Modulation of Viability, Proliferation, and Stemness by Rosmarinic Acid in Medulloblastoma Cells: Involvement of HDACs and EGFR
Pediatric medulloblastoma (MB) is the most common malignant pediatric brain tumor. Due to its unique molecular and clinical characteristics, the treatment of this type of tumor has been a focus of clinical research. Existing treatments mainly include maximal surgical resection, radiotherapy, and chemotherapy, but these treatments often lead to long-term poor quality of life for patients, including cognitive, motor, and neuropsychiatric deficits. Therefore, it is urgent to find new therapeutic drugs that can effectively combat tumors while maintaining patients’ quality of life.
In this context, the author team conducted a series of studies on rosmarinic acid (RA), a compound derived from plants. Rosmarinic acid has been studied to have certain effects on various diseases, including cancer. In this study, the authors explored the mechanism of action of rosmarinic acid on medulloblastoma for the first time.
Research Team and Publication
This article was co-authored by Alice Laschuk Herlinger, Gustavo Lovatto Michaelsen, Marialva Sinigaglia, and several other researchers. The research institutions involved include the Cancer and Neurobiology Laboratory at the Federal University of Rio Grande do Sul, Brazil, the National Institute of Science and Technology for Pediatric Oncology and Childhood Cancer Biology, Brazil, and the Bioinformatics Graduate Program at the Federal University of Northern Rio Grande, Brazil. The paper was published in the journal “Neuromolecular Medicine” on September 23, 2023.
Research Background and Objectives
This study aimed to explore the effects of rosmarinic acid in two human medulloblastoma cell lines (DAOY and D283) and its potential molecular mechanisms. The DAOY cell line represents the SONIC HEDGEHOG (SHH) subgroup, while the D283 cell line represents the Group 3 molecular subgroup.
Research Process
1. Cell Culture
DAOY (HTb186™) and D283 (HTb185™) cell lines were obtained from the American Type Culture Collection (ATCC) and authenticated to ensure their identity and absence of contamination. Cells were cultured in low-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) containing 2% (W/V) L-glutamine and 10% (V/V) fetal bovine serum (FBS).
2. Drug Treatment
Rosmarinic acid (RA) was purchased from Sigma-Aldrich and diluted to a concentration of 100mM. Dimethyl sulfoxide (DMSO) was used as a solvent for dilution, and fresh preparations were made before experiments.
3. Cell Viability Assessment
Cell viability was assessed using the trypan blue cell counting method. Cell survival rates were calculated after 48 hours of treatment with different concentrations of rosmarinic acid, and IC50 values were estimated through non-linear regression.
4. Clonogenic Assay
After 48 hours of rosmarinic acid treatment, cells were re-cultured at low density, and the number and area of colony formation were observed.
5. Cell Proliferation Assessment
The effect of rosmarinic acid on cell proliferation was evaluated by estimating the cumulative population doubling (CPD) of cells over 12 days.
6. Neurosphere Formation Assessment
Cancer stem cell (CSC) proliferation ability was evaluated using neurosphere formation experiments, including the area of neurospheres and expression of CSC markers after rosmarinic acid treatment.
7. Quantitative Reverse Transcription Polymerase Chain Reaction (qPCR)
Total RNA was extracted from cells, and mRNA levels of target genes were measured by qPCR.
8. Western Blot
Western blot was performed to evaluate the expression levels of target proteins after 48 hours of rosmarinic acid treatment.
9. Survival Analysis
Public gene expression datasets were used to analyze overall survival rates and molecular subgroups of medulloblastoma patients.
10. Statistical Analysis
Various statistical analysis methods were used to verify the significance of experimental results.
Main Results
Cytotoxicity of Rosmarinic Acid on Cell Lines: Rosmarinic acid significantly reduced the survival rates of DAOY and D283 cells, with IC50 values of 168μM and 334μM, respectively.
Clonogenic Ability: Rosmarinic acid significantly reduced the area of clone formation in DAOY cells but had no significant effect on D283 cells.
Cell Proliferation: The CPD of DAOY cells was significantly reduced after rosmarinic acid treatment, while the compound had little effect on the proliferation of D283 cells.
Neurosphere Formation and CSC Markers: Rosmarinic acid significantly reduced the area of neurospheres formed by both cell lines and downregulated the expression of CSC markers (PROM1/CD133).
Molecular Mechanism: In DAOY cells, rosmarinic acid achieved cell cycle arrest and reduced cell survival by downregulating HDAC1 expression, increasing H3K9Ac and CDKN1A/P21 expression, inhibiting SOX2, and reducing the activity of EGFR and ERK/AKT signaling pathways.
Conclusion
This study reveals for the first time the cytotoxic and cytostatic effects of rosmarinic acid on medulloblastoma cells, as well as its inhibitory effect on cancer stem cell characteristics, without toxicity to healthy brain cells. Rosmarinic acid regulates the proliferation and stemness of MB cells by affecting HDAC and EGFR signaling pathways. In summary, the research value and application prospects of rosmarinic acid as a potential therapeutic drug for MB are worth further exploration.
Significance and Value
This study confirms the potential anti-cancer effect of rosmarinic acid in medulloblastoma and proposes its molecular mechanism of achieving cell cycle arrest and cell death through inhibition of HDAC and EGFR signaling pathways. Moreover, the low toxicity and high efficacy of rosmarinic acid demonstrated in basic research and preclinical studies of other cancers also enhance its potential as a novel anti-tumor drug.
This research provides new ideas and data support for future drug development and clinical applications. Especially in the context of seeking safer and more effective tumor treatment options, the study of rosmarinic acid will undoubtedly receive more attention.