Hepatocellular carcinoma is a solid tumor that exhibits severe angiogenesis, cell cycle dysregulation, and evasion of apoptosis. Late diagnosis in a large proportion of patients is making the carcinomas inoperable, and various chemotherapeutic agents are used. Due to the numerous side effects of known chemotherapeutic agents, intensive research is performed on the use of natural products and their synthetic analogues. The authors of the presented study investigated the effect of carboxymethyl chitosan oligosaccharides on the growth of hepatocellular tumors in mice.

To prepare carboxymethyl chitosan oligosaccharides (CM-COS), carboxymethylation of chitosan from snow crab (95% deacetylation degree, molecular weight 195 kDa) followed by enzymatic hydrolysis with chitosanases was performed. In vitro cell toxicity was investigated by MTT method on L-02 (normal liver cells) and BEL-7402 cells (hepatoma carcinoma) at different CM-COS concentrations (100, 500 and 2000 µg/ml). The effect of CM-COS on solid tumors (H22) was investigated in Kunming mice. Aseptic H22 cells were injected subcutaneous into mice for tumor implantation. Mice were divided into three treatment groups and one control group with 10 animals each. For 14 days, animals in the treatment groups were orally administered 50, 100, and 200 mg CM-COS per kg, respectively. On day 15, the animals were sacrificed and tumor, spleen, and thymus tissues were collected and examined. The VEGF and MMP-9 levels in the serum of the mice were determined. In addition, the in vivo and in vitro effects of CM-COS on the phagocytosing capacity of murine peritoneal macrophage cells were tested.


  • Strong inhibition of proliferation of hepatocellular carcinoma cells BEL-7402 by CM-COS in vitro
  • No toxicity towards normal liver cells (L-02) in vitro
  • Dose-dependent inhibition of hepatoma-22 growth, faster tumor growth in control group
  • Increase of spleen and thymus index in mice with tumors
  • Down-regulation of VEGF (induction of angiogenesis) and MMP-9 (induction of metastasis)
  • Induction of necrosis of tumor cells
  • Activation of expression of caspase-3 in tumor tissue and IL-2 in spleen tissue
  • Increase in in vitro cell viability, phagocytic capabilities and nitric oxide production of mouse peritoneal macrophages
  • Increase in in vivo phagocytic capacity of peritoneal macrophages from Kunming mice


The results of this study demonstrate that carboxymethyl chitosan oligosaccharides inhibit liver tumor growth in vitro and in vivo via different mechanisms. The CM-COS inhibit proliferation, induce immunological functions and cell apoptosis and necrosis without being toxic towards normal liver cells. Continued studies are necessary to investigate the potential of carboxymethyl chitosan in cancer immunotherapy further.