Lactonic Sophorolipids Increase Tumor Burden in Apc^min+/- Mice

<div><p>Sophorolipids (SL) are amphiphilic biosurfactant molecules consisting of a disaccharide sophorose with one fatty acid at the C1 position and optional acetylation at the C6’and C6” positions. They exist in a closed ring lactonic (LSL) or open acidic (ASL) structure Sophorolipids are produced in crude mixtures in economically viable amounts by the yeast <i>Starmerella bombicola</i> and used in a variety of consumer products. Varying levels of anti- proliferative and anti-cancer activity of crude sophorolipid mixtures are described in a number of tumor cell lines <i>in vitro</i>. However, significant inter-study variation exists in the composition of sophorolipid species as well as other biologically active compounds in these mixtures, which makes interpretation of <i>in vitro</i> and <i>in vivo</i> studies difficult. We produced a 96% pure C18:1 lactonic sophorolipid that dose-dependently reduces the viability of colorectal cancer, as well as normal human colonic and lung cell lines <i>in vitro</i>. Oral administration of vehicle-only; or lactonic sophorolipids (50 mg/kg for 70 days), to Apc^min+/- mice resulted in an increase in the number (55.5 ± 3.3 <i>vs</i> 70.50 ± 7.8: p < 0.05) and size (modal size 2mm vs 4mm) of intestinal polyps. Lactonic administration resulted in a systematic effect via reduced hematocrit (49.5 ± 1.0 vs 28.2 ± 2.0 <i>vs</i>: p<0.03) and splenomegaly (0.56 ± 0.03g vs 0.71 ± 0.04g; p<0.01) confirming exacerbation of disease progression in this model.</p></div

Topological Connection between Vesicles and Nanotubes in Single-Molecule Lipid Membranes Driven by Head–Tail Interactions

Lipid nanotube–vesicle networks are important channels for intercellular communication and transport of matter. Experimentally observed in neighboring mammalian cells but also reproduced in model membrane systems, a broad consensus exists on their formation and stability. Lipid membranes must be composed of at least two molecular components, each stabilizing low (generally a phospholipid) and high curvatures. Strong anisotropy or enhanced conical shape of the second amphiphile is crucial for the formation of nanotunnels. Anisotropic driving forces generally favor nanotube protrusions from vesicles. In this work, we report the unique case of topologically connected nanotubes–vesicles obtained in the absence of directional forces, in single-molecule membranes, composed of an anisotropic bolaform glucolipid, above its melting temperature, Tm. Cryo-TEM and fluorescence confocal microscopy show the interconnection between vesicles and nanotubes in a single-phase region, between 60 and 90 °C under diluted conditions. Solid-state NMR demonstrates that the glucolipid can assume two distinct configurations, head–head and head–tail. These arrangements, seemingly of comparable energy above the Tm, could explain the existence and stability of the topologically connected vesicles and nanotubes, which are generally not observed for classical single-molecule phospholipid-based membranes above their Tm

LSL administration causes a dose-dependent effect on cell viability and morphology.

<p>(a) Following 24h of treatment with LSL we observed a significant reduction in O.D.570 values at doses between 20–100μg/ml (p<0.0001) in the control cell lines CCD-841-CoN and MRC5. (b) There was a significant reduction in O.D.570 values at doses between 30–100μg/ml in HCT116, Caco2 and LS180 (p < 0.05) and at doses above 60μg/ml inn HT29 and HT115 cells (p < 0.001). Graphs show a representative data set from three independent experimental replicates. Values indicate mean ± SEM (n = 6). Statistical significance was assessed one-way ANOVA (*p < 0.05; **p < 0.001; ***p < 0.0001). (c) Light micrographs from CCD-841-CoN colonic epithelial cells (top) treated with vehicle-only control (left) where cells show a typical bipolar morphology. After treatment with 40μg/ml (central) or 70μg/ml (right) LSL the remaining adherent cells are rounded and there are large regions devoid of cells. In HT29 cultures treated with vehicle-only (bottom right) the cells are densely packed with a cobblestone-like morphology. Following treatment with 40μg/ml LSL (middle), there are no discernable changes in cell morphology, however treatment with 70μg/ml LSL (right) leads to cell rounding and conspicuous cell free areas. Scale bar = 50μm for all images.</p

Purification of LSL produced by S. bombicola.

<p>HPLC-ELSD and LC-MS analysis of the LSL precipitate from the bioreactor. (a) The MW 688 g/mol corresponds to diacetyl C18:1 LSL. (b) HPLC-ELSD analysis following purification of the LSL precipitate. The MW 706 g/mol = diacetyl C18:1 ASL, which is the result of minor hydrolysis of the MW 688 compound.</p

LSL treatment increases tumour number and size in Apc^min+/- mice.

<p>PCR and restriction enzyme analysis of genomic DNA from wt or Apc^{min+/- littermates} (a); wt band: 111bp, heterozygous Apc^min+/- mouse—111 & 123bp. Mice were fed either LSL or vehicle-only every other day for 70 days. Examination of low power images of intestinal segments from wt mice treated with vehicle or 50mg/kg LSL revealed no discernible differences in gross morphology (top). However, in Apc^min+/- (bottom), those treated with vehicle-only (left) showed evidence of occult bleeding (arrow) as well as numerous polyps (c arrow heads) with a modal diameter of 2mm (d). In Apc^min+/- mice treated with LSL, there was evidence of recent hemorrhage (arrows) as well as significant increases in both polyp number (c;*p <0.01) and size (d;**p < 0.001) compared to vehicle-only controls. Values represent mean ± SEM (n = 10/group). Statistical significance was determined by one-way ANOVA (c) or a student T-tests (d). (e) Histological sections from wt (top) or Apc^min+/- (bottom) stained with Haematoxylin and Eosin. Distinct villi in wt treated with vehicle (left) or 50mg/kg LSL are seen. In Apc^min+/- littermate mice villous structure is grossly perturbed within polyps in both vehicle-treated mice (bottom left) as well as those treated with 50mg/kg (bottom right).</p

Lactonic Sophorolipid.

<p>The structure of C18:1 diacetylated LSL.</p

LSL treatment increases spleen size and red pulp area in Apc^min+/- mice.

<p>a; Photographs of dissected spleen from a wt mouse and Apc^min+/- fed with vehicle or LSL. b; Apc^min+/- mice had a significantly greater spleen weight compared to wt mice (**** p<0.00001). LSL administration resulted in a significant increase in splenic weights compared to the vehicle control mice (* p<0.01). Splenic histology from wt mice (6c top), Apc^min+/- mice fed vehicle control only (6c middle) and Apc^{min+/- fed} LSL (6c bottom). WP = White pulp, RP = Red pulp, BV = Blood vessel. Apc^min+/- mice demonstrated altered splenic pathology with increased red pulp regions (hematopoietic rich tissue). LSL fed mice showed a further change in histopathology (<i>c</i>.<i>f</i> 6c middle vs bottom) with spleens demonstrating a significant increase in red pulp (* p<0.05). Graphs represented of mean ± SEM. Animals per group n = 10. Statistical significance was determined by students T-test.</p

LSL induced both necrosis and apoptosis <i>in vitro</i>.

<p>Photomicrographs of acridine orange and ethidium bromide stained cultures following treatments with 0 (a-e), 20 (f-j) or 70μg/ml (k-o) LSL and quantification of live, apoptotic or necrotic cells (p-t). The vast number of CCD-841-CoN (a) and cancer cells treated with vehicle control are morphologically viable with a small number showing condensed nuclei (apoptotic). 20μg/ml LSL resulted in necrosis (red/orange clusters) in all cell lines, although CCD-841-CoN (k.p ****p<0.0001) and Caco2 cells (j.o ** p<0.001) were more susceptible. At 70μg/ml, very few adherent CCD-841-CoN cells were observed (k), remaining adhered cells were either necrotic or apoptotic (p). HT29 (l), HT115 (m), HCT116 (n) and Caco2 (o) cells exposed to 70μg/ml LSL all had 50% of cells with morphological features of cells death (q-t) and statistically significant increases in either the numbers of necrotic (*** p<0.0001) or apoptotic cells (**p<0.001) as compared with vehicle only controls.</p