The schematic represents the concept of how fenretinide and exogenously administered sphingosine 1 phosphate (S1P) affect the adult lung structure maintenance.

<p>Administration of fenretinide changes the ceramide/S1P ratio by increasing the generation of ceramide, which in turn decreases HIF-1α and VEGF expression in the lung. Reduction of HIF-1alpha and VEGF -both central to the adult lung structure maintenance program- causes emphysema (A). Exogenously administered S1P increases the amount of intracellular S1P via sphingosine kinase 1 (Sphk1) activation (B), thus protecting against lung cell apoptosis.</p

Immunohistochemical analysis of HIF-1α.

<p>The number of the HIF-1α positive cells is counted in vehicle control, S1P, fenretinide, and fenretinide with S1P treated rat lungs. Then they were referenced to the total length of the alveolar perimeters (A). Representative images of immunohistochemical staining of HIF-1α are shown (B). Bars = 50 µm, Original Magnification x100 Data are expressed as mean ± SEM. C = Control, F = Fenretinide, S = S1P, TLAP =  total length of the alveolar perimeters.</p

Effect of exogenous S1P on phospho-sphingosine kinase 1 (pSphk1) and the balance of dihydroceramide and dihydro-S1P (n = 4, each for group).

<p>Densitometric data and representative protein bands are shown. Phospho-Sphk1 was adjusted to Sphk1 (A). The balance of dihydroceramide/dihydro-S1P was investigated by mass spectrometric analysis (B). Data are expressed as mean ± SEM. C = Control, F = Fenretinide, S = S1P.</p

Morphological analysis of the lungs from fenretinide challenged rats treated with or without sphingosine 1-phosphate (S1P).

<p>When compared to control lungs (A), the low power magnification shows air-space enlargement in the chronic fenretinide treated rat lungs (B). Examples of data based on concurrent S1P administration and S1P alone treatment are shown in (C) and (D), respectively. Quantitative analysis is shown in (E). Data are expressed as mean ± SEM. C = Control, F = Fenretinide, S = S1P Bars = 250 µm, Original Magnification x40.</p

Mass spectrometric analysis of dihydroceramide and long chain ceramide species.

<p>The concentrations of dihydroceramide in the rat lungs are shown (A). The individual data of the dihydroceramide species are depicted graphically and numerically (B). Data are expressed as mean ± SEM. C = Control, F = Fenretinide, S = S1P.</p

Western blot analysis of cytoplasmic and nuclear proteins (n = 4, for each group).

<p>Densitometric data and representative protein bands are shown. HIF-1α, HDAC2 and Nrf2 expression was normalized to a housekeeping protein Lamin B (A, C and D, respectively). VEGF expression was normalized to β actin (B). Data are expressed as mean ± SEM. C = Control, F = Fenretinide, S = S1P.</p

K145 suppresses the growth of U937 tumors in nude mice by oral administration.

<p>BALB/c-nu mice (n = 7) with palpable U937 xenograft were treated daily with vehicle, tamibarotene (20 mg/kg), or <b>K145</b> (50 mg/kg) for 15 days by oral gavage. After treatment, animals were sacrificed and tumors were removed, weighed and images were taken. A) Tumor weight and TGI comparison; B) Images of tumor samples from control and treatment groups (n = 7 for each group) after the experiments; C) Tumor volumes were measured every other day; D) Animal weights were measured every other day. Data are expressed as mean value ± SEM. *P<0.05 compared to control group.</p

HINT scores of the docked molecules into SphK1 and SphK2.

a<p>Previous studies have shown that ∼515 score units correspond to ΔΔG = −1.0 kcal/mol <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056471#pone.0056471-Kellogg1" target="_blank">[49]</a>. In the absence of a reference point from a calibration for this specific biomolecular system, the HINT score <i>differences</i> between ligands and/or between SphK1 and SphK2 are more meaningful than their specific values.</p

K145 inhibits SphK2 but not SphK1.

<p>A) SphK1 and SphK2 activities were measured with 5 µM sphingosine in the absence or presence of the indicated concentrations of <b>K145</b> or 10 µM DMS. Data are expressed as percentage SphK activity in the absence of inhibitor; B) SphK2 activity was measured with increasing concentrations of sphingosine and the indicated concentrations of <b>K145</b>. Lineweaver-Burk analysis revealed a Vmax of 10820±210 pmol/min per mg of protein, and a K_i of 6.4±0.7 µM for SphK2; C) Overlay of <b>K145</b> with sphingosine; D) CERK activities were measured with 12.5 µM ceramide in the absence or presence of the indicated concentrations of <b>K145</b> or 100 µM of DMS and sphingosine; E) Effect of <b>K145</b> (10 µM) on activity of the indicated enzymes was tested by SelectScreen Kinase Profiling from Invitrogen. Data are expressed as percentage of control activity averaged from 2 independent experiments. Data are expressed as mean value ± SEM.</p

K145 suppresses the growth of JC xenograft in BALB/c mice.

<p>BALB/c mice (n = 8) with palpable JC xenograft were treated daily with vehicle or <b>K145</b> (20 mg/kg and 35 mg/kg) for 15 days by i.p. injection. A) Tumor volumes were measured every other day; B) After treatment, animals were sacrificed and tumors were removed and weighed; C) The S1P and K145 levels in the tumor samples from vehicle and treatment (20 mg/kg) groups (n = 4) were measured by ESI-MS/MS; D) Images of tumor samples from control and treatment groups (n = 7 for each group) after the experiments; E) Tumor samples (20 mg/kg and control groups) were analyzed by Western blot. Data are expressed as mean value ± SEM. *P<0.05 compared to control group.</p