Abstract
Gliomas are the most common and aggressive of brain tumors in adults. Cancer stem cells (CSC) contribute to chemoresistance in many solid tumors including gliomas. The function of prostate apoptosis response-4 (Par-4) as a pro-apoptotic protein is well documented in many cancers; however, its role in CSC remains obscure. In this study, we aimed to explore the role of Par-4 in drug-induced cytotoxicity using human glioma stem cell line - HNGC-2 and primary culture (G1) derived from high grade glioma. We show that among the panel of drugs- lomustine, carmustine, UCN-01, oxaliplatin, temozolomide and tamoxifen (TAM) screened, only TAM induced cell death and up-regulated Par-4 levels significantly. TAM-induced apoptosis was confirmed by PARP cleavage, Annexin V and propidium iodide staining and caspase-3 activity. Knock down of Par-4 by siRNA inhibited cell death by TAM, suggesting the role of Par-4 in induction of apoptosis. We also demonstrate that the mechanism involves break down of mitochondrial membrane potential, down regulation of Bcl-2 and reduced activation of Akt and ERK 42/44. Secretory Par-4 and GRP-78 were significantly expressed in HNGC-2 cells on exposure to TAM and specific antibodies to these molecules inhibited cell death suggesting that extrinsic Par-4 is important in TAM-induced apoptosis. Interestingly, TAM decreased the expression of neural stem cell markers - Nestin, Bmi1, Vimentin, Sox2, and Musashi in HNGC-2 cell line and G1 cells implicating its potential as a stemness inhibiting drug. Based on these data and our findings that enhanced levels of Par-4 sensitize the resistant glioma stem cells to drug-induced apoptosis, we propose that Par-4 may be explored for evaluating anti-tumor agents in CSC.
Highlights
High grade gliomas (HGG) or malignant gliomas are the most common of brain tumors in adults
We examined the sensitivity of glioma derived stem cell line - human neuroglial culture (HNGC)-2 and primary culture derived from glioma tumor samples that express neural stem cell markers (G1) to a panel of drugs including- lomustine, carmustine, UCN-01, oxaliplatin, temozolomide, tamoxifen (TAM) and the association of prostate apoptosis response-4 (Par-4) with drug-induced apoptosis
HNGC-2 cells were treated with increasing doses of lomustine, carmustine, UCN-01, oxaliplatin, temozolomide, and tamoxifen (TAM) for 24 h and cell viability was measured by MTT assay
Summary
High grade gliomas (HGG) or malignant gliomas are the most common of brain tumors in adults. Understanding and unraveling the biological basis of tumor formation and progression in gliomas is important for devising improved therapeutic strategies. Recent reports have shed light on a subpopulation of cells termed ‘cancer stem cells’ (CSC) within solid tumors that compel tumor formation and growth [3,4,5]. We have recently reported a model system comprising of a longterm in vitro culture of human neuroglial culture (HNGC)-1 and an established cell line, HNGC-2, derived from the same human adult glioma tissue [9]. We have earlier reported detailed characterization of this cell line encompassing the essential features of cancer stem cells, which include the ability of self-renewal, the capacity to form CD133-positive neurospheres and develop intracranial tumors. HNGC-2 cell line serves as an ideal tool for studying glioma stem cells [10]
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