Abstract
BackgroundDifferentiation therapy has been shown effective in treatment of several types of cancer cells and may prove to be effective in treatment of glioblastoma multiforme, the most common and most aggressive primary brain tumor. Although extensively used as a reagent to inhibit protein synthesis in mammalian cells, whether cycloheximide treatment leads to glioma cell differentiation has not been reported.MethodsC6 glioma cell was treated with or without cycloheximide at low concentrations (0.5-1 μg/ml) for 1, 2 and 3 days. Cell proliferation rate was assessed by direct cell counting and colony formation assays. Apoptosis was assessed by Hoechst 33258 staining and FACS analysis. Changes in several cell cycle regulators such as Cyclins D1 and E, PCNA and Ki67, and several apoptosis-related regulators such as p53, p-JNK, p-AKT, and PARP were determined by Western blot analysis. C6 glioma differentiation was determined by morphological characterization, immunostaining and Western blot analysis on upregulation of GFAP and o p-STAT3 expression, and upregulation of intracellular cAMP.ResultsTreatment of C6 cell with low concentration of cycloheximide inhibited cell proliferation and depleted cells at both G2 and M phases, suggesting blockade at G1 and S phases. While no cell death was observed, cells underwent profound morphological transformation that indicated cell differentiation. Western blotting and immunostaining analyses further indicated that changes in expression of several cell cycle regulators and the differentiation marker GFAP were accompanied with cycloheximide-induced cell cycle arrest and cell differentiation. Increase in intracellular cAMP, a known promoter for C6 cell differentiation, was found to be elevated and required for cycloheximide-promoted C6 cell differentiation.ConclusionOur results suggest that partial inhibition of protein synthesis in C6 glioma by low concentration of cycloheximide induces cell cycle arrest at G1 and M phases and cAMP-dependent cell differentiation.
Highlights
Differentiation therapy has been shown effective in treatment of several types of cancer cells and may prove to be effective in treatment of glioblastoma multiforme, the most common and most aggressive primary brain tumor
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
Unlike high concentration of CHX that provokes C6 cell death in a few hours, LCC is not lethal to C6 cell even after several days of treatment; it causes cell cycle arrest at G1 and S phases during which large amount of proteins, and many of them being critical for cell cycle progression, are synthesized
Summary
Differentiation therapy has been shown effective in treatment of several types of cancer cells and may prove to be effective in treatment of glioblastoma multiforme, the most common and most aggressive primary brain tumor. Extensively used as a reagent to inhibit protein synthesis in mammalian cells, whether cycloheximide treatment leads to glioma cell differentiation has not been reported. 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 [2]. Cholera toxin was reported to induce malignant glioma cell differentiation via the PKA/CREB pathway [8]. 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
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