Alginate Oligosaccharide DP5 Exhibits Antitumor Effects in Osteosarcoma Patients following Surgery

Abstract

Osteosarcoma is a malignant musculoskeletal tumor that has high-rate morbidity and mortality worldwide. Alginate oligosaccharide (AOS), a natural product, has antitumor activities and may have therapeutic effects in osteosarcoma, the molecular mechanisms of which remain unclear. AOS was prepared from alginate sodium using alginate lyase. The fractions of AOS were further isolated by size-exclusion chromatography and verified by electrospray ionization mass spectrometry (ESI-MS). Osteosarcoma patients were enrolled in the study and assigned into two groups: AOS (AG, oral administration of 10-mg AOS daily) and control groups (CG, placebo). Preoperative and postoperative clinical data were investigated and analyzed. Four different degrees of polymerizations (DPs) were isolated and denominated as DP2, DP3, DP4, and DP5. Among these polymers, only DP5 showed antitumor functions on osteosarcoma cells. Before surgery and the outcome of primary end point after surgery, no significant differences were observed for clinical data and tumor size between the AG and CG groups (P > 0.05). After 2-year therapy, the mean tumor volume was 214.6 ± 145.7 c.c. in AG and 467.2 ± 225.3 c.c in CG (P < 0.01). The rate of local recurrence was 44.9 and 68.7% in AG and CG, respectively (P < 0.01). AOS treatment resulted in the increase in serum levels of SOD, GSH, HDL-C, and reduction in the levels of interleukin-1 (IL-1) beta and IL-6; the ratios of AST/ALT; and triglycerides, total cholesterol (TC), low-density lipoprotein cholesterol LDL-C, and malondialdehyde (MDA) (P < 0.05). AOS reduces osteosarcoma progression, which is associated with improvement in antioxidant and anti-inflammatory capacities of patients, and may be used as a potential drug for osteosarcoma therapy.

Keywords: alginate oligosaccharide; anti-inflammatory; antioxidant; local recurrence; osteosarcoma; tumor volume.

Figure 1

Gel chromatograms of the fractions from digested AOS. A 6 × 60 cm Superdex 75 prep grade column was eluted by 50 mm sodium phosphate buffer, pH 7.5 and 300 mM at a linear flow rate of 0.5 cm/min. Each fraction was measured at 280 nm via ultraviolet absorption.

Figure 2

ESI MASS spectrometry analysis of the polymer degrees of digested AOS from alginate sodium under the conditions that produced mass spectra with M + H⁺. (A) mass spectra were visualized following the separation of DP2 ([M + H]⁺ = 415 Da); (B) mass spectra were visualized following the separation of DP3 ([M + H]⁺ = 613 Da); (C) mass spectra were visualized following the separation of DP4 ([M + H]⁺ = 811 Da); and (D) mass spectra were visualized following the separation of DP5 ([M + H]⁺ = 1,009 Da).

Figure 3

Real-time analysis for the effects of AOS on the growth of MG-63 osteosarcoma cells. (A) The effects of AOS DP2 on the growth of MG-63 cells. (B) The effects of AOS DP3 on the growth of MG-63 cells. (C) The effects of AOS DP4 on the growth of MG-63 cells. (D) The effects of AOS DP5 on the growth of MG-63 cells. (E) The effects of polymer mixture on the growth of MG-63 cells.

Figure 4

MRI images of OS at different stages. (A) OS at G1 grade. (B) OS at G2 grade. (C) OS at G3 grade. (D) OS at G4 grade.

Figure 5

H&E staining analysis of OS samples at different stages. (A) The OS consists of an atypical round to cell proliferation with osteoid deposition at G1 stage (H&E stain ×200). (B) OS cells shape variable with chromatic nuclei and mitosis fields at G2 stages. (C) Spindle cell neoplasm has high cellularity, abnormal mitotic characters and atypical nuclear at G3 stage. (D) The OS forms a highly-cancerous and high-mortality bone tumor at G4 stage.

Figure 6

Histopathological examination of intraosseous vascular granulation tissue at different stages of OS (H&E stain ×200). (A) Internal hemorrhage were observed within the bone tissues at G1 stage. (B) Granulation tissue and responsive hyperosteogeny increased at G2 stage. (C) Granulation tissue and responsive hyperosteogeny increased further at G3 stage. (D) Giant granulation tissue and responsive hyperosteogeny at G4 stage.

Figure 7

The concentrations of IL-1 beta and IL-6 in blood samples. (A) The serum level of IL-1 beta. (B) The serum level of IL-6. All data were presented as mean values ± SD. There are statistically significant differences if P < 0.05.

Figure 8

Cartoon illustrations of alginate oligosaccharide (AOS) inhibiting the growth of osteosarcoma and recurrence. AOS may bind the receptor on the membrane of osteosarcoma cells and activate cancer suppressors, which improve antioxidant and anti-inflammatory properties of OS patients.