Glioma-associated protein CHI3L2 suppresses cells viability and induces G1/S transition arrest

Aim. To analyze the effect of the CHI3L2 protein on malignant and non-malignant cell viability, and determined the CHI3L2 impact on the cell cycle and signaling pathways involved in the cell cycle regulation. Methods. MTT-based cell proliferation assay, FACS, western blot analysis. Results. The CHI3L2 protein inhibits the glioma cells viability and potentiates the effect of anti-cancer cytotoxic agents. The CHI3L2 treatment results in the G1/S transition arrest. CHI3L2 provoked a dramatic reduction of pRB phosphorylation and a signifi cant decrease in the cyclin D1 expression, whereas the p53 and p21 expression levels were substantially increased. Conclusions. The CHI3L2 protein, which is overexpressed in human gliomas, is a negative regulator of the glioma cells viability. The reduced cell viability after the CHI3L2 treatment could be due to the activation of pRB and p53 and the downregulation of cyclin D.


Introduction
Almost half of the intracranial tumors are gliomas, the majority of which are astrocytic gliomas.They are considered to be the most lethal among all types of tumors.We have previously compared the gene expression in glioblastoma, the most common and aggressive malignant brain tumor, and normal adult human brain by Serial Analysis of Gene Expression (SAGE) to identify those involved in gliomagenesis and found that that 44 genes were expressed at a signifi cantly higher level in the tumors compared to the normal brain cells [1].Chitinase 3-like 2 (CHI3L2) is one of the genes expressed in glyoblastoma; it encodes a 39 kDa secreted chitinase-like protein.
CHI3L2 is a member of the 18 glycosyl hydrolase family [1].It is closely related to another mammalian chitinase-like protein (CLP) CHI3L1.CHI3L1 is overexpressed in glioblastoma and possesses oncogenic properties [2].CHI2L1 and 2 may have different functions since the molecular structures of these homologous proteins have signifi cant differences [3].We have previously demonstrated that the activation of ERK1/2 phosphorylation by CHI3L2 inhibits the cell mitogenesis and proliferation.In contrast, the activation of ERK1/2 phosphorylation by CHI3L1 leads to the proliferation [4].Here we have reported that CHI3L2 downregulates the glioma cells viability and potentiates the effect of cytotoxic agents; the CHI3L2-mediated growth suppression is mediated by G1/S transition arrest.

Materials and Methods
Cell proliferation assay (MTT-assay) was performed as described in [4].The CHI3L2 protein was obtained using Bac-toBac expression system (Invitro-gen, USA).Bradykinin (BK) antagonist BKM-570 was kindly provided by Dr. Lajos Gera (University of Colorado Denver).Temozolomide was purchased from Abcam (UK).
Cell cycle analysis.293 cells were incubated with the CHI3L2 protein or transfected with pcDNA4/ TO_CHI3L2.After 48 hours, the cells were fi xed with cold 70 % ethanol, and resuspended in 1 ml of hypotonic buffer (0.1 % sodium citrate, 0.1 % Triton X-100, 5 mg/ml PI (Sigma, USA), 20 mg/ml RNase A (Thermo Scientifi c, USA) and 40 mg/ml propidium iodide (Sigma) for 1 hour.The cells were then analyzed using the BD Accuri C6 machine (Becton Dickinson, USA) according to the manufacturer's instructions.The data were analyzed using the software package CFlow (Becton Dickinson) and Flow-Jo (De Novo Software, USA).

Results and Discussion
Although the investigation of human CLPs was mainly focused on the expression patterns in a number of pathological conditions [5], CHI3L2 remains poorly characterized.To explore an effect of the CHI3L2 protein on the cells viability, we performed the MTT test with the human glioma cells U251 and non-transformed 293 cells.The CHI3L2 protein exhibits cytotoxic properties in both U251 and 293 cells: an addition of 100 ng/ml CHI3L2 to the cell culture medium led to ~ 40% reduction of the cell viability as compared to the control group (0 ng/ml) (Fig. 1 a).The ability of CHI3L2 to inhibit the viability of malignant and non-transformed cells lines may suggest that a mode of its action is cell-type independent.The proteins with cytotoxic properties could increase the effi cacy of anticancer chemotherapy when applied in combinations [6].Taking into account that CHI3L2 is overexpressed in glioblastoma, we aimed to see the outcome of the combination of CHI3L2 with the fi rstline anti-gliomic drug temozolomide, as well as with a highly potent anti-cancer agent, bradykinin antagonist BKM-570, possessing a signifi cant cytotoxic activity against the glioma cell lines [7].We did not observe any potentiation of the temozolomide activity but the combination of CHI3L2 with BKM-570 led to an increased cytotoxic effect (Fig. 1 b), suggesting that CHI3L2 could potentially be used as a component of a combo approach for the glioma treatment.To elucidate the molecular mechanisms of the observed cell viability reduction, we studied the impact of CHI3L2 on cell cycle.While no apoptotic action of CHI3L2 was observed, CHI3L2 induced the G1/S transition arrest.The proportion of cells in G1 phase increased after treatment by exogenous CHI3L2 as well as after its ectopic expression by 19 % and 13,7 %, correspondingly (Fig. 2).To explore how CHI3L2 affects the specifi c signal cascades involved in the cell cycle regulation, we have studied the state of some proteins essential for the G1/S cell cycle transition.The CHI2L2 treatment or ectopic expression leads to a reduction of pRB phosphorylation, as well as to a decrease of the cyclin D1 expression level (Fig. 3).Furthermore, the expression levels of p53 and its transcriptional target p21 were signifi cantly upregulated.Thus, the G1/S transition arrest through changes in the cell cycle regulation cascades is a putative component of the molecular mechanism underlying the cytotoxic effect of CHI3L2 protein.The blockage of cell cycle in a certain phase is the mechanism of action described for a set of cytotoxic agents [8], however, it has never been reported for the chitinase-like proteins.CHI3L1, the most investigated protein in this family, is also overexpressed in gliomas and was shown to be an oncogen [2].CHI3L2 has a high level of homology to CHI3L1, however, a set of structural differences exists between them giving a ground to distinct functions [3].CHI3L2 is not a glycoprotein and does not bind heparin, a component of the important receptor complexes [3].Nevertheless nothing is know about a function of CHI3L2 in brain issue, the results described in this paper is a step forward shedding a new light on molecular pathways involved by these similar proteins to realize their function.
The complex application of chemotherapeutic agents and cytotoxic proteins is a promising approach to treat cancer [6].Taking into account the cytotoxic properties of CHI3L2 and its overexpression in glioblastomas, one could speculate that at least in a certain part of patients the CHI3L2 protein could potentiates the action of chemotherapeutics.
Further investigations are needed to clarify a role of the chitinase-like proteins in the human gliomas.

Conclusions
In summary, we have demonstrated that CHI3L2 acts as a negative regulator of the cell growth and affects the cell cycle regulation machinery, resulting in the G1/S transition blockage.