Leptin Reverts Pro-Apoptotic and Antiproliferative Effects of α-Linolenic Acids in BCR-ABL Positive Leukemic Cells: Involvement of PI3K Pathway

It is suspected that bone marrow (BM) microenvironmental factors may influence the evolution of chronic myeloid leukaemia (CML). In this study, we postulated that adipocytes and lipids could be involved in the progression of CML. To test this hypothesis, adipocytes were co-cultured with two BCR-ABL positive cell lines (PCMDS and K562). T cell (Jurkat) and stroma cell (HS-5) lines were used as controls. In the second set of experiments, leukemic cell lines were treated with stearic, oleic, linoleic or α-linolenic acids in presence or absence of leptin. Survival, proliferation, leptin production, OB-R isoforms (OB-Ra and OB-Rb), phosphoinositide 3-kinase (PI3k) and BCL-2 expression have been tested after 24h, 48h and 72h of treatment. Our results showed that adipocytes induced a decrease of CML proliferation and an increase in lipid accumulation in leukemic cells. In addition, CML cell lines induced adipocytes cell death. Chromatography analysis showed that BM microenvironment cells were full of saturated (SFA) and monounsaturated (MUFA) fatty acids, fatty acids that protect tumor cells against external agents. Stearic acid increased Bcl-2 expression in PCMDS, whereas oleic and linoleic acids had no effects. In contrast, α-linolenic acid decreased the proliferation and the survival of CML cell lines as well as BCL-2 and OB-R expression. The effect of α-linolenic acids seemed to be due to PI3K pathway and Bcl-2 inhibition. Leptin production was detected in the co-culture medium. In the presence of leptin, the effect of α-linolenic acid on proliferation, survival, OB-R and BCl-2 expression was reduced

In Search of Small Molecule Inhibitors Targeting the Flexible CK2 Subunit Interface

Protein kinase CK2 is a tetrameric holoenzyme composed of two catalytic (α and/or α’) subunits and two regulatory (β) subunits. Crystallographic data paired with fluorescence imaging techniques have suggested that the formation of the CK2 holoenzyme complex within cells is a dynamic process. Although the monomeric CK2α subunit is endowed with a constitutive catalytic activity, many of the plethora of CK2 substrates are exclusively phosphorylated by the CK2 holoenzyme. This means that the spatial and high affinity interaction between CK2α and CK2β subunits is critically important and that its disruption may provide a powerful and selective way to block the phosphorylation of substrates requiring the presence of CK2β. In search of compounds inhibiting this critical protein–protein interaction, we previously designed an active cyclic peptide (Pc) derived from the CK2β carboxy-terminal domain that can efficiently antagonize the CK2 subunit interaction. To understand the functional significance of this interaction, we generated cell-permeable versions of Pc, exploring its molecular mechanisms of action and the perturbations of the signaling pathways that it induces in intact cells. The identification of small molecules inhibitors of this critical interaction may represent the first-choice approach to manipulate CK2 in an unconventional way

Coculture of FLFCs and PCMDS: Leptin production and OB-R expression.

<p><b>a</b> leptin production assessed by enzyme-linked immunosorbent assay in direct contact (co) and transwell (trw) coculture of PCMDS with FLFCs or HS-5 stromal cell lines after one week (<i>black columns</i> no contact; <i>hatched columns</i> direct end transwell contact). ***<i>P</i><0.001 versus PCMDS in control condition (Student <i>t</i> test). <b>b</b> after 48h, leptin receptor (OBR) expression was analysed by flow cytometry after immunostaining of PCMDS cultured in control conditions (<i>black columns</i>) or in co-culture with FLFCs or HS-5 (<i>hatched columns</i>).*<i>P</i><0.05 versus PCMDS in control condition (Student <i>t</i> test). <i>Columns</i>, mean of at least three independent determinations, <i>bars</i>. SD.</p

α-Linolenic acid inhibit AKT phosphorylation and induce caspase activation.

<p><b>a</b> Akt phosphorylation in PCMDS stimulated by α-linolenic acid 24 hours with and without leptin (<i>black columns</i> PCMDS without stimulation; <i>hatched columns</i> PCMDS with stimulation) measured by western blot. *<i>P</i><0.05 versus in control condition. <b>b</b> PhosphoAkt (<i>black columns</i>) and BCL-2 (<i>hatched columns</i>) level in PCMDS after α-linolenic acid contact with and without leptin. Akt phosphorylation and BCL-2 were measured by western blot. *<i>P</i><0.05 versus in control condition (Student <i>t</i> test).<i>Columns</i>, mean of at least three independent determinations, <i>bars</i>. SD. <b>c</b> PCMDS survival in control, with α-linolenic acid or with α-linolenic acid and caspase inhibitor after 24 hours.</p

Coculture of FLFCs and PCMDS for 48h: Influence on PCMDS.

<p><b>a</b> PCMDS proliferation in direct contact (co) and in transwell (trw) conditions with FLFCs and HS-5 stromal cell lines vs K562 and Jurkat cells in direct contact with FLFCs. **<i>P</i><0.01 versus control cells (<i>black columns</i>) (Student <i>t</i> test). <i>Columns</i>, mean of at least three independent determinations, <i>bars</i>. SD. <b>b</b> lipid accumulation was observed by Oil Red O staining in PCMDS and in K562 in direct contact and in transwell condition (not show) but not in Jurkat cells (magnification, x20) → shows intracytoplasmic lipid droplets. <b>c</b> cell-cell direct contact with PCMDS induce morphological alterations in FLFCs that are not observed in transwell condition (magnification, x20).</p

Fatty acid composition of bone marrow aspirates and biopsies.

<p>Fatty acids was analysed by gas lipid chromatography as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025651#s2" target="_blank">Materials and methods</a>”<b>.</b> Values expressed as mean ± SD (standard deviation)</p

Leptin disturbed the effect of α-linolenic acids.

<p><b>a</b> effect of 200 µM α-linolenic acids alone or with leptin (300pg/ml) on membranous OB-R expression in PCMDS after 24 hours (<i>black columns</i> PCMDS without stimulation; <i>hatched columns</i> PCMDS with stimulation). OB-R expression was analysed by flow cytometry. *<i>P</i><0.05 and ***<i>P</i><0.001 versus in control condition (Student <i>t</i> test)<b>. b</b> effect of 200 µM α-linolenic acids alone or with leptin (300pg/ml) on BCL-2 expression in PCMDS after 24 hours (<i>black columns</i> PCMDS without stimulation; <i>hatched columns</i> PCMDS with stimulation) measured by western blot. *<i>P</i><0.05 versus in control condition (Student <i>t</i> test). <b>c</b> effet of α-linolenic acids with or without leptin (300 pg/ml) on PCMDS survival after 48 hours. *<i>P</i><0.05 and ***<i>P</i><0.001 versus in control condition (Student <i>t</i> test) <i>Columns</i>, mean of at least three independent determinations, <i>bars</i>. SD.</p

PCMDS, K562 and Jurkat cells cultured with stearic, oleic, linoleic and α-linolenic acids.

<p><b>a</b> lipid accumulation in PCMDS, K562 and Jurkat cell in contact with saturated (SFA) and polyunsaturated (PUFA) fatty acids (magnification, x20 and x40) → shows intracytoplasmic lipid droplets. <b>b</b> effects of α-linolenic acid on PCMDS, K562 and Jurkat cells proliferation after 48 hours. *<i>P</i><0.05 and ***<i>P</i><0.001 versus in control condition (Student <i>t</i> test). <b>c</b> effects of α-linolenic acid on PCMDS, K562 and Jurkat cells survival after 48 hours. **<i>P</i><0.01 and ***<i>P</i><0.001 versus in control condition (Student <i>t</i> test). <b>d</b> effects of 200 µM of α-linolenic, linoleic, oleic and stearic acids on Bcl-2 expression (<i>black columns</i> no lipids stimulation; <i>hatched columns</i> lipids stimulation) measured by western blot. *<i>P</i><0.05 versus PCMDS in control condition (Student <i>t</i> test). <b>e</b> effects of 200 µM of α-linolenic, linoleic, oleic and stearic acids after 24 hours on OB-R (<i>black columns)</i> no lipids stimulation; <i>hatched columns</i> lipids stimulation) and on OB-Ra (<i>black columns</i>) and OB-Rb (<i>hatched columns</i>) mRNA expression in PCMDS. *<i>P</i><0.05 and ***<i>P</i><0.001 versus in control condition <b>(</b>Student <i>t</i> test) <i>Columns</i>, mean of at least three independent determinations, <i>bars</i>. SD.</p