[Synergistic effect of polysaccharide from Trichoderma pseudokoningii and oxaliplatin on colorectal cancer cells in vitro].

OBJECTIVE: To explore the synergistic inhibitory effect of polysaccharide from Trichoderma pseudokoningii (EPS) and oxaliplatin (Oxa) on colorectal cancer (CRC) HCT116 cells. OBJECTIVE: HCT116 cells were treated with 8 µg/mL Oxa and 100 µg/mL EPS alone or in combination, and the changes in cell viability was assessed with CCK-8 assay. CompuSyn software was used for fitting the Fa-CI curve to evaluate the combined effect of the two agents. Flow cytometry was performed to analyze cell apoptosis and cell cycle changes, and wound healing assay and Transwell assay were used to examine the migration ability of the treated cells. Oxa- and EPS-related genes and CRC-related genes were intersected for protein-protein interaction (PPI) analysis and GO and KEGG enrichment analyses. OBJECTIVE: Treatment with Oxa alone or in combination with EPS significantly inhibited the viability of HCT116 cells in a dose- and time-dependent manner, and the two agents exhibited a significant synergistic effect (CI < 1). The combined treatment with Oxa and EPS resulted in a significantly higher total cell apoptosis rate and a higher percentage of cells in S phase than Oxa alone and the control treatment (P < 0.05). EPS and Oxa alone both inhibited the migration of HCT116 cells, and their combination produced a stronger inhibitory effect. GO enrichment analysis of the key genes related with Oxa, EPS and CRC suggested that these genes were involved mainly in such biological processes as exogenous apoptosis signaling, cell response to chemical stress, and reactive oxygen metabolism; KEGG analysis showed that these genes were involved in the pathways of drug resistance, apoptosis and angiogenesis. OBJECTIVE: EPS and Oxa can synergistically inhibit the proliferation of HCT116 cells possibly through the PI3K-Akt, MAPK, VEGF, and p53 signaling pathways.

Effect of exopolysaccharide from Trichoderma pseudokoningii on proliferation and apoptosis of human colon cancer cell line HCT116 / 中国药理学通报

;Aim To study the effect of exopolysaccharide from Trichoderma pseudokoningii (EPS) on the proliferation and apoptosis of human colon cancer cell line HCT116. Methods The proliferation of HCT116 cells treated with EPS was examined by CCK-8 assay. The effect of EPS on the clone formation of HCT116 cells was detected by crystal violet staining. Cell apoptotic rate was determined by flow cytometry. The changes of mitochondrial membrane potential were observed by JC-1. The expressions of apoptosis-related proteins Bcl-2 and Bax were analyzed by Western blot, and caspase-9, caspase-8 and caspase-3 expressions were detected by the kit. Results EPS dose- and time-dependently inhibited the proliferation of the HCT116 cells in the range of 0 -800 mg • L " 1 . With increase of EPS concentration, the colony-formation ability of HCT116 cells decreased; the proportion of apoptotic cells increased and mitochondrial membrane potential decreased; the expression of Bcl-2 protein decreased, while the expression of Bax protein increased; the ratio of Bcl-2/Bax decreased gradually; caspase-9, caspase-8 and caspase-3 activities significantly increased. Conclusions EPS can inhibit proliferation of HCT116 and induce its apoptosis by up-regulating expression of Bax, caspase-9,caspase-8, caspase-3 and down-regulating the expression of Bcl-2.

Purification and characterization of α-amylase from Trichoderma pseudokoningii.

BACKGROUND: Previous studies have demonstrated that members of Trichoderma are able to generate appreciable amount of extracellular amylase and glucoamylase on soluble potato starch. In this study the α-amylase was purified and characterized from Trichoderma pseudokoningii grown on orange peel under solid state fermentation (SSF). RESULTS: Five α-amylases A1-A5 from Trichodrma pseudokoningii were separated on DEAE-Sepharose column. The homogeneity of α-amylase A4 was detected after chromatography on Sephacryl S-200. α-Amylase A4 had molecular weight of 30 kDa by Sephacryl S-200 and SDS-PAGE. The enzyme had a broad pH optimum ranged from 4.5 to 8.5. The optimum temperature of A4 was 50 °C with high retention of its activity from 30 to 80 °C. The thermal stability of A4 was detected up to 50 °C and the enzyme was highly stable till 80 °C after 1 h incubation. All substrate analogues tested had amylase activity toward A4 ranged from 12 to 100% of its initial activity. The Km and Vmax values of A4 were 4 mg starch/ml and 0.74 µmol reducing sugar, respectively. The most of metals tested caused moderate inhibitory effect, except of Ca2+ and Mg2+ enhanced the activity. Hg2+ and Cd+ 2 strongly inhibited the activity of A4. EDTA as metal chelator caused strong inhibitory effect. CONCLUSIONS: The properties of the purified α-amylase A4 from T. pseudokoningii meet the prerequisites needed for several applications.

Exopolysaccharide from Trichoderma pseudokoningii induces macrophage activation.

In this study, we evaluated the immunomodulatory activity of an exopolysaccharide (EPS) derived from Trichoderma pseudokoningii and investigated the molecular mechanism of EPS-mediated activation of macrophages. Results revealed that EPS could significantly induce the production of nitric oxide (NO), tumor necrosis factor (TNF)-α and interleukin (IL)-1ß and enhance phagocytic activity in RAW 264.7 cells. Immunofluorescence staining indicated that EPS promoted the nuclear translocation of nuclear factor (NF)-κB p65 subunit. Western blot analysis showed that EPS increased the expression of inducible nitric oxide synthase (iNOS) protein, the degradation of IκB-α and the phosphorylation of mitogen-activated protein kinases (MAPKs). Furthermore, pretreatment of RAW 264.7 cells with specific inhibitors of NF-κB and MAPKs significantly attenuated EPS-induced TNF-α and IL-1ß production. EPS also induced the inhibition of cytokine secretion by special antibodies against Toll-like receptor-4 (TLR4) and Dectin-1. These data suggest that EPS from Trichoderma pseudokoningii activates RAW 264.7 cells through NF-κB and MAPKs signaling pathways via TLR4 and Dectin-1.

Exopolysaccharide from Trichoderma pseudokoningii promotes maturation of murine dendritic cells.

Dendritic cells (DCs) are the key regulators of immune responses. In this study, the effect of an exopolysaccharide (EPS) from the culture broth of Trichoderma pseudokoningii on the phenotypic and functional maturation of murine DCs and its underlying molecular mechanisms were investigated. It showed that EPS induced the morphological changes of DCs and the enhanced expression of DCs featured surface molecules CD11c, CD86, CD80 and major histocompatibility complex II (MHC-II). Flow cytometry analysis showed that the treatment with EPS could reduce FITC-dextran uptake by DCs. Sequentially, the results of ELISA indicated that EPS could increase the production of interleukin-12p70 (IL-12p70) in culture supernatant of DCs. Immunofluorescence staining and western blot analysis further revealed that EPS significantly prompted nuclear factor (NF)-κB subunit p65 translocation, IκB-α protein degradation, and p38 mitogen-activated protein kinase (MAPK) phosphorylation. And the production of IL-12p70 was significantly decreased in condition of the inhibition of p38 or NF-κB signaling pathway. These findings suggested that EPS could induce DCs maturation through both p38 MAPK and NF-κB signaling pathways.

Exopolysaccharide from Trichoderma pseudokoningii induces the apoptosis of MCF-7 cells through an intrinsic mitochondrial pathway.

In this study, we reported the anticancer efficacy of exopolysaccharide (EPS) derived from Trichoderma pseudokoningii, on human breast cancer MCF-7 cells. Our results showed that EPS inhibited the proliferation of MCF-7 cells and induced lactic dehydrogenase release by inducing apoptosis and cell arrest at S phase. Further study revealed that EPS-induced apoptosis of MCF-7 cells was associated with alteration of nuclear morphology, disruption of mitochondrial membrane potential and accumulation of intracellular reactive oxygen species. Sequentially, EPS increased the activation of caspase-9 and caspase-3 in a dose-dependent manner; however, caspase-8 remained intact. Western blot analysis revealed that EPS increased the ratio of Bax/Bcl-2 and promoted the release of cytochrome c into the cytoplasm. Taken together, these findings provided evidence that EPS induced the apoptosis of MCF-7 cells through an intrinsic mitochondrial apoptotic pathway and that EPS may therefore be considered as an effective adjuvant agent against human breast cancer.

Limited vegetative compatibility as a cause of somatic recombination in Trichoderma pseudokoningii

With the aim of a better characterization of the somatic recombination process in Trichoderma pseudokoningii, a progeny from crossings between T. pseudokoningii strains contrasting for auxotroph markers was characterized by RAPD markers and PFGE (electrophoretic karyotype). Cytological studies of the conidia, conidiogenesis and heterokaryotic colonies were also performed. The genotypes of the majority of the recombinant strains analyzed were similar to only one of the parental strains and the low frequency of polymorphic RAPD bands suggested that the nuclear fusions may not occur into the heterokaryon. In some heterokaryotic regions the existence of intensely staining hyphae might be related to cell death. We proposed that a mechanism of somatic recombination other than parasexuality might occur, being related to limited vegetative compatibility after postfusion events, as described for other Trichoderma species.

Limited vegetative compatibility as a cause of somatic recombination in Trichoderma pseudokoningii.

With the aim of a better characterization of the somatic recombination process in Trichoderma pseudokoningii, a progeny from crossings between T. pseudokoningii strains contrasting for auxotroph markers was characterized by RAPD markers and PFGE (electrophoretic karyotype). Cytological studies of the conidia, conidiogenesis and heterokaryotic colonies were also performed. The genotypes of the majority of the recombinant strains analyzed were similar to only one of the parental strains and the low frequency of polymorphic RAPD bands suggested that the nuclear fusions may not occur into the heterokaryon. In some heterokaryotic regions the existence of intensely staining hyphae might be related to cell death. We proposed that a mechanism of somatic recombination other than parasexuality might occur, being related to limited vegetative compatibility after postfusion events, as described for other Trichoderma species.