Radiation and photodynamic therapies used in anti-tumor treatment generate only reactive oxygen species (ROS). At the same time, it has been shown that high concentrations of nitric oxide (NO) induce apoptosis of tumor cells, suggesting that nitrogen-dependent stress may be one of the decisive factors in anti-tumor therapy. Cold atmospheric plasma (CAP) is a highly reactive ionized physical state that causes various biological effects. The processes of ionization, dissociation, excitation, and recombination of atoms and molecules in low-temperature plasma (LTP) lead to the formation of a large number of reactive oxygen and nitrogen species. This review presents research results that reveal the mechanism of the anti-tumor action of LTP, its effects on various tumor cell lines, and describes the outcomes of tumor treatment in animal models. Suggestions are made regarding the use of LTP in the therapy of malignant neoplasms.
Recently, much attention has been paid to studying the influence of reactive oxygen and nitrogen species (ROS and RNS) on tumor cell emergence, tumor progression, and treatment. The mechanism of action of radiation therapy and chemotherapy is often mediated by the generation of ROS and RNS, which act directly on tumor cells. Plasma is a partially ionized gas containing ions, electrons, and neutral particles (atoms, molecules, and radicals). Plasma can be of two types: hot and non-hot or low-temperature (cold) atmospheric (LTP), which at the point of contact has a temperature below 104°F, i.e., room temperature. Various gases (helium, argon, nitrogen, a helium-oxygen mixture, air) are used to produce LTP. Depending on the LTP source, the composition and concentration of individual ROS and RNS components vary. The processes of ionization, dissociation, excitation, and recombination of atoms and molecules in plasma lead to the formation of a large number of reactive oxygen species (ROS): atomic oxygen (O), hydroxyl (OH), superoxide (O2−), singlet delta oxygen (1O2), and hydrogen peroxide (H2O2), and, depending on the gas (gas mixture) and plasma geometry, a large number of reactive nitrogen species (RNS): atomic nitrogen (N), nitric oxide (NO), peroxynitrite (ONOO−), and other active forms of the NOx family. It is known that radiation and photodynamic therapies used in anti-tumor treatment generate only ROS. At the same time, it has been shown that high concentrations of NO induce apoptosis of tumor cells, suggesting that nitrogen-dependent stress may be one of the decisive factors in anti-tumor therapy. The involvement of ROS in the initiation and progression of tumors and their therapeutic potential have been studied for many years. Small amounts of ROS are well tolerated by any cell and are neutralized by special enzymes, including superoxide dismutase and catalase. The innate increased metabolic activity in malignant cells (the Warburg effect) may represent a therapeutic target, as tumor cells are essentially already at the limit of ROS tolerance compared to normal cells. This is why, for many years, various methods that generate large amounts of ROS (radiation and photodynamic therapy, some chemotherapeutic agents) have been used in anti-tumor therapy, leading to the death of malignant cells. The ability to generate ROS and RNS allows cold atmospheric plasma to be considered a highly effective candidate for inclusion in anti-tumor therapy.
The aim of the study is to summarize current knowledge about the mechanisms of the biological action of LTP on tumor cells and to present possible directions for the clinical application of LTP as part of anti-tumor treatment. The effect of LTP on cells and tissues is a multiphase process that begins directly during plasma generation, followed by the afterglow phase, leading to diffuse interaction with a liquid-like layer or environment. The liquid medium can be represented either by the treatment of cell cultures in laboratory experiments or by physiological fluid inside and around the tumor during the clinical application of LTP. It is precisely the plasma-modified liquid medium that affects the cells and tissues around it. T. Murakami et al. proposed a global model describing this process, involving more than 60 different active species and about 1,000 different reactions. A.M. Hirst et al. schematically presented this process with an approximate time scale for various phenomena in plasma and liquid phases and subsequent biological interaction. Further research results decipher the complex chemical process at the gas-liquid-tissue phase boundaries, allowing a more accurate assessment of the mechanism of LTP effects on tumor cells.
The biological action of LTP on tumor cells has been studied on various cell lines. M. Vandamme et al. presented the results of one of the first experiments on the effect of LTP on tumors in vivo. U87-Luc glioma tumor cells (4×106 cells in 0.1 ml of saline) were subcutaneously injected into female mice (athymic nude Balb/c and C57bl6 mice). Treatment with LTP pulses (3 times for 2 minutes at 100 Hz with 1-minute intervals) was started as soon as the tumor volume reached 150 ± 50 mm3. The authors showed that 24 hours after the first pulse, all experimental mice exhibited a 1.3-fold increase in tumor bioluminescence, which may indicate increased activity in tumor cells due to a reoxygenation effect associated with a large amount of ROS, commonly observed with low-dose irradiation. This effect increases the sensitivity of tumor cells to further exposure, which was observed after 5 days of LTP pulse treatment: a decrease in bioluminescence intensity by 54−88% and a reduction in tumor volume by 33%, with no changes observed in healthy tissues.
A sufficient number of studies, both in vitro and in vivo, have been conducted on the effects of LTP on melanoma. Thanks to these studies, the model of apoptotic death of tumor cells has been detailed, involving apoptosis through activation of the TNF-ASK1, ATM/p53, and MAPK signaling pathways. It has been recorded that ROS are generated in cells exposed to stress conditions such as hypoxia, chemical exposure, UV radiation, etc., which cause damage to intracellular organelles and membranes, proteins, DNA, and lipids, leading to cell death by apoptosis.
Recent works have explored various mechanisms of CAP action in cancer cells, including activation of p53 protein genes and p21 CDK inhibitor, cell cycle arrest in G2/M and S phases, ROS-mediated cell cycle arrest, and apoptosis due to mitochondrial dysfunction. K. Panngom et al. demonstrated decreased mitochondrial enzyme activity and membrane potential in tumor cells after LTP exposure. Moreover, it has been experimentally proven that LTP can control intracellular ROS and RNS and peroxide content. The main pathways of cell signaling and protein functions can be disrupted or completely damaged as a result of strong and prolonged redox signaling disturbances under the influence of LTP.
Overall, the anti-tumor mechanisms of LTP are diverse: DNA damage due to intracellular accumulation of ROS and RNS, reduced cell viability and clonogenicity, decreased proliferation, cell cycle arrest, the phenomenon of natural cell aging, and non-apoptotic tumor cell death, all with a dose-dependent effect. The key molecules of the anti-tumor effect of LTP have been identified as H2O2 and NO. Many researchers have shown that solutions treated with LTP can have effects on tumor cells similar to direct LTP exposure.
H. Tanaka et al. studied the effect of LTP-treated Ringer’s lactate solution on various cell lines (human glioblastoma U251SP, human mammary epithelial MCF10A, human cervical cancer SiHa, human ovarian cancer SK-OV-3, and human neonatal keratinocytes). The studies showed that LTP irradiation of the L-lactate in the solution leads to the formation of large amounts of H2O2, which provides an anti-tumor effect both in cell culture and in a mouse xenograft model. Additionally, under the influence of LTP, lactate forms groups similar to acetic and pyruvic acids, which also have a pronounced anti-tumor effect. The authors demonstrated different sensitivities of tumor cells to LTP-treated solution.
D. Yan et al., in experiments with glioblastoma (U87MG), pancreatic adenocarcinoma (PA-TU-8988T), and breast adenocarcinoma (MDA-MB-231) cell lines, convincingly demonstrated the anti-tumor effect of LTP-treated solutions. Glioblastoma and pancreatic adenocarcinoma cells were the most sensitive, and the effect on glioblastoma cells did not depend on the dilution of LTP-treated solutions. The authors explained the different sensitivities of cell lines to LTP-treated solutions by differences in the amounts of ROS and RNS generated in tumor cells in response to the specific environment containing H2O2.
D. Gümbel et al. suggest that intraoperative treatment with LTP directly on the tumor (osteosarcoma, gallbladder cancer, prostate adenocarcinoma) and tissues in the resection zone may serve as a basis for improving treatment outcomes (reducing the risk of metastasis, recurrence, infection). Furthermore, the pronounced resistance of healthy cells to various concentrations of ROS and RNS generated by LTP compared to tumor cells makes the use of LTP in anti-tumor therapy a promising direction.
Conclusion:
The level of recurrence-free survival in the treatment of operable malignant neoplasms depends primarily on the most complete removal of the tumor within healthy tissues and the prevention of dissemination of tumor cells and contamination of the surgical wound. One of the promising applications of cold atmospheric plasma in oncology is the selective eradication of cancer cells. Based on the characteristics of LTP, improved local control with minimal surgical side effects can be achieved with intraoperative LTP exposure. Further clinical studies are needed to evaluate the effectiveness of cold atmospheric plasma (LTP) in the treatment of malignant tumors.