Acetone Fraction of the Red Marine Alga Laurencia papillosa Reduces the Expression of Bcl-2 Anti-apoptotic Marker and Flotillin-2 Lipid Raft Marker in MCF-7 Breast Cancer Cells.

Marine macroalgae have attracted much attention in recent years as a valuable source of bioactive metabolites. The cytotoxic potential of the Laurencia papillosa red alga collected from the Lebanese coast has been investigated on human breast cancer cells MCF-7. The crude extract of Laurencia papillosa (L. papillosa) was fractionated by column chromatography using a series of increasingly polar solvents (methylene chloride, acetone and methanol). Cytotoxicity of the crude extract and fractions was determined by MTT assay in MCF-7 cells. Apoptosis was detected by annexin V/propidium iodide assay and by measurement of Bcl-2 expression. Flotillin-2 expression was examined using RT-qPCR and Western blot. The crude extract, and the fractions of CH2Cl2 and acetone exhibited a dose-dependent cytotoxic effect on MCF-7 cells. Apoptosis was specifically induced by one of the acetone fractions having the highest cytotoxicity. It has been demonstrated by an increase in late phase apoptotic cell populations, and a decrease in Bcl-2 anti-apoptotic marker expression on mRNA and protein levels in a dose- and time- dependent manner. Furthermore, this active fraction decreased Flotillin-2 expression associated with cancer progression. Our data suggest that L. papillosa is an important source of cytotoxic metabolites. Further studies are needed for the chemical characterization of the metabolite associated with observed biological activities.

4-cholesten-3-one decreases breast cancer cell viability and alters membrane raft-localized EGFR expression by reducing lipogenesis and enhancing LXR-dependent cholesterol transporters.

BACKGROUND: The alteration of lipid metabolism in cancer cells is recognized as one of the most important metabolic hallmarks of cancer. Membrane rafts defined as plasma membrane microdomains enriched in cholesterol and sphingolipids serve as platforms for signaling regulation in cancer. The main purpose of this study was to evaluate the effect of the cholesterol metabolite, 4-cholesten-3-one, on lipid metabolism and membrane raft integrity in two breast cancer cell lines, MCF-7 and MDA-MB-231. Its ability to reduce cell viability and migration has also been investigated. METHODS: RT-qPCR was performed to evaluate the expression of enzymes involved in lipogenesis and cholesterol synthesis, and ABCG1 and ABCA1 transporters involved in cholesterol efflux. Its effect on cell viability and migration was studied using the MTT assay, the wound healing assay and the Transwell migration assay, respectively. The effect of 4-cholesten-3-one on membrane rafts integrity was investigated by studying the protein expression of flotillin-2, a membrane raft marker, and raft-enriched EGFR by western blot. RESULTS: Interestingly, we found that 4-cholesten-3-one treatment decreased mRNA expression of different enzymes including ACC1, FASN, SCD1 and HMGCR. We further demonstrated that 4-cholesten-3-one increased the expression of ABCG1 and ABCA1. We also found that 4-cholesten-3-one decreased the viability of MCF-7 and MDA-MB-231 cells. This effect was neutralized after treatment with LXR inverse agonist or after LXRß knockdown by siRNA. As a result, we also demonstrated that 4-cholesten-3-one disrupts membrane rafts and cell migration capacity. CONCLUSION: Our results show that 4-cholesten-3-one exerts promising antitumor activity by altering LXR-dependent lipid metabolism in breast cancer cells without increasing lipogenesis.

Lithocholic bile acid inhibits lipogenesis and induces apoptosis in breast cancer cells.

BACKGROUND: It has amply been documented that mammary tumor cells may exhibit an increased lipogenesis. Biliary acids are currently recognized as signaling molecules in the intestine, in addition to their classical roles in the digestion and absorption of lipids. The aim of our study was to evaluate the impact of lithocholic acid (LCA) on the lipogenesis of breast cancer cells. The putative cytotoxic effects of LCA on these cells were also examined. METHODS: The effects of LCA on breast cancer-derived MCF-7 and MDA-MB-231 cells were studied using MTT viability assays, Annexin-FITC and Akt phosphorylation assays to evaluate anti-proliferative and pro-apoptotic properties, qRT-PCR and Western blotting assays to assess the expression of the bile acid receptor TGR5 and the estrogen receptor ERα, and genes and proteins involved in apoptosis (Bax, Bcl-2, p53) and lipogenesis (SREBP-1c, FASN, ACACA). Intracellular lipid droplets were visualized using Oil Red O staining. RESULTS: We found that LCA induces TGR5 expression and exhibits anti-proliferative and pro-apoptotic effects in MCF-7 and MDA-MB-231 cells. Also, an increase in pro-apoptotic p53 protein expression and a decrease in anti-apoptotic Bcl-2 protein expression were observed after LCA treatment of MCF-7 cells. In addition, we found that LCA reduced Akt phosphorylation in MCF-7 cells, but not in MDA-MB-231 cells. We also noted that LCA reduced the expression of SREBP-1c, FASN and ACACA in both breast cancer-derived cell lines and that cells treated with LCA contained low numbers of lipid droplets compared to untreated control cells. Finally, a decrease in ERα expression was observed in MCF-7 cells treated with LCA. CONCLUSIONS: Our data suggest a potential therapeutic role of lithocholic acid in breast cancer cells through a reversion of lipid metabolism deregulation.

LXR Activation Down-regulates Lipid Raft Markers FLOT2 and DHHC5 in MCF-7 Breast Cancer Cells.

BACKGROUND/AIM: Lipid rafts are cholesterol-enriched microdomains of the plasma membrane. Recent studies have underlined that their integrity is critical for cancer cell survival. Liver X receptor (LXR) has a central role in cellular cholesterol homeostasis and its stimulation inhibits proliferation of several cancer cell lines. This study investigated whether LXR could modulate lipid rafts integrity and consequently alter proliferation of the MCF-7 breast cancer cell line. MATERIALS AND METHODS: Effect of LXR agonist T0901317 on integrity of MCF-7 lipid rafts was examined by studying the expression of rafts marker flotillin-2 (FLOT2) and DHHC5, which palmitoylates FLOT2, and by studying the expression of phospho-Akt. RESULTS: We demonstrated that LXR stimulation decreases mRNA and protein expression of FLOT2 and DHHC5 in MCF-7 cells. LXR stimulation also reduces Akt phosphorylation and its localization at the plasma membrane. CONCLUSION: We showed, for the first time, that LXR regulates transcription of specific proteins of lipid rafts in a breast cancer model.

IRES-dependent translation of the long non coding RNA meloe in melanoma cells produces the most immunogenic MELOE antigens.

MELOE-1 and MELOE-2, two highly specific melanoma antigens involved in T cell immunosurveillance are produced by IRES-dependent translation of the long « non coding ¼ and polycistronic RNA, meloe. In the present study, we document the expression of an additional ORF, MELOE-3, located in the 5' region of meloe. Data from in vitro translation experiments and transfection of melanoma cells with bicistronic vectors documented that MELOE-3 is exclusively translated by the classical cap-dependent pathway. Using a sensitive tandem mass spectrometry technique, we detected the presence of MELOE-3 in total lysates of both melanoma cells and normal melanocytes. This contrasts with our previous observation of the melanoma-restricted expression of MELOE-1 and MELOE-2. Furthermore, in vitro stimulation of PBMC from 6 healthy donors with overlapping peptides from MELOE-1 or MELOE-3 revealed a very scarce MELOE-3 specific T cell repertoire as compared to the abundant repertoire observed against MELOE-1. The poor immunogenicity of MELOE-3 and its expression in melanocytes is consistent with an immune tolerance towards a physiologically expressed protein. In contrast, melanoma-restricted expression of IRES-dependent MELOE-1 may explain its high immunogenicity. In conclusion, within the MELOE family, IRES-dependent antigens represent the best T cell targets for immunotherapy of melanoma.

Cucurbitacin-D-induced CDK1 mRNA up-regulation causes proliferation arrest of a non-small cell lung carcinoma cell line (NSCLC-N6).

Despite progress in chemotherapeutic agents, non-small cell lung cancers (NSCLC) still have a poor survival rate. Thus, development of new therapeutic strategies, specifically against cancer cells is still required. For this purpose, we treated the non-small cell lung cancer cell line NSCLC-N6 with the natural product cucurbitacin D (CucD) - extracted from the plant Ecballium elaterium in order first to assess its in vitro cytotoxicity, but also to study the genetic changes that it could bring out. CucD has shown a blocking in the G1 phase of the cell cycle in NSCLC-N6 cells prior to apoptotic cell death. The reverse transcriptase-polymerase chain reaction-differential display (RT-PCR-DD) technique was also applied on treated cells to elucidate the genetic mechanisms involved. We revealed an overexpression of Cyclin-dependent kinase 1 (CDK1) mRNA after treatment and, with the use of antisense oligonucleotides, an effective role in the proliferation arrest of NSCLC-N6 cells. The present study provides new insights about the mechanisms of proliferation arrest in tumor cells and open new ways of treatment to target tumor growth.

The melanoma antigens MELOE-1 and MELOE-2 are translated from a bona fide polycistronic mRNA containing functional IRES sequences.

Our previous studies on melanoma antigens identified two new polypeptides, named MELOE-1 and MELOE-2, that are involved in immunosurveillance. Intriguingly, these antigens are coded by distinct open reading frames (ORF) of the meloe mRNA which is significantly expressed only in the melanocytic lineage. In addition, MELOE-1 and -2 specific T cell clones recognized melanoma cells but very poorly normal melanocytes suggesting differential translation of meloe in normal vs tumor cells. This prompted us to elucidate the mechanisms of translation of these antigens in melanoma cells. We first demonstrated that no splicing event or cryptic promoter could generate shorter meloe transcripts containing only one of the two ORFs. Triggering meloe RNA degradation with a siRNA close to the ORF coding for MELOE-2 abrogated expression of both MELOE-1 and MELOE-2, thus confirming that the two ORFs are always associated. Next we showed, in a bicistronic reporter system, that IRES activities could be detected upstream of MELOE-1 and MELOE-2 and finally confirmed their translation from full length meloe cDNA in melanoma cells with eGFP constructs. In conclusion, meloe is a polycistronic mRNA that generates both MELOE-1 and MELOE-2 antigens through IRES-dependent translation in melanoma cells and that may explain their tumor specificity.

Synthesis of N-aryl-3-(indol-3-yl)propanamides and their immunosuppressive activities.

N-aryl-3-(indol-3-yl)propanamides were synthesized and their immunosuppressive activities were evaluated. This study highlighted the promising potency of 3-[1-(4-chlorobenzyl)-1H-indol-3-yl]-N-(4-nitrophenyl)propanamide 15 which exhibited a significant inhibitory activity on murine splenocytes proliferation assay in vitro and on mice delayed-type hypersensitivity (DTH) assay in vivo.

A novel indole-3-propanamide exerts its immunosuppressive activity by inhibiting JAK3 in T cells.

We previously identified an indole-3-propanamide derivative, 3-[1-(4-chlorobenzyl)indol-3-yl]-N-(pyridin-4-yl)propanamide (AD412), as a potential immunosuppressive agent. Here, we document that AD412 inhibited the proliferative response of CD3/CD28-stimulated human T cells without inhibiting their interleukin 2 (IL-2) production and also inhibited the proliferation of CTL-L2 cells in response to IL-2. These results prompted us to analyze the effect of our compound on the three main signaling pathways coupled to the IL-2 receptor. We provide evidence that AD412 inhibited the JAK1/3-dependent phosphorylations of Akt, STAT5a/b, and ERK1/2 in IL-2-stimulated CTL-L2 cells. In contrast, AD412 had little effect on the JAK1/2-dependent INF-gamma-induced phosphorylation of STAT1 in U266 cells. This suggested a preferential inhibition of JAK3 over JAK1 or JAK 2 activities by AD412 that was confirmed by in vitro kinase assays with purified JAK2 and JAK3 kinases. In addition, we provide evidence that the inhibition of IL-2 response by AD412 was not due to inhibition of IL-2Ralpha up-regulation because neither AD412 nor JAK3 inhibitors described previously [4-[(3-bromo-4-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline (WHI-P154) and alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamid (AG-490)] significantly inhibited IL-2-induced IL-2Ralpha overexpression. Finally, we further document the immunosuppressive activity of AD412 in vivo by showing that its administration per os significantly prolonged heart allograft graft survival. This molecule may thus represent an interesting lead compound to develop new immunosuppressive agents in the field of transplantation and autoimmune diseases.

Synthetic N-pyridinyl(methyl)-indol-3-ylpropanamides as new potential immunosuppressive agents.

Several N-pyridinyl(methyl)-indol-3-ylpropanamides were synthesized and pharmacological evaluations of their immunosuppressive potential were performed. Among thirteen compounds tested in vitro on murine T proliferation, three showed interesting inhibiting activity. For the most active compound (propanamide 18), immunosuppressive activity was documented both in vitro on human T lymphocytes proliferation and in vivo on mice delayed-type hypersensitivity. These experimental data demonstrated that these compounds hold potential as immunosuppressive agents.

A new carboxamide compound exerts immuno-suppressive activity by inhibiting dendritic cell maturation.

The immunosuppressive properties of a benzamide derivative, JM34, previously characterized as an anti-inflammatory compound are described. The immunosuppressive potential of JM34 was evidenced by inhibition of PBMC proliferation in vitro with an IC50 of 20 microM. In contrast with classical immunosuppressive drugs, JM34 affected neither cytokine production nor IL-2R expression from activated T cell clones, and displayed only moderate inhibition of IL-2-induced or anti-CD3/anti-CD28-induced proliferation. We investigated its effects on dendritic cells (DC) in vitro. Addition of JM34 during DC maturation inhibited the expression of some maturation markers: specifically, MHC molecule up-regulation was totally inhibited and CD83 expression was significantly reduced, while up-regulation of CD86, CD80 or CD40 was less affected. Moreover, JM34-treated DC showed impaired IL-12 but not IL-10 secretion, and a markedly reduced ability to present antigens to naive T lymphocytes in vitro. We provide evidence that these JM34-induced alterations of DC were associated with a marked inhibition of NF-kappaB nuclear translocation. Finally, JM34 inhibited delayed type hypersensitivity dose dependently in mice. In conclusion, our data suggest that JM34 inhibited T lymphocyte activation mainly by targeting DC, and thus may represent a new class of therapeutic agents in the fields of transplantation and autoimmune diseases.

Identification of a novel putative non-coding RNA involved in proliferation arrest of a non-small cell lung carcinoma cell line treated with an original chemical substance, methyl-4-methoxy-3-(3-methyl-2-butanoyl) benzoate.

Non-small cell lung cancers remain particularly refractory to current treatments. Thus, characterisation of new molecular targets whose expression during chemotherapy could stop tumour growth, is required. In order to identify these new targets, we applied RT-PCR differential display (RT-PCR-DD) to a non-small cell lung cancer line (NSCLC-N6) treated by an original chemical substance, VT1, capable of arresting the proliferation of NSCLC-N6 cells in G1 phase. This study enabled us to identify a novel RNA, which has a strong homology with a DNA clone (GenBank accession no.: AY166681). This RNA resides in 6p24-p25 within intron 2 of the HEF1 gene, has no apparent open reading frame and may consists of a single large exon. Antisense oligonucleotides indicated that this RNA is involved in the proliferation arrest induced with VT1 treatment in NSCLC-N6 cells. The structure of this novel RNA resembles that of the previous identified extremely long non-coding RNAs which seem to regulate gene expression. Thus, this novel B2 transcript may belong to this new expanding non-coding RNA family.

Synthesis and evaluation of disubstituted N1- and N3-imidazolidin-2-ones acting as potential immunosuppressive agents.

New N1-mono and N1, N2-disubstituted imidazolidin-2-one with a significant immunosuppressive activity have been discovered. Among the 17 synthesized and tested compounds, five of them showed maximal inhibition of proliferation of concanavallin A (Con A)- stimulated splenocytes at 90 microM, identical to that obtained with cyclosporin A (CsA) at 5 microM, an optimal concentration.