P53 UPREGULATED MODULATOR OF APOPTOSIS SENSITIZES DRUG-RESISTANT U251 GLIOBLASTOMA STEM CELLS TO TEMOZOLOMIDE THROUGH ENHANCED APOPTOSIS

Introduction: Malignant glioma is a highly aggressive brain tumor with a dismal prognosis. Temozolomide (TMZ), a DNA-alkylating agent, is currently the first-line chemotherapeutic for glioblastoma (GBM), known for improving overall survival rates and progression-free survival. However, the emergence of drug resistance, particularly in glioblastoma stem cells (GSCs), limits TMZ's clinical efficacy. Resistance mechanisms in GSCs remain unclear, necessitating further research to enhance therapeutic outcomes. This study investigates whether the p53 upregulated modulator of apoptosis (PUMA) can sensitize TMZ-resistant U251 GBM stem cells to TMZ, aiming to elucidate the underlying molecular mechanisms.

Key Findings: This study developed a TMZ-resistant U251 GBM cell line, U251R, over six months by progressively increasing TMZ concentration. These cells were further subdivided into CD133+ and CD133− subpopulations using magnetic-activated cell sorting (MACS). The CD133+ U251R cells exhibited significantly more excellent resistance to TMZ-induced growth inhibition and apoptosis than CD133− U251R and parental U251MG cells. Specifically, CD133+ U251R cells had a 14.8-fold increase in the half-maximal inhibitory concentration (IC50) compared to a 10.6-fold increase in U251R cells, indicating a much higher resistance level. Despite no significant differences in O6-methylguanine-DNA-methyltransferase (MGMT) expression between CD133+ and CD133− U251R cells, the CD133+ U251R cells were notably more resistant to TMZ. This suggests that additional resistance mechanisms beyond MGMT-mediated pathways are at play.

PUMA expression was significantly lower in CD133+ U251R cells than in U251MG, U251R, and CD133− U251R cells. Introducing adenoviruses expressing wild-type PUMA (Ad-PUMA) into CD133+ U251R cells significantly increased their sensitivity to TMZ. This was evident by a marked elevation in the Bcl-2-associated X protein (Bax)/B-cell lymphoma-2 (Bcl-2) ratio, a key marker of apoptosis, without altering MGMT expression. The IC50 of TMZ in CD133+ U251R cells decreased by three- to eight-fold following Ad-PUMA infection, highlighting the potential of PUMA to overcome drug resistance in these cells. Additionally, annexin-V/propidium iodide double-staining assays revealed a significant increase in apoptosis in Ad-PUMA-treated cells, especially when combined with TMZ.

Innovative Tools: The researchers employed several advanced methodologies to ensure the accuracy and reliability of their findings. Magnetic-activated cell sorting (MACS) was used to isolate CD133+ and CD133− subpopulations, allowing for precise glioblastoma stem cells (GSCs) characterization. Flow cytometry (FACS) was utilized to assess the proportion of CD133+ cells and evaluate apoptotic rates following various treatments. Western blotting and reverse transcription polymerase chain reaction (RT-PCR) were conducted to analyze the expression levels of MGMT, PUMA, Bax, and Bcl-2, providing insights into the molecular mechanisms underlying drug resistance and apoptosis. The annexin-V/propidium iodide double-staining assay was crucial in quantifying apoptosis in the treated cells, further validating the effectiveness of Ad-PUMA in sensitizing TMZ-resistant cells.

Role of PUMA in Overcoming Drug Resistance: The study demonstrated that PUMA plays a pivotal role in overcoming TMZ resistance in glioblastoma stem cells. Wild-type p53 protein is a potent tumor suppressor, but p53 mutations, common in GBM, often result in a loss of function and acquisition of oncogenic properties. PUMA, a downstream molecule of p53, has a pro-apoptotic role independent of p53 status. This study found that exogenous expression of PUMA inhibited cell growth and significantly increased apoptotic rates in CD133+ U251R cells. In vitro experiments showed that Ad-PUMA combined with TMZ induced substantial growth inhibition and apoptosis in these cells. In vivo experiments using a nude mouse subcutaneous glioma model further confirmed that the combination of Ad-PUMA and TMZ had a synergistic effect in suppressing tumor growth, demonstrating the potential of this combination therapy in overcoming TMZ resistance in gliomas.

Therapeutic Implications: The study's findings suggest that targeting PUMA could be a promising strategy for enhancing the efficacy of TMZ in treating glioblastoma, particularly in drug-resistant cases. The ability of PUMA to induce apoptosis through increasing the Bax/Bcl-2 ratio, independent of MGMT, highlights its potential as a therapeutic target. The in vitro and in vivo data indicate that combining Ad-PUMA and TMZ could effectively suppress tumor growth and overcome drug resistance, offering a new avenue for glioma treatment. Future research should further elucidate the molecular mechanisms of PUMA-mediated apoptosis and explore the clinical potential of PUMA-based therapies in glioblastoma patients.

Conclusions: This study underscores the significance of PUMA in sensitizing drug-resistant glioblastoma stem cells to TMZ, primarily through apoptosis induction and increasing the Bax/Bcl-2 ratio. The findings highlight that targeting PUMA can effectively overcome TMZ resistance in glioblastoma, offering a potential therapeutic approach. The combination of Ad-PUMA and TMZ shows promising synergistic effects, both in vitro and in vivo, suggesting a viable strategy for improving clinical outcomes in GBM treatment. Further research is warranted to fully explore the therapeutic potential of PUMA and develop effective treatments for glioblastoma patients who are resistant to conventional therapies.

Join the Discussion: We invite you to share your thoughts on the potential of PUMA as a therapeutic target in overcoming drug resistance in glioblastoma. How do you think targeting apoptotic pathways could revolutionize the treatment of resistant cancers? What other strategies could complement the use of PUMA in enhancing chemotherapy efficacy? Join the conversation in the comments below and share your insights and ideas.

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Original Research: The original research, "p53 upregulated modulator of apoptosis sensitizes drug-resistant U251 glioblastoma stem cells to temozolomide through enhanced apoptosis," can be found on PubMed here.