Viacheslav S¹, Jorge S.¹, Perez C.², Prajapati J.¹, Diaz A.J.¹, Wang T.^{1, 3,}

Temozolomide (TMZ) is an alkylating agent central to glioblastoma therapy. Its activity is classically attributed to DNA methylation. Still, the reactive methyl-diazonium ion it generates is an electrophile with the potential to modify multiple cellular nucleophiles, and growing evidence points to a protein-level dimension of TMZ's action—including changes in histone methylation and in the activity of repair-associated enzymes. Because this protein-level activity appears cell-type-specific, treating resistance as a purely DNA-level event may overlook a mechanistic layer relevant to why tumor types differ in their sensitivity to the drug. In this course-based undergraduate research experience (CURE) in Clinical Chemistry, we evaluated the cytotoxic response of three cancer cell lines — MCF-7 (estrogen receptor-positive breast cancer), MDA-MB-231 (triple-negative breast cancer), and U87 (glioblastoma) to TMZ. Cells were treated for 48 hours across a 50–200 µM concentration range, and the MTS assay quantified viability. The Wilcoxon signed-rank test was used to assess significance relative to untreated controls, and four-parameter logistic (4PL) regression was used to model dose-response and estimate IC₅₀ values. Both breast cancer lines were sensitive to TMZ, with IC₅₀ values of 59.0 µM (MCF-7) and 46.4 µM (MDA-MB-231), whereas U87 glioblastoma cells were markedly less responsive (nominal IC₅₀ 213.7 µM). Because only half of the U87 dataset reached statistical significance, its IC₅₀ is considered preliminary. The greater resistance of glioblastoma is consistent with MGMT-mediated repair of O6-methylguanine, but is best interpreted alongside a protein-level dimension of TMZ's action observed in our prior work: in glioma cells, increasing TMZ concentrations were associated with a decrease in histone methylation and a concentration-dependent, bimodal change in the activity of the histone demethylase LSD1 (KDM1A) — largely preserved at 100 µM or below and reduced at higher concentrations — an enzyme itself linked to DNA repair. Considered together, these DNA- and protein-level effects offer a fuller account of TMZ sensitivity than DNA damage alone. Beyond these biological findings, the project gave undergraduates hands-on training in experimental design, statistical analysis, and scientific communication, illustrating the value of embedding authentic research within the undergraduate curriculum.

CUREs, temozolomide, cytotoxicity, breast cancer and glioma