{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T04:36:57Z","timestamp":1787027817471,"version":"build-2736575974"},"reference-count":47,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2023,8,21]],"date-time":"2023-08-21T00:00:00Z","timestamp":1692576000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"D\u2019Youville University","award":["P30CA16056"],"award-info":[{"award-number":["P30CA16056"]}]},{"name":"D\u2019Youville University School of Pharmacy","award":["P30CA16056"],"award-info":[{"award-number":["P30CA16056"]}]},{"name":"Haniva Technology, LLC","award":["P30CA16056"],"award-info":[{"award-number":["P30CA16056"]}]},{"name":"Center for Advanced Technology grant from the University at Buffalo","award":["P30CA16056"],"award-info":[{"award-number":["P30CA16056"]}]},{"name":"NCI","award":["P30CA16056"],"award-info":[{"award-number":["P30CA16056"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Although melanoma accounts for only 5.3% of skin cancer, it results in &gt;75% of skin-cancer-related deaths. To avoid disfiguring surgeries on the head and neck associated with surgical excision, there is a clear unmet need for other strategies to selectively remove cutaneous melanoma lesions. Mohs surgery is the current treatment for cutaneous melanoma lesions and squamous and basal cell carcinoma. While Mohs surgery is an effective way to remove melanomas in situ, normal tissue is also excised to achieve histologically negative margins. This paper describes a novel combination therapy of nonthermal plasma (NTP) which emits a multitude of reactive oxygen species (ROS) and the injection of a pharmaceutical agent. We have shown that the effects of NTP are augmented by the DNA-damaging prodrug, tirapazamine (TPZ), which becomes a free radical only in conditions of hypoxemia, which is often enhanced in the tumor microenvironment. In this study, we demonstrate the efficacy of the combination therapy through experiments with B16-F10 and 1205 Lu metastatic melanoma cells both in vitro and in vivo. We also show the safety parameters of the therapy with no significant effects of the therapy when applied to porcine skin. We show the need for the intratumor delivery of TPZ in combination with the surface treatment of NTP and present a model of a medical device to deliver this combination therapy. The importance of functional gap junctions is indicated as a mechanism to promote the therapeutic effect. Collectively, the data support a novel therapeutic combination to treat melanoma and the development of a medical device to deliver the treatment in situ.<\/jats:p>","DOI":"10.3390\/cells12162113","type":"journal-article","created":{"date-parts":[[2023,8,21]],"date-time":"2023-08-21T08:53:31Z","timestamp":1692608011000},"page":"2113","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["The Development of Nonthermal Plasma and Tirapazamine as a Novel Combination Therapy to Treat Melanoma In Situ"],"prefix":"10.3390","volume":"12","author":[{"given":"Matthew","family":"Yehl","sequence":"first","affiliation":[{"name":"D\u2019Youville University School of Pharmacy, 320 Porter Avenue, Buffalo, NY 14201, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dominik","family":"Kucharski","sequence":"additional","affiliation":[{"name":"D\u2019Youville University School of Pharmacy, 320 Porter Avenue, Buffalo, NY 14201, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michelle","family":"Eubank","sequence":"additional","affiliation":[{"name":"D\u2019Youville University School of Pharmacy, 320 Porter Avenue, Buffalo, NY 14201, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brandon","family":"Gulledge","sequence":"additional","affiliation":[{"name":"D\u2019Youville University School of Pharmacy, 320 Porter Avenue, Buffalo, NY 14201, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0611-9646","authenticated-orcid":false,"given":"Gamal","family":"Rayan","sequence":"additional","affiliation":[{"name":"D\u2019Youville University School of Pharmacy, 320 Porter Avenue, Buffalo, NY 14201, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-7235-2082","authenticated-orcid":false,"given":"Md Gias","family":"Uddin","sequence":"additional","affiliation":[{"name":"D\u2019Youville University School of Pharmacy, 320 Porter Avenue, Buffalo, NY 14201, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Genevieve","family":"Remmers","sequence":"additional","affiliation":[{"name":"IDEXX Laboratories, 1 IDEXX Drive, Westbrook, ME 04092, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9310-7557","authenticated-orcid":false,"given":"Eugene S.","family":"Kandel","sequence":"additional","affiliation":[{"name":"Roswell Park Comprehensive Cancer Center, 665 Elm Street, Buffalo, NY 14203, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Douglas P.","family":"DuFaux","sequence":"additional","affiliation":[{"name":"Alfie Technology Corporation, 227 Thorn Avenue, Orchard Park, NY 14127, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1630-874X","authenticated-orcid":false,"given":"Timothy C.","family":"Hutcherson","sequence":"additional","affiliation":[{"name":"D\u2019Youville University School of Pharmacy, 320 Porter Avenue, Buffalo, NY 14201, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandra","family":"Sexton","sequence":"additional","affiliation":[{"name":"Roswell Park Comprehensive Cancer Center, 665 Elm Street, Buffalo, NY 14203, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shoshanna N.","family":"Zucker","sequence":"additional","affiliation":[{"name":"D\u2019Youville University School of Pharmacy, 320 Porter Avenue, Buffalo, NY 14201, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,8,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"495","DOI":"10.1001\/jamadermatol.2022.0160","article-title":"Global burden of cutaneous melanoma in 2020 and projections to 2040","volume":"158","author":"Arnold","year":"2022","journal-title":"JAMA Dermatol."},{"key":"ref_2","unstructured":"American Cancer Society (2023, June 28). Cancer Facts and Figures 2023. Available online: https:\/\/www.cancer.org\/content\/dam\/cancer-org\/research\/cancer-facts-and-statistics\/annual-cancer-facts-and-figures\/2023\/2023-cancer-facts-and-figures.pdf."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"35","DOI":"10.2147\/ITT.S134842","article-title":"Melanoma treatment in review","volume":"7","author":"Domingues","year":"2018","journal-title":"ImmunoTargets Ther."},{"key":"ref_4","unstructured":"American Cancer Society (2023, June 28). Chemotherapy for Melanoma Skin Cancer. Available online: https:\/\/www.cancer.org\/cancer\/types\/melanoma-skin-cancer\/treating\/chemotherapy.html."},{"key":"ref_5","unstructured":"American Cancer Society (2023, June 28). Radiation Therapy for Melanoma Skin Cancer. Available online: https:\/\/www.cancer.org\/cancer\/types\/melanoma-skin-cancer\/treating\/radiation-therapy.html."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1","DOI":"10.2478\/v10019-010-0008-x","article-title":"Role of radiotherapy in melanoma management","volume":"44","author":"Strojan","year":"2010","journal-title":"Radiol. Oncol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1304","DOI":"10.1002\/hed.25562","article-title":"Patterns of recurrence and retreatment outcomes among clinical stage I and II head and neck melanoma patients","volume":"41","author":"Namin","year":"2019","journal-title":"Head. Neck"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1016\/j.fsc.2017.03.002","article-title":"Mohs micrographic surgery for the management of cutaneous malignancies","volume":"25","author":"Mansouri","year":"2017","journal-title":"Facial Plast. Surg. Clin."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"767","DOI":"10.1016\/j.jaad.2019.05.057","article-title":"Mohs micrographic surgery for melanoma: A prospective multicenter study","volume":"81","author":"Ellison","year":"2019","journal-title":"J. Am. Acad. Dermatol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1533033818791795","DOI":"10.1177\/1533033818791795","article-title":"Photodynamic therapy for metastatic melanoma treatment: A review","volume":"17","author":"Naidoo","year":"2018","journal-title":"Technol. Cancer Res. Treat."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1120","DOI":"10.1016\/j.cell.2017.07.024","article-title":"Distinct cellular mechanisms underlie anti-CTLA-4 and anti-PD-1 checkpoint blockade","volume":"170","author":"Wei","year":"2017","journal-title":"Cell"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"S26","DOI":"10.1038\/d41586-018-02483-3","article-title":"Bringing down the cost of cancer treatment","volume":"555","author":"Dolgin","year":"2018","journal-title":"Nature"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"642","DOI":"10.1016\/j.ejca.2015.01.009","article-title":"Vemurafenib for BRAF V600 mutated advanced melanoma: Results of treatment beyond progression","volume":"51","author":"Scholtens","year":"2015","journal-title":"Eur. J. Cancer"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1248","DOI":"10.1016\/S1470-2045(16)30122-X","article-title":"Cobimetinib combined with vemurafenib in advanced BRAF(V600)-mutant melanoma (coBRIM): Updated efficacy results from a randomised, double-blind, phase 3 trial","volume":"17","author":"Ascierto","year":"2016","journal-title":"Lancet Oncol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"954","DOI":"10.1016\/S1470-2045(14)70301-8","article-title":"Combination of vemurafenib and cobimetinib in patients with advanced BRAF(V600)-mutated melanoma: A phase 1b study","volume":"15","author":"Ribas","year":"2014","journal-title":"Lancet Oncol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Curl, P., Vujic, I., van \u2018t Veer, L.J., Ortiz-Urda, S., and Kahn, J.G. (2014). Cost-effectiveness of treatment strategies for BRAF-mutated metastatic melanoma. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0107255"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2185","DOI":"10.1002\/ijc.26252","article-title":"ROS implication in a new antitumor strategy based on non-thermal plasma","volume":"130","author":"Vandamme","year":"2012","journal-title":"Int. J. Cancer"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"9269","DOI":"10.1021\/acsami.7b18653","article-title":"Adaptation of operational parameters of cold atmospheric plasma for in vitro treatment of cancer cells","volume":"10","author":"Gjika","year":"2018","journal-title":"ACS Appl. Mater. Interfaces"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1299","DOI":"10.4161\/cbt.21787","article-title":"Preferential induction of apoptotic cell death in melanoma cells as compared with normal keratinocytes using a non-thermal plasma torch","volume":"13","author":"Zucker","year":"2012","journal-title":"Cancer Biol. Ther."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"29020","DOI":"10.1038\/srep29020","article-title":"Anti-cancer efficacy of nonthermal plasma dissolved in a liquid, liquid plasma in heterogeneous cancer cells","volume":"6","author":"Nguyen","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"e1056","DOI":"10.1038\/cddis.2014.33","article-title":"Nonthermal plasma induces head and neck cancer cell death: The potential involvement of mitogen-activated protein kinase-dependent mitochondrial reactive oxygen species","volume":"5","author":"Kang","year":"2014","journal-title":"Cell Death Dis."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"915785","DOI":"10.3389\/fcell.2022.915785","article-title":"Cold atmospheric plasma induces apoptosis in human colon and lung cancer cells through modulating mitochondrial pathway","volume":"10","author":"Wang","year":"2022","journal-title":"Front. Cell Dev. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"3443","DOI":"10.18632\/oncotarget.27732","article-title":"Novel combination therapy for melanoma induces apoptosis via a gap junction positive feedback mechanism","volume":"11","author":"Bagati","year":"2020","journal-title":"Oncotarget"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Canady, J., Murthy, S.R.K., Zhuang, T., Gitelis, S., Nissan, A., Ly, L., Jones, O.Z., Cheng, X., Adileh, M., and Blank, A.T. (2023). The first cold atmospheric plasma phase I clinical trial for the treatment of advanced solid tumors: A novel treatment arm for cancer. Cancers, 15.","DOI":"10.3390\/cancers15143688"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"9916796","DOI":"10.1155\/2021\/9916796","article-title":"Cold physical plasma in cancer therapy: Mechanisms, signaling, and immunity","volume":"2021","author":"Faramarzi","year":"2021","journal-title":"Oxid. Med. Cell Longev."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Korbecki, J., Simi\u0144ska, D., Gassowska-Dobrowolska, M., Listos, J., Gutowska, I., Chlubek, D., and Baranowska-Bosiacka, I. (2021). Chronic and cycling hypoxia: Drivers of cancer chronic inflammation through HIF-1 and NF- \u03baB activation: A review of the molecular mechanisms. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms221910701"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"71","DOI":"10.2174\/157488406775268192","article-title":"Tirapazamine: From bench to clinical trials","volume":"1","author":"Marcu","year":"2006","journal-title":"Curr. Clin. Pharmacol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"736","DOI":"10.1158\/0008-5472.CAN-03-2488","article-title":"Selective potentiation of the hypoxic cytotoxicity of tirapazamine by its 1-N-oxide metabolite SR 4317","volume":"64","author":"Siim","year":"2004","journal-title":"Cancer Res."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"780","DOI":"10.1038\/bjc.1995.151","article-title":"Molecular mechanisms of tirapazamine (SR 4233, Win 59075)-induced hepatocyte toxicity under low oxygen concentrations","volume":"71","author":"Khan","year":"1995","journal-title":"Br. J. Cancer"},{"key":"ref_30","unstructured":"Zucker, S. (2017). Combination Therapy for Treating Cancer and Method for Treating Cancer Using a Combination Therapy. (9,586,056), U.S. Patent."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"579","DOI":"10.1038\/nrd2803","article-title":"Targeting cancer cells by ROS-mediated mechanisms: A radical therapeutic approach?","volume":"8","author":"Trachootham","year":"2009","journal-title":"Nat. Rev. Drug Discov."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"481782","DOI":"10.1155\/2015\/481782","article-title":"Cellular mechanisms of oxidative stress and action in melanoma","volume":"2015","author":"Venza","year":"2015","journal-title":"Oxid. Med. Cell Longev."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Remigante, A., Spinelli, S., Marino, A., Pusch, M., Morabito, R., and Dossena, S. (2023). Oxidative stress and immune response in melanoma: Ion channels as targets of therapy. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24010887"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"32074","DOI":"10.1038\/srep32074","article-title":"A micromechanical comparison of human and porcine skin before and after preservation by freezing for medical device development","volume":"6","author":"Ranamukhaarachchi","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1016\/j.molimm.2014.10.023","article-title":"The immunology of the porcine skin and its value as a model for human skin","volume":"66","author":"Summerfield","year":"2015","journal-title":"Mol. Immunol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1159\/000401274","article-title":"The skin of domestic mammals as a model for the human skin with special reference to the domestic pig","volume":"7","author":"Meyer","year":"1978","journal-title":"Curr. Probl. Dermatol."},{"key":"ref_37","unstructured":"Zucker, S.N., and DuFaux, D.P. (2022). Method and Apparatus for Administering a Cancer Drug. (Application No. 2022\/0399096), U.S. Patent."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"853","DOI":"10.1002\/jcp.24235","article-title":"A dominant negative Cx43 mutant differentially affects tumorigenic and invasive properties in human metastatic melanoma cells","volume":"228","author":"Zucker","year":"2013","journal-title":"J. Cell Physiol."},{"key":"ref_39","unstructured":"(2023, June 28). Registry of Industrial Toxicology Animal-Data. Available online: https:\/\/reni.item.fraunhofer.de\/reni\/trimming."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"700407","DOI":"10.3389\/fonc.2021.700407","article-title":"Targeting hypoxia: Hypoxia-activated prodrugs in cancer therapy","volume":"11","author":"Li","year":"2021","journal-title":"Front. Oncol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"7994","DOI":"10.1074\/jbc.M506533200","article-title":"Mutation of a conserved threonine in the third transmembrane helix of alpha- and beta-connexins creates a dominant-negative closed gap junction channel","volume":"281","author":"Beahm","year":"2006","journal-title":"J. Biol. Chem."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"894","DOI":"10.1007\/s12325-016-0338-1","article-title":"Clinical and biological principles of cold atmospheric plasma application in skin cancer","volume":"33","author":"Ruano","year":"2016","journal-title":"Adv. Ther."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1295","DOI":"10.1038\/bjc.2011.386","article-title":"Cold plasma selectivity and the possibility of a paradigm shift in cancer therapy","volume":"105","author":"Keidar","year":"2011","journal-title":"Br. J. Cancer"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"264","DOI":"10.1002\/ppap.200900080","article-title":"Antitumor effect of plasma treatment on U87 glioma xenografts: Preliminary results","volume":"7","author":"Vandamme","year":"2010","journal-title":"Plasma Processes Polym."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1400","DOI":"10.1002\/ppap.201500140","article-title":"Reaction chemistry generated by nanosecond pulsed dielectric barrier discharge treatment is responsible for the tumor eradication in the B16 melanoma mouse model","volume":"12","author":"Chernets","year":"2015","journal-title":"Plasma Process Polym."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"11937","DOI":"10.1073\/pnas.1613466113","article-title":"Hypoxia-activated cytoxic agent tirapazamine enhances hepatic artery ligation-induced killing of liver tumor in HBx transgenic mice","volume":"113","author":"Lin","year":"2016","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"699","DOI":"10.1021\/acs.chemrestox.6b00417","article-title":"Cytotoxicity of tirapazamine (3-Amino-1,2,4-benzotriazine-1,4-dioxide)-induced DNA damage in chicken DT40 cells","volume":"30","author":"Moriwaki","year":"2017","journal-title":"Chem. Res. Toxicol."}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/12\/16\/2113\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:38:31Z","timestamp":1760128711000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/12\/16\/2113"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,8,21]]},"references-count":47,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2023,8]]}},"alternative-id":["cells12162113"],"URL":"https:\/\/doi.org\/10.3390\/cells12162113","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,8,21]]}}}