THE USE OF MANDAMUS TO COMPEL EDUCATIONAL INSTITUTIONS TO CONFER DEGREES

Hispolon is an active phenolic compound of <i>Phellinus igniarius</i>, a mushroom that was recently shown to have antioxidant and anticancer activities in various solid tumors. Here, the molecular mechanisms by which hispolon exerts anticancer effects in acute myeloid leukemia (AML) cells was investigated. The results showed that hispolon suppressed cell proliferation in the various AML cell lines. Furthermore, hispolon effectively induced apoptosis of HL-60 AML cells through caspases-8, -9, and -3 activations and PARP cleavage. Moreover, treatment of HL-60 cells with hispolon induced sustained activation of JNK1/2, and inhibition of JNK by JNK1/2 inhibitor or JNK1/2-specific siRNA significantly abolished the hispolon-induced activation of the caspase-8/-9/-3. In vivo, hispolon significantly reduced tumor growth in mice with HL-60 tumor xenografts. In hispolon-treated tumors, activation of caspase-3 and a decrease in Ki67-positive cells were observed. Our results indicated that hispolon may have the potential to serve as a therapeutic tool to treat AML

Critical role of NF-kB in NCTD-induced transcriptional inhibition of MMP-9 and u-PA in Huh7 cells.

<p>Huh7 cells were treated with NCTD 10 µM for 24 h and then the nuclear fraction was prepared as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031055#s4" target="_blank"><i>Materials and Methods</i></a>”. (A and B) ChIP analysis of the association of various transcription factors with the MMP-9 and u-PA promoter region in Huh7 cells. (C and D) Representative results of NF-KB protein levels and phosphorylation of IkBα by Western blot analysis. Quantitative results of NF-kB protein levels, which were adjusted with the internal control Fibrillian or GAPDH protein level. The values represented the means ± SD of at least three independent experiments. <i>*P</i><0.05 as compared with the vehicle group.</p

Effect of NCTD on cell viability, <i>in vitro</i> wound closure, cell migration and invasion in Huh7 cells.

<p>(A) Huh7 cells were treated with NCTD (0, 2.5, 5, 10 and 20 µM) for 24 h before being subjected to a MTT assay for cell viability. The values represented the means ± SD of at least three independent experiments. (B) Huh7 cells were wounded and then treated with vehicle (DMSO) or NCTD (2.5, 5 and 10 µM) for 0 h, 12 h and 24 h in 0.5% FBS-containing medium. At 0, 12 and 24 h, phase-contrast pictures of the wounds at three different locations were taken. (C & D) Figure C&D showing the cell migration and invasion were measured using a Boyden chamber for 16 h and 24 h with polycarbonate filters respectively. The migration and invasion abilities of Huh7 cells were quantified by counting the number of cells that invaded to the underside of the porous polycarbonate as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031055#s4" target="_blank"><i>Materials and Methods</i></a> section. The values represented the means ± SD of at least three independent experiments. <i>*P</i><0.05 as compared with the vehicle group.</p

Effect of NCTD and Erk inhibitor (U0126) on MMP-9, u-PA expression, <i>in vitro</i> wound closure, cell migration and invasion in Huh7 cells.

<p>(A–B) Huh7 cells were pre-treated with U0126 for 30 min and then incubated in the presence or absence of NCTD for 24 h, and then the cell lysates were subjected to SDS–PAGE followed by western blots with anti-MMP-9, anti-u-PA, anti-Erk1/2 (total and phosphorylated) antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031055#s4" target="_blank">Materials and Methods</a>. (C–E) Huh7 cells were pre-treated with U0126 for 30 min and then incubated in the presence or absence of NCTD for 24, Huh7 cells were then subjected to in vitro wound closure, cell migration and invasion assay. The migration and invasion abilities of Huh7 cells were quantified by counting the number of cells that invaded to the underside of the porous polycarbonate as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031055#s4" target="_blank"><i>Materials and Methods</i></a> section. The values represented the means ± SD of at least three independent experiments. <i>*P</i><0.05 as compared with the vehicle group.</p

Effects of NCTD on the MAPKs pathway and PI3K/Akt signalings.

<p>Huh7 cells were cultured in various concentrations of NCTD (0, 2.5, 5, 10 µM) for 24 hours, and then the cell lysates were subjected to SDS–PAGE followed by western blots with (A) anti-ERK1/2, (B) anti-JNK, (C) anti-p38 and (D) anti-PI3K and anti-Akt (total and phosphorylated) antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031055#s4" target="_blank">Materials and Methods</a>. Determined activities of these proteins were subsequently quantified by densitometric analyses with that of control being 100% as shown just below the gel data. The values represented the means ± SD of at least 3 independent experiments. <i>*P</i><0.05 as compared with the vehicle group.</p

Effects of NCTD on the activity and protein level of MMP-9, u-PA and the protein level of the endogenous inhibitor TIMP-1 and PAI-1.

<p>(A) Huh7 cells were treated with NCTD (0, 2.5, 5 and 10 µM) for 24 h and then subjected to gelatin and casein zymography to analyze the activity of MMP-9 and u-PA respectively. (B) Huh7 cells were treated with NCTD (0, 2.5, 5 and 10 µM) for 24 h and then subjected to western blotting to analyze the protein levels of MMP-9, u-PA, PAI-1 and TIMP-1. (C&D) Quantitative results of MMP-9, u-PA, PAI-1 and TIMP-1 protein levels which were adjusted with β-actin protein level. The values represented the means ± SD of at least three independent experiments. <i>*P</i><0.05 as compared with the vehicle group.</p

NCTD suppresses MMP-9 and u-PA expression at a transcriptional level.

<p>Huh7 cells were treated with NCTD (0, 2.5, 5 and 10 µM) for 24 h and then subjected to (A) Reverse Transcription-PCR and (B) quantitative real-time PCR to analyze the mRNA expression of MMP-9, or u-PA. (C&D) MMP-9 and u-PA promoter reporter assay to analyze the promoter activity of MMP-9 and u-PA respectively. Luciferase activity, determined in triplicates, was normalized to β-galactosidase activity. The values represented the means ± SD of at least three independent experiments. <i>*P</i><0.05 as compared with the vehicle group.</p

Cytotoxic effect of glabridin in four AML cell lines (HL-60, MV4-11, U937, and THP-1) and two human normal cells (WI-38 and HOK).

<p>(A) Structure of glabridin. (B) Cell viability analysis of four AML cell lines (HL-60, MV4-11, U937, and THP-1) cultured in presence of glabridin for 24 h by MTT assay. (C) Cell viability analysis of two normal cells (WI-38 and HOK) cultured in presence of glabridin for 24 h by MTT assay. Data represent mean of 3 determinations per condition repeated 3 times. Results are shown as mean ± SE.</p

Hispolon Induces Apoptosis through JNK1/2-Mediated Activation of a Caspase-8, -9, and -3-Dependent Pathway in Acute Myeloid Leukemia (AML) Cells and Inhibits AML Xenograft Tumor Growth in Vivo

Hispolon is an active phenolic compound of <i>Phellinus igniarius</i>, a mushroom that was recently shown to have antioxidant and anticancer activities in various solid tumors. Here, the molecular mechanisms by which hispolon exerts anticancer effects in acute myeloid leukemia (AML) cells was investigated. The results showed that hispolon suppressed cell proliferation in the various AML cell lines. Furthermore, hispolon effectively induced apoptosis of HL-60 AML cells through caspases-8, -9, and -3 activations and PARP cleavage. Moreover, treatment of HL-60 cells with hispolon induced sustained activation of JNK1/2, and inhibition of JNK by JNK1/2 inhibitor or JNK1/2-specific siRNA significantly abolished the hispolon-induced activation of the caspase-8/-9/-3. In vivo, hispolon significantly reduced tumor growth in mice with HL-60 tumor xenografts. In hispolon-treated tumors, activation of caspase-3 and a decrease in Ki67-positive cells were observed. Our results indicated that hispolon may have the potential to serve as a therapeutic tool to treat AML

Association of <i>RECK</i> genotypic frequencies with HCC laboratory status.

<p>Mann-Whitney U test was used between two groups.</p>a<p>Mean ± S.E.</p>*<p><i>p</i> value<0.05.</p