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

doi:10.21203/rs.3.rs-7942436/v1

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

https://doi.org/10.21203/rs.3.rs-7942436/v1

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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.

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).

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

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.

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.

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.

Author Contribution

Thomas Cesario: Principle investigator, authorMartina Berger: Investigator, Reviewed and edited manuscriptKeyvan Khosh Abady: Reviewed manuscript, Intellectual contributionsPeter Rentzepis: Reviewed manuscript, intellectual contributions

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Tables are available in the Supplementary Files section.

No competing interests reported.

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

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