All-Trans Retinoic Acid Induces Differentiation and Downregulates Stemness Markers and MGMT Expression in Glioblastoma Stem Cells

Background: Glioblastoma (GBM) remains a devastating brain malignancy characterized by cellular heterogeneity and therapy resistance. Cancer stem-like cells (CSCs) within GBM are implicated as drivers of tumor progression and resistance to the standard-of-care chemotherapy, temozolomide (TMZ). O⁶-methylguanine-DNA methyltransferase (MGMT) expression mediates resistance by repairing TMZ-induced DNA damage. Differentiation therapy offers a potential strategy to reduce the aggressive stem-like phenotype. All-trans retinoic acid (ATRA) is a known differentiating agent. We hypothesized that ATRA treatment could induce differentiation-associated changes in established GBM cell lines, leading to downregulation of stemness markers and the resistance factor MGMT. Methods: Two established human GBM cell lines, U87-MG and A172, were cultured under neurosphere-promoting conditions to enrich for potential stem-like subpopulations. Cells were treated with either 1 µM ATRA or vehicle control (DMSO) for 5 days. Total RNA was extracted, and cDNA was synthesized. Quantitative Real-Time PCR (qPCR) assessed relative mRNA expression levels of key stemness transcription factors (SOX2, NES) and the DNA repair gene MGMT. Gene expression was normalized to the geometric mean of two validated housekeeping genes (GAPDH, ACTB). Relative quantification was calculated using the ΔΔCt method, and statistical significance was determined using Student's t-tests. Results: ATRA treatment (1 µM, 5 days) resulted in a statistically significant downregulation of stemness marker expression in both GBM cell lines compared to vehicle controls. Specifically, SOX2 mRNA levels decreased by 3.7-fold (p=0.0008) in U87-MG and 2.9-fold (p=0.0041) in A172 cells. NES mRNA levels decreased by 4.1-fold (p=0.0005) in U87-MG and 3.3-fold (p=0.0028) in A172 cells. Crucially, ATRA treatment also led to a significant reduction in MGMT mRNA expression. MGMT levels decreased by 2.6-fold (p=0.0065) in U87-MG and 2.2-fold (p=0.0132) in A172 cells following ATRA exposure. Conclusion: Our findings demonstrate that ATRA treatment reduces the expression of stemness-associated genes SOX2 and Nestin in established GBM cell lines cultured under neurosphere conditions. Importantly, ATRA concurrently downregulates MGMT mRNA. These results provide a molecular rationale for exploring ATRA-induced differentiation as a strategy to potentially modulate TMZ resistance pathways in GBM models, warranting further investigation into its therapeutic potential, particularly in combination therapies.