Halting pro-survival autophagy by TGFß inhibition in bone marrow fibroblasts overcomes bortezomib resistance in multiple myeloma patients.

Bortezomib (bort) has improved overall survival in patients with multiple myeloma (MM), but the majority of them develop drug resistance. In this study, we demonstrate that bone marrow (BM) fibroblasts (cancer-associated fibroblasts; CAFs) from bort-resistant patients are insensitive to bort and protect the RPMI8226 and patients' plasma cells against bort-induced apoptosis. Bort triggers CAFs to produce high levels of interleukin (IL)-6, IL-8, insulin-like growth factor (IGF)-1 and transforming growth factor (TGF) ß. Proteomic studies on CAFs demonstrate that bort resistance parallels activation of oxidative stress and pro-survival autophagy. Indeed, bort induces reactive oxygen species in bort-resistant CAFs and activates autophagy by increasing light chain 3 protein (LC3)-II and inhibiting p62 and phospho-mammalian target of rapamycin. The small-interfering RNA knockdown of Atg7, and treatment with 3-methyladenine, restores bort sensitivity in bort-resistant CAFs and produces cytotoxicity in plasma cells co-cultured with CAFs. In the syngeneic 5T33 MM model, bort-treatment induces the expansion of LC3-II(+) CAFs. TGFß mediates bort-induced autophagy, and its blockade by LY2109761, a selective TßRI/II inhibitor, reduces the expression of p-Smad2/3 and LC3-II and induces apoptosis in bort-resistant CAFs. A combination of bort and LY2109761 synergistically induces apoptosis of RPMI8226 co-cultured with bort-resistant CAFs. These data define a key role for CAFs in bort resistance of plasma cells and provide the basis for a novel targeted therapeutic approach.

Comparative proteomic profiling of Hodgkin lymphoma cell lines.

Classical Hodgkin lymphoma (cHL) is a malignancy with complex pathogenesis. The hallmark of the disease is the presence of large mononucleated Hodgkin and bi- or multinucleated Reed/Sternberg (H/RS) cells. The origin of HRS cells in cHL is controversial as these cells show the coexpression of markers of several lineages. Using a proteomic approach, we compared the protein expression profile of cHL models of T- and B-cell derivation to find proteins differentially expressed in these cell lines. A total of 67 proteins were found differentially expressed between the two cell lines including metabolic proteins and proteins involved in the regulation of the cytoskeleton and/or cell migration, which were further validated by western blotting. Additionally, the expression of selected B- and T-cell antigens was also assessed by flow cytometry to reveal significant differences in the expression of different surface markers. Bioinformatics analysis was then applied to our dataset to find enriched pathways and networks, and to identify possible key regulators. In the present study, a proteomic approach was used to compare the protein expression profiles of two cHL cell lines. The identified proteins and/or networks, many of which not previously related to cHL, may be important to better define the pathogenesis of the disease, to identify novel diagnostic markers, and to design new therapeutic strategies.

The impact of proteomics in the understanding of the molecular basis of Paclitaxel-resistance in ovarian tumors.

The current therapy for ovarian cancer has advanced from alkylating agents, to a combination of carboplatinum and paclitaxel offering increased survival. Although most patients respond to this first-line therapy, initially, the majority of these patients relapse within 2 years. The mechanisms responsible for acquired drug resistance in ovarian cancer have been elucidated only in part. They include i) enhanced drug export, ii) activation/inhibition of intracellular signalling pathways, iii) molecular alterations in tubulin isotype composition. A better understanding of these mechanisms is needed, in order to develop new approaches, aimed at overcoming resistance to anticancer agents, and to reveal the complexity of causes, which contribute to drug resistance. In this review we offer an updated overview of proteomic studies on the molecular mechanisms of paclitaxel resistance. These proteomic studies also identify potential targets for modulating drug resistance, that could be predictive of response to chemotherapy in ovarian carcinomas.

Characterisation of intestinal peptide transporter of the Antarctic haemoglobinless teleost Chionodraco hamatus.

H(+)/peptide cotransport was studied in brush-border membrane vesicles (BBMV) from the intestine of the haemoglobinless Antarctic teleost Chionodraco hamatus by monitoring peptide-dependent intravesicular acidification with the pH-sensitive dye Acridine Orange. Diethylpyrocarbonate-inhibited intravesicular acidification was specifically achieved in the presence of extravesicular glycyl-L-proline (Gly-L-Pro) as well as of glycyl-L-alanine (Gly-L-Ala) and D-phenylalanyl-L-alanine (D-Phe-L-Ala). H(+)/Gly-L-Pro cotransport displayed saturable kinetics, involving a single carrier system with an apparent substrate affinity (K(m,app)) of 0.806+/-0.161 mmol l(-1). Using degenerated primers from eel and human (PepT1) transporter sequence, a reverse transcription-polymerase chain reaction (RT-PCR) signal was detected in C. hamatus intestine. RT-PCR paralleled kinetic analysis, confirming the hypothesis of the existence of a PepT1-type transport system in the brush-border membranes of icefish intestine. Functional expression of H(+)/peptide cotransport was successfully performed in Xenopus laevis oocytes after injection of poly(A)(+) RNA (mRNA) isolated from icefish intestinal mucosa. Injection of mRNA stimulated D-Phe-L-Ala uptake in a dose-dependent manner and an excess of glycyl-L-glutamine inhibited this transport. H(+)/peptide cotransport in the Antarctic teleost BBMV exhibited a marked difference in temperature optimum with respect to the temperate teleost Anguilla anguilla, the maximal activity rate occurring at approximately 0 degrees C for the former and 25 degrees C for the latter. Temperature dependence of icefish and eel intestinal mRNA-stimulated uptake in the heterologous system (oocytes) was comparable.

Characterisation of the H(+)/peptide cotransporter of eel intestinal brush-border membranes.

H(+)/peptide cotransport in brush-border membrane vesicles (BBMVs) from eel (Anguilla anguilla) intestine was studied by measuring d-[(3)H]-phenylalanyl-l-alanine uptake and by monitoring peptide-dependent intravesicular acidification using the pH-sensitive dye Acridine Orange. d-[(3)H]-phenylalanyl-l-alanine influx was greatly stimulated by an inside-negative membrane potential and enhanced by an inwardly directed H(+) gradient. In parallel, vesicular H(+) influx was significantly increased in the presence of extravesicular d-phenylalanyl-l-alanine or a series of glycyl and l-prolyl peptides. H(+)/peptide cotransport displayed saturable kinetics involving a single carrier system with apparent substrate affinities of 0.9-2.6 mmol l(-1) depending on the particular peptide. All substrates tested competed with this system. Pre-incubation of BBMVs with dipeptides prevented diethylpyrocarbonate inhibition of transport activity, suggesting that the substrates mask histidine residues involved in the catalytic function of the transporter. Using human PepT1-specific primers, a reverse transcription-polymerase chain reaction (RT-PCR) signal was detected in eel intestine. Our results suggest that, in eel intestine, a brush-border membrane 'low-affinity'-type H(+)/peptide cotransport system is present that shares kinetic features with the mammalian intestinal PepT1-type transporters.

Oleic acid inhibits endothelial activation : A direct vascular antiatherogenic mechanism of a nutritional component in the mediterranean diet.

Because oleic acid is implicated in the antiatherogenic effects attributed to the Mediterranean diet, we investigated whether this fatty acid can modulate endothelial activation, ie, the concerted expression of gene products involved in leukocyte recruitment and early atherogenesis. We incubated sodium oleate with human umbilical vein endothelial cells for 0 to 72 hours, followed by coincubation of oleate with human recombinant tumor necrosis factor, interleukin (IL)-1alpha, IL-1beta, IL-4, Escherichia coli lipopolysaccharide (LPS), or phorbol 12-myristate 13-acetate for a further 6 to 24 hours. The endothelial expression of vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and intercellular adhesion molecule-1 was monitored by cell surface enzyme immunoassays or flow cytometry, and steady-state levels of VCAM-1 mRNA were assessed by Northern blot analysis. At 10 to 100 micromol/L for >24 hours, oleate inhibited the expression of all adhesion molecules tested. After a 72-hour incubation with oleate and a further 16-hour incubation with oleate plus 1 microg/mL LPS, VCAM-1 expression was reduced by >40% compared with control. Adhesion of monocytoid U937 cells to LPS-treated endothelial cells was reduced concomitantly. Oleate also produced a quantitatively similar reduction of VCAM-1 mRNA levels on Northern blot analysis and inhibited nuclear factor-kappaB activation on electrophoretic mobility shift assays. Incubation of endothelial cells with oleate for 72 hours decreased the relative proportions of saturated (palmitic and stearic) acids in total cell lipids and increased the proportions of oleate in total cell lipids without significantly changing the relative proportions of polyunsaturated fatty acids. Although less potent than polyunsaturated fatty acids in inhibiting endothelial activation, oleic acid may contribute to the prevention of atherogenesis through selective displacement of saturated fatty acids in cell membrane phospholipids and a consequent modulation of gene expression for molecules involved in monocyte recruitment.