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BackgroundSimultaneous 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® and TRIzol®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.ResultsTRIzol®LS was used for RNA isolation from 284 human colon cancer samples, including normal colon mucosa, tubulovillous adenomas, and colon carcinomas with proficient and deficient mismatch repair system. TRIzol® was used for RNA isolation from human colon cancer cells, from brains of transgenic Alzheimer’s disease mice model, and from cultured mouse cortical neurons. Following RNA extraction, the TRIzol®-chloroform fractions from human colon cancer samples and from mouse hippocampus and frontal cortex were stored for 2 years and 3 months, respectively, at −80°C until used for protein isolation.Simple modifications to the TRIzol® manufacturer’s protocol, including Urea:SDS solubilization and sonication, allowed improved protein recovery yield compared to the TRIzol® manufacturer’s protocol. Following SDS-PAGE and Ponceau and Coomassie staining, recovered proteins displayed wide molecular weight range and staining pattern comparable to those obtainable with commonly used protein extraction protocols. We also show that nuclear and cytosolic proteins can be easily extracted and detected by immunoblotting, and that posttranslational modifications, such as protein phosphorylation, are detectable in proteins recovered from TRIzol®-chloroform fractions stored for up to 2 years at −80°C.ConclusionsWe provide a novel approach to improve protein recovery from samples processed for nucleic acid extraction with TRIzol® and TRIzol®LS compared to the manufacturer`s protocol, allowing downstream immunoblotting and evaluation of steady-state relative protein expression levels. The method was validated in large sets of samples from multiple sources, including human colon cancer and brains of transgenic Alzheimer’s disease mice model, stored in TRIzol®-chloroform for up to two years. Collectively, we provide a faster and cheaper alternative to the TRIzol® manufacturer`s protein extraction protocol, illustrating the high relevance, and wide applicability, of the present protein isolation method for the immunoblot evaluation of steady-state relative protein expression levels in samples from multiple sources, and following prolonged storage.

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.

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 (P<0.01) and adenocarcinomas (P<0.05), where increased ERK5 expression correlated with the acquisition of more invasive and metastatic potential (P<0.05). Interestingly, we observed a significant correlation between ERK5 expression and NF-κB activation in human adenocarcinomas (P<0.001). We also showed that ERK5 overactivation significantly accelerated cell cycle progression (P<0.05) and increased cell migration (P<0.01). Furthermore, cells with overactivated ERK5 displayed increased NF-κB nuclear translocation and transcriptional activity (P<0.05), together with increased expression of the mesenchymal marker vimentin (P<0.05). We further demonstrated that increased NF-κB activation was associated with increased IκB phosphorylation and degradation (P<0.05). 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.

The MEK5/ERK5 signaling pathway is emerging as an important contributor to colon cancer onset, progression and metastasis; however, its relevance to chemotherapy resistance remains unknown. Here, we evaluated the impact of the MEK5/ERK5 cascade in colon cancer cell sensitivity to 5-fluorouracil (5-FU). Increased ERK5 expression was correlated with poor overall survival in colon cancer patients. In colon cancer cells, 5-FU exposure impaired endogenous KRAS/MEK5/ERK5 expression and/or activation. In turn, MEK5 constitutive activation reduced 5-FU-induced cytotoxicity. Using genetic and pharmacological approaches, we showed that ERK5 inhibition increased caspase-3/7 activity and apoptosis following 5-FU exposure. Mechanistically, this was further associated with increased p53 transcriptional activation of p21 and PUMA. In addition, ERK5 inhibition increased the response of HCT116 p53+/+ cells to 5-FU, but failed to sensitize HCT116 p53−/− cells to the cytotoxic effects of this chemotherapeutic agent, suggesting a p53-dependent axis mediating 5-FU sensitization. Finally, ERK5 inhibition using XMD8-92 was shown to increase the antitumor effects of 5-FU in a murine subcutaneous xenograft model, enhancing apoptosis while markedly reducing tumor growth. Collectively, our results suggest that ERK5-targeted in hibition provides a promising therapeutic approach to overcome resistance to 5-FU-based chemotherapy and improve colon cancer treatment.

New ruthenium(II) and iron(II) organometallic compounds of general formula [(η(5)-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 23 organometallic compounds and SAR's data analysis within this library showed an interesting dependency of the cytotoxic activity on the carbohydrate moiety, linker, phosphane coligands, and metal center. More importantly, two compounds, 14Ru and 18Ru, matched oxaliplatin IC50 (0.45 μM), the standard metallodrug 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.

miR-143 and miR-145 are downregulated in colon cancer. Here, we tested the effect of restoring these miRNAs on sensitization to cetuximab in mutant KRAS (HCT116 and SW480) and wild-type KRAS (SW48) colon cancer cells. We evaluated cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC) and the modulation of signaling pathways involved in immune effector cell-mediated elimination of cancer cells. Stable miR-143 or miR-145 overexpression increased cell sensitivity to cetuximab, resulting in a significant increase of cetuximab-mediated ADCC independently of KRAS status. Importantly, HCT116 cells overexpressing these miRNAs triggered apoptosis in result of cetuximab-mediated ADCC, effected by peripheral blood mononuclear cells (p < 0.01). This was associated with increased apoptosis and caspase-3/7 activity, and reduced Bcl-2 protein expression (p < 0.01). In addition, caspase inhibition abrogated cetuximab-mediated ADCC in HCT116 cells overexpressing either miR-143 or miR-145 (p < 0.01). Furthermore, Bcl-2 silencing led to high level of cetuximab-mediated ADCC, compared to control siRNA (p < 0.05). Importantly, granzyme B inhibition, abrogated cetuximab-mediated ADCC, reducing caspase-3/7 activity (p < 0.01). Collectively, our data suggests that re-introduction of miR-143 or miR-145 may provide a new approach for development of therapeutic strategies to re-sensitize colon cancer cells to cetuximab by stimulating cetuximab-dependent ADCC to induce cell death.

MicroRNAs (miRNAs) regulate a wide variety of biological processes, including tumourigenesis. Altered miRNA expression is associated with deregulation of signalling pathways, which in turn cause abnormal cell growth and de-differentiation, contributing to cancer. miR-143 and miR-145 are anti-tumourigenic and influence the sensitivity of tumour cells to chemotherapy and targeted therapy. Comparative proteomic analysis was performed in HCT116 human colon cancer cells stably transduced with miR-143 or miR-145. Immunoblotting analysis validated the proteomic data in stable and transient miRNA overexpression conditions in human colon cancer cells. We show that approximately 100 proteins are differentially expressed in HCT116 human colon cancer cells stably transduced with miR-143 or miR-145 compared to Empty control cells. Further, Gene Ontology and pathway enrichment analysis indicated that proteins involved in specific cell signalling pathways such as cell death, response to oxidative stress, and protein folding might be modulated by these miRNAs. In particular, antioxidant enzyme superoxide dismutase 1 (SOD1) was downregulated by stable expression of either miR-143 or miR-145. Further, SOD1 gain-of-function experiments rescued cells from miR-143-induced oxidative stress. Moreover, miR-143 overexpression increased oxaliplatin-induced apoptosis associated with reactive oxygen species generation, which was abrogated by genetic and pharmacological inhibition of oxidative stress. Overall, miR-143 might circumvent resistance of colon cancer cells to oxaliplatin via increased oxidative stress in HCT116 human colon cancer cells.