Glioblastoma multiforme (GBM) is the most common central nervous system malignancy, whose highly invasive and diffuse nature leaves rare opportunity for cure. Despite aggressive surgical approaches, optimized radiation therapy regimens and the application of cytotoxic chemotherapies, the median survival rates from time of diagnosis range from 12-15 months .
Differentiation, as apoptosis, is a defense mechanism by which mammalian cells guard against tumorigenesis. Differentiation therapy, using agents that promote cancer cell differentiation, has been shown to be effective in vitro and in vivo in treatment of several types of cancer cells . Thus, neuroblastoma cells were shown to undergo terminal differentiation upon elevation of intracellular adenosine 3', 5'-cyclic monophosphate (cAMP) concentration after treatment with cyclic nucleotide phosphodiesterase inhibitors or adenylate cyclase activators [3–7]. Cholera toxin was reported to induce malignant glioma cell differentiation via the PKA/CREB pathway . Differentiation of glioma cells can also be achieved by treatment of cells with Datura
stramonium agglutinin . Notably, all-trans-retinoic acid has been used as an agent to induce cell differentiation in clinical treatment of acute promyelocytic leukemia (APL) [10, 11], demonstrating the remarkable efficacy of differentiation therapy in treatment of cancers.
Cycloheximide (CHX), originally isolated from Streptomyces griseus, has been widely used as an experimental tool to analyze the contribution of protein synthesis to the intracellular signaling and associated cellular function. It inhibits protein translation in mammalian cells at multiple steps, with a particular potency on translation initiation . Protein synthesis consumes enormous energy and resources in cells especially in fast growing cancer cells, and inhibition of protein synthesis by CHX, even in a relatively mild situation, may constitute a major blockade to the cellular functions such as cell proliferation. Indeed, inhibition of synthesis of specific proteins by CHX, such as c-Myc , p27  has been interpreted as evidence for the necessity of those cell cycle regulators in either cell proliferation or cell cycle arrest. Similarly, CHX has been shown to either block [15–17] or promote [18–20] gene expression-dependent cell death in different types of cells and experimental paradigms. Surprisingly, a possible biological effect of CHX in cancer therapy, to our knowledge, has never been investigated.
In the present study, we show that low concentration of CHX (LCC) potently inhibited C6 cell proliferation and depleted cells at both G2 and M phases, suggesting blockade at G1 and S phases during which massive protein accumulation is required. Surprisingly, treatment of C6 cell with LCC also induced dramatic morphological transformation that is indicative of cellular differentiation; this change was accompanied by upregulation of differentiation marker glial fibrillary acidic protein (GFAP). In addition, we found that intracellular cAMP levels were dramatically increased in C6 cells treated with LCC, and that this elevated intracellular cAMP is responsible for C6 cell differentiation and upregulation of GFAP.