Clitocybe nebularis extract and 5­fluorouracil synergistically inhibit the growth of HT-29 colorectal cancer cells by inducing the S phase arrest.

Although 5-fluorouracil (5-FU) is an important anticancer agent for the treatment of colorectal cancer, drug resistance, and dose-related side effects limit the effectiveness of the treatment. Therefore, developing new pharmaceuticals with effective and low toxicity is critically necessary for cancer therapy. This study aimed to investigate the cytotoxic activity of the Clitocybe nebularis mushroom extract (CN) on HT-29 human colon cancer cells. A series of in vitro experiments were performed on the HT-29, Caco-2, and HEK-293 cells, which includes cytotoxicity, drug interaction, colony formation, cell cycle, and migration assays. In addition, qRT-PCR experiment was also performed to investigate the potential molecular mechanisms of action of CN on the proliferation of colon cancer cell line. Our results show that CN exhibited selective cytotoxic activity on HT-29 and Caco-2 colon cancer cells, whereas no cytotoxic effect was observed on normal HEK-293 cells. With the combination of CN and 5­FU, their cytotoxic activity on HT-29 cells was significantly increased compared to their use alone. In addition, the combination of CN and 5-FU also showed synergistic anticancer activity through cell cycle arrest in the S phase. The results also show that p21, p27, and p53 expression levels increased as a result of CN treatment. Our in vitro findings show that CN has a synergistic effect with 5-FU by inhibiting cell proliferation of colon cancer cells and inducing cell cycle arrest in the S phase.

Comparison of the chemical composition and biological effects of Clitocybe nebularis and Infundibulicybe geotropa.

Clitocybe mushrooms have long been recognized for their various therapeutic potential and medicinal properties. A few members of the genus are considered edible and many others are poisonous. This study investigated the ethanolic extracts obtained from C. nebularis (CN) and I. geotropa (IG) mushrooms for phenolic content and antioxidant, antiproliferative, antimicrobial, and antibiofilm activities. The data from ultra-performance liquid chromatography and Fourier transform infrared spectroscopy analysis of the mushrooms were presented for the first time. According to the results, both ethanolic extracts contain high levels of phenolic (catechin, myricetin, quercetin, rutin, gallic acid, vanillic acid) compounds. Fourier transform infrared spectroscopy results may suggest the presence of clitopycin in CN extract. The ethanol extract of CN scavenged about 79% and the IG 78% of the free 2,2-diphenyl-1-picrylhydrazyl radicals. Additionally, the CN and IG extracts inhibited glutathione-S-transferase by 10%-18% at all concentrations. The CN extract effectively inhibited aldose reductase by 30%-80% at all concentrations. Besides, the CN extract showed promising antiproliferative activity on HT-29 and MCF-7 cell lines. On the other hand, CN and IG extracts displayed inhibitory effects on some multidrug-resistant Gram-positive bacteria and effectively inhibited biofilm production. The obtained results showed that C. nebularis and I. geotropa extracts presented inhibition of biofilm production. Therefore, C. nebularis was demonstrated to be a potential source of natural medicine.

Lectin-Mediated Binding of Engineered Lactococcus lactis to Cancer Cells.

Lectins have been increasingly utilized as carriers for targeted drug delivery based on their specific binding to glycans located on mammalian cells. This study employed two lectins, B subunit of bacterial Shiga holotoxin (Stx1B) and fungal Clitocybe nebularis lectin (CNL), for surface display on the lactic acid bacterium Lactococcus lactis. The specific adhesion of these engineered, lectin-displaying L. lactis to cancer cells was evaluated. The expression and surface display of both lectins on L. lactis were demonstrated by western blotting and flow cytometry, respectively. MTS assays revealed that recombinant Stx1B had no effect on Caco-2 cell viability at concentrations of ≤25 µg/mL, whereas CNL was non-toxic even at relatively high concentrations of ≤250 µg/mL. Stx1B bound to Caco-2, HT-29 and HeLa cells after 1 h of incubation. CNL bound to Caco-2 cells and recognized several glycoproteins in HT-29 and Caco-2 cell homogenates of which a 70 kDa protein predominated. Confocal microscopy revealed adhesion of Stx1B-displaying L. lactis to HeLa, Caco-2, and, to a lesser extent, HT-29 cells; CNL-displaying L. lactis showed a relatively similar level of adherence to HT-29 and Caco-2 cells. Thus, lectin-displaying L. lactis might serve as a carrier in targeted drug delivery when coupled to a therapeutic moiety.

Chemical Constituents of the Fruiting Bodies of Clitocybe nebularis and Their Antifungal Activity.

During a continuing search for antimicrobial substances from Korean native wild mushroom extracts, we found that the methanolic extract of the fruiting body of Clitocybe nebularis exhibited mild antifungal activity against pathogenic fungi. Therefore we evaluated the antifungal substances and other chemical components of the fruiting body of Clitocybe nebularis, which led to the isolation of nebularine, phenylacetic acid, purine, uridine, adenine, uracil, benzoic acid, and mannitol. Nebularine showed mild antifungal activity against Magnaphorthe grisea and Trichophyton mentagrophytes, and phenylacetic acid potently inhibited the growth of Pythium ultiumand displayed moderate antifungal activity against Magnaphorthe grisea, Botrytis cinerea, and Trichophyton mentagrophytes. The other isolated compounds showed no antimicrobial activity.

Chemical Constituents of the Fruiting Bodies of Clitocybe nebularis and Their Antifungal Activity

During a continuing search for antimicrobial substances from Korean native wild mushroom extracts, we found that the methanolic extract of the fruiting body of Clitocybe nebularis exhibited mild antifungal activity against pathogenic fungi. Therefore we evaluated the antifungal substances and other chemical components of the fruiting body of Clitocybe nebularis, which led to the isolation of nebularine, phenylacetic acid, purine, uridine, adenine, uracil, benzoic acid, and mannitol. Nebularine showed mild antifungal activity against Magnaphorthe grisea and Trichophyton mentagrophytes, and phenylacetic acid potently inhibited the growth of Pythium ultiumand displayed moderate antifungal activity against Magnaphorthe grisea, Botrytis cinerea, and Trichophyton mentagrophytes. The other isolated compounds showed no antimicrobial activity.