Photodynamic therapy (PDT) or light-activated treatment is increasingly being employed as adjuvant with other common modalities to treat cancer (1, 2). PDT is considered as a non-invasive strategy to treat various cancer types such as oral, skin, prostate, and head and neck (3, 4). Hence, PDT requires photosensitizer (PS) agent to be activated by light and generates numerous highly reactive molecular species including free radicals, reactive oxygen species (ROS), and singlet oxygen. However, PDT is beneficial and safe for the patient and the physician because it minimizes the interventional surgery, has short recovery periods, and protects organ integrity with a relatively low risk of local and systemic side effects (5–7). The response of PDT is based on the contribution of the three biophysical interactions between the PS, light of suitable wavelength, and oxygen molecules in the cells, which causes the specified effects within pathological tissues (8). In the presence of light, PS is activated and produces ROS. Afterward, ROS are responsible for damaging and killing tumor cells (9). Excitation of the PS with light leads to electron (e) and proton (p) movement to the first singlet excited state. Triplet mode is created after intersystem crossing. The triplet PS transfers energy to triplet oxygen, leading to the assembly and generation of reactive singlet oxygen (¹O₂). ¹O₂ can exert a multitude of actions including direct damaging of cancer cells, destroying the vascular system, and irritating the immune responses (10–13). From the literature on PDT studies, it can be concluded that ROS has various biological effects (14). In fact, it is now well established that PDT can cause tumor destruction by three mechanisms: direct cell death, tumor vascular damage, and immune response induction (15–17). It seems that the mechanisms of PDT in terms of cellular and molecular response requires further investigation. Photochemical reactions trigger various types of cell death mechanisms that lead to tumor tissue destruction, for instance, the shutdown of tumor vascular, leading to necrosis of the tumor via deprivation of oxygen and nutrients (18, 19). For many years, conventional cell death by PDT is divided into three types such as apoptosis, autophagy, and necrosis (20, 21). The molecular mechanisms of photo-killing pathways of PDT-induced apoptosis and autophagy ultimately cause low survival and cytotoxicity effects (22, 23). Moreover, in recent years, the striking progress in molecular techniques greatly expanded, resulting in new forms of cell death being discovered, such as mitotic catastrophe (MC), paraptosis, pyroptosis, parthanatos, necroptosis, and ferroptosis. Additionally, it has been reported that photodamages to the endoplasmic reticulum (ER) and liposomes result in apoptosis and paraptosis cell death (24, 25).

PDT may be more effective if it targets cancer cells at particular times of the cell cycle (CC). For example, targeting cancer cells in the S phase may increase the amount of PS uptake and ROS generation, leading to more efficient cell death via impeding the proliferation and cell cycle arrest (CCA) due to extensive DNA and organelle damages and mitotic and checkpoint failures. To date, various signs of CCA have been reported in cancerous cells under PDT that eventually cause cell damage, apoptosis, mitotic catastrophe, and cell death. Therefore, in this review, we focus on CCA in PDT and how it helps the standardization of PDT protocol and gives new insight to researchers to develop new therapies to target cancer cells at specific phases of the CC.

PDT is a cancer treatment procedure, which employs PSs that are activated under light exposure of certain wavelengths. PDT acts in two stages ( Figure 1 ): (1) PSs absorb light and become excited to higher energy levels, and (2) excited PSs are relaxed and transfer energy to the oxygen molecules, resulting in the generation of ROS, ¹O₂, and other free radicals. This can cause damage to DNA, membrane, and other organelles in cells in two ways. In type I, ³PS• transfers electrons and proton from the nearby molecules such as polyunsaturated fatty acids (PUFAs) in the membrane of a cell. Consequently, the interaction of e and p with cellular oxygen leads to the generation of cytotoxic ROS such as superoxide anion (O₂⁻•), hydroperoxide radical (HOO•), peroxides (H₂O₂, ROOH), and hydroxyl radical (OH•), which trigger free radical chain reactions. Type II involves the transfer of energy from ³PS to molecular oxygen, resulting in the formation of ¹O₂, which is considered a powerful oxidizing agent. Both type I and II reactions can happen simultaneously, and the ratio between them depends mainly on the photo-chemical and photo-physical features of the PS and cellular oxygen. Finally, these reactions trigger various cell death mechanisms in cancer cells in different pathways and lead to the suppression of tumor tissue (19, 26) ( Figure 1 ). Depending on the site of damage, cell death occurs through apoptosis, necrosis, or autophagy. However, several important parameters can influence the PDT performance, including PS properties, the wavelength of light, and amount of oxygen in cells (27).

The normal CC includes G1, S, G2, and M phases. During the G1 phase, the cell grows and prepares to enter the S phase, where DNA replication occurs, resulting in two identical copies of the cells’ DNA. The G2 phase is a period of growth and preparation for cell division. Finally, during the M phase, the cell divides into two same cells through a process called mitosis. After the M phase, the cells may enter a resting state called the G0 phase, where they remain until stimulated to re-enter the CC. Each phase is depending on the type of cell and external factors such as growth factors and stress signals (28). Cyclins and cyclin-dependent kinases (CDKs) are key regulatory proteins that control the progression of cells through the CC. Cyclin D is synthesized during the G1 phase, which binds to CDK4/6 to form an active complex. This complex phosphorylates the retinoblastoma (Rb) protein, which releases transcription factors that drive the expression of genes required for DNA synthesis. In the S phase, DNA replication occurs, and cyclin E is synthesized, which binds to CDK2 to form an active complex. Then, this complex phosphorylates proteins involved in DNA replication and repair. In addition, transforming growth factor beta (TGFβ) can activate cyclin E and CDK2 complex. In the G2 phase, the cell prepares for mitosis by synthesizing various proteins required for cell division. Cyclin A is synthesized during this phase, which binds to CDK1 to form an active complex. This complex drives the cell into mitosis by phosphorylating proteins involved in chromosome condensation and spindle formation. Overall, the regulation of cyclins and CDKs is essential for the progression of CC and genomic stability. Dysregulation of these proteins can lead to uncontrolled cell proliferation and tumor formation (29, 30).

In cancer cells, the CC is often disrupted and uncontrolled, leading to abnormal growth and division. Cancer cells can divide rapidly and continuously without the normal checkpoints that regulate the CC in healthy cells. In some cases, cancer cells may skip the G1 phase and enter directly into DNA replication, leading to an increase in the number of abnormal cells. Additionally, cancer cells may have mutations in genes (cyclins) and oncogenes such as Mdm2, MYC, and E2F that lead to uncontrolled growth and division. As a result of these disruptions, cancer cells may continue to divide and grow, even in the absence of external signals. This uncontrolled growth can lead to the formation of tumors and the spread of cancer. As shown in Figure 2 , in normal cells, several genes control the CC, including the following. First is P53: this gene produces p53 protein to suppress tumor cells by regulating the G1 checkpoint of the CC or promoting apoptosis. Additionally, it aids in preventing the replication and potential mutation-causing effects of cells with damaged DNA. Some proteins such as ATM, BRCA1, 2, MSH2, and MLH1, and the cell conditional such as hypoxia and cell damage cause P53 activation. Second is the RB1: this gene produces a protein called Rb, which helps to regulate the G1 checkpoint. It prevents the activity of E2F transcription factors, preventing them from promoting cell division. Third is the CDK2: this gene produces two proteins, p16 and p14, which also act as tumor suppressors by regulating the G1 checkpoint. Moreover, other proteins such as p17, p18, p19, and p21 play a critical role in this stage. They inhibit the activity of CDK4/6, preventing the phosphorylation of Rb and promoting CCA (31, 32).

The cancer CC is uncontrolled, and this characteristic is crucial for the survival of cancer cells. Unlike normal cells, they undergo cell division only when necessary for growth or repair; cancer cells divide uncontrollably, leading to the formation of tumors. In the G1 phase, the cell grows and prepares for DNA replication. In the S phase, DNA replication occurs, resulting in the formation of two identical copies of DNA. In the G2 phase, essential proteins are synthesized, and the cell prepares for mitosis. Eventually, the cell divides into two daughter cells during the M phase (33–35). However, this paradigm highlights the critical role of checkpoints in cancer to provide a better comprehensive CC control and specific checkpoint functions and create new therapeutic opportunities. Protein kinases are enzymes that play a vital role in regulating the CC checkpoints. They work by adding phosphate groups to other proteins, which can either activate or prevent their function. In the context of CC checkpoints, protein kinases act as gatekeepers that ensure that the cell is ready to proceed to the next phase (36, 37).

Similar to normal cells, cancer cells have four phases in CC such as G1, S, G2, and M (38). Cancer cells divide more rapidly than normal cells and may have mutations that allow them to bypass normal CC checkpoints. This uncontrolled and abnormal cell division leads to the formation of tumors (39, 40). However, all CC phases are important to regulate and progress the CC during G1 and chromosome segregation during M phase ( Figure 3 ).

Cancer cells often have defects in their DNA repair pathways, which allows them to accumulate mutations and grow uncontrollably. Hence, targeting these pathways could be a strategy for cancer therapy. One approach is to use DNA-damaging agents, such as CT or radiation, which induce DNA lesions that cancer cells cannot repair efficiently (85). These modalities remain the most effective for cancer therapy but also induce DNA damage in normal tissue (86). Another approach is to target specific DNA repair enzymes or proteins that are overexpressed or mutated in cancer cells, thereby disrupting their ability to repair DNA damage. In addition to targeting DNA repair pathways directly, cancer therapy also involves regulating the CC, which is tightly controlled to ensure proper DNA replication and segregation. Dysregulation of the CC is a hallmark of cancer, as cancer cells often bypass checkpoints that would normally trigger cell death or repair mechanisms. Therefore, drugs that inhibit specific CC regulators, such as CDKs, have been developed for cancer therapy.

One promising approach for cancer therapy is the combination of PDT with RT or CT. When combined with RT or CT, PDT can enhance the effectiveness of these treatments by increasing DNA damage and inhibiting DNA repair pathways (87, 88) ( Figure 4 ). Research has shown that PDT in combination with RT or CT can improve outcomes in various types of cancers, including lung, head and neck, and pancreas (89–91). This approach has also been shown to reduce side effects and toxicity associated with traditional cancer treatments. However, more research is needed to optimize the timing and dosing of these therapies and to identify biomarkers that can predict response to the treatments. Additionally, the development of more targeted photosensitizing agents and combination therapies tailored to individual patient’s genetic profiles may further improve outcomes in cancer therapy.

Alkaloids and taxanes are classes of CT drugs that have been used in combination with PDT for cancer treatment. Alkaloids, such as vinblastine and vincristine, act by disrupting the cell division process in cancer cells (92–95). Taxanes, such as paclitaxel and docetaxel, also interfere with cell division by stabilizing microtubules (96–98). These CT medications have the ability to improve the efficacy of PDT by boosting the production of ROS and reducing DNA repair pathways. This strategy has shown effective outcomes for breast, ovarian, and prostate cancer. However, as with any cancer treatment, there are potential side effects and toxicity associated with these drugs. Further research is needed to optimize the dosing and timing of these therapies and to identify biomarkers that can predict response to treatment. Additionally, the development of more targeted CT drugs and combination therapies tailored to individual patient’s genetic profiles may further improve outcomes in cancer therapy. Overall, understanding the complex interplay between DNA repair and CC control in cancer cells is crucial for developing effective therapies that can selectively target cancer cells with minimal side effects ( Figure 4 ).

ROS from both internal and external sources has an impact on biology ( Figure 5 ). Radiation, chemotherapeutic medications, pathogenesis, xenobiotics, etc. are some examples of external causes, while the internal elements are including cytokines, inflammation, mitochondria, and peroxisomes (99–101).

Naturally, ROS are produced in cells as a byproduct of normal metabolic processes. ROS plays an important role in cellular signaling and defense against pathogens. In normal cells, ROS production is tightly regulated to maintain a balance between ROS generation and elimination by antioxidant systems. Uncontrolled ROS production induces oxidative stress, which can damage cellular components and contribute to the development of various diseases including cancer and neurodegenerative and cardiovascular diseases. In cancer, the intracellular ROS causes oxidation and mutations in pro-oncogenes such as Ras and p53 as tumor suppressor genes (102).

Indeed, ROS can have both beneficial and harmful (double-edged sword) effects on cells depending on their concentration and location. At low levels, ROS can act as signaling molecules to regulate cell proliferation, differentiation, and apoptosis. However, at high levels, ROS can cause oxidative damage in macromolecules such as lipids, proteins, and DNA, leading to cell death and tissue damage. Therefore, maintaining a balance in ROS levels is crucial for cellular homeostasis and overall health. The high level of ROS in mitochondria leads to the activation of mitogen-activated protein kinase (MAPK) and Ras-ERK. This signal induce cell proliferation, cell survival, cell migration, and epithelial–mesenchymal transition (103, 104).

Edderkaoui and co-workers reported that ROS generation in the extracellular matrix cause the survival of pancreatic cancer cells through 5-lipoxygenase (LOX) and NOX (105). For example, the combination of gemcitabine with trichostatin-A, epigallocate-3-gallate (EGCG), capsaicin, and benzylisothiocyanate (BITC) has been proven to be effective for pancreatic cancer treatment (106). All these drug action are based on the elevation of intracellular ROS levels to trigger apoptosis. Furthermore, it is reported nonsteroidal anti-inflammatory drugs (NSAIDs) like Sulindac enhances the intracellular level of ROS that causes cell death and apoptosis in colon and lung cancer cells that are more sensitive to H₂O₂ (107, 108). Similarly, an anticancer drug Aminoflavone promotes cell death in MCF-7 and MDA-MB-468 breast cancer cells. Aminoflavone induces programmed cell death due to an increased intracellular level of ROS and activation of caspase 3. Some clinical chemotherapeutic agents can cause cell death via mitochondrial DNA damage and increase cellular ROS levels (109, 110). The ROS production and its biological effects are summarized in Figure 5 .

Chemotherapeutic agents such as doxorubicin, epirubicin, bleomycin, and platinum can also induce oxidative stress in cancer cells through the alteration of ROS levels. These drugs target mitochondria and generate ROS (111–113). For decades, RT has been utilized for cancer therapy. RT can induce DNA breaks and apoptosis to indirectly militate against the antitumor treatment by inducing the ROS levels (114, 115). PDT also induces the generation of various ROS, which prompts signaling cascades and cell death in cancer cells when exposed to light of specific wavelength (116, 117). Yokomizo et al. and Sasada et al. reported that thioredoxin could overcome cancer cell resistance via ROS-generating and its antioxidant activity (118, 119). However, Ravi et al. illustrated that thioredoxin in combination with daunomycin increases the cytotoxic effect in MCF-7 breast cancer cells. Daunomycin induces cancer cell death by redox cycling reactions and ROS generation. These processes cause DNA damage and apoptosis of tumor cells ( Figure 5 ) (120, 121). Procarbazine, one of the earlier drugs, produces H₂O₂ via oxidation in an aqueous solution, which is crucial for cytotoxic activity. Subsequently, Mathe and colleagues have reported that procarbazine can be used for the treatment of Hodgkin’s lymphoma and hematolymphoid malignancies (122).

In general, ROS are highly reactive molecules that are generated as byproducts of normal cellular metabolism. They play a crucial role in cellular signaling, but excessive ROS production can lead to oxidative stress and macromolecules damage, such as DNA, proteins, and lipids. ROS-induced oxidative stress causes accumulative damage over time and contributes to the aging process. Overall, understanding the role of ROS in cellular physiology and pathology is an active area of research, with important implications for aging, cancer, and other diseases. The level of ROS produced by internal and external sources cause biological effects such as cell proliferation or prevention of proliferation (CCA), organ, cell damage, and cell death. This article highlights the level of ROS and biological effects that follow from two mathematical models. The first one is linear model (1) and the second one is quadratic linear model (2). In both these models, we have considered the threshold or normal level of ROS (b) because there is an ROS level to regulate signaling pathways and normal cell proliferation in the body, while the damage happens when the level of ROS is above the threshold (123). In summary, Figure 6 depicts the relationship between biological damage (effect) and ROS level, demonstrating how an increase in ROS level causes cancer cells death. These models share some similarities with mathematical dose–response curves used in radiobiology because of the way x-ray radiation and photodynamic treatment cause ROS and DNA damage (124).

where

y= damage or biological effects, x= ROS level, s= slope, and b= normal level of ROS.

The role of PDT in the initiation of MC and CCA was first observed and studied with the emergence of second-generation PSs in PDT; most of them are in preclinical and clinical trials ( Table 1 ) (12). Hence, molecular pathways of cell death in PDT and these PSs provide new insight for cancer therapy and modified the treatment strategy. As discussed in Figure 1 , the PSs triggering cell death may have different cellular localizations. Since the range travel of ¹O₂ is only approximately 10–20 nm, the most damage and destruction is at the site of the PS during PDT.

The CCA occurs at various phases as a result of the PDT-induced microtubule damage or distortion. Interestingly, mitotic block can happen in PDT, with or without PS photoinduction. For instance, the findings of Cenklová et al. has shown that α, β, χ, δ porphyrin-Tetrakis (1-methylpyridinium-4-yl) p-toluenesulfonateporphyrin (TMPyP) causes increased mitotic index and mitotic arrest in HeLa/G361 cells (126). Moreover, two separate studies by Csík et al. and Pizova et al. reported that TMPyP can cause DNA damage and protein interactions in the nucleosomes of G361 and MCF-7 cells, resulting in the alterations in DNA structure and gene expression (157, 158). Some porphyrins, by binding to tubulins, prevent their polymerization during combination therapy with PDT or single therapy (159). A study has shown the mechanism action of hypericin-based PDT that induces damage of microtubules and the mitotic spindle, resulting in CCA at the M phase (160). Dysfunction of some centrosomal proteins including AuroraA, ninein, TOG, and TACC3, which have an important role in microtubule arrangement, promotes apoptotic-like cell death (161). Mascaraque and colleagues reported that slight effects on spindle morphology lead to the distorting of microtubular of γ-tubulin and CCA in the mitotic phase in HeLa cells (125). The atomic force microscopy method by Jung et al. illustrated that chlorin under PDT disrupts the cytoskeleton in J82 bladder cancer cells and activation of apoptosis or programmable cell death (162). Furthermore, novel PS, DTPP leads to disruption of the cytoskeletal structure, cleavage of proteins, and CCA in the S phase in A549 cells following the mitochondria and lysosomes damaged by PDT-generated ROS (129, 163).

CCA and cell synchronizing refer to the process of temporarily stopping or synchronizing the growth and division of cells. This can be used in cancer therapy to improve the effectiveness of treatments and reduce their toxicity on normal healthy cells. By targeting cancer cells during specific phases of the CC, such as during DNA replication or cell division, therapies can be optimized for individual patients and may help to reduce the development of treatment resistance. Additionally, studying the relationship between the CC and cancer biology can provide insights into new treatment approaches and therapies. There are some potential advantages of cell synchronization in cancer therapy:

However, from the results in Table 1 , it seems that the mechanism of actions of PDT on CCA depends on the type of cell line, PS agent, and ROS level. Consequently, the organelles and DNA damages lead to CCA. The CCA prevents cell progression and synchronization. We come to the conclusion that even though CCA has been documented in phases like G0/G1, S, and G2/M, the highest percentage of CCA has been reported in G2/M phase under PDT. DNA is considered as the sensitive target to ROS action directly and indirectly. From Table 1 , among 33 studies that have been investigated on CCA in PDT, approximately 45.45% of them have reported that the G2/M phase is the most sensitive phase of CC to be arrested for cancer therapeutic application ( Figures 7 , 8 ). In summary, PDT can arrest CCA at different stages of CC, which can act as a strong cancer cells progression barrier, and this mechanism could help to improve treatment efficacy via different doses of PS and light irradiation.

The standardization of PDT protocol remains one of the critical challenges to resolve. To overcome this challenge, the biological response of PDT needs to be investigated. CCA is common in apoptosis induced by PDT; it seems that some of the PS has their own special action without PDT. This review highlights the opportunities to develop PDT standardization protocol and combinatorial treatments. Authors introduced two mathematical models of ROS level and its effects in cells. Furthermore, effects of ROS level on oxidative stress, the mitotic checkpoint, and delaying or arresting CC progression have been reviewed. We have suggested new strategies that PDT could be used alone or in combination with immunotherapy, RT, or chemo-therapeutic drugs in cancer therapy because the mechanism of action of PDT is similar to chemo-radiotherapy in some ways. However, they can have synergistic effects on cancer treatment. One of the important issues in combination therapy is the decision on the prioritization of the modalities in order to achieve high treatment efficiency. Hence, understanding the CC control in cancer cells could open therapeutic opportunities to improve treatment outcomes.

The ideal PDT protocol should provide some requirements including the following: first is predicting tumor sensitivity to PDT; second is the choice of an appropriate PS with low toxicity and high photochemical activity on CCA; third, targeting and concentration of PS and irradiation dose should be tailored to the tumor; fourth, the evaluation of oxygen level in the tumor microenvironment; fifth, the reparation of PS and radiation dose after detecting CCA can be more effective; and finally, PDT should activate the immune system.

To sum up, standardization is one of the challenges in PDT. There is currently no standardized protocol for PDT, which can lead to variability in treatment outcomes and hinder its widespread adoption. Developing standardized guidelines for patient selection, dosing, and treatment parameters is essential for improving the consistency and reproducibility of PDT. However, some research has suggested that targeting cancer cells during specific phases of the CC may enhance the effectiveness of PDT. For example, targeting cancer cells in the S phase (DNA synthesis phase) may increase the amount of PS uptake and ROS generation, leading to more efficient cell death pathways. However, further research is needed to understand the potential benefits of synchronizing cancer cells in PDT. In addition, as a suggestion, most PS drug and nano-photosensitizers have the potential as radiosensitizers, so they can be used in combination with PDT and RT in cancer therapy. This suggestion helps in highly effective treatment and prevention of cell proliferation via CCA. Furthermore, stopping CC with repetition PDT regime promotes to more cell damage and cell death. From a prospective view, the CC studies in vitro can be confirmed by in vivo testing with repeated PS administration and irradiation to confirm better treatment efficiency. Moreover, as we reviewed, CCA in specific phases can be a specific feature to determine cell line and PS drug, so this feature could be considered in tailoring therapy in PDT.

Conceptualization, KM and BG. Writing—original draft preparation, KM. Writing—review and editing, BG and HA. Supervision, BG and HA. These authors contributed equally to this work. All authors contributed to the article and approved the submitted version.