Cancer Letters

Cancer Letters

Volume 278, Issue 1, 8 June 2009, Pages 9-16
Cancer Letters

Low molecular weight hyaluronan inhibits colorectal carcinoma growth by decreasing tumor cell proliferation and stimulating immune response

https://doi.org/10.1016/j.canlet.2008.12.029Get rights and content

Abstract

Hyaluronan modulates cancer progression by multiple mechanisms; nevertheless, its effects remain controversial. In this work, low molecular weight (LMW) hyaluronan but not high molecular weight (HMW) was found to significantly reduce colorectal carcinoma (CRC) growth in vitro and in vivo. Both survival and proliferation of CT26 tumor cells were affected by treatment with low doses of LMW HA, with involvement of Akt signaling mechanisms. We show for the first time that splenocytes isolated from LMW HA-treated animals present significantly higher proliferative capacity upon stimulation with dendritic cells (DCs) pulsed with tumor lysate. Consistently, expression of MHC class II and costimulatory molecules were increased in DCs isolated from the spleen of LMW HA-treated mice. Besides, increased tumor infiltrating lymphocytes was observed in animals treated with LMW HA. Our results suggest that LMW HA in a model of CRC triggers an activation of immune system, which is likely involved in the observed tumor growth inhibition. LMW HA is suggested as a candidate molecule for therapeutic adjuvant treatments in CRC immunotherapy.

Introduction

Colorectal carcinoma is the second most common cancer worldwide [1], [2] and surgery remains the primary treatment. However it can only be performed in 20% of patients with metastatic disease, with a 5-year survival rate of 25–40%, despite adjuvant chemotherapy [3]. Therefore, new therapeutic options are urgently needed for the advanced disease. At the moment, most of research is focused on the development of new therapeutic strategies, such as blockage/inhibition of signal transduction mechanisms (i.e. targeting the epidermal growth factor receptor or Ras), sequestering vascular endothelial growth factor receptor ligands and/or enhancing immune response mechanisms [4], [5]. Immunotherapeutic strategies hold promise; however, they still remain as an experimental discipline. Despite that CRC has been considered poorly immunogenic, it has been found that the presence of lymphocyte infiltration in tumors is correlated with patient survival, showing higher survival rate in comparison with those with low density of T-cell infiltrates [6]. For this reason it is necessary to find an immune adjuvant or to artificially up-regulate the immune response in order to allow a curative effect.

Hyaluronan (HA) is a lineal, large and ubiquitous glycosaminoglycan found in almost all tissues and especially in those undergoing cell proliferation, regeneration and repair, such as embryonic, inflammated and tumor stroma tissues [7]. It is well established that HA functions are size-dependent: in normal tissues HMW HA (≈106 Da) is the predominant form and has a homeostatic role in regulating cell behavior. During the repair, inflammation and tumor processes, there is a predominance of LMW HA fragments (≈105 Da) which have been shown to promote cell proliferation, adhesion and motility [8]. However, smaller HA fragments (≈2.5 × 103) were found to disrupt HA-CD44 interaction and to induce tumor cell death [7]. The effects of HA and its fragments have been shown to be mediated by the PI3K/Akt/NFkB pathway in different tumors such as breast cancer, melanoma, colorectal carcinoma and lymphoma [9], [10], [11].

During the inflammation process, HMW HA can be depolymerized to LMW fragments via oxygen radicals and enzymatic degradation by hyaluronidase, glucuronidase, and hexosaminidase [12]. LMW HA form has been shown to up-regulate the expression of inflammatory genes in epithelial, endothelial and DCs as well as in fibroblasts and macrophages. Genes induced by LMW HA include members of the chemokine family, cytokines (IL-8, IL-12 and TNF-α) and inducible NO synthase [13], [14]. In addition, LMW HA or its fragments were shown to enhance T cell responses by activating and up-regulating costimulatory molecules on DCs [15], [16]. On the contrary, recent evidence suggests that HMW HA has an immunosuppressive role instead [17], [18].

Several studies have shown association of HA biosynthesis alteration with cancer progression (including CRC), as well as an increase in HA serum levels and tumor stroma deposition [19], [20], [21], [22]. It is assumed that the predominance of LMW HA in tumors is a result of tissue injury and that it is caused by a lack of balance in between HA synthases (Has) and hyaluronidases (Hyal) activities. The LMW HA found within tumor microenvironment is considered to be a tumor growth promoter [8], [23], [24]. Nonetheless, few studies have shown the exact molecular weight of HA in tumors and its effect on the antitumoral immune response [15], [19]. Therefore, assessment of the exact HA molecular weight form and its role in immune regulation during cancer progression remains largely unexplored.

In this work we have addressed the in vivo effects of exogenous application of both LMW- and HMW-HA into a murine model of CRC.

Section snippets

Reagents

HA recombinant of definite size (HMW 1.5–1.8 × 106 Da; LMW 1–3 × 105 Da; from CPN spol.s.r.o. Czech Republic) was kindly supplied by Farmatrade (Bs. As., Argentina). Oligosaccharides were obtained by enzymatic digestion from HMW-HA, as previously described [25]. This method render fragments ranging from HA4 tetrasaccharides (two disaccharide units in length: 700 Da) to HA14 oligosaccharides (seven disaccharide units in length: 2600 Da). HA-FITC from bovine trachea (≈1 × 105 Da) was purchased to Calbiochem.

Results and discussion

It has been previously described that HA modulate the behavior of different tumors by multiple mechanisms [7], [19], [31]. These effects are dependent on HA molecular weight, concentration and interaction with other extracellular matrix components present in tumor microenvironment [7], [8], [31]. Thus, we decided to examine in vitro and in vivo the pro- or anti tumoral effect of different types of HA in an experimental CRC model. In order to analyze if HA could directly modify CT26 behavior, we

Conflict of interest statement

All the authors declare that they do not have conflict of interest to disclose and they did not receive honoraria and support from any pharmaceutical company.

Acknowledgments

This work was supported by grants from Mizutani Foundation for Glycoscience (80072), Universidad Austral and Agencia Nacional de Promoción Científica y Tecnológica (PICT-2006-1882; PICT-2005-34788, PICTO-CRUP 2005-31179), CTE-06 and AECI 2006. We acknowledge the continuous support from Inés Bemberg. We thank Soledad Arregui and Guillermo Gastón for expert technical assistance.

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