While the task of developing effective cancer treatment has convened scientists, there still exist multiple drawbacks in current cancer therapies limiting the definitive ability to improve the patient’s survival and quality of life. Solely causing almost 10 million deaths globally in 2020 [1], cancer is the second most lethal family of disease and is also recognized as one of the most challenging in the medical field due to the inadvertent harmful side effects of brought about by cancer treatments [2] and increased resistance to established therapeutics [3].

However, significant findings have arisen in recent decades regarding cancer biology that not only allows a better understanding of cancer cells but also suggest effective methods for the treatment of cancer. Among the discovered hallmarks of cancer cells was elevated levels of reactive oxygen species (ROS) compared to non-tumor cell lines [4]. ROS are two-electron reduction byproducts of cellular metabolism and oxygen consumption. They are comprised of superoxide anions, hydrogen peroxides, hydroxyl radicals, and other highly unstable oxygen free radicals [5]. When ROS are prevented from building up in excessive amounts via antioxidant defenses, they are utilized in controlling cell proliferation and differentiation. As observed in cancer cells, abnormally elevated ROS are thought to be pro-tumorigenic by inducing excessive oxidative stress, deregulating the anti-oxidative systems, and contributing to abnormal cell growth, pro-survival signaling pathways, and resistance to apoptosis [6,7]. Nonetheless, excessive accumulation of ROS in cancer cells leads to toxic levels of oxidative stress which can trigger cancer cell death [8-10]. Therefore, scientists are currently focusing on this paradoxical role ROS plays in cancer cells and investigating possible treatments to affect the cancer cells’ regulatory mechanisms of ROS to induce cellular death.

One type of treatment that is currently being studied to alter ROS levels in cancer cells is cold physical plasma. Cold physical plasma, also referred to as plasma, contains multiple common ROS that can be applied to cancer cells to induce oxidative stress and activate desired pathways within cells [11-13]. While methods of plasma’s therapeutic applications vary between dielectric barrier discharges (DBDs) and plasma jets, both are similar in the capability of having free electrons and radicals, ions, and neutral molecules in constant interaction, resulting in ROS as the plasmas’ biological effect’s main active agents [14]. The plasma’s efficiency and pattern in ROS production heavily depend on several factors such as the gases used (with their type differing for each technology), applied voltage, gas flow rate, and distance to the target [15]. Notwithstanding, challenges exist with utilizing plasma, such as producing toxic substances like ozone and being a multi-component system composed of neutral particles and electric fields that could potentially affect the target cells [16].

Another technology recently gaining attention in its potential application for treating cancer is photobiomodulation (PBM). PBM utilizes red or near-infrared light (600-1,000 nm) from coherent or non-coherent light to stimulate cellular processes with relatively limited safety concerns [17]. Light-emitting diode (LED) arrays are known to be capable of delivering sufficient energy densities to considerable tissue surface area while mixing different wavelengths [18], allowing users to test specific parameters in great detail. Furthermore, PBM was observed to be capable of antitumor action by increasing ROS to excessive levels [19-21], highlighting its potency as the technology that can efficiently trigger tumor cell death. Clinical usage of PBM has yet to gain widespread acceptance, as the molecular, cellular, and tissue mechanisms of laser action on cancer cells remain poorly known.

This paper aims to examine and present an overview of the utilization of plasma therapy and PBM as feasible cancer treatments through excessive ROS generation in cancer cells and identify optimal treatment methods that induce tumor cell death efficiently and accurately (Fig. 1).

Figure 1. Plasma treatment and photobiomodulation through either laser or LED can be utilized to generate excessive ROS in cancer cells and induce mitochondrial DNA damage which can positively contribute to activation of apoptotic mechanisms. LED, light emitting diode; ROS, reactive oxygen species; H2O2, hydrogen peroxide; OH, hydroxyl radical; O2-, superoxide anion radical.

Human cells have developed to generate biologically relevant ROS from endogenous metabolism and scavenge from exogenous environments. Two cellular components mainly carry out the endogenous generation of ROS: the mitochondrial electron transport chain (ETC) and the transmembrane NADPH oxidases, commonly referred to as the NOX family [22]. Both endogenous mechanisms are responsible for the inherent supply of ROS. Exogenous generation of cells’ ROS is induced by exposure to particulate matter like air pollutants and metals or radiation [23].

Once these ROS are created and/or acquired by the cell, they interact with several intracellular biomolecules to stimulate an array of cellular mechanisms. These interactions are thought to include gene transcription and moderation of protein expression related to inflammation and cell survival. Along with appropriate signaling pathways, they act as secondary messengers that have the function to carry out a broad range of cellular functions from cell homeostasis to cell death depending on surrounding distributions and variable concentrations [24]. While each cell on average is thought to experience 1.5 × 105 oxidative hits, if the ROS production is greatly increased or the reduction of ROS levels is impaired by some cause, the cells are expected to experience oxidative stress, a condition known to be implicated in cancer cells’ pathophysiology [25,26]. Specifically, increased ROS levels by the Warburg effect’s aerobic glycolysis can stimulate oncogene activity, induce genetic instability, and damage various molecules inside the cell, including lipids, proteins, and DNA. For example, oxidative stress increased free radicals and causes unfavorable changes to the cell membrane’s lipid bilayer when interacting with lipids, creating electron leakage in the mitochondrial intermembrane space [27,28]. ROS can also alter redox reactions in cellular pathways via its interaction with proteins, inducing uncontrolled cell proliferation and further oxidative stress-inducing cell death [29,30]. ROS-driven redox reactions could also influence specific transcription factors, making these factors capable of binding to nuclear DNA and interacting with specific DNA responses such as abnormal responses to various stimuli and activating or suppressing embryogenesis and cell proliferation and death [31,32].

Because ROS accumulation can be detrimental to a human cell’s viability, different antioxidant defense mechanisms balance ROS concentrations and maintain redox equilibrium. One intracellular defense mechanism is superoxide dismutases (SODs), which rapidly dismutate superoxide into hydrogen peroxide (H₂O₂) across multiple cellular compartments, which is then converted into H₂O by enzymes such as catalase (CAT) glutathione peroxidase (GPX), and peroxiredoxin (PRX) [33]. CAT converts H₂O₂ in two different mechanisms, using reduction by non-NADPH hydrogen donors, in which the ROS is split into two H₂O or one H₂O molecule and an O₂ molecule, depending on H₂O₂ concentration [34]. GPX performs catalytic activity via a series of redox reactions that consist of a back-and-forth redox reaction between glutathione, glutathione disulfide, and glutathione reductase [35]. PRX scavenges H₂O₂ and converts it into H₂O using high-affinity binding sites via oxidation, and the enzyme is later reduced by thioredoxin [36]. All these enzymes for controlling ROS levels are regulated by nuclear factor E2-related factor 2 (NRF2), which controls the production of NRF2 proteins that activates the transcription of CAT, GPX, and PRX, overall controlling the cell’s ROS levels [37,38].

The effects triggered with increased ROS is dependent on the type of cancer cell, the concentration of ROS, and the cell’s genetic background. Among the most observed effects include enhanced cell proliferation, induced genetic instability, and cell death. Because of its advantageous and disadvantageous effects on cancer cells, ROS is also referred to as a double-edged sword in developing effective cancer treatments [32,39].

ROS has been implicated in cellular pathways inside both normal and cancer cells. Elevated ROS levels have been shown to have a role in tumorigenic signaling and enhance cancer cell survival, growth, and proliferation. High levels of ROS concentrations above the normal balance have been known to support cancer cell development by regulating the mitogen activated-protein kinase (MAPK)/extracellular-regulated kinase (ERK 1/2), phosphoinositide-3-kinase/protein kinase B (PKB [Akt]), and protein kinase D (PKD) signaling pathways. Activation of MAPK/ERK 1/2 pathways and K-Ras stimulated pathways have been associated with increased cell proliferation [40,41]. Heightened levels of metabolic ROS particularly H₂O₂, have been observed to activate the ERK 1/2 pathway and induce cell proliferation in breast tumor cells [42]. Activation of the same pathway has been shown to enhance breast, leukemia, melanoma, and ovarian cancer cell survival, growth, and their anchorage-dependent motility suggesting pro-tumorigenic effects on signaling pathways [40,43,44]. The Akt pathway, responsible for targeting and inactivating proteins, is another cellular pathway activated by increased ROS levels promoting cell survival. The pathway is usually negatively regulated by PTEN and PTP1B, which are inactivated via oxidation induced by H₂O₂, increasing the cancer cell’s survival [45,46]. Specifically, the inactivation of PTEN was observed in a range of cancer types [47], suggesting the ability of ROS to induce signaling pathways favorable to cancer cells. PKD1 signaling, involved in pancreatic ductal adenocarcinomas (PDA), was shown to express oncoprotein K-Ras in most cases, showcasing increased ROS production [48,49]. These PDA cases were then observed with increased antioxidant SOD2 levels and decreased CAT levels [50]. PKD2 and PKD3 were also implicated in cancer cell survival, including breast cancer [51]. These correlations between ROS levels and PKD signaling, adding to ROS effects on other cellular pathways, suggest that cancer cells’ higher ROS accumulation and production leads to pro-tumorigenic signaling, enhancing cancer cells’ cellular survival and proliferation.

ROS’s effects on DNA are suggested to be the main driver for oncogenesis and can also account for genetic diversity across different cancer cell types. Because the typical rate of DNA mutation is insufficient to account for the number of mutations required for oncogenic transformation, cancer cells, unlike normal cells, require some form of genetic instability [52]. Excessive ROS can also induce DNA damage, where the DNA strands could undergo depyrimidination, depurination, base modifications, accelerated telomere shortening, and dysfunction [53]. One specific mechanism recently demonstrated was in acute myeloid leukemia (AML) cells, NOX-generated ROS playing a role in the DNA double-strand breaks (DSBs) and DNA oxidation downstream of the FLT3-internal tandem duplication (ITD) oncogene. These FLT3-ITD expressing cells have shown an increase in H₂O₂ concentrations. These AML cells displayed a 100% increase in oxidative stress markers and a 50%-75% increase in DSBs markers, suggesting ROS’s role in this oncogene expression [54]. The downstream ROS has significant implications for cycles of genomic instability and DNA damage via oxidative stress. These effects include deletions, insertions, base-pair mutations, and other deleterious lesions in AML [55,56], which are expected to be a driving force in many cancer cell development. Another method ROS changes DNA strands are by oxidizing DNA strands by damaging bases, especially guanine and adenine, that are on the nascent strand and suppressing DNA methylation [57]. Alternatively, ROS is also capable of inducing hypermethylation. This process enhances cancer cells’ actions if the strands at effect are promoter regions of tumor suppressor genes, resulting in the expression of tumor phenotypes [58-60]. These unrepaired oxidized guanine are thought to be one of the most mutagenic lesions, assisting research by serving as potentially prominent markers for tumorigenesis and ROS-induced mutagenesis [61].

Compared to normal cells, higher concentrations of ROS have been demonstrated in cancer cells, inducing pro-tumorigenic effects. However, if ROS levels reach toxic levels, it could induce cell cycle arrest, apoptosis (type 1 programmed cell death), and senescence [62,63]. Chemotherapy is known to induce excessive ROS levels in cells by depleting their antioxidant proteins [64]. One mechanism is the c-Jun N-terminal kinases (JNK) pathway, in which increased ROS production enhances intrinsic apoptotic signaling, and extrinsic death receptor pathways which causes cell death [65]. These JNK pathways were mutated in multiple cancer cell types [66]. ROS is also connected to one of the most understood hallmarks of apoptosis, caspases, and cysteine-dependent aspartate-directed proteases that induce DNA fragmentation and ultimately result in cell death. These caspase activations are triggered when death receptors (Fas, TNFR1, TRAIL-R1, TRAIL-R2, and more) bind to its ligands, which results in downstream caspase-3 and Bcl-2 protein Bid cleaving, resulting in the mitochondria translocating cytochrome c [67]. This cytoplasmic release of cytochrome c could build the apoptosome by binding to apoptotic protease activating factor 1 and pro-caspase 9 [32]. Some of the ligands that activate these death receptors are TRAIL, Fas ligand, and tumor necrosis factor-α [68], but ROS was shown to play a critical role in this process by activating the death receptors and inducing apoptosis [69]. While the most common method to kill cancer cells is by utilizing their apoptotic pathways, several studies also shed light on alternative methods that focus on toxic ROS levels and their effect on inducing autophagy (type II programmed cell death) and necroptosis (type III programmed cell death). Both mechanisms are also being studied as tumor suppressor mechanisms when ROS levels are increased above the threshold, where the former increases autophagosomes and oxidizes and thereby inhibits negative regulators of the cancer cell death process [70-72], whereas the latter uses ROS generated by increased energy metabolism or formation of ceramide to cause cancer cell death [73-75].

Plasma is a rising treatment method for cancer cells gaining recognition for its ability to increase intracellular ROS levels. Plasma technology is mainly divided into plasma jets and DBD plasma [76]. While the two devices use separate mechanisms in their plasma production, both have been found capable of increasing intracellular ROS levels in several studies. These increased ROS levels were observed to affect various factors in tumor cell survival via internal cell death signaling, mainly decreasing cell proliferation or migration and inducing apoptosis. Several studies have successfully induced anti-tumorigenic reactions with plasma jets in cancer therapy. Kim et al. [77] directly applied plasma (He, O₂) to human tumor cells (HCT-116, SW480 colorectal carcinoma) and observed its effects. Major ROS species generated by the plasma and applied to the cells were reactive radicals O and hydroxyl radical (OH). The results showed plasma-treated cells had considerable inhibition of cell migration and invasion compared to untreated cells accompanied with apoptotic behavior, including nuclear condensation and DNA fragmentation. Furthermore, when the ROS scavengers were treated in the cells, their caspase 3/7 activities were abolished, suggesting that the increased ROS levels play a significant role in plasma-induced apoptosis [77]. Another plasma jet example comes from Xu et al. [78], who applied indirect, He plasma to human tumor cells (RPMI8226 and LP-1 MM cell line), with the major reactive radicals produced by the plasma being OH. The biological consequences of the plasma treatment were an increase in Blimp-1 and XBP-1 and a decrease in early B cell factor, which are expected to be beneficial for chemotherapy, and a reduced expression of MMP-2 and MMP-9 could suppress melanoma cell migration. Furthermore, JNK decreased while eukaryotic translation initiation factor 2A increased with plasma, suggesting that the reactive species triggered stress responses expected to induce mitochondria-associated apoptosis [78]. DBD plasma devices have also brought out similar outcomes in cancer cells. Karki et al [79]. applied indirect DBD plasma on human A549 lung adenocarcinoma epithelial cells to assess the changes induced in their cell detachment, migration, and apoptosis. The results were that plasma inhibited the target cells’ proliferation and migration, and their viability decreased through apoptotic induction. Various ROS species were also observed during the study. The induced apoptosis is thought to be due to DNA damage by increased generation of the observed ROS species because plasma does not directly cause cell disruptions but increases ROS levels that, in turn, trigger cell death mechanisms [79]. While other studies that have applied plasma therapy to human tumor cells have not mentioned or changed some of the sample’s proliferation, migration, and apoptosis, all of these studies have demonstrated plasma therapy increasing their target cells’ ROS levels [80-86]. However, disadvantages follow as the increase of toxic chemicals like ozone in production [87] could prevent plasma therapy from being widely acclaimed as a practical cancer treatment. Table 1 summarizes some studies’ results on plasma therapy’s effects on intracellular ROS levels.

Table 1 . Results of researches on the effects of plasma therapy on intracellular ROS levels

Plasma device	Gas/modality	Human cell or tissue type	ROS level	Reference
Plasma jet	He, O2/direct	HCT-116, SW480 colorectal carcinoma	↑	Kim et al. (2010) [77]
	He/indirect	RPMI8226 and LP-1 MM cell line	↑	Xu et al. (2016) [78]
	He/direct	G361 melanoma	↑	Lee et al. (2009) [83]
	He, O2/direct	BHP10-3 and TPC1 thyroid papillary carcinoma cell line	↑	Chang et al. (2014) [81]
	Ar/indirect	Sk-Mel-147 melanoma cell line	↑	Schmidt et al. (2015) [85]
	Ar/indirect	HEC-1 and GCIY endometrial and gastric cancer	↑	Ikeda et al. (2018) [82]
	Air/indirect	ES2, SKOV3, and WI-38 cell lines	↑	Nakamura et al. (2017) [84]
	Air/direct, indirect	U-87 MG brain cancer cells	↑	Akter et al. (2020) [80]
DBD plasma	Air/indirect	A549 lung adenocarcinoma epithelial cells	↑	Karki et al. (2017) [79]
	Air/direct, indirect	U-87 MG glioblastoma and HCT-116 colorectal carcinoma	↑	Vandamme et al. (2012) [86]

ROS, reactive oxygen species; DBD, dielectric barrier discharge.

PBM is another rising treatment methodology for cancer therapy. PBM consists of applying different wavelengths of light to the target cell using a coherent or non-coherent light source, such as LED and low-level laser (LLL) [18]. Different parameters of the lasers, including wavelength and output power, were shown to induce different reactions for different cell types, providing more opportunities for scientists to test specific parameters and freely explore various effects, diagnostic potential [88] and mechanisms aside from mere tissue ablation and vaporization [89,90].

Studies have noted the importance of taking advantage of exogenous and endogenous photosensitizers capable of absorbing radiation at the wavelength typically of ultraviolet A and near-visible light [89,91,92]. After radiation absorption, the photosensitizers transfer their energy to form singlet oxygens [93] which are expected to last for extended periods [94]. ROS that can be formed by this endogenous mechanism of exogenous stress and regular mitochondrial activity include OH, H₂O₂, and superoxide anion radical [95], highlighting their relationship with applied light and potentially PBM. Chang et al. [20] demonstrated that when mid-infrared light (MIR) was applied to A549 cells, it stimulated their DNA damage checkpoint pathway leading to DNA damage and induced G2/M cell cycle arrest. They specifically examined proteins that contribute to the DNA damage-signaling pathway: the tumor suppressor p53 binding protein 1 and γ-H2AX. When the cells went through PBM, the two protein types responded by forming numerous distinguished subnuclear foci colocalized in the cells’ nuclei. This colocalization of damage markers was diminished when ROS scavenger N-acetylcysteine was treated in the cells, suggesting that the mechanism of MIR causing cell cycle arrests are associated with ROS-mediated DNA damage [20].

Similarly, Schroeder et al. [96] used near-infrared on human skin fibroblasts and observed that irradiation involved ROS generation. The irradiation increased intracellular ROS production and induced heat shock protein 27 [96], providing more examples of how PBM could induce higher ROS levels in different cells. Light-induced ROS levels’ relations to antitumor action are gaining recognition too. Infrared light inducing mitochondrial ROS damaged human mitochondrial DNA (mtDNA), altering the cell’s respiratory chain function [97]. Since mtDNA is located adjacent to the ETC, where ROS is generated the most during PBM, studies suggest that infrared-induced ROS will likely damage mtDNA and trigger apoptotic mechanisms [98]. Light-induced ROS could generate other outcomes, including reduced cell migration activity, reorganization of cellular structures, and suppression of cancer cell proliferation, which are optimal outcomes for ideal cancer treatments [19,98,99].

While plasma therapy and PBM have yet to have clear standardization of procedures for optimal results, they are prospective technologies in the development of effective cancer treatments. In multiple mentioned studies, they have been observed to effectively increase ROS levels in their subject cells, directly inducing multiple mechanisms. Specifically, their ability to increase intracellular ROS levels to toxic concentrations has shown potential in their applicability in cancer treatments, triggering anti-tumorigenic responses in cancer cells, and eventually inducing cell death. However, plasma has shown comparable disadvantages compared to PBM, including plasma creating toxic chemicals like ozone in applying plasma technology [100] and requiring residues of gas, whether that may be ambient air or prepared gas flows [76]. At the same time, PBM is gaining popularity as its process only requires a light source such as LLL or LED, and its absence of safety concerns. LLLs are highly monochromatic and capable of high powers, but their devices can be large and expensive, requiring high maintenance.

On the other hand, LEDs are small, adaptable, and have a low cost compared to LLL, but their low power, thermal effects, and large beam divergence could be considered a disadvantage [101]. However, both coherent and non-coherent light behaves similarly for most medical applications. These technological limitations of LEDs do not pose significant safety concerns [18]. Therefore, proper moderation of LED devices, additional studies about LED’s mechanism and application in intracellular ROS levels, and extensive support from bodies concerned with biomedical science is expected to remedy these disadvantages. Furthermore, other controllable parameters for plasma application are gas flows for DBD plasmas and applied voltage and distance from the target for plasma jets [87], which is a relatively small range of parameters for studying compared to LED, which can accurately change the applied wavelength ranging from visible red to near-infrared, and also moderate its output power and distance [17], allowing various combinations of set parameters. Specifically, for PBM, optimum parameters induce benefits for particular diseases. Its principle is called the biphasic dose-response, or the Arndt-Schulz curve, where if too little or too much energy is applied within the known threshold, optimal biostimulation will not be achieved [102,103]. Therefore, while the number of studies conducted on LED’s effect on cancer cells is significantly smaller than studies using plasma on the same topics, LED’s advantage in safety, accuracy, and efficiency makes it a more prospective technology to conduct further studies regarding its mechanisms and applicability for cancer treatment development.

Previous studies have associated intracellular ROS levels and production as traits cancer cells uniquely possess. Cancer cells have an abnormal higher ROS level that supports or directly induces pro-tumorigenic activities, including enhanced cell proliferation and viability and inducing genetic instability. Increasing ROS levels to toxic concentrations have been shown to induce anti-tumorigenic activities, including triggering cell death, giving attention to technologies and strategies that can induce changes to ROS levels.

Both plasma therapy and PBM have gained attention for their ability to increase intracellular ROS levels externally. However, after evaluating their technology’s mechanisms and range of controllable parameters, it was concluded that LED is the more practical and prospective technology promising in applications to cancer therapy.

However, the literature on the subject is still incipient because of the lack of standardized methodologies and a scarcity of specific scientific evidence and studies on LED’s relationship to cancer cells. Therefore, more studies determining LED’s effect on cancer cells’ ROS levels and its ability to enhance anti-tumorigenic actions both in vitro and in vivo should be conducted, which would set optimal parameters and conditions for LED and thus support its translation as an effective, accurate cancer treatment.

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Conceptualization: KW. Data curation: KW. Formal analysis: all authors. Investigation: all authors. Validation: all authors. Visualization: AP. Writing–original draft: KW. Writing–review & editing: all authors.

Andrew Padalhin is an editorial board member of the journal but was not involved in the review process of this manuscript. Otherwise, there is no conflict of interest to declare.

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