ATRA enhanced differentiation of CD133⁺ hCSCs by down-regulating the Wnt/β-catenin signaling pathway.

<p>(<b>A</b>) ATRA treatment (concentrations from 10⁻⁹ M to 10⁻⁵ M) decreased the percentage of CD133-expressing HepG2 cells at day 5 <i>in vitro</i>. (<b>B</b>) Expression of the CD133 protein decreased in CD133⁺ hCSCs after treatment with 1 × 10⁻⁵ M, 5 × 10⁻⁵ M and 10 × 10⁻⁵ M ATRA for 12 or 48 hours. <i>Control</i>, normal cell culture. (<b>C</b>) ATRA treatment down-regulated the protein expression level of other stem cell markers SOX2, NANOG and OCT4 after 5 days ATRA exposure (concentrations from 10⁻⁹ M to 10⁻⁵ M). (<b>D</b>) ATRA treatment decreased the protein expression level of β-catenin and increased its phosphorylation after 5 days ATRA exposure (concentrations from 10⁻⁹ M to 10⁻⁵ M). (<b>E</b>) ATRA inactivated the PI3K-AKT signaling pathway in CD133⁺ hCSCs after 5 days ATRA exposure (concentrations from 10⁻⁹ M to 10⁻⁵ M). (<b>F-G</b>) Silencing β-catenin mRNA decreased both the mRNA (<b>F</b>) and protein (<b>G</b>) expression of stem cell markers SOX2, NANOG and OCT4. <i>Control</i>, normal cell culture; <i>PLKO</i>.<i>1</i>, cells treated with empty vector PLKO.1; <i>β-catenin shRNA</i>, cells treated with the PLKO.1-β-catenin-shRNA constructed vector. ***p < 0.001; **p < 0.01.</p

ATRA-induced differentiation of CD133⁺ hCSCs increased their sensitivity to docetaxel (DOC) treatment <i>in vitro</i>.

<p>(<b>A-B</b>) Survival and proliferation of CD133⁺ hCSCs after treatment with three concentrations (10⁻⁵ M, 10⁻⁶ M and 10⁻⁷ M) of ATRA. (<b>C-D</b>) Survival and proliferation of CD133⁺ hCSCs after treatment with three different concentrations (10⁻¹⁰ M, 10⁻⁹ M and 10⁻⁸ M) of DOC. (<b>E-F</b>) Combined treatment with ATRA (10⁻⁵ M, 10⁻⁶ M and 0⁻⁷ M) and DOC (10⁻⁸ M) decreased the growth of CD133⁺ hCSCs. (<b>A, B, C</b>) Growth curves of CD133⁺ hCSCs; (<b>D, E, F</b>) Quantitative analyses of CD133⁺ hCSC growth at day 5. ***p < 0.001; **p < 0.01. (<b>G-H</b>) ATRA treatment decreased the survival and proliferation of β-catenin mRNA knockdown-CD133⁺ hCSCs. (<b>I-J</b>) Knockdown of β-catenin mRNA decreased resistance of CD133⁺ hCSCs to DOC treatment (10⁻⁸ M). Concentration of ATRA in <b>G-J</b> is 10⁻⁶ M. (<b>G, I</b>) Growth curves of CD133⁺ hCSCs; (<b>H, J</b>) Quantitative analyses of CD133⁺ hCSC growth at day 3, day 4 and day 5. ***p < 0.001; **p < 0.01. (<b>K</b>) Molecular mechanism of ATRA-mediated differentiation of CD133⁺ hCSCs. Wnt/β-catenin was required to maintain the stemness of hCSCs. Binding of ATRA to RARs induced inactivation of the PI3K-AKT pathway, enhancing GSK-3β-dependent phosphorylation of β-catenin.</p

Gene profiling of dehydrogenases related to RA synthesis in different cancer types.

<p><b>(A)</b> The process of RA synthesis and catalytic enzymes. Three alcohol dehydrogenases (ADH1, ADH3, ADH4), two retinol dehydrogenases (RDH1 and RDH10) and three retinaldehyde dehydrogenases (RALDH1, RALDH2, RALDH3) are involved in the biosynthesis of retinoic acid from retinol. The main catalyzing sites for chemical retinol, retinaldehyde and retinoic acid are highlighted by dashed boxes. <b>(B-D)</b> Gene expression profiling of RALDH1, ADH1 and RDH10 was analyzed using the Oncomine database (Bittner Multi-cancer data set, including 1,911 clinical patients, pathologically classified by 16 different cancer types). (<b>B</b>) RALDH1; (<b>C</b>) ADH1; (<b>D</b>) RDH10. Liver cancer (HCC) group is highlighted by dark blue. (1) Bladder cancer (n = 32); (2) Brain and central nervous system cancer (n = 4); (3) Breast cancer (n = 328); (4) Cervical cancer (n = 35); (5) Colorectal cancer (n = 330); (6) Esophageal cancer (n = 7); (7) Gastric cancer (n = 7); (8) Head and neck cancer (n = 41); (9) Kidney cancer (n = 254); (10) Liver cancer (n = 11); (11) Lung cancer (n = 107); (12) Lymphoma (n = 19); (13) Ovarian cancer (n = 166); (14) Pancreatic cancer (n = 19); (15) Prostate cancer (n = 59); (16) Sarcoma (n = 49).</p

<i>Hrg</i> deletion in mice suppresses syngeneic tumor growth.

<p>Lewis Lung carcinoma cells (<b>A</b>) or B16F1 melanoma cells (<b>B</b>) were injected in the backs of <i>hrg</i> null or wild type C57BL/6 mice (50,000 cells/mouse). Tumor volumes were assessed over 16 days following implantation. *P<0.05.</p

<i>Hrg</i> deletion in mice suppresses Lewis Lung tumor vascularity.

<p>(<b>A</b>) Lewis Lung tumors as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040033#pone-0040033-g003" target="_blank">Figure 3</a> were dissected, sectioned and examined by immunofluorescence microscopy using anti-VEGF receptor antibody (green) to detect blood vessels. DAPI stained nuclei are blue. Magnification bars represent 100 µm. IgG control is shown in bottom panel as negative control. (<b>B</b>) Vessel densities measured as vessels per mm2. Median vessel density: wt 12.33, cd36 null 6.99.</p

<i>Cd36</i> deletion in mice enhances syngeneic tumor growth.

<p>Lewis Lung carcinoma cells (<b>A</b>) or B16F1 melanoma cells (<b>B</b>) were injected in the backs of <i>cd36</i> null or wild type C57BL/6 mice (50,000 cells/mouse). Tumor volumes were assessed over 17 days following implantation. *P<0.05.</p

Thrombospondin-1 expression in LL2 and B16F1 melanoma cells and tumors.

<p>(<b>A</b>) Lewis Lung (LL2) or B16F melanoma cells were cultured in serum free media for 24 hours (1d) at which point proteins in post culture media (CM) were precipitated by TCA, separated under reducing conditions by SDS/PAGE and analyzed by immunoblot using anti-TSP-1 antibody. TSP-1 monomers were detected at 170 kDa in the media conditioned by LL2 cells, but not B16F1 cells. Purified human HRG and TSP were used as controls. (<b>B</b>) B16F1 melanoma tumor tissue was analyzed by western blot analysis for TSP expression. Intact TSP was not observed at 150 kDa, however possible degredation products were observed around 55 kDa. (<b>C</b>) B16F1 and LL2 tumor tissue was analyzed by RT-PCR for expression of TSP. TSP was detected in both tumor types, approximately 7 fold higher in LL2. (<b>D</b>) Conditioned media was collected from 4 different antibiotic resistant clones of TSR transfected B16F melanoma cells and analyzed by immunoblot as in panel A. Clone 11 expressed abundant anti-TSP reactive material at the appropriate molecular weight of recombinant TSR and was utilized for subsequent tumor studies. (<b>E</b>) TSP knockdown efficiency was analyzed by RT-PCR with statistical significance as indicated; **P<0.05; *P = 0.06. In both instances of TSP knockdown 1 (K1 and K2), reductions in TSP message levels were detected as compared with nontargeted control (NT) cells.</p

TSR transfected B16F1 melanoma cells show enhanced tumor growth in <i>cd36</i> null mice and suppressed tumor growth in <i>hrgp</i> null mice.

<p>50,000 cells from a stably transfected B16f1 melanoma cell line (Clone 11) were injected in the backs of <i>cd36</i> null (A) or <i>hrgp</i> null (B) mice. C57BL/6 mice were used as controls. Tumor volumes were assessed at timed points as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040033#pone-0040033-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040033#pone-0040033-g003" target="_blank">3</a>. *P<0.05; **P = 0.08; ***P = 0.06. LL2 cells stabily transfected with nontargeted (NT) or TSP targeted constructs, K1 and K2, constructs were similarly injected subcutaneously onto the backs of wildtype (WT) and cd36 null mice (Null). (<b>C</b>) WT-NT tumors grew smaller than WT-K1 and WT-K2 tumors, with statistically significant differences seen at days 7 and 15 for both NT vs K1 and NT vs K2. (<b>D</b>) No differences were observed between NT, K1 and K1 in cd36 null mice.</p

TSR transfected B16F1 melanoma cells show enhanced tumor vascularity in <i>cd36</i> null mice and suppressed tumor vascularity in <i>hrg</i> null mice.

<p>(<b>A</b>) Tumors from TSR transfected B16F1 melanoma cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040033#pone-0040033-g006" target="_blank">Figure 6</a> were dissected, sectioned and examined by immunofluorescence microscopy using anti-VE-Cadherin antibody (green) to detect blood vessels. DAPI stained nuclei are blue. Magnification bars represent 100 µm. IgG control is shown in bottom panels as negative control. (<b>B</b>). Median vessel density: wt 8.99, cd36 null 16.32. Median vessel density: wt 9.33, hrgp null 5.83. (<b>C</b>) Tumors from lentiviral (NT, K1 and K2) transfected LL2 cells from wildtype and cd36 null mice were examined using anti-VE-Cadherin antibody to detect blood vessels. Vessel densities measured as vessels per mm².</p

Expression of CXCR4 in tree shrews' lymphocytes.

<p>(<b>A</b>) 9.07 % of tree shrew CXCR4-positive lymphocytes were detected by mouse anti-human CXCR4 antibody (left). For the control, 13.30 % of human CXCR4-expressing lymphocytes were detected by the same antibody (right). (<b>B</b>) 0.16 % of tree shrew CXCR4-positive lymphocytes were detected by rat anti-mouse CXCR4 antibody (left). For the control, 8.09 % of mouse CXCR4-expressing lymphocytes were detected using the same antibody (right). Blank (red), isotype (green), anti-human CXCR4 antibody (brown) and anti-mouse CXCR4 antibody (brown).</p