All-Trans Retinoic Acid Induces Differentiation and Downregulates Stemness Markers and MGMT Expression in Glioblastoma Stem Cells
Background: Glioblastoma (GBM) remains almost uniformly fatal, owing in part to therapy-resistant cancer stem-like cells (CSCs) and to temozolomide (TMZ) resistance driven by O<sup>6</sup>-methylguanine-DNA methyltransferase (MGMT). Differentiation therapy with all-trans retinoic acid (A...
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2025-05-01
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| description | Background: Glioblastoma (GBM) remains almost uniformly fatal, owing in part to therapy-resistant cancer stem-like cells (CSCs) and to temozolomide (TMZ) resistance driven by O<sup>6</sup>-methylguanine-DNA methyltransferase (MGMT). Differentiation therapy with all-trans retinoic acid (ATRA) has the potential to attenuate stemness and sensitize GBM to TMZ. We therefore asked whether ATRA reduces expression of key CSC markers and MGMT in established GBM lines. 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 and corresponding protein levels were measured by an Enzyme-Linked Immunosorbent Assay (ELISA). 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 <i>t</i>-tests. Results: ATRA markedly suppressed stemness and MGMT in both lines. In U87-MG, SOX2 mRNA fell 3.7-fold (<i>p</i> = 0.0008) and protein 2.99-fold (148.3 ± 6.0 → 49.7 ± 2.7 pg µg<sup>−1</sup>; <i>p</i> = 0.0002); Nestin dropped 4.1-fold (<i>p</i> = 0.0005) and 3.51-fold (450.0 ± 17.3 → 128.3 ± 4.4 pg µg<sup>−1</sup>; <i>p</i> = 0.00008). MGMT decreased 2.6-fold at transcript level (<i>p</i> = 0.0065) and 2.11-fold at protein level (81.7 ± 4.4 → 38.7 ± 1.8 pg µg<sup>−1</sup>; <i>p</i> = 0.0005). In A172, SOX2 was reduced 2.9-fold (<i>p</i> = 0.0041) and 2.31-fold (<i>p</i> = 0.0007); Nestin 3.3-fold (<i>p</i> = 0.0028) and 2.79-fold (<i>p</i> = 0.00009). MGMT declined 2.2-fold (<i>p</i> = 0.0132) and 1.82-fold (<i>p</i> = 0.0015), respectively. Conclusions: Five-day exposure to ATRA diminishes SOX2, Nestin, and MGMT at both mRNA and protein levels in stem-enriched GBM cultures, supporting the premise that ATRA-induced differentiation can concurrently blunt CSC traits and TMZ-resistance mechanisms. These data provide a molecular rationale for testing ATRA in combination regimens aimed at improving GBM therapy. |
| format | Article |
| id | doaj-art-e6de484ca2ed45c582a8b21bc89f02ed |
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| language | English |
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| spelling | doaj-art-e6de484ca2ed45c582a8b21bc89f02ed2025-08-20T03:14:32ZengMDPI AGCells2073-44092025-05-01141074610.3390/cells14100746All-Trans Retinoic Acid Induces Differentiation and Downregulates Stemness Markers and MGMT Expression in Glioblastoma Stem CellsJustin Tang0Raymond Yang1Department of Biomedical Science, University of Guelph, Guelph, ON N1G 2W1, CanadaDepartment of Surveillance & Evaluation, Health Canada, Ottawa, ON K1A 0K9, CanadaBackground: Glioblastoma (GBM) remains almost uniformly fatal, owing in part to therapy-resistant cancer stem-like cells (CSCs) and to temozolomide (TMZ) resistance driven by O<sup>6</sup>-methylguanine-DNA methyltransferase (MGMT). Differentiation therapy with all-trans retinoic acid (ATRA) has the potential to attenuate stemness and sensitize GBM to TMZ. We therefore asked whether ATRA reduces expression of key CSC markers and MGMT in established GBM lines. 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 and corresponding protein levels were measured by an Enzyme-Linked Immunosorbent Assay (ELISA). 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 <i>t</i>-tests. Results: ATRA markedly suppressed stemness and MGMT in both lines. In U87-MG, SOX2 mRNA fell 3.7-fold (<i>p</i> = 0.0008) and protein 2.99-fold (148.3 ± 6.0 → 49.7 ± 2.7 pg µg<sup>−1</sup>; <i>p</i> = 0.0002); Nestin dropped 4.1-fold (<i>p</i> = 0.0005) and 3.51-fold (450.0 ± 17.3 → 128.3 ± 4.4 pg µg<sup>−1</sup>; <i>p</i> = 0.00008). MGMT decreased 2.6-fold at transcript level (<i>p</i> = 0.0065) and 2.11-fold at protein level (81.7 ± 4.4 → 38.7 ± 1.8 pg µg<sup>−1</sup>; <i>p</i> = 0.0005). In A172, SOX2 was reduced 2.9-fold (<i>p</i> = 0.0041) and 2.31-fold (<i>p</i> = 0.0007); Nestin 3.3-fold (<i>p</i> = 0.0028) and 2.79-fold (<i>p</i> = 0.00009). MGMT declined 2.2-fold (<i>p</i> = 0.0132) and 1.82-fold (<i>p</i> = 0.0015), respectively. Conclusions: Five-day exposure to ATRA diminishes SOX2, Nestin, and MGMT at both mRNA and protein levels in stem-enriched GBM cultures, supporting the premise that ATRA-induced differentiation can concurrently blunt CSC traits and TMZ-resistance mechanisms. These data provide a molecular rationale for testing ATRA in combination regimens aimed at improving GBM therapy.https://www.mdpi.com/2073-4409/14/10/746glioblastomacancer stem cellsestablished cell linesU87-MGA172all-trans retinoic acid (ATRA) |
| spellingShingle | Justin Tang Raymond Yang All-Trans Retinoic Acid Induces Differentiation and Downregulates Stemness Markers and MGMT Expression in Glioblastoma Stem Cells Cells glioblastoma cancer stem cells established cell lines U87-MG A172 all-trans retinoic acid (ATRA) |
| title | All-Trans Retinoic Acid Induces Differentiation and Downregulates Stemness Markers and MGMT Expression in Glioblastoma Stem Cells |
| title_full | All-Trans Retinoic Acid Induces Differentiation and Downregulates Stemness Markers and MGMT Expression in Glioblastoma Stem Cells |
| title_fullStr | All-Trans Retinoic Acid Induces Differentiation and Downregulates Stemness Markers and MGMT Expression in Glioblastoma Stem Cells |
| title_full_unstemmed | All-Trans Retinoic Acid Induces Differentiation and Downregulates Stemness Markers and MGMT Expression in Glioblastoma Stem Cells |
| title_short | All-Trans Retinoic Acid Induces Differentiation and Downregulates Stemness Markers and MGMT Expression in Glioblastoma Stem Cells |
| title_sort | all trans retinoic acid induces differentiation and downregulates stemness markers and mgmt expression in glioblastoma stem cells |
| topic | glioblastoma cancer stem cells established cell lines U87-MG A172 all-trans retinoic acid (ATRA) |
| url | https://www.mdpi.com/2073-4409/14/10/746 |
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