News Release
Title
Ferroptosis induced by plasma-activated Ringer’s lactate solution prevents
oral cancer progression
Key Points
・ We discovered that the killing effect of plasma-activated Ringer’s lactate(PAL) is specific to OSCC cells and that PAL induces ferroptosis.
・PAL prevents cancer progression and improves the survival rate by decreasing
LOX expression in cancer cells and changing collagen matrix formation.
SummaryThis study aimed to investigate the effect of plasma-activated Ringer’s lactate
solution (PAL) on cancer cells, considering iron metabolism and the tumormicroenvironment.
PAL killed oral squamous cell carcinoma (OSCC) cells at lower concentrations
than epithelial cells and fibroblasts in vitro. The killing effect was prevented bydeferoxamine and ferrostatin-1. Apoptosis, necroptosis and autophagy wereexcluded. After PAL treatment, iron increased in the cells, and lipid peroxidation
occurred. PAL dramatically decreased the lysyl oxidase levels. Furthermore, PAL
inhibited both migration and invasion. PAL reduced the incidence rate of SCC
and improved the survival rate without causing severe side effects in vivo. PAL
treatment resulted in lower expression of GPx4 and higher expression of
8-OHdG and 4-HNE. The type of cell death was ferroptosis. Furthermore, thestudy results indicate that PAL decreased lysyl oxidase (LOX) expression incancer cells and the amount of collagen. To our knowledge, this is the first study
to evaluate the effect of PAL on oral cancer with a focus on iron and collagenous
matrix formation.
PAL could provide a sustainable and effective therapy for oral cancer patients.
These data support the further clinical development of PAL treatment for oralcancer.
Research BackgroundThe number of patients with oral cancer has been increasing worldwide, andthe 5-year survival rate of patients with tongue cancer is 67.1%. This rate has
not improved since 2010, even though the prognosis is relatively good if
patients receive prompt and early treatment. Surgery is the first choice oftreatment. However, some patients cannot undergo surgery because of theirgeneral condition or medical history, and chemotherapy and radiotherapy arethus performed instead. These treatments are particularly effective atpreserving tissue but can cause severe side effects in some patients,
necessitating treatment discontinuation. Furthermore, preventing recurrence
after primary treatment is important to maintain the quality of life of patientsand increase their survival rates. Although the morbidity associated with oral

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cancer, which affects approximately 2% of patients, is not as high as that ofother cancers, the reduction in quality of life after treatment is severe.
Therefore, developing sustainable, effective and harmless novel treatments isimportant as a key research effort.
Nonthermal plasma (NTP), or low-temperature plasma, is a contradictory
condition with high energy at near-body temperature that was invented via
engineering in the 1990s. NTP is currently being used in preclinical experiments
in various fields. NTP can be applied in two ways: directly on lesions or indirectly
using Ringer’s lactate solution irradiated with NTP, which is called plasmaactivated Ringer’s lactate solution (PAL). Recently, some researchers haveshown that PAL is effective against ovarian and pancreatic cancers in vivo.
It has been considered that cancer cells contain Fe2+ more than nontumorouscells and that is a key factor of the killing effect of PAL. In addition, someresearchers have reported that collagen created around cancer cell is closely
related with cancer metastasis.

Scheme of PAL production and application method (Figure 1)：PAL is made fromapplying plasma to Ringer’s lactate solution

Research Results
We used three OSCC cell lines, one keratinocyte cell line and two fibroblastlines, and cell viability assays, immunoblotting, flow cytometry and transmission
electron microscopy were performed to evaluate the effect and type of celldeath. The effect of PAL treatment on lysyl oxidase (LOX) expression wasinvestigated in vitro and in vivo. Tamoxifen-inducible Mob1a/b double-knockout
mice were used for the in vivo experiment. We found that PAL specifically kills
OSCC cells and that the type of cell death is ferroptosis. Furthermore, theresults indicate that PAL prevents cancer progression and improves the survival
rate by decreasing LOX expression in cancer cells and changing collagen matrix
formation.

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Schematic of the plasma-activated Ringer’s lactate solution (PAL) treatment of oral
cancer cells.
PAL upregulates catalytic Fe2+ in cancer cells, influencing iron metabolism and lipid
peroxidation and leading to ferroptosis. Furthermore, PAL downregulates LOX in cancer
cells, leading to the suppression of cancer progression. Fe, ferrum; TF, transferrin; TfR,transferrin receptor; FPN, ferroportin; ●OH, hydroxyl radical; GPx4, glutathione
peroxidase 4; LOX, lysyl oxidase; PAL, plasma-activated Ringer’s lactate solution.

Research Summary and Future Perspective
We found that PAL specifically kills OSCC cells and induces ferroptosis.
Furthermore, the results indicate that PAL prevents cancer progression andimproves the survival rate by decreasing LOX expression in cancer cells andchanging collagen matrix formation (Figure 2). PAL could provide a sustainable
and effective therapy for patients with oral cancer. These data support thefurther clinical development of PAL treatment for oral cancer.
Publication
Kotaro Sato, Ming Yang, Kae Nakamura, Hiromasa Tanaka, Masaru Hori,
Miki Nishio, Akira Suzuki, Hideharu Hibi, Shinya Toyokuni. Ferroptosis inducedby plasma-activated Ringer’s lactate solution prevents oral cancerprogression. Oral Diseases, December 4, 2023 (Version of Record online)
DOI: 10.1111/ODI.14827
URL: https://doi.org/10.1111/odi.14827

Japanese ver.
https://www.med.nagoya-u.ac.jp/medical_J/research/pdf/Ora_231205.pdf

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