Study on Anti-Acute Mononuclear Leukemia Activity and Mechanism of Lentinus Edodes Derived β-Glucan/ Selenium Nanoparticles Composites

Objective: Acute monocytic leukemia (AMoL) is the M5 subtype of acute myeloid leukemia (AML) which has the characteristics of high degree of malignancy, prone to extramedullary infiltration, poor efficacy of conventional chemotherapy, easy recurrence and so on. Besides, chemotherapy has large side effects and high cost. Therefore, it is urgent to find natural, non-toxic and cheap drugs for the treatment of acute monocytic leukemia. We evaluated the anti-acute mononuclear leukemia activities of selenium nanoparticles embedded in lentinan in vivo and in vitro, and explored the mechanism of how AMoL cells react to it. It is expected to provide a new method and basis for the treatment of acute mononuclear leukemia.

Methods: Utilizing the aggregation and assembly behavior of Lentinus edodes extracted rigid tri-helical β-glucan (Lentinan, LNT) in aqueous solution, cooperative research group prepared Lentinus β-glucan/Selenium nanoparticle complex called LNT-Se by encapsulating selenium nanoparticles (SeNPs) in the cavity of LNT. CCK-8 and flow cytometry were used to detect cytotoxicity, apoptosis rate and cell cycle changes after LNT-Se treatment. Single cell microfluidics chip was used to observe the fluorescence intensity of green fluorescent protein (GFP) of single cell treated by drug. Transmission electron microscopy (TEM) was used to detect ultrastructural changes of THP-1. Coumarin 6 (C6) was used to modify LNT-Se, flow cytometry and confocal microscopy were used to detect drug uptake and primary targeting organelles in AML-M5 cells treated with C6-LNT-Se. The changes of reactive oxygen species (ROS) levels in AML-M5 cells after LNT-Se treatment were detected by flow cytometry. Oxidation-related indexes were further detected, such as malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px). The RNA extracted from untreated and LNT-Se-treated THP-1 cells was sequenced by mRNA-seq. AML-M5 patient-derived tumor xenograft (PDX) leukemia mouse model was established to evaluate the efficacy of LNT-Se in vivo. Serum was collected for detecting liver and kidney functions. Tissue sections were stained by hematoxylin-eosin staining (HE). Specific molecules on the surface of human leukemia cells were detected by immunohistochemistry (IHC) to investigate the efficacy of LNT-Se.

Results: SeNPs were successfully embedded into LNT hollow nanotubes and were stably dispersed in water. LNT-Se could significantly inhibit the proliferation of AML-M5 cell lines and had no obvious toxic effect on normal cell lines. LNT-Se could promote the apoptosis of AML-M5 cells and block the cell cycle in the G2/M phase. 50μg/ mL LNT-Se could promote more than 40% apoptosis rate in AML-M5 patient cells, while only about 10% in healthy volunteer cells. At the single cell microfluidic level, the green fluorescence of THP-1 GFP ⁺ cells decreased rapidly after 2mg/mL LNT-Se treatment for 2h. After LNT-Se treatment, THP-1 cells showed obvious apoptotic vacuolation, nuclear condensation, chromatin agglutination under TEM. AML-M5 cells could take up C6-LNT-Se and reached the highest uptake at 2h, and C6 fluorescence sites mainly overlapped with lysosomal fluorescence. LNT-Se could decrease intracellular ROS level and MDA content, and increase the activity of GPX in supernatant, but the MDA content and SOD activity in supernatant had no significant changes. Differential gene analysis of transcriptome showed that the first 12 genes were all mitochondrial coding genes. Enrichment analysis showed that TNF, MAPK and NF-κB pathways were up-regulated and oxidative phosphorylation pathways were down-regulated. The experimental results of leukemia PDX animal model showed that the weight loss of mice was slowed down after LNT-Se treatment, and the tumor burden was reduced. Compared with normal mice, there was no significant difference in liver and kidney function after LNT-Se administration, and no pathological changes were observed in viscera.

Conclusions: LNT-Se had a good anti-acute mononuclear leukemia effect in vitro and in vivo, and could be absorbed by cells, and the primary target organelle was lysosome. Both in vivo and in vitro experiments suggested that LNT-Se was less toxic. LNT-Se had antioxidant activity and might exert anti-AML-M5 activity through TNF, MAPK and NF-κB pathways. These results indicated that LNT-Se could be used as an effective anti-AML-M5 drug.