Effect of Silver Nitrate and Colloidal Silver With and Without Agents to Augment Their Action on Two Transitional Cell Bladder Carcinoma Cell Lines

Effect of Silver Nitrate and Colloidal Silver With and Without Agents to Augment Their Action on Two Transitional Cell Bladder Carcinoma Cell Lines | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effect of Silver Nitrate and Colloidal Silver With and Without Agents to Augment Their Action on Two Transitional Cell Bladder Carcinoma Cell Lines Thomas C Cesario, Martina Berger, Keyvan Khosh Abady, Peter Rentzepis This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7942436/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract We determined the effect of silver as both silver nitrate and colloidal silver on UM-UC-3 and J 82 bladder carcinoma cell lines. We found both forms of silver are potent agents in destroying both cancer cell lines as might be anticipated but we went on to show, using silver as an agent itself, but also as a potential surrogate for other agents, that the effect we observed was enhanced by addition of a source of oxygen radicals, hydrogen peroxide. Further the effect of this combination was augmented by pretreatment with ethacrynic acid to try to decrease glutathione levels in the cells. This three agent model could be used topically in bladder and possibly other superficial carcinomas and should be tested further in other models. Introduction Silver is known to have lethal effects on living cells. It has been shown to have fatal influences on bacteria (Chernousova,2013 ), fungi (Kim, 2009) and cancer cells (Barua, 2017). Silver renders its effects by the generation of toxic oxygen radicals (Zhang, 2019), membrane injury (Zhang, 2019), and damage to genetic material (Hackenberg, 2011). It has been shown to effect breast cancer (Ulagesan, 2021 ) and bladder cancer cells (Ferreira,2020 ). It can be applied topically, has been instilled into the bladder to treat bladder hemorrhage (Kumar,1976)and tried as a salvage therapy after treatment of bladder cancer (Flamm, 1984). We enhanced the effects of silver nitrate and colloidal silver by providing a source of oxygen radicals, hydrogen peroxide, to increase oxidative stress in the hypoxic environments of cancer cells (Zhang 2019, Lin 2018 ). We augmented the antineoplastic activity of silver/hydrogen peroxide by trying to compromise the ability of the cells to mitigate the effects of reactive oxygen using ethacrynic acid (EA) which has antineoplastic activities (Yu, 2023), can inhibit the action of glutathione transferase (Musdal,2023) and may reduce levels of glutathione( 23 ). EA may be useful in the treatment of noninvasive bladder neoplasms as it appears to be excreted in the urinary system (Jackson,2018) and has been used clinically (Alisky, 2003). Materials and Methods Colloidal silver, gold, copper and zinc were purchased from Purest Colloids (Westampton NJ; Catalog numbers not used) containing silver particles 65 nanometers or less (Product brocheur). Silver nitrate was obtained from Flinn Scientific, (Batavia, Ill; Catalog number SO 434). Hydrogen peroxide was purchased from Rite Aid (Camp Hill Pa). Ethacrynic acid (EA) was obtained from MilliporeSigma (Burlington, Mass; Catalog number SML 1083). MTT reagent was obtained from Biotium (Fremont Calif.; Catalog number 30006). UM-UC-3 (Catalog number CRL 1749) and J 82 (Catalog number HTB 1) transitional cell bladder carcinoma cells were purchased from American Type Culture Collection (Manassas, Va) and grown in Minimal Essential Media (MEM) from Mediatech (Manassas,Va; Catalog number 10-022-CV) with fetal bovine serum (FBS) purchased from JR Scientific (Woodland, Calif,; Catalog number 35–073–CV). Assays were performed in 96 well microtiter plates purchased from Genesee Scientific (El Cajon, Calif. Catalog number 22-553V) on confluent cells. Assays were performed by emptying the plates by inversion, adding the reagents in the appropriate concentration in phosphate buffered saline ( PBS) containing either BFS or bovine serum albumin (BSA) obtained from MilliporeSigma (Burlington Mass.; Catalog number 810683). Incubation was carried out for three hours in a 37 centigrade degree incubator in the presence of 5% CO2. Subsequently, the plates were emptied, the wells washed twice with PBS, and then MTT reagent was added and the plates incubated a further two hours. DMSO ( MilliporeSigma, Burlington Mass.; Catalog number 472301) was added and the plates read at 570 and 630 nanometers. EA was used diluted in McCoys Media obtained from ATCC (Manassas, Va,; Catalog number 30-2007) or MEM, supplemented as above, and incubated for three hours in a 5% CO2 incubator. The plates were then emptied, fresh EA plus the appropriate concentration of other reagents were added, and the assayed carried out as above. The percentage of surviving cells was calculated from unexposed control wells. EA was stored frozen until used. Glutathione was assayed using a colorimetric detection kit from Arbor Assays (Ann Arbor Mich.; Catalog number K006-H1) on cells grown as above. Statistical comparisons were performed by t test ( 13 ). Protocols were reviewed and approved by the UCI IRB before commencement of the study. Data can be made available by contacting the lead investigator Results We determined the minimal concentration of silver nitrate, colloidal silver and hydrogen peroxide required to effect cell survival for both cell lines. It took five to ten times as much colloidal silver to kill equivalent amounts of cells as did silver nitrate related to the likelihood that ionic silver in colloidal silver is responsible for cell death. We also demonstrated the time required to effect cell viability in the presence of these reagents. We determined the effect of the combination of hydrogen peroxide with both silver nitrate (Table 1) and colloidal silver (Table 2) on both cell lines. Using either form of silver, the effect of the combination on cell survival was greater than either of the agents tested alone in every case. Concentrations of EA from 110 micromoles to 27.5 preserved sufficient cell viability to assess effect and were used to test the result of the combination of EA with silver nitrate or colloidal silver (Table 3). The combination of the two agents resulted in lower percentages of cell survival than either agent used alone. Using hydrogen peroxide with EA did not result in such an effect. We tested silver nitrate, hydrogen peroxide and EA on the viability of UM-UC-3 and J 82 cells employing minimum concentrations to demonstrate an effect. Tables 4 and 5 show these results. In 8 of the 8 cases tested, UM-UC-3 cell survival was lower with the triple combination than any single or dual combination of agents (t < .05) tested. For J 82 cells a greater effect of the combination of EA, silver and hydrogen peroxide was recorded only with the higher concentration of EA tested (55 microM) compared to any dual combination or single agent tested alone (t < .05). We compared the effect of the silver forms used here with other agents (Table 6). We measured glutathione in the presence and absence of EA concentrations that we determined to preserve viability in our cultures but at these concentrations we could not significantly show the low glutathione levels existent in J 82 cells were reduced by the EA (Table 7). We attempted to do this in UM-UC -3 cells but could not get a sufficient number of cells to draw a conclusion as to the effects of EA on glutathione levels in those cells. Discussion Silver nanoparticles have been shown to reduce the viability of tumor cell lines from breast (Ulagesan, 2021), lung (Pallavi, 2022), cervical (Barua, 2017), bone (Michalakis, 2021) and leukemia (Guo, 2014) neoplasms. Ferreira (Ferreira, 2020) found silver nanoparticles reduced cell viability in bladder cancer cells. Castiglioni (Castiglioni, 2025)showed that the cytotoxic effects of silver nanoparticles on T24 cells was dependent on ERK activation and Flamm and Grof (Flamm, 1984) tried to use silver nitrate installation following surgery for bladder tumors. We have shown that the combination of either colloidal silver or silver nitrate and hydrogen peroxide yields greater cancer cell loss than any of these agents used alone. Choudhary et al (Choudhary, 2011) reported hydrogen peroxide cooperated with oncogenic H-Ras and FK 228 to activate the erk pathway inducing J82 cell death. We found the combination of silver forms and hydrogen peroxide is augmented by EA in the probable hypoxic environment of these cells (Zhang,2019, Lin 2018). We have done this using concentrations and exposure times previously used (Fletcher, 2015, Shen, 1990). EA has been used with other agents (Nagouney, 1990) but not in bladder cells. The BSA and FBS used here may have diminished the effects we report here by binding silver (Shahabadi, 2019, Gnanadhas, 2019) . While the mean levels of glutathione were lower in treated cells we could not verify the reduction was significant due to the low levels of this agent in the J 82 cells. It is possible the levels of glutathione were reduced enough to account for the effects we observed but the effect of EA we report may be due to other properties (Yu,2023) of EA. Conclusion The antineoplastic effects of silver compounds are enhanced by hydrogen peroxide and augmented further by pretreatment with EA. This combination should be further tested in live models and EA should be studied further in bladder cancers using other chemotherapeutic agents. The combination of silver and other agents as we have used here should be studied further in the treatment of other superficial cancers where treatments could be applied topically. Declarations Author Contribution Thomas Cesario: Principle investigator, authorMartina Berger: Investigator, Reviewed and edited manuscriptKeyvan Khosh Abady: Reviewed manuscript, Intellectual contributionsPeter Rentzepis: Reviewed manuscript, intellectual contributions References Alisky J and Tuttle T. “Ethacrynic acid can be effective for refractory congestive heart failure and ascites” South Med J. 2003; 96:1148–50. Https://doi 1097/01.smj.0000082004.40613.07. Barua, S, Banerjee P, Sadhu A, et al. “Silver nanoparticles as antibacterial and anticancer materials against human breast, cervical and oral cancers cells.” Nanosci and Nanotechnol. 2017: 17:968–976. Https://doi 10.1166/jnn.2017.12636 . Castiglioni s, Cazzaniga, Perotta C, Maier J. “Silver nanoparticles induced cytotoxicity requires ERK activation in human bladder carcinoma cells” Toxicol Lett. 2025 237:237 – 43. Https://doi 10.1016/j.toxlet.2015.06.1707 . Chen Z, Han F, Du Y, Shi H, Zhou W. “Hypoxic microenvironment in cancer: Molecular mechanisms and therapeutic interventions” Signal Transduction Target Ther 2023; 8: 70. Https://doi10.1038/s41392-023-01332-8. Chernousova S and Epple M. “Silver as antibacterial agent: Ion, nanoparticle and metal” Angew. Chem Int. Ed. 2013; 52:1636–1653. Https://doi10.1002/anie.201205923. Choudhary S, Wang K and Wang H. “Oncogenic H-Ras, FK228 and exogenous H2O2 cooperatively activate the erk pathway in selective induction of human bladder cancer J82 cell death” Molecular Carcinogenesis. 2011; 50:215–219. Https://doi:10.1002/mc.20708 Ferreira A, Garcia-Fossa F, Radaic A, Duran N, Favero W andde Jesus M. “ Biogenic silver nanoparticles in vitro and in vivo antitumor activity in bladder cancer” Eur J Pharm Biopharm. 2020; 151:162–170, Https://doi10.1016/j.ejpb.2020.04.012 Flamm J and Grof F. “Can the persistence rate of transurethrally resected bladder tumors be lowered by postoperatively instillation of silver nitrate” Z Urol Nephrol 1984;77:1–6. (Article in German). Fletcher M, Boshier P, Wakabayashi K, et al.“ Influence of glutathione S transferase (GST) inhibition on lung epithelial cell injury: role of oxidative stress and metabolism” Am J Physiol Lung Cell Mol Physiol. 2015; 308:1274–85. Https://doi10.1152/ajplung.00220.2014 Gnanadhas D, Thomas M, Thomas R and Raichur A. “Interaction of silver nanoparticles with serum proteins affects their antimicrobial activity in vivo” Antimicrob Agents Chemother. 2013; 57: 4945–4955. Https://doi.1128/aac.00152 – 13 Guo D, Zhao Y, Zhang Y, et al. “The cellular uptake and cytotoxic effect of silver nanoparticles on chronic myeloid leukemia cells” J Biomed Nanotech 2014; 10:669–78. Https://doi.1166/jbn.2014.1625. Hackenberg S, Scherzed A, Kessler M et al. “Silver nanoparticles: Evaluation of DNA damage, toxicity and functional impairment in human mesenchymal cells” Toxicol Lett 2011; 201:27–33. Https://doi10.1016/j.toxlet.2010.12.001. Hill A B. “Problems of sampling; Differences between averages” in Principles of Medical Statistics. A. B. Hill, 1991; Twelfth edition: 143–161, Oxford University Press, New York . Jackson E. “Drugs affecting renal excretory function” In The Pharmacological Basis of Therapeutics editors Brunton L, Hilal-Danda R and Knollman B, New York 2018, pp445-470. Kim K, Sung W, Suh K, Moon S, Choi J, Kim J, Lee D. “Antifungal activity of silver nanoparticles on Candida albicans” Biometals 2009;22:235–242. Https://doi 10.1007/s10534-008-9159-2 . Kumar A, Wrenn E, Jayalakshmamma B, Conrad L, Quinn P and Cox C.”Silver irrigation to control bladder hemorrhage in children receiving cancer chemotherapy”J Urol 1976; 116:85–86. Https://doi10.1016/s0022-5347(17)58690-5 Lin T, Zhao X, Zhao S et al.“O2-generating MnO2 nanoparticles for enhanced photodynamic therapy of bladder cancer by ameliorating hypoxia” Theranostics 2018; 8:990–1004. Https://doi10.7150/thno.22465 Michalakis K, Bakopoulou A, Papachristou E, et al.“Evaluation of response of HOS and Saos-2 osteosarcoma cell lines when exposed to different sizes and concentrations of silver nanoparticles” Biomed Res Int. 2021 Open Access. Https://doi 10.1155/2021/5013065 . Musdal Y. Hegagazy U, Aksoy Y, Mannervik B. “ FDA approved drugs and other compounds tested as inhibitors glutathione transferase P1” Chem Biol Interact. 2023;205:53–62. Https://doi 10.1016/j.cbi.2013.06.003 . Nagourney R, Messenger J, Kern D and Weisenthal L. “Enhancement of anthracycline and alkylator cytotoxicity by ethacrynic acid in primary cultures of human tissues” Cancer Chemother and Pharmacol. 1990; 26:318–322. Https://doi10.1007/BF02897285. Pallavi S, Rudayni H, Bepari A, Niazi S and Nayaka S. “ Green synthesis of silver nanoparticles using Streptomyces hirsutus strain SNPGA-8 and their characterization, antimicrobial activity and anticancer activity against human lung carcinoma cell line A549” Saudi Biol Sci. 2022; 29:228–238. Https://10.1016/jsbs.2021.08.084 . Rizzardini M, Lupi M, Bernasconi S, Mangolini A, Cantoni L. “Mitochondrial dysfunction and death in motor neurons exposed to the glutathione-depleting agent ethacrynic acid” J Neurol Sci 2003;207:51–58. Https://doi10.1016/s0022-510x(02)00357-x Shahabadi N, Maghsudi M, Ahmadipour Z. “ Study on the interaction of silver complexes with bovine serum albumin by spectroscopic tehniques” Spectrochim Acta A Mol Biomol Spectrosc. 2019; 92:184–188. Https://doi 10.1016/j.saa.2012.02.071 Shen H, Ranganathan S, Kuzmich S and Tew K. “Influence of ethacrynic acid in glutathione s-transferase and protein half lives in human colon cancer cells” Biochemical Pharmacology “ Cancer Chemotherapy and Pharmacology. 1990; 50:1233–1238. Https://doi10.1016/0006-2952(95)00263-y Ulagesan S, Nam J, Choi Y. “ Cytotoxicity against breast carcinoma cells of silver nanoparticles biosynthesized using Capsosiphon fulvescens extract” Bioprocess Biosyst Eng 2021; 44:901–911. Https://doi10.1007/s00449-020-02498-2 Yu L, Lee H, Rho S, Park M and Lee C. “Ethacrynic acid: A promising candidate for drug repurposing as an anticancer agent” International Journal of molecular Sciences. 2023; 24–6712 - doi.org/10.3390/ijms24076712 Zhang L, Wu L, Yazhu M, Youbin S. “Silver nanoparticles induced cell apoptosis, membrane damage of Azotobacteria vinelandia and Nitosomonas europaea via generation of reactive oxygen species” Bull Environ Contam Toxicol 2019;103:181–186. Https://doi 10.1007/s00128-019-02622-0 . Tables Tables are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Tables.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 23 Dec, 2025 Reviewers agreed at journal 17 Nov, 2025 Reviewers invited by journal 12 Nov, 2025 Editor assigned by journal 25 Oct, 2025 Submission checks completed at journal 25 Oct, 2025 First submitted to journal 24 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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It has been shown to have fatal influences on bacteria (Chernousova,2013 ), fungi (Kim, 2009) and cancer cells (Barua, 2017). Silver renders its effects by the generation of toxic oxygen radicals (Zhang, 2019), membrane injury (Zhang, 2019), and damage to genetic material (Hackenberg, 2011). It has been shown to effect breast cancer (Ulagesan, 2021 ) and bladder cancer cells (Ferreira,2020 ). It can be applied topically, has been instilled into the bladder to treat bladder hemorrhage (Kumar,1976)and tried as a salvage therapy after treatment of bladder cancer (Flamm, 1984).\u003c/p\u003e\u003cp\u003eWe enhanced the effects of silver nitrate and colloidal silver by providing a source of oxygen radicals, hydrogen peroxide, to increase oxidative stress in the hypoxic environments of cancer cells (Zhang 2019, Lin 2018 ). We augmented the antineoplastic activity of silver/hydrogen peroxide by trying to compromise the ability of the cells to mitigate the effects of reactive oxygen using ethacrynic acid (EA) which has antineoplastic activities (Yu, 2023), can inhibit the action of glutathione transferase (Musdal,2023) and may reduce levels of glutathione(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). EA may be useful in the treatment of noninvasive bladder neoplasms as it appears to be excreted in the urinary system (Jackson,2018) and has been used clinically (Alisky, 2003).\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eColloidal silver, gold, copper and zinc were purchased from Purest Colloids (Westampton NJ; Catalog numbers not used) containing silver particles 65 nanometers or less (Product brocheur). Silver nitrate was obtained from Flinn Scientific, (Batavia, Ill; Catalog number SO 434). Hydrogen peroxide was purchased from Rite Aid (Camp Hill Pa). Ethacrynic acid (EA) was obtained from MilliporeSigma (Burlington, Mass; Catalog number SML 1083). MTT reagent was obtained from Biotium (Fremont Calif.; Catalog number 30006). UM-UC-3 (Catalog number CRL 1749) and J 82 (Catalog number HTB 1) transitional cell bladder carcinoma cells were purchased from American Type Culture Collection (Manassas, Va) and grown in Minimal Essential Media (MEM) from Mediatech (Manassas,Va; Catalog number 10-022-CV) with fetal bovine serum (FBS) purchased from JR Scientific (Woodland, Calif,; Catalog number 35\u0026ndash;073\u0026ndash;CV). Assays were performed in 96 well microtiter plates purchased from Genesee Scientific (El Cajon, Calif. Catalog number 22-553V) on confluent cells.\u003c/p\u003e\u003cp\u003eAssays were performed by emptying the plates by inversion, adding the reagents in the appropriate concentration in phosphate buffered saline ( PBS) containing either BFS or bovine serum albumin (BSA) obtained from MilliporeSigma (Burlington Mass.; Catalog number 810683). Incubation was carried out for three hours in a 37 centigrade degree incubator in the presence of 5% CO2. Subsequently, the plates were emptied, the wells washed twice with PBS, and then MTT reagent was added and the plates incubated a further two hours. DMSO ( MilliporeSigma, Burlington Mass.; Catalog number 472301) was added and the plates read at 570 and 630 nanometers.\u003c/p\u003e\u003cp\u003eEA was used diluted in McCoys Media obtained from ATCC (Manassas, Va,; Catalog number 30-2007) or MEM, supplemented as above, and incubated for three hours in a 5% CO2 incubator. The plates were then emptied, fresh EA plus the appropriate concentration of other reagents were added, and the assayed carried out as above. The percentage of surviving cells was calculated from unexposed control wells. EA was stored frozen until used.\u003c/p\u003e\u003cp\u003eGlutathione was assayed using a colorimetric detection kit from Arbor Assays (Ann Arbor Mich.; Catalog number K006-H1) on cells grown as above.\u003c/p\u003e\u003cp\u003eStatistical comparisons were performed by t test (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eProtocols were reviewed and approved by the UCI IRB before commencement of the study.\u003c/p\u003e\u003cp\u003eData can be made available by contacting the lead investigator\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eWe determined the minimal concentration of silver nitrate, colloidal silver and hydrogen peroxide required to effect cell survival for both cell lines. It took five to ten times as much colloidal silver to kill equivalent amounts of cells as did silver nitrate related to the likelihood that ionic silver in colloidal silver is responsible for cell death. We also demonstrated the time required to effect cell viability in the presence of these reagents.\u003c/p\u003e\u003cp\u003eWe determined the effect of the combination of hydrogen peroxide with both silver nitrate (Table\u0026nbsp;1) and colloidal silver (Table\u0026nbsp;2) on both cell lines. Using either form of silver, the effect of the combination on cell survival was greater than either of the agents tested alone in every case.\u003c/p\u003e\u003cp\u003eConcentrations of EA from 110 micromoles to 27.5 preserved sufficient cell viability to assess effect and were used to test the result of the combination of EA with silver nitrate or colloidal silver (Table\u0026nbsp;3). The combination of the two agents resulted in lower percentages of cell survival than either agent used alone. Using hydrogen peroxide with EA did not result in such an effect.\u003c/p\u003e\u003cp\u003eWe tested silver nitrate, hydrogen peroxide and EA on the viability of UM-UC-3 and J 82 cells employing minimum concentrations to demonstrate an effect. Tables\u0026nbsp;4 and 5 show these results. In 8 of the 8 cases tested, UM-UC-3 cell survival was lower with the triple combination than any single or dual combination of agents (t\u0026thinsp;\u0026lt;\u0026thinsp;.05) tested.\u003c/p\u003e\u003cp\u003eFor J 82 cells a greater effect of the combination of EA, silver and hydrogen peroxide was recorded only with the higher concentration of EA tested (55 microM) compared to any dual combination or single agent tested alone (t\u0026thinsp;\u0026lt;\u0026thinsp;.05).\u003c/p\u003e\u003cp\u003eWe compared the effect of the silver forms used here with other agents (Table\u0026nbsp;6).\u003c/p\u003e\u003cp\u003eWe measured glutathione in the presence and absence of EA concentrations that we determined to preserve viability in our cultures but at these concentrations we could not significantly show the low glutathione levels existent in J 82 cells were reduced by the EA (Table\u0026nbsp;7). We attempted to do this in UM-UC -3 cells but could not get a sufficient number of cells to draw a conclusion as to the effects of EA on glutathione levels in those cells.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSilver nanoparticles have been shown to reduce the viability of tumor cell lines from breast (Ulagesan, 2021), lung (Pallavi, 2022), cervical (Barua, 2017), bone (Michalakis, 2021) and leukemia (Guo, 2014) neoplasms. Ferreira (Ferreira, 2020) found silver nanoparticles reduced cell viability in bladder cancer cells.\u003c/p\u003e\u003cp\u003eCastiglioni (Castiglioni, 2025)showed that the cytotoxic effects of silver nanoparticles on T24 cells was dependent on ERK activation and Flamm and Grof (Flamm, 1984) tried to use silver nitrate installation following surgery for bladder tumors.\u003c/p\u003e\u003cp\u003eWe have shown that the combination of either colloidal silver or silver nitrate and hydrogen peroxide yields greater cancer cell loss than any of these agents used alone. Choudhary et al (Choudhary, 2011) reported hydrogen peroxide cooperated with oncogenic H-Ras and FK 228 to activate the erk pathway inducing J82 cell death.\u003c/p\u003e\u003cp\u003eWe found the combination of silver forms and hydrogen peroxide is augmented by EA in the probable hypoxic environment of these cells (Zhang,2019, Lin 2018). We have done this using concentrations and exposure times previously used (Fletcher, 2015, Shen, 1990). EA has been used with other agents (Nagouney, 1990) but not in bladder cells.\u003c/p\u003e\u003cp\u003eThe BSA and FBS used here may have diminished the effects we report here by binding silver (Shahabadi, 2019, Gnanadhas, 2019) .\u003c/p\u003e\u003cp\u003eWhile the mean levels of glutathione were lower in treated cells we could not verify the reduction was significant due to the low levels of this agent in the J 82 cells. It is possible the levels of glutathione were reduced enough to account for the effects we observed but the effect of EA we report may be due to other properties (Yu,2023) of EA.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe antineoplastic effects of silver compounds are enhanced by hydrogen peroxide and augmented further by pretreatment with EA. This combination should be further tested in live models and EA should be studied further in bladder cancers using other chemotherapeutic agents. The combination of silver and other agents as we have used here should be studied further in the treatment of other superficial cancers where treatments could be applied topically.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThomas Cesario: Principle investigator, authorMartina Berger: Investigator, Reviewed and edited manuscriptKeyvan Khosh Abady: Reviewed manuscript, Intellectual contributionsPeter Rentzepis: Reviewed manuscript, intellectual contributions\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlisky J and Tuttle T. \u0026ldquo;Ethacrynic acid can be effective for refractory congestive heart failure and ascites\u0026rdquo; South Med J. 2003; 96:1148\u0026ndash;50. 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Https://doi10.1007/s00449-020-02498-2\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYu L, Lee H, Rho S, Park M and Lee C. \u0026ldquo;Ethacrynic acid: A promising candidate for drug repurposing as an anticancer agent\u0026rdquo; International Journal of molecular Sciences. 2023; 24\u0026ndash;6712\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e- doi.org/10.3390/ijms24076712\u003c/span\u003e\u003cspan address=\"- 10.3390/ijms24076712\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhang L, Wu L, Yazhu M, Youbin S. \u0026ldquo;Silver nanoparticles induced cell apoptosis, membrane damage of Azotobacteria vinelandia and Nitosomonas europaea via generation of reactive oxygen species\u0026rdquo; Bull Environ Contam Toxicol 2019;103:181\u0026ndash;186. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003eHttps://doi 10.1007/s00128-019-02622-0\u003c/span\u003e\u003cspan address=\"Https://doi 10.1007/s00128-019-02622-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biometals","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biom","sideBox":"Learn more about [BioMetals](http://link.springer.com/journal/10534)","snPcode":"10534","submissionUrl":"https://submission.nature.com/new-submission/10534/3","title":"BioMetals","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7942436/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7942436/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe determined the effect of silver as both silver nitrate and colloidal silver on UM-UC-3 and J 82 bladder carcinoma cell lines. We found both forms of silver are potent agents in destroying both cancer cell lines as might be anticipated but we went on to show, using silver as an agent itself, but also as a potential surrogate for other agents, that the effect we observed was enhanced by addition of a source of oxygen radicals, hydrogen peroxide. Further the effect of this combination was augmented by pretreatment with ethacrynic acid to try to decrease glutathione levels in the cells. This three agent model could be used topically in bladder and possibly other superficial carcinomas and should be tested further in other models.\u003c/p\u003e","manuscriptTitle":"Effect of Silver Nitrate and Colloidal Silver With and Without Agents to Augment Their Action on Two Transitional Cell Bladder Carcinoma Cell Lines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-24 11:15:50","doi":"10.21203/rs.3.rs-7942436/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2025-12-23T20:13:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"187384574729641188705629361376556827661","date":"2025-11-17T19:41:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-12T07:39:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-25T08:32:22+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-25T08:30:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"BioMetals","date":"2025-10-24T16:52:18+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biometals","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"biom","sideBox":"Learn more about [BioMetals](http://link.springer.com/journal/10534)","snPcode":"10534","submissionUrl":"https://submission.nature.com/new-submission/10534/3","title":"BioMetals","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"cfb3f34e-de81-4354-8500-b5f21782c63e","owner":[],"postedDate":"November 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-11-24T11:15:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-24 11:15:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7942436","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7942436","identity":"rs-7942436","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}