Induction of granulocytic differentiation in acute promyelocytic leukemia cells (HL-60) by water-soluble chitosan oligomer

Abstract

Water-soluble chitosan oligomer (WSCO) has been reported to have anticancer activity, immuno-enhancing effect and antimicrobial activity. However, other biological activities are unknown. Herein, we have shown that WSCO is able to inhibit proliferation of human leukemia HL-60 cells and induce these cells to differentiate. Treatment with WSCO for 4 days resulted in a concentration-dependent reduction in HL-60 cell growth as measured by cell counting and MTT assay. This effect was accompanied by a marked increase in the proportion of G₀/G₁ cells as measured by flow cytometry. WSCO also induced differentiation of the cells as measured by phorbol ester-dependent reduction of NBT, morphological changes as examined by Wright-Giemsa staining and expression of CD11b but not of CD14 as analysed by flow cytometry, indicating differentiation of HL-60 cells toward granulocyte-like cells. A combination of low dose of WSCO with all-trans retinoic acid, a differentiating agent toward granulocyte-like cells, exhibited a synergistic effect on the differentiation. In addition, treatment of HL-60 cells with WSCO for 6 or 8 days resulted in the induction of apoptosis as assayed qualitatively by agarose gel electrophoresis and quantitatively by Annexin V technique using flow cytometry. Collectively, there is a potential for WSCO in the treatment of myeloid leukemia.

Introduction

The conventional approach to treatment of acute myeloid leukemia (AML) has been the use of chemotherapy, which is cytotoxic to malignant clones, is also cytodestructive to normal cells. In addition, some leukemic cells develop resistance to chemotherapy and are therefore difficult to eradicate. Differentiation therapy, which is associated with less adverse effects, has been tested as a leukemia treatment modality. Differentiation therapy has been successful as a treatment for acute promyelocytic leukemia [1]. Several compounds including dimethyl sulfoxide, retinoic acid, phorbol ester and 1,25-dihydroxy vitamin D3 induce AML cells to differentiate toward mature cells. Among them, retinoic acid induces AML cells to differentiate toward granulocytes [2], whereas 1,25-dihydroxy vitamin D3 induces AML cells to differentiate toward monocytes [2].

Chitin (Fig. 1(a)), a polymer of N-acetylglucosamine, is cellulose-like biopolymer present in the exoskeleton of crustaceans and in cell walls of fungi, insects and yeast. Chitosan (Fig. 1(b)) is derived from chitin by deacetylation in the presence of alkali. Chitin and chitosan oligomers can be obtained by either chemical or enzymatic hydrolysis of chitin and chitosan [3]. They have lower viscosity, low molecular weights and short chains and are soluble in neutral aqueous solutions. Subsequently, they seem to be readily absorbed in vivo. Chitin and chitosan oligomers are known to have various biological activities including antitumor activities [4], [5], immuno-enhancing effects [6], increased protective effects against infection with some pathogens [7], antifungal activities [8], and antimicrobial activities [8]. To our knowledge, there is no report on the effect of water-soluble chitosan oligomer (WSCO) on leukemia cell proliferation and differentiation. Thus, the aim of the present investigation was to examine the differentiation-inducing effect of WSCO on the human myeloid leukemia HL-60 cells. To our knowledge, this is the first time it has been shown that WSCO is able to inhibit proliferation of HL-60 cells and induce these cells to differentiate.