Down‐regulation of lipocalin 2 contributes to chemoresistance in glioblastoma cells

Abstract

Malignant gliomas are the most common primary brain tumor and have a poor clinical prognosis. 1, 3‐Bis (2‐chloroethyl)‐1‐nitrosourea (BCNU) is an alkylating agent that is commonly used in glioma therapy. However, BCNU chemotherapy often fails due to drug resistance. To gain better understanding of molecular mechanisms underlying the drug resistance of glioma, a BCNU‐resistant variant (C6R) of C6 rat glioma cells was selected and characterized. The established C6R cells were resistant to BCNU‐induced cell death and cell cycle arrest as confirmed by 3‐(4, 5‐dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide reduction assay and flow cytometric analysis of DNA content. C6R cells showed an increased expression of common drug resistance‐related genes such as O6‐methylguanine‐DNA methyltransferase and multiple drug resistance 1. In contrast, C6R cells showed a decreased expression of glial fibrillary acidic protein, therefore, displaying shorter cellular processes compared with parental C6 cells. More importantly, in conjunction with the morphological changes, the expression of lipocalin‐2 (lcn2), a 25‐kDa secreted proapoptotic protein, was markedly reduced in the BCNU‐resistant C6R cells. However, there was no significant change in the expression of lcn2 receptors. Addition of recombinant LCN2 protein or introduction of lcn2 cDNA significantly increased the sensitivity of C6 cells and human glioma cells to BCNU or other anticancer drugs, while knockdown of lcn2 expression by antisense cDNA transfection decreased the sensitivity. When lcn2 was re‐expressed in C6R cells, the BCNU sensitivity was restored. Lcn2 enhanced BCNU‐induced Akt dephosphorylation providing a molecular basis of apoptosis sensitization. These results suggest that LCN2 protein may be involved in glioma drug resistance and may provide a new approach to sensitizing glioblastoma to chemotherapy.

Lipocalin-2 inhibits osteoclast formation by suppressing the proliferation and differentiation of osteoclast lineage cells

“…Recombinant mouse LCN2 protein was prepared as described previously [32,33]. Briefly, recombinant LCN2 protein was expressed as a glutathione S-transferase (GST) fusion protein in BL21 strain E. coli, which does not synthesize siderophore.…”

Apolipoprotein D mediates autocrine protection of astrocytes and controls their reactivity level, contributing to the functional maintenance of paraquat-challenged dopaminergic systems

“…Interestingly, another Lipocalin known to be induced upon stress in the vertebrate nervous system, Lcn2, mediates astrocyte reactivity. Over-expressing or adding Lcn2 to astrocytes sensitizes them to cytotoxic stimuli and induces astrogliosis , while decreasing Lcn2 correlates with decreased astrogliosis (Zheng et al, 2009). Turning on and off glial reactivity can be therefore accomplished by the complementary actions of the two Lipocalins.…”

Proteomic Analysis of Glioma Chemoresistance

“…In order to investigate the proteome-based mechanism of glioma chemoresistance, a variant of C6 rat glioma cells that is resistant to chemotherapeutic agents was established, and its proteome profile was compared with that of drug-sensitive parental cells [38]. C6 glioma cells were cultured in vitro in the presence of low concentrations of BCNU for a long period of time to induce phenotypic changes (Fig.…”

“…Among numerous proteins identified by the proteomic comparison of drug-sensitive and resistant glioma cells, lipocalin 2 (LCN2) [38] and integrin β3 (ITGB3) [39] played a central role in regulating glioma chemosensitivity (Fig. 5 ).…”

“…LCN2 is involved in diverse biological processes such as iron delivery, the innate immune response to bacterial infection, cell death/survival, and many types of human cancers [48-51]. Our previous study showed that LCN2 gene expression was significantly down-regulated in the phenotypically selected BCNU-resistant C6 glioma cells [38]. Further studies such as recombinant LCN2 protein treatment, forced expression, or knockdown of LCN2 gene expression in glioma cells revealed that LCN2 downregulation played a key role in the BCNU resistance of glioma cells.…”