Cultivar-specific markers, mutations, and chimerisim of Cavendish banana somaclonal variants resistant to Fusarium oxysporum f. sp. cubense tropical race 4.

BACKGROUND: The selection of tissue culture-derived somaclonal variants of Giant Cavendish banana (Musa spp., Cavendish sub-group AAA) by the Taiwan Banana Research Institute (TBRI) has resulted in several cultivars resistant to Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), a destructive fungus threatening global banana production. However, the mutations in these somaclonal variants have not yet been determined. We performed an RNA-sequencing (RNA-seq) analysis of three TBRI Foc TR4-resistant cultivars: 'Tai-Chiao No. 5' (TC5), 'Tai-Chiao No. 7' (TC7), and 'Formosana' (FM), as well as their susceptible progenitor 'Pei-Chiao' (PC), to investigate the sequence variations among them and develop cultivar-specific markers. RESULTS: A group of single-nucleotide variants (SNVs) specific to one cultivar were identified from the analysis of RNA-seq data and validated using Sanger sequencing from genomic DNA. Several SNVs were further converted into cleaved amplified polymorphic sequence (CAPS) markers or derived CAPS markers that could identify the three Foc TR4-resistant cultivars among 6 local and 5 international Cavendish cultivars. Compared with PC, the three resistant cultivars showed a loss or alteration of heterozygosity in some chromosomal regions, which appears to be a consequence of single-copy chromosomal deletions. Notably, TC7 and FM shared a common deletion region on chromosome 5; however, different TC7 tissues displayed varying degrees of allele ratios in this region, suggesting the presence of chimerism in TC7. CONCLUSIONS: This work demonstrates that reliable SNV markers of tissue culture-derived and propagated banana cultivars with a triploid genome can be developed through RNA-seq data analysis. Moreover, the analysis of sequence heterozygosity can uncover chromosomal deletions and chimerism in banana somaclonal variants. The markers obtained from this study will assist with the identification of TBRI Cavendish somaclonal variants for the quality control of tissue culture propagation, and the protection of breeders' rights.

WSG, a Glucose-Rich Polysaccharide from Ganoderma lucidum, Combined with Cisplatin Potentiates Inhibition of Lung Cancer In Vitro and In Vivo.

Lung cancer has the highest global mortality rate of any cancer. Although targeted therapeutic drugs are commercially available, the common drug resistance and insensitivity to cisplatin-based chemotherapy, a common clinical treatment for lung cancer, have prompted active research on alternative lung cancer therapies and methods for mitigating cisplatin-related complications. In this study, we investigated the effect of WSG, a glucose-rich, water soluble polysaccharide derived from Ganoderma lucidum, on cisplatin-based treatment for lung cancer. Murine Lewis lung carcinoma (LLC1) cells were injected into C57BL/6 mice subcutaneously and through the tail vein. The combined administration of WSG and cisplatin effectively inhibited tumor growth and the formation of metastatic nodules in the lung tissue of the mice. Moreover, WSG increased the survival rate of mice receiving cisplatin. Co-treatment with WSG and cisplatin induced a synergistic inhibitory effect on the growth of lung cancer cells, enhancing the apoptotic responses mediated by cisplatin. WSG also reduced the cytotoxic effect of cisplatin in both macrophages and normal lung fibroblasts. Our findings suggest that WSG can increase the therapeutic effectiveness of cisplatin. In clinical settings, WSG may be used as an adjuvant or supplementary agent.

Identification of MaWRKY40 and MaDLO1 as Effective Marker Genes for Tracking the Salicylic Acid-Mediated Immune Response in Bananas.

Bananas are among the world's most important cash and staple crops but are threatened by various devastating pathogens. The phytohormone salicylic acid (SA) plays a key role in the regulation of plant immune response. Tracking the expression of SA-responsive marker genes under pathogen infection is important in pathogenesis elucidation. However, the common SA-responsive marker genes are not consistently induced in different banana cultivars or different organs. Here, we conducted transcriptome analysis for SA response of a banana cultivar, 'Pei-Chiao' (Cavendish, AAA genome), and identified three genes, MaWRKY40, MaWRKY70, and Downy Mildew Resistant 6 (DMR6)-Like Oxygenase 1 (MaDLO1) that are robustly induced upon SA treatment in both the leaves and roots. Consistent induction of these three genes by SA treatment was also detected in both the leaves and roots of bananas belonging to different genome types such as 'Tai-Chiao No. 7' (Cavendish, AAA genome), 'Pisang Awak' (ABB genome), and 'Lady Finger' (AA genome). Furthermore, the biotrophic pathogen cucumber mosaic virus elicited the expression of MaWRKY40 and MaDLO1 in infected leaves of susceptible cultivars. The hemibiotrophic fungal pathogen Fusarium oxysporum f. sp. cubense tropical race 4 (TR4) also consistently induced the expression of MaWRKY40 and MaDLO1 in the infected roots of the F. oxysporum f. sp. cubense TR4-resistant cultivar. These results indicate that MaWRKY40 and MaDLO1 can be used as reliable SA-responsive marker genes for the study of plant immunity in banana. Revealing SA-responsive marker genes provides a stepping stone for further studies in banana resistance to pathogens.

Effects of WSG, a polysaccharide from Ganoderma lucidum, on suppressing cell growth and mobility of lung cancer.

WSG is a water soluble polysaccharides isolated from Ganoderma lucidum. In this study, we showed that WSG, a glucose-rich polysaccharide with an average molecular mass of approximately 1000 kDa, effectively inhibited cell viability and mobility of lung cancer cells. Functional studies revealed that WSG reduced phosphorylation of ERK1/2 in cells upon either EGF or TGFß stimulation. WSG also inhibited phosphorylation of multiple intracellular signaling molecules such as FAK, AKT and Smad2. Mechanistically, we demonstrated that WSG induced degradation of TGFß and EGF receptors via proteasome and lysosome, respectively. Moreover, we found that WSG significantly suppressed lung tumor growth, reduced the size of metastatic nodules in the lungs and prolonged the survival of LLC1-bearing mice. Our findings suggested that WSG may have potential as a therapeutic intervention for treatment of lung cancer.

Widespread Exon Junction Complex Footprints in the RNA Degradome Mark mRNA Degradation before Steady State Translation.

Exon junction complexes (EJCs) are deposited on mRNAs during splicing and displaced by ribosomes during the pioneer round of translation. Nonsense-mediated mRNA decay (NMD) degrades EJC-bound mRNA, but the lack of suitable methodology has prevented the identification of other degradation pathways. Here, we show that the RNA degradomes of Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), worm (Caenorhabditis elegans), and human (Homo sapiens) cells exhibit an enrichment of 5' monophosphate (5'P) ends of degradation intermediates that map to the canonical EJC region. Inhibition of 5' to 3' exoribonuclease activity and overexpression of an EJC disassembly factor in Arabidopsis reduced the accumulation of these 5'P ends, supporting the notion that they are in vivo EJC footprints. Hundreds of Arabidopsis NMD targets possess evident EJC footprints, validating their degradation during the pioneer round of translation. In addition to premature termination codons, plant microRNAs can also direct the degradation of EJC-bound mRNAs. However, the production of EJC footprints from NMD but not microRNA targets requires the NMD factor SUPPRESSOR WITH MORPHOLOGICAL EFFECT ON GENITALIA PROTEIN7. Together, our results demonstrating in vivo EJC footprinting in Arabidopsis unravel the composition of the RNA degradome and provide a new avenue for studying NMD and other mechanisms targeting EJC-bound mRNAs for degradation before steady state translation.

Fucoidan induces Toll-like receptor 4-regulated reactive oxygen species and promotes endoplasmic reticulum stress-mediated apoptosis in lung cancer.

Fucoidan, a sulfated polysaccharide extracted from brown algae, exhibits anti-cancer activity. However, the effects and mechanism of fucoidan-induced apoptosis via endoplasmic reticulum (ER) stress is unclear. In this study, we demonstrated that fucoidan prevents tumorigenesis and reduces tumor size in LLC1-xenograft male C57BL/6 mice. Fucoidan induces an ER stress response by activating the PERK-ATF4-CHOP pathway, resulting in apoptotic cell death in vitro and in vivo. Furthermore, ATF4 knockdown abolishes fucoidan-induced CHOP expression and rescues cell viability. Specifically, fucoidan increases intracellular reactive oxygen species (ROS), which increase ATF4 and CHOP in lung cancer cells. Using the ROS scavenger N-acetyl-l-cysteine (NAC), we found that ROS generation is involved in fucoidan-induced ER stress-mediated apoptosis. Moreover, via Toll-like receptor 4 (TLR4) knockdown, we demonstrated that fucoidan-induced ROS and CHOP expression were attenuated. Our study is the first to identify a novel mechanism for the antitumor activity of fucoidan. We showed that fucoidan inhibits tumor viability by activating the TLR4/ROS/ER stress axis and the downstream PERK-ATF4-CHOP pathway, leading to apoptosis and suppression of lung cancer cell progression. Together, these results indicate that fucoidan is a potential preventive and therapeutic agent for lung cancer that acts via activation of ROS-dependent ER stress pathways.

Induction of Cbl-dependent epidermal growth factor receptor degradation in Ling Zhi-8 suppressed lung cancer.

Activating mutation of epidermal growth factor receptor (EGFR) is correlated with malignant lung tumor. In our study, we demonstrated that recombinant LZ-8 (rLZ-8), a medicinal mushroom Ganoderma lucidum protein, induced cell cycle arrest and apoptosis by downregulating the expression of wild-type and mutated EGFR and inhibiting EGFR downstream effectors, AKT and ERK1/2 in lung cancer cells. We showed that rLZ-8 effectively inhibited lung cancer progression and suppressed EGFR expression of lung tumor lesions in mouse model. Functional studies revealed that rLZ-8 reduced the amount of EGFR in cell membranes by altering EGFR localization to enhance the EGF-induced degradation of EGFR. Mechanistically, we demonstrated that rLZ-8 bound to EGFR to induce EGFR autophosphorylation at tyrosine1045 and trigger ubiquitination by inducing the formation of EGFR/Cbl complexes, resulting in the degradation of EGFR; however, Cbl-shRNA abolished rLZ-8-induced EGFR degradation. We provide the first evidence showing that rLZ-8 inhibits growth and induces apoptosis of lung cancer cells by promoting EGFR degradation. The current findings therefore suggest a novel anti-cancer function of rLZ-8 that targeting EGFR overexpression or mutation as well as EGFR-dependent processes in cancer cells.

Fucose-containing fraction of Ling-Zhi enhances lipid rafts-dependent ubiquitination of TGFß receptor degradation and attenuates breast cancer tumorigenesis.

Ganoderma lucidum exerts antitumor activity, but the mechanism of G. lucidum polysaccharides on cancer is unclear. Here, we demonstrated that a fucose-containing fraction of Ling-Zhi (FFLZ) reduced tumor size and suppressed metastasis in vivo. Furthermore, FFLZ inhibited breast cancer cell migration and altered the epithelial-to-mesenchymal transition (EMT) phenotype. Transforming growth factor-ß receptor (TGFR) pathways act as key mediators to promote tumor progression and metastasis. We found that FFLZ down-regulated TGFR and downstream signaling pathways, including the phosphorylation of Smad2/3 and the expression of Smad4. In an investigation of the underlying mechanisms, we found that FFLZ enhanced the Smurf2-dependent ubiquitination of TGFR by disrupting the balance of the lipid rafts, promoted the "re-localization" of the TGFR to the caveolae, and facilitated the degradation of TGFR. Together, our data indicated that FFLZ is associated with the inhibition of EMT and the prevention of metastasis by promoting ubiquitination-dependent TGFR degradation and abolishing TGFR signaling pathways. Moreover, the combination of FFLZ and trastuzumab synergistically inhibited the viability of certain trastuzumab-resistant human breast cancer cells. In summary, our current findings indicate that FFLZ is a potential therapeutic or dietary supplemental agent for cancer patients and that it functions via the caveolin-1/Smad7/Smurf2-dependent ubiquitin-mediated degradation of TGFR.

Fucoidan inhibition of lung cancer in vivo and in vitro : role of the Smurf2-dependent ubiquitin proteasome pathway in TGFß receptor degradation.

Fucoidan, a polysaccharide extracted from brown seaweeds, reduces tumor cell proliferation. In this study, we demonstrate that fucoidan reduces tumor size in LLC1-xenograft male C57BL/6 mice. Moreover, we found that LLC1-bearing mice continuously fed fucoidan showed greater antitumor activity than mice with discontinuous feeding. Fucoidan inhibited the in vitro growth of lung cancer cells. Transforming growth factor ß (TGFß) receptors (TGFRs) play important roles in the regulation of proliferation and progression, and high TGFRI expression in lung cancer specimens is associated with a worse prognosis. Herein, using lung cancer cells, we found that fucoidan effectively reduces TGFRI and TGFRII protein levels in vivo and in vitro. Moreover, fucoidan reduces TGFR downstream signaling events, including those in Smad2/3 and non-Smad pathways: Akt, Erk1/2, and FAK phosphorylation. Furthermore, fucoidan suppresses lung cancer cell mobility upon TGFß stimulation. To elucidate how fucoidan decreases TGFR proteins in lung cancer cells, we found that fucoidan enhances the ubiquitination proteasome pathway (UPP)-mediated degradation of TGFRs in A549 and CL1-5 cells. Mechanistically, fucoidan promotes Smurf2 and Smad7 to conjugate TGFRs, resulting in TGF degradation; however, Smurf2-shRNA abolishes fucoidan-enhanced UPP-mediated TGFR degradation. Our study is the first to identify a novel mechanism for the antitumor activity of fucoidan, namely decreasing tumor growth by modulating the TGFR/Smad7/Smurf2-dependent axis, leading to TGFR protein degradation and inhibition of lung cancer cell progression in vitro and in vivo. Our current findings indicate that fucoidan is a potential therapeutic agent or dietary supplementation for lung cancer, acting via the Smurf2-dependent ubiquitin degradation of TGFß receptors.

Immunization of fucose-containing polysaccharides from Reishi mushroom induces antibodies to tumor-associated Globo H-series epitopes.

Carbohydrate-based vaccines have shown therapeutic efficacy for infectious disease and cancer. The mushroom Ganoderma lucidum (Reishi) containing complex polysaccharides has been used as antitumor supplement, but the mechanism of immune response has rarely been studied. Here, we show that the mice immunized with a l-fucose (Fuc)-enriched Reishi polysaccharide fraction (designated as FMS) induce antibodies against murine Lewis lung carcinoma cells, with increased antibody-mediated cytotoxicity and reduced production of tumor-associated inflammatory mediators (in particular, monocyte chemoattractant protein-1). The mice showed a significant increase in the peritoneal B1 B-cell population, suggesting FMS-mediated anti-glycan IgM production. Furthermore, the glycan microarray analysis of FMS-induced antisera displayed a high specificity toward tumor-associated glycans, with the antigenic structure located in the nonreducing termini (i.e., Fucα1-2Galß1-3GalNAc-R, where Gal, GalNAc, and R represent, respectively, D-galactose, D-N-acetyl galactosamine, and reducing end), typically found in Globo H and related tumor antigens. The composition of FMS contains mainly the backbone of 1,4-mannan and 1,6-α-galactan and through the Fucα1-2Gal, Fucα1-3/4Man, Fucα1-4Xyl, and Fucα1-2Fuc linkages (where Man and Xyl represent d-mannose and d-xylose, respectively), underlying the molecular basis of the FMS-induced IgM antibodies against tumor-specific glycans.

Reishi Protein LZ-8 Induces FOXP3(+) Treg Expansion via a CD45-Dependent Signaling Pathway and Alleviates Acute Intestinal Inflammation in Mice.

LZ-8, an immunomodulatory protein isolated from Ganoderma lucidum (also known as Ling-Zhi or Reishi), has been shown to promote cell proliferation and IL-2 production in T cells. In this study, we show that LZ-8 induces the expansion of both murine and human CD4(+) T cells into FOXP3(+) regulatory T (Treg) cells. LZ-8 treatment was found to stimulate a 4-fold and a 10-fold expansion in the Treg populations of murine and human primary CD4(+) T cells, respectively. In addition, the expression of CTLA-4 and IL-10 was induced in LZ-8-treated CD4(+) T cells. Using neutralizing antibodies and gene-deficient T-cell lines, we also found that LZ-8 promotes Treg expansion through a CD45-mediated signaling pathway and that the CD18-dependent induction of IL-2 was involved in Treg formation and IL-10 production. The suppressive activity of LZ-8 was confirmed using a murine model of DSS-induced colitis; the disease was alleviated by the adoptive transfer of LZ-8-treated CD4(+) T cells. In conclusion, a new regulatory function for LZ-8 was identified, and the molecular mechanisms underlying this function were elucidated.

Fucoidan induces changes in the epithelial to mesenchymal transition and decreases metastasis by enhancing ubiquitin-dependent TGFß receptor degradation in breast cancer.

Fucoidan, a polysaccharide extracted from brown seaweeds, reduces tumor cell proliferation. Fucoidan inhibits the growth of breast cancer cells such as 4T1 and MDA-MB-231 and decreases their cell colony formation. Moreover, fucoidan reduces metastatic lung nodules in 4T1 xenograft female Balb/c mice. The molecular network of transforming growth factor ß (TGFß) receptors (TGFRs) plays an important role in the regulation of the epithelial to mesenchymal transition (EMT) in cancer cells. Using 4T1 and MDA-MB-231 cells, we found that fucoidan effectively reverses TGFR-induced EMT morphological changes, upregulates epithelial markers, downregulates mesenchymal markers and decreases the expression of transcriptional repressors Snail, Slug and Twist. Moreover, fucoidan inhibits migration and invasion during the EMT, suggesting the involvement of TGFR-mediated signaling in breast cancer cells. Fucoidan decreases TGFRI and TGFRII proteins and affects downstream signaling molecules, including Smad2/3 phosphorylation and Smad4 expression. In order to elucidate how fucoidan decreases TGFRI and TGFRII proteins in MDA-MB-231 cells, we investigated ubiquitination activity downregulation of TGFRs. It was found that fucoidan enhances proteasome-mediated degradation/ubiquitination of TGFR. This study is the first to identify a novel mechanism for fucoidan antitumor activity, namely regulation of the EMT via modulation of TGFR/Smad-dependent signaling, which leads to an inhibition of breast cancer cell growth in vitro and in vivo. Our current findings indicate that fucoidan is a potential therapeutic agent for breast cancer and acts via an ubiquitin-dependent degradation pathway that affects the TGFR/Smad/Snail, Slug, Twist and EMT axes.