Autophagy regulates Selumetinib (AZD6244) induced-apoptosis in colorectal cancer cells.

OBJECTIVE: As Selumetinib is a MEK1/2 inhibitor that has gained interest as an anti-tumor agent, the present study was designed to investigate autophagy involvement on Selumetinib-induced apoptosis in colorectal cancer (CRC) cells. METHODS: CRC cells death and cycle studies were assessed by AnnexinV-FITC and PI staining, respectively. Autophagy flux was analysed by Western Blot (LC3II and p62 protein levels) and retroviral infection of SW480 cells for siBecn1 RNA interference experiments. Confocal microscopy was used to determine mCherry-EGFP-LC3 distribution. KEY FINDINGS: The Selumetinib effects were concentration-dependent in SW480 cell line. Whereas 1 µM exerted an arrest in the cell cycle (G1 phase), higher concentrations (10 µM) induced cell death, which was accompanied by autophagy blockage in its last stages. Autophagy induction by Rapamycin (RAPA) increased cell survival, whereas pharmacology autophagy inhibition by Bafilomycin A1 (BAF), Chloroquine (CQ) or 3-Methyladenine (3-MA) increased Selumetinib-induced CRC cells death. CONCLUSIONS: Altogether, these results suggest that autophagy plays a fundamental role in CRC cells response to Selumetinib. In addition, the combination of Selumetinib with autophagy inhibitors may be a useful therapeutic strategy to enhance its activity against colorectal tumours.

HGUE-C-1 is an atypical and novel colon carcinoma cell line.

BACKGROUND: Colorectal carcinoma is a common cause of cancer. Adjuvant treatments include: 5-fluorouracil administered together with folinic acid, or more recently, oral fluoropyrimidines such as capecitabine, in combination with oxaliplatin or irinotecan. Metastatic colorectal cancer patients can benefit from other additional treatments such as cetuximab or bevacizumab. METHODS: Using cell culture techniques, we isolated clonal populations from primary cultures of ascitic effusion derived from a colon cancer patient and after several passages an established cell line, HGUE-C-1, was obtained. Genetic analysis of HGUE-C-1 cells was performed by PCR of selected exons and sequencing. Cell proliferation studies were performed by MTT assays and cell cycle analyses were performed by flow cytometry. Retinoblastoma activity was measured by luciferase assays and proteins levels and activity were analysed by Western blot or immunohistochemistry. RESULTS: We have established a new cell line from ascitic efussion of a colon cancer patient who did not respond to 5-fluorouracil or irinotecan. HGUE-C-1 cells did not show microsatellite instability and did not harbour mutations in KRAS, BRAF, PI3KCA or TP53. However, these cells showed loss of heterozygosity affecting Adenomatous Polyposis Coli and nuclear staining of ß-catenin protein. The HGUE-C-1 cell line was sensitive to erlotinib, gefitinib, NVP-BEZ235, rapamycin and trichostatin, among other drugs, but partially resistant to heat shock protein inhibitors and highly resistant to AZD-6244 and oxaliplatin, even though the patient from which this cell line was derived had not been exposed to these drugs. Molecular characterization of this cell line revealed low expression levels and activity of Retinoblastoma protein and elevated basal levels of Erk1/2 activity and p70S6K expression and activity, which may be related to chemoresistance mechanisms. CONCLUSIONS: HGUE-C-1 represents a novel and peculiar colon carcinoma model to study chemoresistance to chemotherapeutic agents and to novel anti-neoplasic drugs that interrupt signalling pathways such as the APC/ßcatenin, Ras/Raf/Mek/Erk, PI3K/mTOR/p70S6K pathways as well as histone regulation mechanisms.

Liver damage and caspase-dependent apoptosis is related to protein malnutrition in mice: effect of methionine.

This study aimed to determine whether the effects on the mouse liver caused by three periods of feeding a protein-free diet for 5 days followed by a normal complete diet for 5 days (3PFD-CD) are prevented by a constant methionine supply (3PFD+Met-CD). The expressions of carbonic anhydrase III (CAIII), fatty acid synthase (FAS), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and glutathione S-transferase P1 (GSTP1) were assessed by proteomics and reverse transcriptase-polymerase chain reactions. The liver redox status was examined by measuring the activities of superoxide dismutase (SOD) and catalase (CAT), as well as protein carbonylation. Because oxidative stress can result in apoptosis, the activity and content of caspase-3, as well as the x-linked inhibitor of the apoptosis protein (XIAP) and mitochondrial caspase-independent apoptosis inducing factor (AIF) contents were assessed. In addition, the liver histomorphology was examined. Compared to the controls fed a normal complete diet throughout, feeding with 3PFD-CD increased the FAS content, decreased the CAIII content, decreased both the SOD and CAT activities, and increased protein carbonylation. It also activated caspase-3, decreased the XIAP content, decreased the AIF content, increased the number of GSTP1-positive foci and caspase-3-positive cells, and caused fatty livers. Conversely, the changes were lessened to varying degrees in mice fed 3PFD+Met-CD. The present results indicate that a regular Met supply lessens the biochemical changes, damage, and caspase-dependent apoptosis provoked by recurrent dietary amino acid deprivation in the mouse liver.

Effect of rosemary polyphenols on human colon cancer cells: transcriptomic profiling and functional enrichment analysis.

In this work, the effect of rosemary extracts rich on polyphenols obtained using pressurized fluids was investigated on the gene expression of human SW480 and HT29 colon cancer cells. The application of transcriptomic profiling and functional enrichment analysis was done via two computational approaches, Ingenuity Pathway Analysis and Gene Set Enrichment Analysis. These two approaches were used for functional enrichment analysis as a previous step for a reliable interpretation of the data obtained from microarray analysis. Reverse transcription quantitative-PCR was used to confirm relative changes in mRNA levels of selected genes from microarrays. The selection of genes was based on their expression change, adjusted p value, and known biological function. According to genome-wide transcriptomics analysis, rosemary polyphenols altered the expression of ~4 % of the genes covered by the Affymetrix Human Gene 1.0ST chip in both colon cancer cells. However, only ~18 % of the differentially expressed genes were common to both cell lines, indicating markedly different expression profiles in response to the treatment. Differences in induction of G2/M arrest observed by rosemary polyphenols in the two colon adenocarcinoma cell lines suggest that the extract may be differentially effective against tumors with specific mutational pattern. From our results, it is also concluded that rosemary polyphenols induced a low degree of apoptosis indicating that other multiple signaling pathways may contribute to colon cancer cell death.

Effect of dietary polyphenols on K562 leukemia cells: a Foodomics approach.

In this work, a global Foodomics strategy has been applied to study the antiproliferative effect of dietary polyphenols from rosemary on two human leukemia lines, one showing a drug-sensitive phenotype (K562), and another exhibiting a drug-resistant phenotype (K562/R). To this aim, whole-transcriptome microarray together with an MS-based nontargeted analytical approach (via CE-TOF MS and UPLC-TOF MS) have been employed to carry out transcriptomics and metabolomics analyses, respectively. Functional enrichment analysis was done using ingenuity pathway analysis (IPA) software as a previous step for a reliable interpretation of transcriptomic and metabolomic profiles. Rosemary polyphenols altered the expression of approximately 1% of the genes covered by the whole transcriptome microarray in both leukemia cell lines. Overall, differences in the transcriptional induction of a number of genes encoding phase II detoxifying and antioxidant genes, as well as differences in the metabolic profiles observed in the two leukemia cell lines suggest that rosemary polyphenols may exert a differential chemopreventive effect in leukemia cells with different phenotypes. IPA predictions on transcription factor analysis highlighted inhibition of Myc transcription factor function by rosemary polyphenols, which may explain the observed antiproliferative effect of rosemary extract in the leukemia cells. Metabolomics analysis suggested that rosemary polyphenols affected differently the intracellular levels of some metabolites in two leukemia cell sublines. Integration of data obtained from transcriptomics and metabolomics platforms was attempted by overlaying datasets on canonical (defined) metabolic pathways using IPA software. This strategy enabled the identification of several differentially expressed genes in the metabolic pathways modulated by rosemary polyphenols providing more evidences on the effect of these compounds.

Acquisition of MDR phenotype by leukemic cells is associated with increased caspase-3 activity and a collateral sensitivity to cold stress.

The acquisition of a multidrug-resistant (MDR) phenotype by tumor cells that renders them unsusceptible to anti-neoplasic agents is one of the main causes of chemotherapy failure in human malignancies. The increased expression of P-glycoprotein (MDR1, P-gp, ABCB1) in tumor cells contributes to drug resistance by extruding chemotherapeutic agents or by regulating programmed cell death. In a study of MDR cell survival under cold stress conditions, it was found that resistant leukemic cells with P-gp over-expression, but not their sensitive counterparts, are hypersensitive to cold-induced cell death when exposed to temperatures below 4 °C. The transfection of parental cells with a P-gp-expressing plasmid makes these cells sensitive to cold stress, demonstrating an association between P-gp expression and cell death at low temperatures. Furthermore, we observed increased basal expression and activity of effector caspase-3 at physiological temperature (37 °C) in MDR cells compared with their parental cell line. Treatment with a caspase-3 inhibitor partially rescues MDR leukemic cells from cold-induced apoptosis, which suggests that the cell death mechanism may require caspase-3 activity. Taken together, these findings demonstrate that P-gp expression plays a role in MDR cell survival, and is accompanied by a collateral sensitivity to death induced by cold stress. These findings may assist in the design of specific therapeutic strategies to complement current chemotherapy treatment against cancer.

Uptake of the antileishmania drug tafenoquine follows a sterol-dependent diffusion process in Leishmania.

OBJECTIVES: The present study was designed to elucidate the mechanism of tafenoquine uptake in Leishmania and its sterol dependence. METHODS: Because tafenoquine is a fluorescent compound, spectrofluorimetric analysis allowed us to monitor its uptake by Leishmania promastigotes and intracellular amastigotes, and to evaluate the effect of temperature, energy and H+ gradient on drug entry. The influence of sterols on tafenoquine uptake in Leishmania parasites was determined in experiments using sterol-depleting agents such as methyl-ß-cyclodextrin or cholesterol oxidase. RESULTS: Tafenoquine exhibited fast entry kinetics into Leishmania in an energy-independent, but pH- and temperature-dependent, non-saturable process. Furthermore, sterol depletion decreased tafenoquine uptake. CONCLUSIONS: These findings suggest that Leishmania takes up tafenoquine by a diffusion process and that decreases in membrane sterol content may induce a decrease in drug uptake.

Structural and functional changes induced in the nicotinic acetylcholine receptor by membrane phospholipids.

Ligand-gated ion channels (LGICs) constitute an important family of complex membrane proteins acting as receptors for neurotransmitters (Barnard, 1992; Ortells and Lunt, 1995). The nicotinic acetylcholine receptor (nAChR) from Torpedo is the most extensively studied member of the LGIC family and consists of a pentameric transmembrane glycoprotein composed of four different polypeptide subunits (alpha, beta, gamma, and delta) in a 2:1:1:1 stoichiometry (Galzi and Changeux, 1995; Hucho et al., 1996) that are arranged pseudosymmetrically around a central cation-selective ion channel. Conformational transitions, from the closed (nonconducting), to agonist-induced open (ion-conducting), to desensitized (nonconducting) states, are critical for functioning of the nAChR (Karlin, 2002). The ability of the nAChR to undergo these transitions is profoundly influenced by the lipid composition of the bilayer (Barrantes, 2004). Despite existing information on lipid dependence of AChR function, no satisfactory explanation has been given on the molecular events by which specific lipids exert such effects on the activity of an integral membrane protein. To date, several hypotheses have been entertained, including (1) indirect effects of lipids through the alteration of properties of the bilayer, such as fluidity (an optimal fluidity hypothesis [Fong and McNamee, 1986]) or membrane curvature and lateral pressure (Cantor, 1997; de Kruijff, 1997), or (2) direct effects through binding of lipids to defined sites on the transmembrane portion of the protein (Jones and McNamee, 1988; Blanton and Wang, 1990; Fernández et al., 1993; Fernández-Ballester et al., 1994), which has led to the postulation of a possible role of certain lipids as peculiar allosteric ligands of the protein. In this paper we have reconstituted purified AChRs from Torpedo into complex multicomponent lipid vesicles in which the phospholipid composition has been systematically altered. Stopped-flow rapid kinetics of cation translocation and Fourier transform-infrared (FT-IR) spectroscopy studies have been used to illustrate the lipid dependence of both AChR function and AChR secondary structure, respectively.

Acquisition of multidrug resistance by L1210 leukemia cells decreases their tumorigenicity and enhances their susceptibility to the host immune response.

The use of antineoplastic drugs for cancer treatment is frequently associated with the acquisition of a multidrug-resistant (MDR) phenotype that renders tumoural cells insensitive to antineoplastics. It remains elusive whether the acquisition of the MDR phenotype alters immunological parameters that could influence the cell sensitivity to an eventual host immune response. We report that immunisation of syngeneic mice with gamma-irradiated L1210S (parental line) and L1210R (MDR phenotype) cells results in a significant rejection of subsequently implanted L1210R-based tumours, but not of the L1210S ones. Notably, L1210R tumours display a twofold reduction in vivo proliferative capacity and are less aggressive in terms of mouse survival than their sensitive counterparts. Also, analysis of surface expression of molecules involved in antigen presentation and cytokine activity revealed a slight increase in IFN-gamma receptor expression, a decrease of Fas molecule, and a fourfold up-regulation of MHC class I molecules in L1210R cells. Nonetheless, both cell lines were able to induce a cytotoxic response in syngeneic mice and were equally susceptible to cytotoxicity by splenic cells. Together, these findings indicate that acquisition of drug resistance by L1210 cells is accompanied by pleiotropic changes that result in reduced tumour proliferative capacity and tumorigenicity in syngeneic mice. Hence, immunological studies of MDR tumours may assist in the design of specific therapeutic strategies that complement current chemotherapy treatments.