Apurinic/Apyrimidinic Endonuclease 1 Regulates Inflammatory Response in Macrophages

The multi-functional apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) DNA repair and redox signaling protein has been shown to have a role in cancer growth and survival, however, little has been investigated concerning its role in inflammation. In this study, an APE1 redox-specific inhibitor (E3330) was used in lypopolysaccharide (LPS)-stimulated macrophages (RAW264.7). E3330 clearly suppressed secretion of inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin (IL-6) and IL-12 and inflammatory mediators nitric oxide (NO) as well as prostaglandin E2 (PGE2) from the LPS-stimulated RAW264.7 cells. These data were supported by the down-regulation of the LPS-dependent expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) genes in the RAW264.7 cells. The effects of E3330 were mediated by the inhibition of transcription factors nuclear factor-κB (NF-κB) and activator protein 1 (AP-1) in the LPS-stimulated macrophages, both known targets of APE1. In conclusion, pharmacological inhibition of APE1 by E3330 suppresses inflammatory response in activated macrophages and can be considered as a novel therapeutic strategy for the inhibition of tumor-associated macrophages

The mushroom Ganoderma lucidum suppresses breast-to-lung cancer metastasis through the inhibition of pro-invasive genes.

Breast cancer metastasis is one of the major reasons for the high morbidity and mortality of breast cancer patients. In spite of surgical interventions, chemotherapy, radiation therapy and targeted therapy, some patients are considering alternative therapies with herbal/natural products. In the present study, we evaluated a well-characterized extract from the medicinal mushroom Ganoderma lucidum (GLE) for its affects on tumor growth and breast-to-lung cancer metastasis. MDA-MB-231 human breast cancer cells were implanted into the mammary fat pads of nude mice. GLE (100 mg/kg/every other day) was administered to the mice by an oral gavage for 4 weeks, and tumor size was measured using microcalipers. Lung metastases were evaluated by hematoxylin and eosin (H&E) staining. Gene expression in MDA-MB-231 cells was determined by DNA microarray analysis and confirmed by quantitative PCR. Identified genes were silenced by siRNA, and cell migration was determined in Boyden chambers and by wound-healing assay. Although an oral administration of GLE only slightly suppressed the growth of large tumors, the same treatment significantly inhibited the number of breast-to-lung cancer metastases. GLE also downregulated the expression of genes associated with invasive behavior (HRAS, VIL2, S100A4, MCAM, I2PP2A and FN1) i

Mushroom \u3ci\u3eGanoderma lucidum\u3c/i\u3e Prevents Colitis- Associated Carcinogenesis in Mice

Background: Epidemiological studies suggest that mushroom intake is inversely correlated with gastric, gastrointestinal and breast cancers. We have recently demonstrated anticancer and anti-inflammatory activity of triterpene extract isolated from mushroom Ganoderma lucidum (GLT). The aim of the present study was to evaluate whether GLT prevents colitis-associated carcinogenesis in mice. Methods/Principal Findings: Colon carcinogenesis was induced by the food-borne carcinogen (2-Amino-1-methyl-6- phenylimidazol[4,5-b]pyridine [PhIP]) and inflammation (dextran sodium sulfate [DSS]) in mice. Mice were treated with 0, 100, 300 and 500 mg GLT/kg of body weight 3 times per week for 4 months. Cell proliferation, expression of cyclin D1 and COX-2 and macrophage infiltration was assessed by immunohistochemistry. The effect of GLT on XRE/AhR, PXR and rPXR was evaluated by the reporter gene assays. Expression of metabolizing enzymes CYP1A2, CYP3A1 and CYP3A4 in colon tissue was determined by immunohistochemistry. GLT treatment significantly suppressed focal hyperplasia, aberrant crypt foci (ACF) formation and tumor formation in mice exposed to PhIP/DSS. The anti-proliferative effects of GLT were further confirmed by the decreased staining with Ki-67 in colon tissues. PhIP/DSS-induced colon inflammation was demonstrated by the significant shortening of the large intestine and macrophage infiltrations, whereas GLT treatment prevented the shortening of colon lengths, and reduced infiltration of macrophages in colon tissue. GLT treatment also significantly downregulated PhIP/DSS-dependent expression of cyclin D1, COX-2, CYP1A2 and CYP3A4 in colon tissue. Conclusions: Our data suggest that GLT could be considered as an alternative dietary approach for the prevention of colitis-associated cancer

Anti-inflammatory activity of edible oyster mushroom is mediated through the inhibition of NF-κB and AP-1 signaling

<p>Abstract</p> <p>Background</p> <p>Mushrooms are well recognized for their culinary properties as well as for their potency to enhance immune response. In the present study, we evaluated anti-inflammatory properties of an edible oyster mushroom (<it>Pleurotus ostreatus</it>) <it>in vitro </it>and <it>in vivo</it>.</p> <p>Methods</p> <p>RAW264.7 murine macrophage cell line and murine splenocytes were incubated with the oyster mushroom concentrate (OMC, 0-100 μg/ml) in the absence or presence of lipopolysacharide (LPS) or concanavalin A (ConA), respectively. Cell proliferation was determined by MTT assay. Expression of cytokines and proteins was measured by ELISA assay and Western blot analysis, respectively. DNA-binding activity was assayed by the gel-shift analysis. Inflammation in mice was induced by intraperitoneal injection of LPS.</p> <p>Results</p> <p>OMC suppressed LPS-induced secretion of tumor necrosis factor-α (TNF-α, interleukin-6 (IL-6), and IL-12p40 from RAW264.7 macrophages. OMC inhibited LPS-induced production of prostaglandin E2 (PGE₂) and nitric oxide (NO) through the down-regulation of expression of COX-2 and iNOS, respectively. OMC also inhibited LPS-dependent DNA-binding activity of AP-1 and NF-κB in RAW264.7 cells. Oral administration of OMC markedly suppressed secretion of TNF-α and IL-6 in mice challenged with LPS <it>in vivo</it>. Anti-inflammatory activity of OMC was confirmed by the inhibition of proliferation and secretion of interferon-γ (IFN-γ), IL-2, and IL-6 from concanavalin A (ConA)-stimulated mouse splenocytes.</p> <p>Conclusions</p> <p>Our study suggests that oyster mushroom possesses anti-inflammatory activities and could be considered a dietary agent against inflammation. The health benefits of the oyster mushroom warrant further clinical studies.</p

NAHA, a Novel Hydroxamic Acid-Derivative, Inhibits Growth and Angiogenesis of Breast Cancer In Vitro and In Vivo

BACKGROUND: We have recently synthesized novel N-alkylated amino acid-derived hydroxamate, 2-[Benzyl-(2-nitro-benzenesulfonyl)-amino]-N-hydroxy-3-methyl-N-propyl-butyramide (NAHA). Here, we evaluate the anticancer activity of NAHA against highly invasive human breast cancer cells MDA-MB-231 in vitro and in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Cell growth was evaluated by MTT and soft agar assays. Protein expression was determined by DNA microarray and Western blot analysis. Metastatic potential was evaluated by cell adhesion, migration, invasion, capillary morphogenesis, and ELISA assays. The anticancer activity in vivo was evaluated in mouse xenograft model. NAHA inhibited proliferation and colony formation of MDA-MB-231 cells together with the down-regulation of expression of Cdk2 and CDC20 proteins. NAHA inhibited cell adhesion, migration, and invasion through the suppression of secretion of uPA. NAHA suppressed secretion of VEGF from MDA-MB-231 cells and inhibited capillary morphogenesis of human aortic endothelial cells (HAECs). Finally, NAHA at 50 mg/kg was not toxic and decreased tumor volume and tumor weight in vivo. This suppression of tumor growth was associated with the inhibition of mitotic figures and induction of apoptosis, and the reduction of CD31 and VEGF positive cells in tumors. CONCLUSION: NAHA could be a novel promising compound for the development of new drugs for the therapy of invasive breast cancers

Histology of MDA-MB-231 xenografts.

<p>Tumors dissected from animals were sectioned and stained with (A) H&E, and (B) mitotic figures (arrowheads), and (C) apoptotic bodies (arrows) evaluated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>.</p

Expression of CD31 and VEGF in tumors.

<p>The expression of (A) CD31, and (B) VEGF were quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Data are mean ± SD (n = 6–10), * significantly different from control (p<0.05) by ANOVA.</p

NAHA inhibits growth of breast cancer cells.

<p>(A) Structure of NAHA, <i>2-[Benzyl-(2-nitro-benzenesulfonyl)-amino]-N-hydroxy-3-methyl-N-propyl-butyramide</i>. (B) MDA-MB-231, (C) MCF-7, (D) MCF-10A, (E) HMEC cells were treated with NAHA (0–50 µM). Cell proliferation was determined by the tetrazolium salt method as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Cell viability was determined by trypan blue staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Data are the means ± SD. Similar results were obtained in at least two additional experiments. * p<0.05 for cell proliferation, # p<0.05 for cell viability. (F) Anchorage-independent growth (colony formation) of MDA-MB-231 cells was assessed on 1% agarose after incubation with NAHA (a – 0, b – 10 µM, c -25 µM, d – 50 µM) for 14 days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. (G) MDA-MB-231 cells were treated with NAHA (0–50 µM) for 24 hours and whole cell extracts were subjected to Western blot analysis with anti-Cdk2 and anti-CDC20 antibodies as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. The equal protein loading was verified with anti β-actin antibodies. The rfesults are representative of three independent experiments.</p

NAHA inhibits invasive behavior of breast cancer cells and capillary morphogenesis of endothelial cells.

<p>(A) Cell adhesion. MDA-MB-231 cells were treated with NAHA (0–50 µM) for 24 hours and cell adhesion to vitronectin determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Each bar represents the mean ± SD of three experiments. * p<0.05. (B) Cell migration. Cell migration of MDA-MB-231 cells was determined after 5 hours of incubation in the presence of NAHA (0–50 µM), as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Each bar represents the mean ± SD of three experiments. * p<0.05. (C) Cell invasion. Invasion of MDA-MB-231 cells through Matrigel was determined after 24 hours of incubation in the presence of NAHA (0–50 µM) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Each bar represents the mean ± SD of three experiments. * p<0.05. (D) uPA secretion. MDA-MB-231 cells were treated with NAHA (0–50 µM) for 24 hours, and the expression of uPA detected in conditioned media from the same amount of cells with anti-uPA antibody by Western blot analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. The results are representative of three independent experiments. (E) MDA-MB-231 cells were treated with NAHA (0–50 µM) for 24 hours, media collected and secretion of VEGF determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Each bar represents the mean ± SD of three experiments. * p<0.05. (F) HAECs were treated with NAHA (0–50 µM) for 16 hours. Capillary morphogenesis was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034283#s4" target="_blank"><i>Materials and Methods</i></a>. Each bar represents the mean ± SD of three experiments. * p<0.05.</p