PP suppressed tumor xenograft growth of Huh7 cells in nude mice model.

<p><b>A.</b> Growth curves and representative photographs of nude mice treated with i.p (left panel) or oral administration (right panel) of PP with the vehicle control (water). Point represents the mean tumor size with the equation V = (length×width²)/2. <b>B.</b> The tumors harvested from water-treated or PP-treated mice were fixed, embedded, sectioned, and applied to Ki-67 immunohistochemical staining and quantification. The number of positively stained nuclei in a minimum of six randomly selected fields from representative tumor sections divided by the total number of cells is presented in the right panel. <b>C.</b> The tumor tissues were harvested for western blot. Data are expressed as mean ± SD. Significant differences between the PP-treated and untreated cells were statistically analyzed by ANOVA (Tukey's multiple comparison test; *, <i>P</i><0.01; **, <i>P</i><0.001).</p

PP exerts its anticancer effect through inactivation of PI3K/AKT signaling pathway.

<p><b>A.</b> Huh7 and Hep3B cells were treated with different concentrations of PP (0, 50 or 100 µg/ml) and cell lysis harvested at 48 hr for western blot analysis. PP treatment exerted a significant reduction in expression levels of p-AKT and its downstream targets including p-GSK-3β, Bcl-xL, and survivin. However, PP did not affect expressions of p-ERK and PTEN but VEGF expression was decreased in PP-treated cells. The experiments were repeated 3 times. <b>B.</b> Huh7 cells were transfected with AKT-overexpressing vector or the vector control, then treated with PP (50 µg/ml) and the cell viability measured by MTT at 72 hr. The results revealed an abrogated inhibitory effect in AKT-transfected cells when compared with the control. Data are given as percentage of untreated cells as control which was set at 100%. Data are means ± SD of three independent experiments. <b>C.</b> Huh7 cells were transfected with AKT-overexpressing vector or the vector control and then treated with PP. Cell lysate was harvested and applied to western blot. Significant differences between the PP-treated and untreated cells were statistically analyzed by ANOVA (Tukey's multiple comparison test; *, <i>P</i><0.01; **, <i>P</i><0.001).</p

No obvious adverse effects find in PP-treated mice.

<p><b>A.</b> Hematoxylin and eosin (H&E) staining of lung, liver, kidney from the four indicated groups of mice. <b>B.</b> Body weight (gram) of the mice treated with PP and control.</p

Effects of PP on the proliferation of liver cancer cell lines.

<p><b>A.</b> MTT assay was taken to evaluate the liver cancer cell (Huh7, Hep3B, HepG2 and SMMC-7721) viability after the treatment with different concentrations of PP (0, 25, 50, 100, 200 or 400 µg/ml respectively) for up to 96 hr. Data are means (bars, SD) of three independent experiments. <b>B.</b> The colony-formation assay demonstrated a dose-dependent decrease in liver cancer cell proliferation (Huh7, Hep3B, HepG2 and SMMC-7721) when exposure to PP. <b>C.</b> Western blot was conducted and a dose-dependent reduction of cyclin B1 expression was observed. Actin was involved as a loading control.</p

Extract of <em>Pleurotus pulmonarius</em> Suppresses Liver Cancer Development and Progression through Inhibition of VEGF-Induced PI3K/AKT Signaling Pathway

<div><p>Liver cancer or hepatocellular carcinoma is one of the leading causes of cancer-related deaths. Conventional chemotherapies are limited by the development of drug resistance and various side effects. Because of its non-toxicity and potent biopharmacological activity, metabolites derived from mushrooms have received more attention in cancer therapy. Our previous studies have demonstrated the anticancer effects of polysaccharide-protein complexes derived from the <em>Pleurotus</em> mushrooms. The aim of this study was to investigate the underlying molecular mechanism of the anticancer activity of a hot water extract containing a polysaccharide-protein complex isolated from <em>Pleurotus pulmonarius</em> (PP) in liver cancer cells. Our results indicated that exposure of liver cancer cells to PP not only significantly reduced the <em>in vitro</em> cancer cell proliferation and invasion but also enhanced the drug-sensitivity to the chemotherapeutic drug Cisplatin. Both oral administration and intraperitoneal injection of PP significantly inhibited the tumor growth in xenograft BALB/c nude mice. PP triggered a marked suppression of the PI3K/AKT signaling pathway in liver cancer cells <em>in vitro</em> and <em>in vivo</em>, and overexpression of the constitutively active form of AKT, Myr-AKT, abrogated this effect and the inhibited proliferation and invasion by PP. Both western blot and ELISA results showed that PP-treated liver cancer cells had reduced expression and secretion of vascular endothelial growth factor (VEGF). Addition of recombinant human VEGF attenuated the inhibitory effects of PP on PI3K/AKT pathway and the cancer phenotypes. Our results demonstrated that PP suppressed the proliferation, invasion, and drug-resistance of liver cancer cells <em>in vitro</em> and <em>in vivo</em>, mediated by the inhibition of autocrine VEGF-induced PI3K/AKT signaling pathway. This study suggests the potential therapeutic implication of PP in the treatment of human liver cancer.</p> </div

Inhibition of VEGF expression and secretion by PP mediates its effects on PI3K/AKT pathway.

<p><b>A.</b> Effects of PP on secretion of VEGF in Huh7 and Hep3B cells. After the cells were treated by PP with various concentrations of (0, 50 or 100 µg/ml) for 48 hr, the conditioned medium were collected and VEGF level in the conditioned medium was determined by a human VEGF ELISA Kit. The result demonstrated a suppression of VEGF secretion in a dose-dependent manner upon PP treatment. <b>B.</b> MTT assay was performed to evaluate the proliferation when exposure to PP (50 µg/ml) in presence or absence of rhVEGF (0.6 ng/ml) for 72 hr in Huh7 cells. <b>C.</b> Huh7 cells were treated with PP (50 µg/ml) alone or the combination with rhVEGF (0.6 ng/ml), and cell lysate was harvested and applied to western blot. Data are expressed as mean ± SD. Significant differences between the PP-treated and untreated cells were statistically analyzed by ANOVA (Tukey's multiple comparison test; *, <i>P</i><0.01; **, <i>P</i><0.001).</p

Synergistic effect of PP with cisplatin (DDP) in liver cancer cells.

<p><b>A.</b> Huh7 and Hep3B cell were treated with DDP (up to 10 µM), PP (25 µg/ml), or the combination and then colony formation assay performed. <b>B.</b> Huh7 cells were transfected with AKT-overexpressing vector or the vector control, then treated with low dosage of PP (25 µg/ml), DPP (5 µM), or the combination for 72 hr and the cell viability measured by MTT assay. <b>C.</b> MTT assay was performed to evaluate the proliferation when exposure to low dosage of PP (25 µg/ml), DDP (5 µM) alone or the combination, in the absence or presence of rhVEGF for 72 hr in Huh7 cells. Addition of rhVEGF (0.6 ng/ml) led to an abrogated sensitization. Data are given as percentage of untreated controls which were set at 100%, with mean ± SD of at least three independent experiments. Significant differences between the PP-treated and untreated cells were statistically analyzed by ANOVA (Tukey's multiple comparison test; *, <i>P</i><0.01; **, <i>P</i><0.001).</p

Effect of PP on invasive potential of liver cancer cells.

<p><b>A.</b> Boyden chamber assay was used to determine the invasiveness of Huh7 and Hep3B cells upon treatment with control or PP (50 µg/ml) PP for 24 hr. PP significantly suppressed liver cancer cell invasion. Right panel showed the quantification by submerging the chambers in 1% SDS and measuring the absorbance of the solution at 570 nm. The mean of triplicate assays for each experiment condition was calculated and values normalized to the untreated cells. <b>B.</b> The Huh7 cells transfected with AKT-overexpressing vector, or the vector control were treated with PP, and the cell invasiveness was tested. Right panel shows the quantification. <b>C.</b> Huh7 cells were treated with PP in the absence or presence of 0.6 ng/ml rhVEGF, and the invasive potential determined. Right panel shows the quantification. Data are expressed as mean ± SD. Significant differences between the PP-treated and untreated cells were statistically analyzed by ANOVA (Tukey's multiple comparison test; *, <i>P</i><0.01; **, <i>P</i><0.001).</p

Representative cell cycle distribution by flow cytometry.

<p>Huh7 and Hep3B cells were treated with PP (0, 50 or 100 µg/ml) for 48 hr, harvested and subjected to flow cytometry analysis of their DNA content. Data are means±SD of three independent experiments. Significant differences between the PP-treated and untreated cells were statistically analyzed by ANOVA (Tukey's multiple comparison test; *, <i>P</i><0.05; **, <i>P</i><0.005).</p

Data_Sheet_1_Airway Relaxation Effects of Water-Soluble Sclerotial Extract From Lignosus rhinocerotis.docx

<p>Lignosus rhinocerotis has a long history of use by the indigenous community within East Asia to treat a range of health conditions including asthma and chronic cough. To date, there is limited scientific evidence to support its therapeutic effects in relieving these airways conditions. In this study, we examined the effects of the different molecular weight fractions [high-molecular-weight (HMW), medium-molecular-weight (MMW), and low-molecular-weight (LMW)] obtained from the cold water sclerotial extract (CWE) of L. rhinocerotis on airways patency using airway segments isolated from Sprague Dawley rat in an organ bath set-up. It is demonstrated that the HMW and MMW fractions exhibited higher efficacy in relaxing the pre-contracted airways when compared to the CWE and LMW fraction. In addition, the HMW fraction markedly supressed carbachol-, 5-hydroxytrptamine-, and calcium-induced airway contractions. CWE demonstrated a lower efficacy than the HMW fraction but it also significantly attenuated carbachol- and calcium-induced airway contractions. Results showed that the bronchorelaxation effect of CWE and fractions is mediated via blockade of extracellular Ca²⁺ influx. The composition analysis revealed the following parts of carbohydrate and proteins, respectively: HMW fraction: 71 and 4%; MMW fraction: 35 and 1%; and LMW fraction: 22 and 0.3%. Our results strongly suggest that the polysaccharide–protein complex or proteins found in the HMW and MMW fractions is likely to contribute to the bronchorelaxation effect of CWE.</p