{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T08:51:15Z","timestamp":1785919875759,"version":"3.56.0"},"reference-count":51,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T00:00:00Z","timestamp":1785888000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100005297","name":"Medical University of Bialystok","doi-asserted-by":"publisher","award":["B.SUB.24.317"],"award-info":[{"award-number":["B.SUB.24.317"]}],"id":[{"id":"10.13039\/501100005297","id-type":"DOI","asserted-by":"publisher"}]},{"award":["B.SUB.24.317"],"award-info":[{"award-number":["B.SUB.24.317"]}],"id":[{"id":"https:\/\/ror.org\/00y4ya841","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Glioblastoma multiforme (GBM) remains one of the most aggressive and treatment-resistant brain tumors, characterized by rapid proliferation and poor patient prognosis. Novel therapeutic strategies are urgently needed to improve clinical outcomes. In this study, we investigated the cytotoxic and pro-apoptotic effects of bortezomib (BORT), a proteasome inhibitor, and graphene oxide (GO), a nanomaterial with known anticancer potential, on human glioblastoma cell lines. Treatment with BORT and GO, both individually and in combination, significantly reduced cell viability in a dose-dependent manner, as determined by MTT. In this study, we observed enhanced apoptotic cell death, accompanied by increased activation of both caspase-8 and caspase-9, indicating simultaneous engagement of extrinsic and intrinsic apoptotic pathways. Western blot analysis demonstrated downregulation of anti-apoptotic proteins Bcl-2 and upregulation of pro-apoptotic markers (NOXA, cleaved PARP). A central finding of this work is the pronounced increase in intracellular reactive oxygen species (ROS) levels following BORT\u2013GO treatment. The elevated ROS levels observed in BORT\u2013GO-treated cells compared with free bortezomib therefore suggest that GO-mediated oxidative stress may amplify proteasome inhibition-induced apoptosis, which is particularly visible in the A172 and LN229 cell lines. Notably, the combination of BORT and GO may suggest a potential cooperative mechanism through proteasome inhibition and oxidative stress induction. These findings indicate that graphene oxide may modulate the antitumor efficacy of bortezomib in a cell line-dependent manner and support further investigation of this combination as a promising therapeutic approach for glioblastoma.<\/jats:p>","DOI":"10.3390\/cells15151417","type":"journal-article","created":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T08:14:46Z","timestamp":1785917686000},"page":"1417","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Graphene Oxide Modulates ROS Production and Apoptotic Responses to Bortezomib in Human Glioblastoma Cells: An In Vitro Study"],"prefix":"10.3390","volume":"15","author":[{"given":"Rafa\u0142","family":"Kr\u0119towski","sequence":"first","affiliation":[{"name":"Department of Pharmaceutical Biochemistry, Medical University of Bialystok, Mickiewicza 2A, 15-222 Bialystok, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7711-3017","authenticated-orcid":false,"given":"Agata","family":"Jab\u0142o\u0144ska-Trypu\u0107","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Biology and Biotechnology, Bialystok University of Technology, Wiejska 45A, 15-351 Bialystok, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Natalia","family":"Tyszka","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Biochemistry, Medical University of Bialystok, Mickiewicza 2A, 15-222 Bialystok, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4163-4600","authenticated-orcid":false,"given":"Joanna","family":"Kalita","sequence":"additional","affiliation":[{"name":"Department of Biology and Pharmacognosy, Medical University of Bialystok, Mickiewicza 2A, 15-222 Bialystok, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2806-399X","authenticated-orcid":false,"given":"Marzanna","family":"Cechowska-Pasko","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Biochemistry, Medical University of Bialystok, Mickiewicza 2A, 15-222 Bialystok, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,5]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1016\/j.bbrc.2016.07.080","article-title":"Bortezomib sensitizes human glioblastoma cells to induction of apoptosis by type I interferons through NOXA expression and Mcl-1 cleavage","volume":"478","author":"Wang","year":"2016","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1186\/s40880-019-0424-2","article-title":"Bortezomib inhibits growth and sensitizes glioma to temozolomide via down-regulating the FOXM1\u2013Survivin axis","volume":"39","author":"Tang","year":"2019","journal-title":"Cancer Commun."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"200","DOI":"10.1186\/s12967-020-02359-z","article-title":"Graphene oxide suppresses the growth and malignancy of glioblastoma stem cell-like spheroids via epigenetic mechanisms","volume":"18","author":"Wang","year":"2020","journal-title":"J. Transl. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"112330","DOI":"10.1016\/j.msec.2021.112330","article-title":"Unravelling the potential of graphene in glioblastoma therapy","volume":"128","author":"Foo","year":"2021","journal-title":"Mater. Sci. Eng. C"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"591","DOI":"10.2217\/nnm-2022-0030","article-title":"Graphene oxide induces autophagy and apoptosis via the ROS-dependent AMPK\/mTOR\/ULK-1 pathway in colorectal cancer cells","volume":"17","author":"Shen","year":"2022","journal-title":"Nanomedicine"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e70055","DOI":"10.1002\/btm2.70055","article-title":"Revolutionizing cancer treatment: The multifaceted role of graphene oxide in modern oncology","volume":"11","author":"Wu","year":"2025","journal-title":"Bioeng. Transl. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"e2405511","DOI":"10.1002\/smll.202405511","article-title":"Recent Developments in Glioblastoma-On-A-Chip for Advanced Drug Screening Applications","volume":"21","author":"Maity","year":"2025","journal-title":"Small"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"611","DOI":"10.1186\/s12967-024-05438-7","article-title":"Graphene-Based Hybrid Composites for Cancer Diagnostic and Therapy","volume":"22","author":"Asadi","year":"2024","journal-title":"J. Transl. Med."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"116842","DOI":"10.1016\/j.biopha.2024.116842","article-title":"Perilous paradigm of graphene oxide and its derivatives in biomedical applications: Insight to immunocompatibility","volume":"176","author":"Ayreen","year":"2024","journal-title":"Biomed. Pharmacother."},{"key":"ref_10","first-page":"936","article-title":"Evaluation of a tetrazolium-based semiautomated colorimetric assay: Assessment of chemosensitivity testing","volume":"47","author":"Carmichael","year":"1987","journal-title":"Cancer Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"364","DOI":"10.1016\/S0076-6879(05)98030-0","article-title":"Monitoring activity and inhibition of 26S proteasomes with fluorogenic peptide substrates","volume":"398","author":"Kisselev","year":"2005","journal-title":"Methods Enzymol."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Kr\u0119towski, R., and Cechowska-Pasko, M. (2022). The reduced graphene oxide induces apoptosis, autophagy and cell cycle arrest in breast cancer cells. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23169285"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Kozak, Y., Finiuk, N., Czarnomysy, R., Gornowicz, A., Pinyazhko, R., Lozynskyi, A., Holota, S., Klyuchivska, O., Karkhut, A., and Polovkovych, S. (2025). Juglone-bearing thiopyrano[2,3-d]thiazoles induce apoptosis in colorectal adenocarcinoma cells. Cells, 14.","DOI":"10.3390\/cells14060465"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1016\/0003-2697(85)90442-7","article-title":"Measurement of protein using bicinchoninic acid","volume":"150","author":"Smith","year":"1985","journal-title":"Anal. Biochem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"680","DOI":"10.1038\/227680a0","article-title":"Cleavage of structural proteins during the assembly of the head of bacteriophage T4","volume":"227","author":"Laemmli","year":"1970","journal-title":"Nature"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1133","DOI":"10.1080\/13543784.2020.1803827","article-title":"Proteasome inhibition for the treatment of glioblastoma","volume":"29","author":"Roth","year":"2020","journal-title":"Expert Opin. Investig. Drugs"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"123125","DOI":"10.1016\/j.lfs.2024.123125","article-title":"Bortezomib in cancer therapy: Mechanisms, side effects, and future proteasome inhibitors","volume":"358","author":"Sogbein","year":"2024","journal-title":"Life Sci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"686","DOI":"10.1158\/1535-7163.MCT-04-0338","article-title":"Proteasome inhibitor therapy in multiple myeloma","volume":"4","author":"Chauhan","year":"2005","journal-title":"Mol. Cancer Ther."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2489","DOI":"10.1182\/blood-2007-08-104950","article-title":"Molecular basis of bortezomib resistance: Proteasome subunit beta5 (PSMB5) gene mutation and overexpression of PSMB5 protein","volume":"112","author":"Oerlemans","year":"2008","journal-title":"Blood"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1354","DOI":"10.1007\/s11864-024-01273-6","article-title":"Acquired bortezomib resistance in multiple myeloma: From mechanisms to strategy","volume":"25","author":"Li","year":"2024","journal-title":"Curr. Treat. Options Oncol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"751","DOI":"10.1038\/nnano.2007.387","article-title":"Nanocarriers as an emerging platform for cancer therapy","volume":"2","author":"Peer","year":"2007","journal-title":"Nat. Nanotechnol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"16639","DOI":"10.1074\/jbc.M200360200","article-title":"NF-\u03baB as a therapeutic target in multiple myeloma","volume":"277","author":"Hideshima","year":"2002","journal-title":"J. Biol. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"9676","DOI":"10.3390\/curroncol30110702","article-title":"Proteasome inhibitors against glioblastoma\u2014Overview of molecular mechanisms of cytotoxicity, progress in clinical trials, and perspective for use in personalized medicine","volume":"30","author":"Gozdz","year":"2023","journal-title":"Curr. Oncol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"105780","DOI":"10.1016\/j.phrs.2021.105780","article-title":"Glioblastoma multiforme: An overview of current therapies and mechanisms of resistance","volume":"171","author":"Wu","year":"2021","journal-title":"Pharmacol. Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"563","DOI":"10.1016\/j.ccr.2014.03.015","article-title":"Endoplasmic reticulum stress in malignancy","volume":"25","author":"Clarke","year":"2014","journal-title":"Cancer Cell"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"3496","DOI":"10.1158\/0008-5472.CAN-07-0325","article-title":"GRP78 induction in cancer: Therapeutic and prognostic implications","volume":"67","author":"Lee","year":"2007","journal-title":"Cancer Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1016\/j.ccr.2006.03.029","article-title":"A dual PI3 kinase\/mTOR inhibitor reveals emergent efficacy in glioma","volume":"9","author":"Fan","year":"2006","journal-title":"Cancer Cell"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"448","DOI":"10.1038\/sj.cdd.4401359","article-title":"Role of autophagy in temozolomide-induced cytotoxicity for malignant glioma cells","volume":"11","author":"Kanzawa","year":"2004","journal-title":"Cell Death Differ."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"344","DOI":"10.1038\/sj.onc.1208225","article-title":"Proteasome inhibitor PS-341 causes cell growth arrest and apoptosis in human glioblastoma multiforme","volume":"24","author":"Yin","year":"2005","journal-title":"Oncogene"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"196","DOI":"10.1215\/15228517-4-3-196","article-title":"PTEN signaling in gliomas","volume":"4","author":"Knobbe","year":"2002","journal-title":"Neuro Oncol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1016\/j.ccr.2009.12.020","article-title":"Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1","volume":"17","author":"Verhaak","year":"2010","journal-title":"Cancer Cell"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Sawant, P., Mathew, A., Bensalel, J., Gallego-Delgado, J., and Mandal, P. (2025). Cancer drug bortezomib, a proteasomal inhibitor, triggers cytotoxicity in microvascular endothelial cells via multi-organelle stress. bioRxiv.","DOI":"10.2139\/ssrn.5221086"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"e2201968","DOI":"10.1002\/adhm.202201968","article-title":"Graphene oxide nanoscale platform enhances the anti-cancer properties of bortezomib in glioblastoma models","volume":"12","author":"Sharp","year":"2023","journal-title":"Adv. Healthc. Mater."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1291","DOI":"10.7150\/ijbs.26776","article-title":"Combined inhibition of autophagy and Nrf2 signaling augments bortezomib-induced apoptosis by increasing ROS production and ER stress in pancreatic cancer cells","volume":"14","author":"Li","year":"2018","journal-title":"Int. J. Biol. Sci."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"3201","DOI":"10.1038\/s41388-021-01697-6","article-title":"Generation of TRAIL-resistant cell line models reveals distinct adaptive mechanisms for acquired resistance and re-sensitization","volume":"40","author":"Morova","year":"2021","journal-title":"Oncogene"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"228","DOI":"10.1038\/sj.cgt.7700792","article-title":"Mechanisms of resistance to TRAIL-induced apoptosis in cancer","volume":"12","author":"Zhang","year":"2005","journal-title":"Cancer Gene Ther."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"64","DOI":"10.1016\/j.jconrel.2018.07.034","article-title":"Graphene-based nanomaterials and their potentials in advanced drug delivery and cancer therapy","volume":"286","author":"Liu","year":"2018","journal-title":"J. Control. Release"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"368504241291342","DOI":"10.1177\/00368504241291342","article-title":"Research progress of graphene-based nanomaterials in the diagnosis and treatment of head and neck cancer","volume":"107","author":"He","year":"2024","journal-title":"Sci. Prog."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1007\/s12192-020-01078-0","article-title":"Blockade of HSP70 by VER-155008 synergistically enhances bortezomib-induced cytotoxicity in multiple myeloma","volume":"25","author":"Huang","year":"2020","journal-title":"Cell Stress Chaperones"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Liao, K.F., Chiu, T.L., Chang, S.F., Wang, M.J., and Chiu, S.C. (2021). Hispolon induces apoptosis, suppresses migration and invasion of glioblastoma cells and inhibits GBM xenograft tumor growth in vivo. Molecules, 26.","DOI":"10.3390\/molecules26154497"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"4339","DOI":"10.31557\/APJCP.2022.23.12.4339","article-title":"The effect of caspase 8 and 9 gene polymorphisms on non-Hodgkin lymphoma susceptibility and clinical\/pathological features","volume":"23","author":"Barati","year":"2022","journal-title":"Asian Pac. J. Cancer Prev."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"588","DOI":"10.3892\/ol.2025.15334","article-title":"Bortezomib overcomes TRAIL resistance in Burkitt\u2019s lymphoma by enhancing apoptosis via reactive oxygen species-mediated DR5 upregulation and MAPK pathway activation","volume":"30","author":"Chen","year":"2025","journal-title":"Oncol. Lett."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Kr\u0119towski, R., Szynaka, B., Jab\u0142o\u0144ska-Trypu\u0107, A., Kie\u0142tyka-Dadasiewicz, A., and Cechowska-Pasko, M. (2024). The synergistic effect of reduced graphene oxide and proteasome inhibitor in the induction of apoptosis through oxidative stress in breast cancer cell lines. Int. J. Mol. Sci., 25.","DOI":"10.3390\/ijms25105436"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"12896","DOI":"10.1038\/s41598-017-13396-y","article-title":"Biochemical mechanisms of dose-dependent cytotoxicity and ROS-mediated apoptosis induced by lead sulfide\/graphene oxide quantum dots for potential bioimaging applications","volume":"7","author":"Ayoubi","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"126158","DOI":"10.1016\/j.jhazmat.2021.126158","article-title":"Graphene oxide disrupted mitochondrial homeostasis through inducing intracellular redox deviation and autophagy-lysosomal network dysfunction in SH-SY5Y cells","volume":"416","author":"Feng","year":"2021","journal-title":"J. Hazard. Mater."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"4","DOI":"10.1080\/15548627.2018.1509171","article-title":"Necrotic, apoptotic and autophagic cell fates triggered by nanoparticles","volume":"15","author":"Mohammadinejad","year":"2019","journal-title":"Autophagy"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"66","DOI":"10.1111\/pcmr.13014","article-title":"Neratinib kills B-RAF V600E melanoma via ROS-dependent autophagosome formation and death receptor signaling","volume":"35","author":"Dent","year":"2022","journal-title":"Pigment Cell Melanoma Res."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"e70089","DOI":"10.1111\/phpp.70089","article-title":"Interplay of skin aging: Mitochondrial stress and ultraviolet exposure","volume":"42","author":"Liao","year":"2026","journal-title":"Photodermatol. Photoimmunol. Photomed."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"e36057","DOI":"10.1016\/j.heliyon.2024.e36057","article-title":"Nanoparticle-mediated gene delivery of TRAIL to resistant cancer cells: A review","volume":"10","author":"Habibizadeh","year":"2024","journal-title":"Heliyon"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"116546","DOI":"10.1016\/j.biopha.2024.116546","article-title":"Recent advances in functional nucleic acid decorated nanomaterials for cancer imaging and therapy","volume":"174","author":"Wu","year":"2024","journal-title":"Biomed. Pharmacother."},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Panza, S., Malivindi, R., Caruso, A., Russo, U., Giordano, F., Gy\u0151rffy, B., Gelsomino, L., De Amicis, F., Barone, I., and Conforti, F.L. (2021). Novel Insights into the Antagonistic Effects of Losartan against Angiotensin II\/AGTR1 Signaling in Glioblastoma Cells. Cancers, 13.","DOI":"10.3390\/cancers13184555"}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/15\/1417\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T08:30:57Z","timestamp":1785918657000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/15\/1417"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,5]]},"references-count":51,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["cells15151417"],"URL":"https:\/\/doi.org\/10.3390\/cells15151417","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,5]]}}}