Autophagy is a self-digestive process that degrades the cytoplasmic constituents. Immunomodulatory protein, one major bioactive component of Ganoderma, has antitumor activity. In this study, recombinant fungal immunomodulatory protein, GMI, was cloned from Ganoderma microsporum and purified. We demonstrated that GMI induces lung cancer cell death by activating autophagy, but does not induce apoptotic cell death. On western blot, GMI increased LC3 conversion and decreased p53 expression in a time- and concentration-dependent manner. Cytoplasmic calcium chelator BAPTA-AM was used to prove that GMI promotes autophagy via a calcium-mediated signaling pathway. 3-methyladenine (3-MA), an autophagy inhibitor, enhanced the cytotoxicity of GMI on cell viability assay. Using VZV-G pseudotyped lentivirus-shRNA system for autophagy-related genes silencing, the capabilities of GMI to reduce cell viability and colony formation were abolished in autophagy-defective cells. Furthermore, GMI did not stimulate apoptosis after blocking of autophagy by 3-MA or shRNA knockdown system. In xenograft studies, oral administration of GMI inhibited the tumor growth and induced autophagy significantly in nude mice that had received a subcutaneous injection of A549 cells. This is the first study to reveal the novel function of GMI in activating autophagy. GMI may be a potential chemopreventive agent against non-small cell lung cancer.

Heat-killed lactic acid bacteria not only possess immunomodulatory functions but also provide the advantages of longer product shelf life, easier storage, and more convenient transportation. To establish appropriate heat treatments for the industrial preparation of probiotics with immunomodulatory effects, 4 different heat treatments were used to kill 11 strains of lactic acid bacteria. Comparisons among the strains and with viable forms were carried out in terms of immunomodulatory activity and adhesion to Caco-2 cells. Field-emission scanning electron microscope (FE-SEM) was employed to observe morphological changes in bacteria after heating. Among the 11 viable strains, Lactobacillus gasseri AI-88 was the strongest inducer of interferon-gamma (IFN)-γ and interleukin (IL)-12p70 production. However, after heat treatments its stimulatory ability was attenuated. Heat-killed Enterococcus faecalis YM-73 and Lactobacillus salivarius AP-32 strains showed enhanced stimulation of IFN-γ and IL-12p70 secretion and coincidental decrease in IL-13 production. The adhesion of lactic acid bacteria to Caco-2 cells decreased with increases in temperature. However, heat exposure did not influence immunomodulatory activity. With rising temperature, roughness and unevenness of bacterial cell surfaces increased significantly. The results indicated that heat-killed E. faecalis YM-73 and L. salivarius AP-32 have immunomodulatory ability via increased Th1-associated cytokines and reduced Th2-associated cytokines, switching the immune response from a Th2 toward a Th1 response. These 2 heat-killed strains have the potential for development as commercial products.

Background:There are close links between chemotherapy-induced intestinal mucositis and microbiota dysbiosis. Previous studies indicated that D-methionine was an excellent candidate for a chemopreventive agent. Here, we investigated the effects of D-methionine on cisplatin-induced mucositis.Materials and methods:Male Wistar rats (176–200 g, 6 weeks old) were given cisplatin (5 mg/kg) and treated with D-methionine (300 mg/kg). Histopathological, digestive enzymes activity, oxidative/antioxidant status, proinflammatory/anti-inflammatory cytokines in intestinal tissues were measured. Next-generation sequencing technologies were also performed to investigate the gut microbial ecology.Results:D-methionine administration increased villus length and crypt depth and improved digestive enzyme (leucine aminopeptidase, sucrose and alkaline phosphatase) activities in the brush-border membrane of cisplatin-treated rats (p < 0.05). Furthermore, D-methionine significantly attenuated oxidative stress and inflammatory reaction and increased interleukin-10 levels in cisplatin-induced intestinal mucositis (p < 0.05). Cisplatin administration resulted in high relative abundances of Deferribacteres and Proteobacteria and a low diversity of the microbiota when compared with control groups, D-methionine only and cisplatin plus D-methionine. Cisplatin markedly increased comparative abundances of Bacteroides caccae, Escherichia coli, Mucispirillum schaedleri, Bacteroides uniformis and Desulfovibrio C21-c20, while Lactobacillus was almost completely depleted, compared with the control group. There were higher abundances of Lactobacillus, Lachnospiraceae, and Clostridium butyrium in cisplatin plus D-methionine rats than in cisplatin rats. D-methionine treatment alone significantly increased the number of Lactobacillus reuteri.Conclusion:D-methionine protects against cisplatin-induced intestinal damage through antioxidative and anti-inflammatory effects. By enhancing growth of beneficial bacteria (Lachnospiraceae and Lactobacillus), D-methionine attenuates gut microbiome imbalance caused by cisplatin and maintains gut homeostasis.

FIP-fve is an immunomodulatory protein isolated from Flammulina velutipes that possesses anti-inflammatory and immunomodulatory activities. However, little is known about its anticancer effects. It is suppressed cell proliferation of A549 lung cancer cells on MTT assay following 48 h treatment of FIP-fve. FIP-fve treatment also resulted in cell cycle arrest but not apoptosis on flow cytometry. This immunomodulatory protein was observed to increase p53 expression, as well as the expression of its downstream gene p21, on Western blot. FIP-fve inhibited migration of A549 cells on wound healing assay and decreased filopodia fiber formation on labeling with Texas Red-X phalloidin. To confirm the effect of FIP-fve on the role of Rac1 in filopodia formation, we investigated the activity of Rac1 in A549 cells following FIP-fve treatment. FIP-fve inhibited EGF-induced activation of Rac1. We demonstrated that FIP-fve decreases RACGAP1 mRNA and protein levels on RT-PCR and Western blot. In addition, the reporter activity of RACGAP1 was reduced by FIP-fve on RacGAP1 promoter assay. Silencing of RacGAP1 decreased cell migration, and overexpression of RacGAP1 increased cell migration in A549 cells. In conclusion, FIP-fve inhibits lung cancer cell migration via RacGAP1 and suppresses the proliferation of A549 via p53 activation pathway.

Matrix metalloproteinase 9 (MMP-9) has been implicated in airway injury in chronic obstructive pulmonary disease (COPD), lung inflammation, and lung cancer and plays a major role in tumor necrosis factor-α (TNF-α)-stimulated tumor invasion and lung inflammation. MMP-9 activity is promoted by the pro-inflammatory cytokine TNF-α. GMI, cloned from Ganoderma microsporum and purified, is one of the recombinant fungal immunomodulatory proteins. To understand the molecular mechanisms involved in the suppression of TNF-α-mediated tumor invasion and inflammation, GMI modulation of this pathway was investigated in human alveolar epithelial A549 cells in this study. GMI exhibited an inhibitory effect on TNF-α-induced invasion, with GMI treatment and TNF-α exposure presenting the most anti-invasive properties on Boyden chamber assay. GMI reduced TNF-α-induced MMP-9 activities on gelatin zymography assay through inhibition of MMP-9 transcriptional activity. RT-PCR and MMP-9 promoter luciferase analysis revealed that GMI inhibits the transcription of MMP-9 mRNA. Moreover, in vitro and in vivo binding experiments, an electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation assay (ChIP) demonstrated that GMI suppresses DNA binding of nuclear factor (NF)-κB transcription factors to MMP-9 promoter. Western blot analysis indicated that GMI blocks the phosphorylation and degradation of IκBα, which in turn leads to suppression of the phosphorylation and nuclear translocation of p65. Thus, overall, our results indicated that GMI mediates antitumor invasion and anti-inflammatory effects through modulation of NF-κB/MMP-9 pathways.