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Review

Studies of Applications of Cold Plasma Systems in Cancer Treatment: Mechanisms of Oxidant Stress and Pathway Signaling

by
David Durán Martínez
,
Adriana Valladares Méndez
,
Jesús Rivera Islas
* and
Jessica Nayelli Sánchez-Carranza
*
Faculty of Pharmacy, Autonomous University of the State of Morelos, Av. Universidad 1001, Cuernavaca 62209, Morelos, Mexico
*
Authors to whom correspondence should be addressed.
Stresses 2024, 4(4), 896-915; https://doi.org/10.3390/stresses4040060
Submission received: 6 November 2024 / Revised: 4 December 2024 / Accepted: 10 December 2024 / Published: 12 December 2024
(This article belongs to the Collection Feature Papers in Human and Animal Stresses)
Browse Figures
Figure 1
<p>CAP generation methods.</p> "> Figure 2
<p>Representative scheme of the effects on cells due to exposure to CAP.</p> "> Figure 3
<p>Differences in the effects of CAP applications on cancer cells and normal cells.</p> "> Figure 4
<p>Differentiated effects of CAP depending on the dose: low doses, healing; high doses, apoptosis in cancer cells.</p> "> Figure 5
<p>Representation of the types of cell death induced by CAP and the associated signaling pathways, highlighting the role of ROS and other mechanisms in different types of cancer.</p> ">
Versions Notes

Abstract

:
Cold atmospheric plasma (CAP) has gained attention as a non-invasive therapeutic option in oncology due to its selective cytotoxicity against cancer cells. CAP produces a complex mixture of reactive oxygen and nitrogen species (RONS), which induce oxidative stress, leading to various forms of cell death, including apoptosis, necrosis, autophagy, and ferroptosis. These mechanisms allow CAP to target cancer cells effectively while sparing healthy tissue, making it a versatile tool in cancer treatment. This review explores the molecular pathways modulated by CAP, including PI3K/AKT, MAPK/ERK, and p53, which are crucial in the regulation of cell survival and proliferation. Additionally, in vivo, in vitro, and clinical studies supporting the efficacy of CAP are collected, providing additional evidence on its potential in oncological therapy.

1. Introduction

Cancer treatment is continuously evolving, and researchers are seeking more effective, targeted therapies that minimize damage to healthy tissue and overcome resistance to traditional treatments [1,2]. Despite advances in chemotherapy, radiotherapy, and targeted molecular therapies, many cancers continue to exhibit resistance, and the side effects of these treatments often impose a substantial burden on patients [3]. The emergence of alternative therapies, such as immunotherapy and photodynamic therapy, along with cold atmospheric plasma therapy, brings renewed hope to address these limitations [4,5,6].
CAP, a partially ionized gas generated at room temperature, has emerged as a promising tool in the field of oncology due to its ability to selectively interact with biological tissues and generate therapeutic effects without causing thermal damage. Although its clinical use as an established treatment still requires further investigation, preliminary studies suggest great potential in this area.
The biological effect of CAP is attributed to the production of a complex mixture of RONS, charged particles, and electromagnetic fields, which together induce a state of oxidative stress in exposed cells. This oxidative stress can modulate key molecular pathways involved in processes such as cell proliferation, apoptosis, and DNA repair, suggesting potential applications in cancer therapy [7,8,9].
CAP is generated by various technologies, such as plasma jets, dielectric barrier discharges, and plasma-activated solutions, known as PAW (plasma-activated water) and PASSs (plasma-activated saline solutions). These technologies not only allow for fine-tuning the composition of generated RONS, but also for diversifying their therapeutic applicability, depending on the specific needs of the treatment.
A distinctive feature of CAP is its ability to induce dual effects depending on the administered dose. At low doses, it can stimulate tissue regeneration by activating pathways that promote cell proliferation and migration; at a higher dose, it generates a redox imbalance that can activate programmed cell death mechanisms, such as apoptosis. This range of potential actions positions CAP as a versatile tool, with applications in both regenerative medicine and oncology.
This review aims to provide a comprehensive overview of the current state of re-search on CAP as a cancer treatment, focusing on the molecular mechanisms of action and key signaling pathways involved. It is crucial to understand how oxidative stress and signaling pathways regulate survival, proliferation, and cell death in the context of oncological treatment. In addition, in vitro, in vivo, and clinical studies are analyzed to assess CAP’s efficacy and safety across different stages of research.

2. Fundamentals of CAP

2.1. Definition and Properties of CAP

CAP is a partially ionized gas that operates at room temperature. It consists of a mixture of reactive species, including ions, electrons, neutral atoms, and molecules, combined with electromagnetic fields. When energy is applied to gas, it triggers ionization, leading to the formation of charged particles, excited molecules, and free radicals. Unlike thermal plasmas found in the sun or lightning, CAP functions at near-ambient temperatures, making it particularly suitable for biological applications, as it minimizes thermal damage to tissues and can safely interact with living cells [10,11,12,13,14,15]. The properties of CAP are described below.
  • Temperature Disparity:
CAP exhibits a significant temperature difference among its components. While electron temperatures can exceed 10,000 K, the heavy particles (ions and neutral atoms) remain relatively close to room temperature, approximately 25 °C to 100 °C. This state is known as non-local thermodynamic equilibrium, where distinct temperatures exist among different species within the plasma. This disparity enables selective and controlled interactions with biological targets, minimizing the risk of thermal damage to surrounding tissues [10,11,12,13,14,15].
  • Ionization and Reactive Species:
The application of energy to a neutral gas in CAP results in ionization, producing a variety of reactive species, including ROS and RNS. Together, these are commonly referred to as RONS, which play a crucial role in various applications, such as sterilization, cancer treatment, and material surface modification. Further discussion on the role and mechanisms of RONS in these applications will be provided in subsequent sections [10,11,12,13,14,15].
  • Low Power Requirement:
Unlike thermal plasmas, which require high power inputs (up to 50 MW), CAP (a type of non-thermal plasma) can be generated with significantly lower power levels, typically in the range of a few watts to kilowatts. This substantial difference in power requirements makes CAP a more cost-effective and practical option for a wide range of applications, as it eliminates the need for complex, high-powered equipment [16].
  • Operation Under Ambient Conditions:
CAP can be generated at room temperature and atmospheric pressure, providing practical advantages for real-world applications. This ease of generation is particularly beneficial for use in fields such as medicine, food safety, and environmental science, where the ability to operate in ambient conditions enhances its utility.
  • Surface Interaction and Modification:
CAP can modify the properties of surfaces, including altering surface chemistry and improving adhesion. This is especially advantageous in biomedical applications, such as the preparation of implants and tissue engineering, where enhanced surface compatibility can significantly improve outcomes.

2.2. CAP Generation and Technology

The generation of CAP, recognized for its wide range of applications in fields such as medicine, materials science, and industry, can be classified based on two main aspects: the discharge mode and the electric field used to generate plasma. Figure 1 details this classification [13].
Each method exhibits unique characteristics, making them suitable for specific applications. For example, DBD is widely used to produce plasma at low temperatures, while plasma jet systems offer high precision for localized treatments. Similarly, the choice of electric field, whether radio frequency (RF), microwave, or direct current (DC), plays a critical role in determining the energy density and uniformity of the plasma [17,18].
This classification and the underlying principles of CAP generation are essential to understanding how plasma can be tailored for applications such as sterilization, material modification, and anti-tumor therapies. Table 1 provides a comprehensive comparison of the various plasma generation methods, highlighting their key features and practical applications.
Despite their differences, all plasma generation methods share a fundamental principle: the ionization of the gas through the application of electrical energy. This process involves:
  • The acceleration of free electrons by the electric field;
  • The collision of these electrons with atoms or molecules, resulting in the release of additional electrons and the formation of charged particles, excited atoms, and chemically reactive species.
This plasma state, composed of electrons, ions, and reactive species, is key to its applications in various fields.

2.3. Plasma-Activated Liquids: Advances in Plasma-Activated Water (PAW) and Plasma-Activated Saline Solutions (PASSs)

Other promising technologies in the field of CAP include plasma-activated water (PAW) and plasma-activated saline solutions (PASSs), both of which are gaining attention for their diverse applications. In both PAW and PASS technologies, plasma is brought into direct contact with the liquid solution, facilitating the transfer of reactive species from the plasma to the liquid, which imparts bioactive properties to the solution [16,19].
A flow of ionized gas generates cold plasma, which is then brought into contact with a liquid solution, such as water. This interaction transfers various RONS into the liquid, endowing it with bioactive properties. PAW has potential applications in areas such as disinfection, cancer treatment, and other biomedical fields that will be discussed in detail later.
Also, PASSs are generated by treating salt solutions with plasma, which produces a variety of reactive chlorine/oxygen–chlorine species (RCS).
In recent years, PAW has also been confirmed to possess outstanding biological activity in biomedical and agricultural sectors [20]. Typically employed as an antimicrobial or disinfectant solution, it is particularly beneficial for heat-sensitive samples, as it excludes heat, electric fields, and UV rays associated with direct plasma treatment. Furthermore, the reactivity and antimicrobial properties of PAW can remain stable over time, contingent upon the storage conditions [21,22].
PASSs have several advantages. First, they offer great application versatility, such as cells, tissues, and biomaterials, without the need for direct contact with plasma. Furthermore, these solutions are easy to store and transport, which facilitates their implementation in clinical settings. Finally, they are generally considered biocompatible, which reduces the risk of adverse side effects, contributing to their safety in biomedical applications [23,24].

3. Mechanisms of Action of CAP in Cancer Therapy: RONS Generation and Application Strategies

CAP has emerged as a versatile and transformative technology with broad applications spanning medicine [25,26,27], industry [28,29,30], and environmental science [31]. Its unique ability to generate RONS underpins its multifunctionality. These highly reactive compounds interact with biological and non-biological systems, inducing changes that have therapeutic, antimicrobial, and material processing benefits.
In medicine, CAP has gained significant attention as a therapeutic tool due to its ability to influence cell signaling, modulate oxidative stress, and alter microbial and cellular processes.
Among its medical applications, CAP’s role in oncology is particularly promising. By producing reactive oxygen and RONS, including superoxide anions (O2−), hydrogen peroxide (H2O2), hydroxyl radicals (·OH), and nitrogen dioxide (NO2), CAP selectively induces oxidative stress and cellular damage in cancer cells while preserving healthy tissues, positioning it as a potent tool to target tumors [32,33,34].
There are two main strategies for applying CAP: direct plasma application (direct CAP) and treatment with plasma-activated solutions (PAW) [16,19].
  • Direct CAP: In this approach, plasma is applied directly onto cancer cells. The RONS generated include short-lived species, such as hydrogen peroxide (H2O2), nitric oxides (NO2−), and superoxide anions (O2−), which induce cell damage mainly through direct interaction with cell membranes and intracellular components, causing apoptosis and cell death.
Although direct treatment with CAP is highly effective, its main limitation lies in the rapid dissipation of these reactive species in the extracellular environment, which may reduce its effectiveness on deeper cells or larger tumors.
  • Indirect treatment with PAW:
Unlike direct treatment, indirect treatment uses plasma-activated solutions, in which the reactive species generated in plasma are transferred to biological solutions, such as culture media or saline solutions. In this context, longer-lived species, such as H2O2, are the ones that play a crucial role in the therapy, as they persist longer in solution and continue their action on tumor cells even after initial exposure to plasma.
This approach allows for a more sustained release of reactive species and opens the possibility of applying the treatment in a more controlled and less invasive manner.
Additionally, innovative modalities like plasma-treated hydrogels are being explored. These hydrogels offer localized delivery of reactive species, paving the way for new clinical applications of CAP, which will be discussed further.

3.1. Oxidative Stress Induced by CAP: Cellular Damage and Triggered Pathways

The effects of CAP are mediated by RONS generated during its application. These RONS are responsible for inducing a state of oxidative stress in exposed cells, which in turn triggers several pathways of cellular damage. Figure 2 shows a schematic of the effects of CAP exposure on cells, highlighting the cascade of events triggered by the generation of RONS, which in turn causes a state of oxidative stress in the cells [35]. This oxidative stress results in three main pathways of cellular damage:
  • Lipid Peroxidation: RONS triggers the peroxidation of cell membrane lipids, particularly polyunsaturated fatty acids (PUFAs), which are highly susceptible to oxidative attack due to their multiple double bonds. The reaction begins when RONS, such as hydroxyl radicals (·OH) or superoxide anions (O2−), abstract a hydrogen atom from the methylene group adjacent to the double bond in PUFAs, leading to the formation of lipid peroxyl radicals (LOO·). These radicals can then react with oxygen to form lipid peroxides (LOOH), which further degrade and propagate the damage. This lipid peroxidation process disrupts the integrity of the cell membrane by increasing its permeability, as the formation of lipid peroxides compromises the bilayer structure and leads to the generation of membrane fragments. Loss of membrane fluidity and structural integrity significantly alters the functionality of membrane proteins, including receptors, ion channels, and transporters, affecting cell signaling, nutrient uptake, and ion homeostasis. Furthermore, the accumulation of lipid peroxides can trigger the formation of cytotoxic aldehydes, such as malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE), which further contribute to cellular dysfunction by forming adducts with proteins and DNA, exacerbating oxidative damage. The resulting cycle of continued oxidative damage creates a positive feedback loop, where damage to the membrane allows for increased entry of RONS into the cell, intensifying oxidative stress. This sustained damage ultimately leads to cellular apoptosis or necrosis through the activation of several signaling pathways, including those involving mitochondrial dysfunction, endoplasmic reticulum stress, and the activation of cell death proteins such as caspases. Therefore, lipid peroxidation not only destabilizes membrane function but also plays a crucial role in executing cell death, which is particularly relevant in the selective targeting of cancer cells by CAP treatment [35].
  • Protein Oxidation: RONS initiate a variety of oxidative modifications in cellular proteins. These reactive species primarily target sulfur-containing amino acids such as cysteine (Cys) and methionine (Met), leading to the oxidation of these amino acid residues. A key modification is the formation of protein carbonyls, which are generated through reactions with lipid peroxidation products such as malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE). These products are highly reactive and cause further protein damage through cross-linking, aggregation, and disruption of protein folding. These oxidative modifications lead to important structural and functional alterations in proteins. For example, oxidation can impair enzymatic activity by altering the active site configuration, hampering molecular recognition, or inducing conformational changes that affect protein stability and interactions. Furthermore, oxidative stress activates cellular responses aimed at controlling misfolded proteins through the unfolded protein response (UPR) in the endoplasmic reticulum (ER). The UPR is a protective mechanism that attempts to restore protein homeostasis by enhancing protein folding capacity or degrading misfolded proteins. However, if the damage is too extensive or prolonged, ER stress becomes overwhelming, disrupting cellular functions and activating signaling pathways that ultimately lead to apoptosis. This cascade of events not only results in protein inactivation, but also contributes to cell dysfunction and death, a crucial mechanism in the therapeutic effects of CAP, particularly in cancer treatment [35].
  • DNA Damage: RONS generated by CAP can inflict significant genotoxic damage to DNA. The most common oxidative DNA lesion is the formation of 8-oxoguanine (8-oxoG), a product of guanine oxidation. Guanine is particularly susceptible to oxidative stress due to its low oxidation potential. 8-oxoG is highly mutagenic because it can mispair with adenine during DNA replication, leading to G:C to T:A transversion mutations, which are a hallmark of genomic instability associated with cancer development. Among the main mechanisms of DNA damage are the following:
    (a)
    Direct oxidation of bases: RONS, such as hydroxyl radicals (·OH) and superoxide anions (O2−), directly interact with DNA bases, particularly guanine, causing oxidation and structural alterations. This results in lesions such as 8-oxoG and single-strand breaks (SSBs).
    (b)
    Indirect damage through lipid peroxidation products: Lipid peroxidation products, such as malondialdehyde (MDA) and 4-hydroxy-2-nonenal (HNE), can form adducts with DNA, further amplifying the genotoxic impact.
    (c)
    Telomeric DNA vulnerability: Telomeres, due to their guanine-rich repetitive sequences, are hot spots for oxidative stress.

3.2. Selective Induction of Oxidative Stress in Cancer Cells

CAP exposure differentially affects normal and cancer cells, which can be exploited for a selective therapeutic approach in cancer treatment [36].
Cancer cells, characterized by their higher metabolic and proliferation rates, typically exhibit elevated basal levels of RONS compared to normal cells. This difference in ROS levels is a crucial factor underlying the selectivity of CAP toward cancer cells. Figure 3 illustrates a comparison of RONS susceptibility between cancer and normal cells under CAP exposure.
Cancer cells generate higher levels of ROS under basal conditions, and CAP exposure increases ROS levels beyond a critical threshold, causing DNA damage, cell cycle arrest, and apoptosis. In contrast, normal cells, with lower basal ROS levels, can better handle CAP-induced oxidative stress, which reduces their rate of cell death [37,38,39]. Also, normal cell membranes contain higher levels of cholesterol, which acts as a barrier and reduces permeability to RONS and sensitivity to CAP. In cancer cells, membrane cholesterol levels are lower, which increases their permeability and sensitivity to CAP, allowing greater entry of RONS and promoting more significant damage. In addition, cancer cells have higher expression of aquaporins, which facilitates the absorption of H2O2 generated by CAP and amplifies intracellular oxidative stress [40,41,42,43].
Catalase expression: Catalase is an enzyme that breaks down H2O2 and protects cells from oxidative damage. Normal cells typically have normal or high expression of catalase, which allows them to neutralize H2O2 and better resist oxidative stress. In contrast, cancer cells typically have reduced expression of catalase, making them more vulnerable to H2O2-induced damage generated by CAP [44,45,46,47].

3.3. Modulation of Survival and Apoptotic Pathways

Several studies have highlighted the differential activation of these pathways in response to cold atmospheric CAP treatment in cancerous versus normal cells. CAP could modulate various molecular signaling pathways crucial to cancer progression, offering promising prospects for innovative oncological therapies [48].
One of the most relevant pathways is the PI3K/AKT/mTOR pathway, which regulates essential processes such as proliferation, survival, and drug resistance. It has been observed that CAP can reduce the phosphorylation of AKT and mTOR, decreasing the activity of this pathway in tumor cells and promoting apoptosis [48,49,50]. This effect translates into the inhibition of tumor growth, as reported in lung and colon cancer models.
In a complementary manner, CAP affects the MAPK pathway, where dual effects are observed. On the one hand, it inhibits ERK1/2, a subpathway associated with cell proliferation and survival, and, on the other, it activates stress-related proteins, such as p38 and JNK, which promote apoptosis through the activation of c-Jun and other proapoptotic molecules [51]. This differential impact reflects the ability of CAP to induce selective oxidative stress, primarily affecting tumor cells while sparing normal cells.
The p53 pathway, known for its role as a tumor suppressor, is also modulated by CAP. In cells with partial mutations or residual p53 functionality, CAP can restore its activity, promoting cell cycle arrest, DNA repair, and apoptosis. This effect has been demonstrated in vitro models of glioblastoma and cervical carcinoma. Likewise, CAP regulates NF-κB signaling, a key transcription factor in inflammation and tumor resistance. Studies have shown that CAP inhibits NF-κB activation, which reduces the expression of genes associated with chronic inflammation and cell survival, particularly in chemoresistant cancers [52].
Furthermore, nuclear factor erythroid 2-related factor 2 (Nrf2) is a key pathway that is also modulated by CAP. Nrf2 regulates the expression of antioxidant and cellular protective genes, contributing to defense against oxidative stress. It has been shown that CAP can activate Nrf2, promoting an antioxidant response that protects normal cells from oxidative damage while increasing oxidative stress in tumor cells, inducing their death [53].
These effects are mediated by RONS generated by CAP, which induce a redox imbalance in exposed cells. This imbalance alters key signaling cascades, such as those mentioned, favoring cell death in malignant cells while minimizing damage in normal cells. These findings underline the potential of CAP not only as an independent therapeutic tool, but also as an adjunct to conventional therapies, opening the door to more effective and targeted combinatorial strategies.
Table 2 summarizes the varying effects of CAP on key signaling pathways involved in cell survival and apoptosis in both normal and cancer cells. The activation of these pathways is critical in shaping cellular responses to the oxidative stress induced by CAP.

4. Dual Applications of CAP: From Tissue Regeneration to Apoptosis Induction in Cancer Cells

The application of CAP produces RONS, which have profound effects on cellular behavior, depending on exposure time and dosage. Prolonged exposure to CAP generates significant redox imbalances that can hinder cell proliferation or lead to cell destruction, an effect beneficial in preventing tumor regrowth [53].
Conversely, shorter exposure times of CAP can stimulate cell proliferation, enhance motility and migration, and activate inflammatory signaling pathways. These effects are particularly advantageous in healthy skin and immune cells, playing a crucial role in wound healing and tissue regeneration [54].
RONS have been shown to effectively regulate various biological processes, including antibacterial actions, apoptosis induction in cancer cells, and promotion of wound healing [55]. The ability of CAP to elicit a spectrum of biological effects is influenced by the dosage and exposure duration, which allows for its versatile application across multiple medical fields, from dermatology to oncology.
In the context of wound healing and cancer treatment, CAP exerts differential effects on cells, a phenomenon explained by the principle of “hormesis”. Low and controlled doses of CAP can enhance cell regeneration and facilitate tissue repair in healthy tissues. In contrast, in malignant tissues, CAP can induce oxidative stress and activate mechanisms that lead to cancer cell apoptosis [56].
The therapeutic efficacy of CAP primarily stems from the generation of RONS, including free radicals, neutral molecules, and electromagnetic radiation such as UV light. These reactive species inflict direct damage on critical cellular components, including lipids, proteins, and DNA, thereby promoting the destruction of tumor cells. In wound healing applications, the ROS and RONS produced help modulate the cellular environment, enhancing the healing response and reducing microbial load [57,58,59].
The generation and concentration of these reactive species during CAP treatment depend on several factors: equipment configuration, gas type, power settings, exposure mode, and the distance between the plasma discharge and the target tissue. The electron energy distribution function (EEDF) is another crucial parameter, directly influencing plasma chemistry and, consequently, the type and quantity of ROS and RNS generated [34,60].
These differentiated effects of CAP are pivotal for its clinical applications, enabling the customization of dose and exposure time according to the therapeutic goals. This maximization of benefits in healthy tissues, alongside selective targeting for the destruction of malignant cells, underscores the significance of precise dose control in optimizing treatment efficacy. Figure 4 illustrates these effects and clinical applications, highlighting the importance of the hormesis principle in the therapeutic landscape of CAP.

5. Preclinical Evidence of CAP in Cancer Treatment

5.1. In Vitro Studies on the Anti-Cancer Effects of CAP

Numerous in vitro and in vivo studies have explored CAP’s ability to selectively induce cell death (apoptosis, for example) in cancer cells while sparing healthy tissues, aiming to establish its efficacy and safety profile for clinical applications. Table 3 presents a compilation of in vitro studies examining the effect of CAP on different types of cancer, revealing its ability to induce selective cytotoxicity and enhance the effectiveness of conventional treatments.
As for the results presented in Table 3, the therapeutic potential of CAP is highlighted. This not only induces direct DNA damage through the generation of RONS, but also activates signaling pathways such as p53 and PI3K/Akt, promoting apoptosis and modulating the tumor microenvironment. Furthermore, the versatility of CAP in its induction of four types of cell death—apoptosis, autophagy, pyroptosis, and ferroptosis—positions it as a promising strategy in the treatment of cancer (Figure 5).

5.2. In Vivo Studies on the Anti-Cancer Effects of CAP

To validate these findings, it is essential to move towards in vivo studies that evaluate the efficacy and safety of CAP in animal models. Table 4, below, presents a compilation of in vivo studies investigating the effects of CAP on various animal models of cancer.
In vivo studies have shown promising results in animal models. However, for CAP to become a viable therapeutic option in clinical practice, it is essential to conduct clinical trials that evaluate its efficacy and safety in human patients.
Table 5, below, presents a compilation of clinical studies that investigate the use of CAP in the treatment of different types of cancer.
The clinical studies described herein suggest the potential of CAP as an alternative in cancer treatment, highlighting its ability to induce selective death in tumor cells and preserve healthy tissue. Devices such as the INPen and CHCP have shown clinical benefits, including tumor size reduction, pain relief, and control of residual cells in surgical margins, suggesting that CAP could be an excellent complementary tool in oncological treatment protocols. Furthermore, its application in precancerous lesions, such as in the case of the treatment of CIN, opens the possibility of using CAP in a preventive and therapeutic context to preserve tissue and reduce the invasive impact of other procedures.
However, further research is required in larger-scale and long-term studies to fully understand the molecular mechanisms of CAP and establish its effectiveness and safety in combined treatment protocols.

6. Advances in CAP Delivery Systems

One of the most promising advances in CAP therapy is the development of plasma-treated hydrogels, which allow for localized and sustained delivery of RONS. These hydrogels, treated with CAP, act as reservoirs for reactive species, overcoming limitations such as the rapid dissipation of RONS in biological environments. This innovative approach improves the precision of CAP-based treatments, minimizes damage to healthy tissues, and expands their clinical applications [90,91,92,93].
Plasma-treated hydrogels effectively encapsulate RONS, releasing them in a controlled manner over extended periods. This sustained release prolongs their cytotoxic activity against cancer cells and ensures that therapeutic effects are localized to the target area. This feature is particularly beneficial for solid tumors and hard-to-reach regions, where direct application of CAP can be difficult [90,91,92].
These hydrogels, often composed of biocompatible materials such as alginate, maintain their structural integrity even after plasma treatment. They exhibit minimal systemic toxicity, making them suitable for integration into clinical settings. Biocompatibility ensures that the hydrogels do not elicit adverse immune responses, further supporting their use in long-term therapeutic applications [92,93].
Studies have shown that plasma-treated hydrogels not only retain RONS but also maintain their bioactivity, exhibiting potent cytotoxic effects on cancer cells. For example, CAP-treated alginate hydrogels have shown significant potential to target bone cancer cells, providing localized cytotoxicity while preserving surrounding healthy tissue [92,93].
Plasma-treated hydrogels address several challenges associated with traditional CAP therapy:
  • Rapid RONS diffusion: By encapsulating reactive species, these hydrogels mitigate the rapid loss of RONS in biological fluids, improving their therapeutic efficacy.
  • Precise delivery: Localized release of RONS minimizes off-target effects, reducing potential damage to non-cancerous tissues.
  • Adaptability: Their modular design allows for customization based on specific tumor environments or therapeutic needs.
The integration of plasma-treated hydrogels into CAP therapy is paving the way for new cancer treatments. These biomaterials are particularly valuable for:
  • Treating solid tumors: Hydrogels provide a means to deliver RONS directly to tumors that would otherwise be difficult to treat with direct CAP.
  • Regenerative medicine: Beyond oncology, plasma-treated hydrogels show promise in wound healing and tissue engineering, where controlled oxidative stress can stimulate repair and regeneration.

7. Prospects for CAP Use in Oncology

Device and Dosing Optimization: Future research should focus on optimizing CAP dosing and application parameters tailored to specific cancer types. This includes adjusting the intensity, duration, and frequency of treatment, as well as tailoring devices to maximize safety and efficacy in specific tissues.
Research into Combination Therapies: Combining CAP with conventional treatments such as chemotherapy, radiotherapy, and immunotherapy promises to improve cancer cell response and reduce drug resistance. Future research could focus on how to effectively integrate CAP into current protocols, enhancing clinical benefits and reducing side effects.
Long-Term Studies and Advanced Clinical Trials: Although preliminary studies are encouraging, advanced-phase clinical trials evaluating the long-term safety and effectiveness of CAP in cancer patients are needed. These studies will help establish guidelines for its use in clinical practice.
Understanding Molecular Mechanisms: Delving deeper into the mechanisms by which CAP induces different types of cell death (such as apoptosis, autophagy, pyroptosis, and ferroptosis) will allow us to identify new therapeutic targets and personalize the use of CAP according to tumor and patient characteristics.
Preventive Applications and Use in Early Cancer: Since CAP has been shown to be effective in precancerous lesions such as cervical CIN, the possibility of using CAP in preventive contexts and in early stages of cancer is open. This could contribute to avoiding tumor progression and reducing the risk of cancer in high-risk patients.

8. Conclusions

This review has analyzed the molecular mechanisms modulated by cold atmospheric plasma (CAP) systems in the treatment of cancer, highlighting their therapeutic potential through the controlled generation of reactive oxygen and nitrogen species (RONS). The analyzed studies demonstrated that CAP could induce selective oxidative stress, modulating key signaling pathways such as PI3K/AKT, MAPK, and p53, which generates specific effects on tumor cells, including the induction of apoptosis, inhibition of proliferation, and decreased metastatic capacity.
The versatility of CAP is evidenced by its ability to generate dual effects: at low doses, it promotes regenerative processes in normal tissues, while at higher doses it activates programmed cell death mechanisms in malignant cells. This balance offers opportunities for its application in both oncology and regenerative medicine.
However, the transition from in vitro and in vivo studies to clinical applications requires further research, particularly in the standardization of parameters, the evaluation of long-term effects, and the optimization of combinations with conventional therapies.
Finally, CAP presents a promising approach for cancer treatment with applications that go beyond oncology. However, its integration into clinical practice will depend on future research validating its efficacy, safety, and scalability. A deeper understanding of the molecular mechanisms modulated by CAP, together with advances in application technologies, will be crucial to consolidate its role in modern medicine.

Author Contributions

Conceptualization, D.D.M. and A.V.M.; visualization, J.R.I. and J.N.S.-C.; formal analysis, D.D.M. and A.V.M.; writing—original draft preparation, D.D.M. and J.N.S.-C.; writing—review and editing, J.N.S.-C. and J.R.I.; supervision, J.N.S.-C. and J.R.I. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding and APC was funded by Stresses MDPI.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. CAP generation methods.
Figure 1. CAP generation methods.
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Figure 2. Representative scheme of the effects on cells due to exposure to CAP.
Figure 2. Representative scheme of the effects on cells due to exposure to CAP.
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Figure 3. Differences in the effects of CAP applications on cancer cells and normal cells.
Figure 3. Differences in the effects of CAP applications on cancer cells and normal cells.
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Figure 4. Differentiated effects of CAP depending on the dose: low doses, healing; high doses, apoptosis in cancer cells.
Figure 4. Differentiated effects of CAP depending on the dose: low doses, healing; high doses, apoptosis in cancer cells.
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Figure 5. Representation of the types of cell death induced by CAP and the associated signaling pathways, highlighting the role of ROS and other mechanisms in different types of cancer.
Figure 5. Representation of the types of cell death induced by CAP and the associated signaling pathways, highlighting the role of ROS and other mechanisms in different types of cancer.
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Table 1. Classification and applications of cold atmospheric plasma by discharge mode and electric field.
Table 1. Classification and applications of cold atmospheric plasma by discharge mode and electric field.
Classification Key CharacteristicsNotable Applications
By Discharge Mode [17,18]DBD
-
Uses a dielectric between electrodes
-
Generates plasma at low temperatures
-
Medical treatments
-
Microorganism inactivation
-
Contaminant degradation
Plasma Jet
-
Cold and controlled plasma
-
Operates at atmospheric pressure and low temperatures
-
Localized precision
-
Medicine: wound healing, localized anti-tumor therapy, disinfection
-
Industry: surface cleaning, material modification
-
Biotechnology: sterilization of medical devices
Gliding Arc Discharge
-
Plasma in an arc form extended by gas flow
-
Welding
-
Coating deposition
Corona Discharge
-
Without or partial dielectric
-
Plasma generated by high voltage gradients
-
Ozonation
-
Air purification
-
Surface treatments
By Electric Field [17,18]RF
-
Frequency: 3 kHz–300 MHz
-
Uniform plasma with controlled applications
-
Semiconductor etching
-
Surface treatments
-
Thin-film deposition
Microwaves
-
Frequency: 1–300 GHz
-
High energy density in small volumes
-
Nanomaterial synthesis
-
Water purification
-
Advanced catalysis
DC
-
Continuous voltage
-
Produces stable plasmas
-
Welding
-
Material cutting
-
High-pressure plasma generation
Table 2. Effects of CAP on key signaling pathways involved in normal and cancer cells.
Table 2. Effects of CAP on key signaling pathways involved in normal and cancer cells.
PathwayNormal CellsCancer CellsReference
PI3K/Akt
-
Transient activation of the PI3K/Akt pathway, favoring cell survival and proliferation
-
CAP does not significantly impact normal cell survival signaling
-
Inhibition of the PI3K/Akt pathway by cold plasma reduces AKT phosphorylation, promoting apoptosis and decreasing proliferation
-
Synergistic effects with chemotherapy enhance drug-induced apoptosis
[50]
MAPK
-
Less pronounced or transient activation of MAPK pathways, minimizing detrimental effects
-
Activation of stress-related kinases (e.g., JNK and p38) while inhibiting ERK1/2, leading to increased apoptosis and reduced proliferation
-
CAP-induced activation of JNK promotes apoptotic cell death in cancer cells
[51]
p53 Activation
-
Typically unaltered in normal cells, maintaining regulatory functions of cell cycle and apoptosis
-
CAP treatment can restore p53 function, increasing expression and activation of apoptotic pathways
-
Enhanced p53 activation leads to DNA damage response, promoting cell death
[52]
Nrf2
-
Activation of the Nrf2 pathway, a master regulator of the antioxidant response
-
Promotes cell survival and enhances the ability to manage oxidative stress
-
Suppression of the Nrf2 pathway, compromising antioxidant capacity
-
Increases sensitivity to oxidative stress, predisposing to DNA damage and apoptosis
[53]
Table 3. Compilation of in vitro studies examining the effect of CAP on mechanisms (ROS and others) in different types of cancer.
Table 3. Compilation of in vitro studies examining the effect of CAP on mechanisms (ROS and others) in different types of cancer.
Study TypeCancer TypeStudy DescriptionMechanism (ROS, Apoptosis, Others)Specific Signaling PathwayReference
In VitroGlioblastomaCAP increased the cytotoxicity of temozolomide in glioblastoma cells, suggesting chemosensitizationROS, apoptosis, direct DNA damagep53, PI3K/Akt[61,62,63]
Colon cancerInduction of cell death by oxidative stress via CAP; potential use as an adjuvant therapyROS, apoptosis, stress on the endoplasmic reticulumCaspasa-9, caspasa-3, PARP y Bax/Bcl-2[63,64,65]
Breast cancerAntiproliferative and apoptosis-inducing effect; potential for chemotherapy sensitizationApoptosis, signaling pathway alterationIncreased Bax/Bcl-2 ratio and cleavage of PARP-1[66,67,68,69]
Sensitization by epidermal growth factor (EGF) enhances the response of triple-negative breast cancer (TNBC) cells to CAP coldThis activation increases the production of reactive ROS and apoptotic signalingEGFR(Y992/1173)[70]
Lung cancerReduction in viable cells and anti-metastatic activity observedROS, apoptosis, microenvironment modulationp38 MAPK, PI3/Akt[71]
Inhibition of proliferation, reduced migrationROS, ferroptosis,Downregulation of HOXB9/SLC7A11K[72]
Cell death in PC9 tumor cells expressing high levels of Gasdermin E (GSDME) in a dose-dependent mannerROS, pyroptosisJNK/cytochrome c/caspase-9/caspase-3[73]
Pancreatic cancerReduction in metabolic activity and cell migration; favorable modulation of inflammatory profileROS, inflammatory regulationNF-κB, IL-6[74,75]
MelanomaCAP combined with nanoparticles enhanced selective toxicity towards cancer cells without damaging normal cellsROS, microenvironment modulationUPR signalling, Notch, Wnt/β-catenin[76]
Table 4. Compilation of in vivo studies examining the effects of CAP on different models of cancer.
Table 4. Compilation of in vivo studies examining the effects of CAP on different models of cancer.
Study TypeCancer TypeDescriptionMechanism of Action and Signaling PathwaysReference
In vivo studiesGlioblastomaCAP increased ROS production, sensitizing tumor cells to chemotherapy with temozolomide.ROS, apoptosis, p53, PI3K/Akt pathways; significant reduction in tumor growth[77]
Colon cancerCAP promoted danger signal release and stimulated adaptive immune response in mouse models.ROS, immune activation; specific T cell response against GUCY2C[78,79]
Myeloid leukemiaCAP blocked three key cancer survival pathways: redox deregulation, glycolysis, and AKT/mTOR/HIF-1α signaling.ROS, apoptosis, AKT/mTOR, HIF-1α pathways; reduced tumor growth and improved survival[80]
Multiple myelomaCAP inhibited tumor implantation in mice, significantly prolonging survival time.ROS, apoptosis, Notch pathway inhibition; reduced tumor cell proliferation[81]
Pancreatic cancerA plasma-activated lactated Ringer’s solution was developed to evaluate its antitumor effects.ROS, cytotoxic effects derived from activated lactic acid; tumor volume reduction[82]
CholangiocarcinomaCAP induced DNA damage and apoptosis in subcutaneous xenografts of cancer cells.ROS, DNA damage, apoptosis; activation of CHK1, p53, and 8-oxoguanine accumulation[83]
Head and neck cancerCAP induced apoptosis and reduced cell viability in head and neck cancer models.ROS, apoptosis; mitochondrial membrane potential modification and MAPK pathway activation[84]
Table 5. Compilation of in clinical studies examining the effect of CAP application on different types of cancer.
Table 5. Compilation of in clinical studies examining the effect of CAP application on different types of cancer.
Cap Application DeviceStudy DescriptionResultReference
kINPenThe study demonstrated that CAP treatment delivered using the kINPen MED device is safe, well tolerated, and effective in reducing tumor size in patients with head and neck cancer. CAP induced selective tumor cell death through oxidative stress without damaging surrounding healthy tissues.Tumor size reduction in head and neck cancer[85]
Plasma jet, kINPen(®) MED (neoplas tools GmbH, Greifswald, Germany)This study concluded that the use of a cold plasma device, specifically a dielectric barrier discharge (DBD) system, in patients with head and neck cancer showed visible responses on the tumor surface and significant apoptotic cell death. The treatment was well tolerated, with a favorable safety profile and no significant adverse effects.Induction of apoptotic death in head and neck cancer[86]
Canady Helios Cold Plasma (CHCP)The CHCP device was investigated in the first phase I clinical study, primarily to demonstrate safety. Preliminary findings were encouraging, showing that CHCP can control residual disease and improve patient survival. Ex vivo experiments on patient tissue samples confirmed CHCP-induced cancer cell death without harming normal cells, indicating its potential to control residual cancer cells at surgical margins.Control of residual tumor cells in surgical margins in combination with surgery[87]
kINPenThis study concluded that CAP use in advanced head and neck cancer patients is safe and may induce positive clinical responses, such as pain reduction and improved quality of life. Two patients achieved partial remission, suggesting CAP’s potential as an effective therapeutic option; however, further research is needed to fully understand its long-term mechanisms and efficacy.Improving quality of life and reducing pain in patients with advanced head and neck cancer[88]
VIO3/APC3 (Erbe Elektromedizin)This study concluded that non-invasive physical plasma (NIPP) is a safe and effective method for treating cervical intraepithelial neoplasia (CIN) grades 1 and 2. Using the cold plasma device, VIO3/APC3, with precise application control, the treatment preserved tissue while inducing lesion regression, making it a promising alternative to current excisional and ablative treatments.Conservative treatment of CIN in women[89]
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Durán Martínez, D.; Valladares Méndez, A.; Rivera Islas, J.; Sánchez-Carranza, J.N. Studies of Applications of Cold Plasma Systems in Cancer Treatment: Mechanisms of Oxidant Stress and Pathway Signaling. Stresses 2024, 4, 896-915. https://doi.org/10.3390/stresses4040060

AMA Style

Durán Martínez D, Valladares Méndez A, Rivera Islas J, Sánchez-Carranza JN. Studies of Applications of Cold Plasma Systems in Cancer Treatment: Mechanisms of Oxidant Stress and Pathway Signaling. Stresses. 2024; 4(4):896-915. https://doi.org/10.3390/stresses4040060

Chicago/Turabian Style

Durán Martínez, David, Adriana Valladares Méndez, Jesús Rivera Islas, and Jessica Nayelli Sánchez-Carranza. 2024. "Studies of Applications of Cold Plasma Systems in Cancer Treatment: Mechanisms of Oxidant Stress and Pathway Signaling" Stresses 4, no. 4: 896-915. https://doi.org/10.3390/stresses4040060

APA Style

Durán Martínez, D., Valladares Méndez, A., Rivera Islas, J., & Sánchez-Carranza, J. N. (2024). Studies of Applications of Cold Plasma Systems in Cancer Treatment: Mechanisms of Oxidant Stress and Pathway Signaling. Stresses, 4(4), 896-915. https://doi.org/10.3390/stresses4040060

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