Chitosan Oligosaccharides Inhibit Tumor Progression and Induce Autophagy Through Activation of the p53/mTOR Pathway in Osteosarcoma

Abstract

Background: The use of chitosan oligosaccharides (COS) as a drug carrier is an emerging new strategy for cancer therapy. However, the application of COS in osteosarcoma has not been reported. Methods: Structure and compositions of COS were identified by High-performance liquid chromatography (HPLC) and Electrospray ionization-mass spectrometry (ESI-MS). We investigated the function of COS in inhibiting osteosarcoma by in vitro Cell Counting Kit-8 (CCK-8), colony formation assay, wound healing assay, and transwell assays. Cell cycle and apoptosis induced by COS were analyzed by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assays. The mRFP-GFP-LC3 adenovirus construct and TEM (transmission electron microscopy) were used to directly demonstrate autophagosome formation. RNA-seq analysis, quantitative real-time polymerase chain reaction (QRT-PCR), and western blotting were used to identify the molecular changes caused by COS treatment. The effect of COS treatment on osteosarcoma tumorigenesis in vivo was studied in a xenograft model. Findings: COS fractions with different degrees of polymerization (DP) ranging from 2-5 were identified, which were composed of dimers (GlcNAc-GlcN, 15.7%), trimers (GlcNAc-(GlcN)2, 30.4%), tetramers (GlcNAc-(GlcN)3, 27.2%), pentamers (GlcNAc-(GlcN)4, 14.2%), and pentamers ((GlcNAc)3-(GlcN)2, 5.6%). Cell viability assays demonstrated that COS showed cytotoxic effects on osteosarcoma cells. The 48-hour 50% inhibitory concentrations (IC50) of COS for three osteosarcoma cell lines were 3.65-11.37 mg/ml. Generally, treatment with COS had significant effects on cell cycle arrest, metastasis inhibition, apoptosis and autophagy induction in osteosarcoma cells compared with the control group. COS treatment triggered pro-apoptosis autophagy through p53/mTOR signaling in osteosarcoma cells. Moreover, p53 inhibitor Pifithrin-μ restored the autophagy promoted by COS. The COS also inhibited tumor growth and metastasis in an osteosarcoma xenograft model. More importantly, our results showed that COS could increase sensitivity to chemotherapy of cisplatin (DDP) in vitro. Interpretation: Our data confirmed COS repressed osteosarcoma cell growth, induced apoptosis, and triggered pro-apoptosis autophagy via p53/mTOR signaling in osteosarcoma. And indicated that COS can serve as a potential novel therapeutic candidate for the treatment of osteosarcoma. Funding Statement: The work is supported by the grants from the National Natural Science Foundation of China on Qing-cheng Yang (81872182) and Dong-dong Cheng (81802685), and the Shanghai Sailing Program on Dong-dong Cheng (18YF1418700). Declaration of Interests: The authors declare that they have no conflicts of interest. Ethics Approval Statement: All animal experiments were performed in accordance with a protocol approved by the State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine. All experiments were subject to approval by the Animal Care and Use Committee of Shanghai Cancer Institute (Shanghia, China).