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J. Mol. Sci.","IJMS"],"abstract":"<jats:p>The prognosis of glioblastoma (GBM) patients remains dismal due to chemoresistance. Repurposing of natural and endogenous compounds, such as the pineal hormone melatonin (MLT) and minor phytocannabinoids like cannabinol (CBN) or cannabigerol (CBG), represents a promising strategy. This study investigates the cytotoxic potential of combining these phytocannabinoids with MLT, evaluating their efficacy both alone and synergistically with temozolomide (TMZ) to overcome drug resistance. To achieve this, cytotoxicity, synergy (Bliss model), and selectivity were evaluated in U87, T98, and U251 GBM lines and normal astrocytes. Mechanisms of damage were characterized via Western blot (\u03b3H2AX and PARP-1), flow cytometry using fluorescent dyes\/probes (DCFDA, JC-1, MitoBright, BODIPY, PI, and Annexin-V), or the protein marker COX IV and confocal analysis. The results demonstrated that CBN-MLT and CBG-MLT regimens exerted synergistic cytotoxicity while sparing healthy astrocytes. Notably, combining these regimens (U87: MLT 0.3 mg\/mL + CBN 25 \u00b5M; MLT 0.2 mg\/mL + CBG 15 \u00b5M. T98: MLT 0.7 mg\/mL + CBN 25 \u00b5M; MLT 0.6 mg\/mL + CBG 30 \u00b5M. U251: MLT 0.4 mg\/mL + CBN 20 \u00b5M; MLT 0.5 mg\/mL + CBG 35 \u00b5M) with TMZ significantly enhanced chemotherapeutic efficacy, overcoming baseline effects of TMZ in these cell lines. The combinations induced necrotic cell death characterized by severe double-strand DNA damage. This was driven by an early accumulation of intracellular ROS, which triggered mitochondrial depolarization, loss of organelle mass, and lipid peroxidation. CBN combinations consistently triggered more robust biochemical alterations than CBG-based treatments. Taken together, this study provides a strong preclinical basis for utilizing minor cannabinoids combined with MLT in GBM management. Crucially, this co-treatment emerges as a promising approach to potentiate TMZ efficacy, offering a novel and potentially effective therapeutic strategy to counter GBM resilience.<\/jats:p>","DOI":"10.3390\/ijms27156774","type":"journal-article","created":{"date-parts":[[2026,7,29]],"date-time":"2026-07-29T08:16:52Z","timestamp":1785313012000},"page":"6774","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Synergistic In Vitro Effects of Minor Phytocannabinoids and Melatonin Combinations Against Human Glioblastoma Cells"],"prefix":"10.3390","volume":"27","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4614-5649","authenticated-orcid":false,"given":"Maria Beatrice","family":"Morelli","sequence":"first","affiliation":[{"name":"Department of Experimental Medicine and Public Health, School of Pharmacy, University of Camerino, Via Madonna delle Carceri, 62032 Camerino, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giorgio","family":"Cameli","sequence":"additional","affiliation":[{"name":"Department of Experimental Medicine and Public Health, School of Pharmacy, University of Camerino, Via Madonna delle Carceri, 62032 Camerino, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-8306-0700","authenticated-orcid":false,"given":"Martina","family":"Giangrossi","sequence":"additional","affiliation":[{"name":"Department of Experimental Medicine and Public Health, School of Pharmacy, University of Camerino, Via Madonna delle Carceri, 62032 Camerino, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6196-9673","authenticated-orcid":false,"given":"Laura","family":"Zeppa","sequence":"additional","affiliation":[{"name":"Department of Experimental Medicine and Public Health, School of Pharmacy, University of Camerino, Via Madonna delle Carceri, 62032 Camerino, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5209-7885","authenticated-orcid":false,"given":"Margherita","family":"Luongo","sequence":"additional","affiliation":[{"name":"\u201cMaria Guarino\u201d Foundation-AMOR No Profit Association, 80078 Pozzuoli, Italy"},{"name":"School of Medicine and Surgery, Luigi Vanvitelli University of Campania, 81100 Naples, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8711-9941","authenticated-orcid":false,"given":"Consuelo","family":"Amantini","sequence":"additional","affiliation":[{"name":"Department of Experimental Medicine and Public Health, School of Biosciences and Veterinary Medicine, University of Camerino, Via Gentile III da Varano, 62032 Camerino, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0165-1385","authenticated-orcid":false,"given":"Massimo","family":"Nabissi","sequence":"additional","affiliation":[{"name":"Department of Experimental Medicine and Public Health, School of Pharmacy, University of Camerino, Via Madonna delle Carceri, 62032 Camerino, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,29]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Kr\u00f3likowska, K., B\u0142aszczak, K., \u0141awicki, S., Zajkowska, M., and Gudowska-Sawczuk, M. 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