancer treatment.
New ruthenium(II) and iron(II) organometallic compounds of general formula [(η5-C5H5)M(PP)Lc][PF6... more New ruthenium(II) and iron(II) organometallic compounds of general formula [(η5-C5H5)M(PP)Lc][PF6], bearing carbohydrate derivative ligands (Lc) were prepared and fully characterized, and the crystal structures of five of those compounds were determined by X-ray diffraction studies. Cell viability of colon cancer HCT116 cell line was determined for a total of twenty-three organometallic compounds and SAR's data analysis within this library showed an interesting dependency of the cytotoxic activity on the carbohydrate moiety, linker, phosphane co-ligands and metal center. More importantly, two compounds, 14Ru and 18Ru, matched oxaliplatin IC50 (0.45 μM), the standard metallo-drug used in CC chemotherapeutics, and our leading compound 14Ru was shown to be significantly more cytotoxic than oxaliplatin to HCT116 cells, triggering higher levels of caspase 3 and -7 activity, and apoptosis in a dose-dependent manner.
MicroRNAs (miRs) are increasingly associated with metabolic liver diseases. We have shown that ur... more MicroRNAs (miRs) are increasingly associated with metabolic liver diseases. We have shown that ursodeoxycholic acid, a hydrophilic bile acid, counteracts the miR-34a/sirtuin 1 (SIRT1)/p53 pathway, activated in the liver of nonalcoholic steatohepatitis (NASH) patients. In contrast, hydrophobic bile acids, particularly deoxycholic acid (DCA), activate apoptosis and are increased in NASH. We evaluated whether DCA-induced apoptosis of rat hepatocytes occurs via miR-34a-dependent pathways and whether they connect with c-Jun N-terminal kinase (JNK) induction. DCA enhanced miR-34a/SIRT1/p53 proapoptotic signaling in a dose-and time-dependent manner. In turn, miR-34a inhibition and SIRT1 overexpression significantly rescued targeting of the miR-34a pathway and apoptosis by DCA. In addition, p53 overexpression activated the miR-34a/SIRT1/p53 pathway, further induced by DCA. DCA increased p53 expression as well as p53 transcriptional activation of PUMA and miR-34a itself, providing a functional mechanism for miR-34a activation. JNK1 and c-Jun were shown to be major targets of DCA, upstream of p53, in engaging the miR-34a pathway and apoptosis. Finally, activation of this JNK1/miR-34a proapoptotic circuit was also shown to occur in vivo in the rat liver. These results suggest that the JNK1/p53/miR-34a/SIRT1 pathway may represent an attractive pharmacological target for the development of new drugs to arrest metabolism-and apoptosis-related liver pathologies.
MicroRNAs (miRNAs) are pivotal post-transcriptional gene expression regulators. These endogenous ... more MicroRNAs (miRNAs) are pivotal post-transcriptional gene expression regulators. These endogenous small non-coding RNAs aberrantly expressed in cancer have significant roles in tumorigenesis and progression. Currently, miRNAs are being pursued as diagnostic and prognostic biomarkers, and as therapeutic tools in cancer. miRNA modulation provides the unique ability to fine-tune multiple genes simultaneously, thereby regulating relevant signaling pathways involved in cell differentiation, proliferation and survival. This unique miRNA feature shifts the traditional one drug one target paradigm to a novel one drug multiple targets paradigm. We herein review in vivo strategies of miRNA modulator (mimic and/or inhibitor) delivery in cancer models, a subject that remains the key challenge to the establishment of this novel class of RNA therapeutics.
Background: Simultaneous isolation of nucleic acids and proteins from a single biological sample ... more Background: Simultaneous isolation of nucleic acids and proteins from a single biological sample facilitates meaningful data interpretation and reduces time, cost and sampling errors. This is particularly relevant for rare human and animal specimens, often scarce, and/or irreplaceable. TRIzol W and TRIzol W LS are suitable for simultaneous isolation of RNA, DNA and proteins from the same biological sample. These reagents are widely used for RNA and/or DNA isolation, while reports on their use for protein extraction are limited, attributable to technical difficulties in protein solubilisation.
This study was designed to evaluate MEK5 and ERK5 expression in colon cancer progression and to a... more This study was designed to evaluate MEK5 and ERK5 expression in colon cancer progression and to ascertain the relevance of MEK5/ERK5 signalling in colon cancer. Expression of MEK5 and ERK5 was evaluated in 323 human colon cancer samples. To evaluate the role of MEK5/ERK5 signalling in colon cancer, we developed a stable cell line model with differential MEK5/ERK5 activation. Impact of differential MEK5/ERK5 signalling was evaluated on cell cycle progression by flow cytometry and cell migration was evaluated by wound healing and transwell migration assays. Finally, we used an orthotopic xenograft mouse model of colon cancer to assess tumour growth and progression. Our results demonstrated that MEK5 and ERK5 are overexpressed in human adenomas (Po0.01) and adenocarcinomas (Po0.05), where increased ERK5 expression correlated with the acquisition of more invasive and metastatic potential (Po0.05). Interestingly, we observed a significant correlation between ERK5 expression and NF-κB activation in human adenocarcinomas (Po0.001). We also showed that ERK5 overactivation significantly accelerated cell cycle progression (Po0.05) and increased cell migration (Po0.01). Furthermore, cells with overactivated ERK5 displayed increased NF-κB nuclear translocation and transcriptional activity (Po0.05), together with increased expression of the mesenchymal marker vimentin (Po0.05). We further demonstrated that increased NF-κB activation was associated with increased IκB phosphorylation and degradation (Po0.05). Finally, in the mouse model, lymph node metastasis was exclusively seen in orthotopically implanted tumours with overactivated MEK5/ERK5, and not in tumours with inhibited MEK5/ERK5. Our results suggested that MEK5/ERK5/NF-κB signalling pathway is important for tumour onset, progression and metastasis, possibly representing a novel relevant therapeutic target in colon cancer treatment.
New ruthenium(II) and iron(II) organometallic compounds of general formula [(η5-C5H5)M(PP)Lc][PF6... more New ruthenium(II) and iron(II) organometallic compounds of general formula [(η5-C5H5)M(PP)Lc][PF6], bearing carbohydrate derivative ligands (Lc) were prepared and fully characterized, and the crystal structures of five of those compounds were determined by X-ray diffraction studies. Cell viability of colon cancer HCT116 cell line was determined for a total of twenty-three organometallic compounds and SAR's data analysis within this library showed an interesting dependency of the cytotoxic activity on the carbohydrate moiety, linker, phosphane co-ligands and metal center. More importantly, two compounds, 14Ru and 18Ru, matched oxaliplatin IC50 (0.45 μM), the standard metallo-drug used in CC chemotherapeutics, and our leading compound 14Ru was shown to be significantly more cytotoxic than oxaliplatin to HCT116 cells, triggering higher levels of caspase 3 and -7 activity, and apoptosis in a dose-dependent manner.
MicroRNAs (miRs) are increasingly associated with metabolic liver diseases. We have shown that ur... more MicroRNAs (miRs) are increasingly associated with metabolic liver diseases. We have shown that ursodeoxycholic acid, a hydrophilic bile acid, counteracts the miR-34a/sirtuin 1 (SIRT1)/p53 pathway, activated in the liver of nonalcoholic steatohepatitis (NASH) patients. In contrast, hydrophobic bile acids, particularly deoxycholic acid (DCA), activate apoptosis and are increased in NASH. We evaluated whether DCA-induced apoptosis of rat hepatocytes occurs via miR-34a-dependent pathways and whether they connect with c-Jun N-terminal kinase (JNK) induction. DCA enhanced miR-34a/SIRT1/p53 proapoptotic signaling in a dose-and time-dependent manner. In turn, miR-34a inhibition and SIRT1 overexpression significantly rescued targeting of the miR-34a pathway and apoptosis by DCA. In addition, p53 overexpression activated the miR-34a/SIRT1/p53 pathway, further induced by DCA. DCA increased p53 expression as well as p53 transcriptional activation of PUMA and miR-34a itself, providing a functional mechanism for miR-34a activation. JNK1 and c-Jun were shown to be major targets of DCA, upstream of p53, in engaging the miR-34a pathway and apoptosis. Finally, activation of this JNK1/miR-34a proapoptotic circuit was also shown to occur in vivo in the rat liver. These results suggest that the JNK1/p53/miR-34a/SIRT1 pathway may represent an attractive pharmacological target for the development of new drugs to arrest metabolism-and apoptosis-related liver pathologies.
MicroRNAs (miRNAs) are pivotal post-transcriptional gene expression regulators. These endogenous ... more MicroRNAs (miRNAs) are pivotal post-transcriptional gene expression regulators. These endogenous small non-coding RNAs aberrantly expressed in cancer have significant roles in tumorigenesis and progression. Currently, miRNAs are being pursued as diagnostic and prognostic biomarkers, and as therapeutic tools in cancer. miRNA modulation provides the unique ability to fine-tune multiple genes simultaneously, thereby regulating relevant signaling pathways involved in cell differentiation, proliferation and survival. This unique miRNA feature shifts the traditional one drug one target paradigm to a novel one drug multiple targets paradigm. We herein review in vivo strategies of miRNA modulator (mimic and/or inhibitor) delivery in cancer models, a subject that remains the key challenge to the establishment of this novel class of RNA therapeutics.
Background: Simultaneous isolation of nucleic acids and proteins from a single biological sample ... more Background: Simultaneous isolation of nucleic acids and proteins from a single biological sample facilitates meaningful data interpretation and reduces time, cost and sampling errors. This is particularly relevant for rare human and animal specimens, often scarce, and/or irreplaceable. TRIzol W and TRIzol W LS are suitable for simultaneous isolation of RNA, DNA and proteins from the same biological sample. These reagents are widely used for RNA and/or DNA isolation, while reports on their use for protein extraction are limited, attributable to technical difficulties in protein solubilisation.
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Papers by Diane Pereira