The β-glucan from Lentinus edodes suppresses cell proliferation and promotes apoptosis in estrogen receptor positive breast cancers

Breast cancer is now the most common cancer in worldwide women, and novel interventions are needed to overcome the resistance occurring in the estrogen-targeted endocrine therapy. Herein, we demonstrate that the β-glucan from Lentinus edodes (LNT) exhibited a profound inhibition ratio of ∼53% against estrogen receptor positive (ER+) MCF-7 tumor growth in nude mice similar to the positive control of cisplatin. Immunohistochemistry images showed that LNT evidently suppressed cell proliferation and promoted apoptosis in MCF-7 tumor tissues. The Western blotting analysis indicated that LNT up-regulated the tumor suppressor p53, phosphorylated extracellular signal-regulated kinase1/2 (p-ERK1/2), cleaved-Caspase 3 and poly [ADP (ribose)] polymerase 1 (PARP 1) protein levels, and reduced the expression of mouse double minute 2 (MDM2), telomerase reverse transcriptase (TERT), nuclear factor-kappa B (NF-κB) p65, B-cell lymphoma-2 (Bcl-2), estrogen receptor α (ERα), etc. in tumor tissues. Moreover, LNT significantly suppressed phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (p-Akt) and mammalian target of rapamycin (mTOR) protein levels. It was thus proposed that LNT inhibited MCF-7 tumor growth through suppressing cell proliferation and enhancing apoptosis possibly via multiple pathways such as PI3K/Akt/mTOR, NF-κB-, ERK-, ERα-, caspase- and p53-dependent pathways. Interestingly, the cell viability assay, siRNA transfection, Western blotting and flow cytometric analysis suggested that LNT targeted p53/ERα to only suppress cell proliferation via cell cycle arrest at G2/M phase without apoptosis in vitro. The big difference between in vivo and in vitro data suggested that the immune responses triggered by the polysaccharide should mainly contribute to the apoptotic effect in vivo. Overall, this work provides a novel strategy to treat ER+ breast cancers by using a naturally occurring β-glucan from mushrooms.


RNA processing, microarray hybridization and data analysis
Raw and normalised gene expression files are available from BGI Tech (China). Total RNA was extracted from tumor tissues using RNAeasy mini kit (Qiagen). There were two methods to treat total RNA. Oligo (dT) magnetic beads were used to select mRNA with polyA tail, or hybridized the rRNA with DNA probe and digested the DNA/RNA hybrid strand, followed by DNase I reaction to remove DNA probe. Then the target RNA after purification was obtained. Fragmented the target RNA and reversed transcription to double-strand cDNA (dscDNA) by N6 random primer. Ended repair the dscDNA with phosphate at 5' end and stickinessed 'A' at 3' end, then ligate and adaptor with stickiness 'T' at 3' end to the dscDNA. Two specific primers were used to amplify the ligation product. The PCR product was denatured by heat and the single strand DNA was cyclized by splint oligo and DNA ligase. Arrays were scanned using an Illumina iScan (Illumina). After getting raw data, each bioinformitics analysis were done as the client appoints on contract. We used Bowtie2 [1] to map clean reads to reference gene and use HISAT [2] to reference genome. Gene Quantification was analyzed by RSEM [3]. Differential gene expression analysis was performed using NOISeq method [4] between control and treated groups. Functional enrichment analysis of differentially expressed genes was performed using KEGG [5]. Heatmaps of differentially expressed genes belonging to clusters enriched were generated using log2 fold change expression values calculated between control and treated conditions. Two samples of each group were used to experiment.

Confocal microscopy
MCF-7 cells were seeded into the confocal dish at a density of 2×10 5 cells/dish for 24 h, followed by incubation at 4°C for 40 min with PE-conjugated antidectin-1 antibody (RH1; Bio-legend; San Diego, USA), and PE-conjugated anti-CD11b (M1/70; Biolegend; San Diego, USA). At the end of incubation, the medium was removed, and the cells were rinsed three times with PBS.
The nuclei were then stained with Hoechst 33342 (10 μg/ mL) for 20 min at room temperature in the dark. Then, the cells were washed two times with PBS followed by fixing with 1 mL of 4% paraformaldehyde for 10 min. Finally, the stained cells were subjected to confocal microscopy observation after washing with PBS using laser excitation at 405, 488 and 543 nm.

Western blotting
The cell or tumor tissue lysates were mixed with 4 × SDS sample buffer and denatured in boiling water for 5 min. Aliquots of 20~60 μg of denatured total proteins were separated by SDS-PAGE on a 12% or 10% polyacrylamide gel and then electrically transferred onto a PVDF membrane (0.45 μm, Millipore). After blocking with 5% (w/v) BSA in TBS (10 mM Tris-HCl (pH 8.0) and 150 mM NaCl) containing 0.1% Tween 20 at room temperature for 1 h, the membranes were then incubated with the corresponding specific primary antibodies including phosphorylated NF-κB p65 (p-p65), p-JNK and p-p38 overnight at 4°C. The antibodies against β-Actin (I-19) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as the loading control. The reactive bands were visualized with a horseradish peroxidase (HRP)-conjugated secondary antibody (Biosharp) for 50 min via enhanced chemiluminescence (ECL) Western blotting detection reagent on a ChemiDoc-It TM imaging system (UVP, America) according to the manufacturer's instructions.

Anti-breast cancer assay in vivo
Exponentially growing MCF-7 cells suspended in PBS were injected subcutaneously into the left flanks of nude mice (5×10 6 cells in 100 μL). When the size of established tumors reached about 200 mm 3 (around two weeks after tumor cells were inoculated), 15 mice were randomized into three groups (n = 5) followed by receiving a daily intraperitoneal injection of 0.9% NaCl (negative group, 15 days), 1 mg/kg LNT (LNT group) and zs-LNT (clinically used LNT injections, zs-LNT group) in a 200 μL volume, respectively. Tumor sizes of mice were measured every two days before sacrifice. The tumor volume was calculated by the formula of tumor volume (mm 3 ) = length × width 2 /2. The mice were finally killed and the local tumors were removed carefully for weighing.