Cancer poses a major challenge in our times, as it affects individuals all over the world, regardless of age, sex, race or socioeconomic status. Advances in research have led to a reduction of cancer-related deaths thanks to early detection methods and the improvement of treatments. However, cancer still poses a challenge to current medicine due to the limitations of therapies like toxicity and the fact that they occasionally become ineffective since tumors can develop resistance to them over time. In this context, radiotherapy and chemotherapy are the most extended remedies, which help to control tumor growth by destroying cancer cells, although they cause toxicity and weakness, resulting in a deterioration of patient’s health status. Additionally, less invasive methods are available, such as immunotherapy and targeted therapy, which reinforce and restore the immune system and target specific molecules, respectively. Still, these treatments can also cause side effects and tumors can develop resistance to them (Sarmento-Ribeiro et al., 2019; Labrie et al., 2022). Thus, further research is needed to develop more efficacious and less harmful treatments. As an alternative to prevent resistance and toxicity, combinations of several compounds are being administered to patients, which work together against cancer, each bringing their own unique strengths to bear on the disease (Woodring et al., 2023; Vladu et al., 2022). In this context, plant extracts obtained from seeds, leaves, roots or fruits are currently under investigation due to their demonstrated antitumor properties that target both cancer cells and the tumor microenvironment (Choudhari et al., 2020; Hosseini and Ghorbani, 2015; Pan et al., 2019; Rana et al., 2021; Xu et al., 2016; Khan et al., 2019; Masuelli et al., 2021). In fact, countless works report the capacity of flavonoids, polyphenols, vitamins, minerals and many other bioactive compounds to reduce tumor growth through numerous mechanisms, such as promoting apoptosis, blocking cell cycle checkpoints, inhibiting cell proliferation, reducing the migratory capacity, modulating the immune response and reducing the secretion of pro-tumoral factors, among others (Esmeeta et al., 2022; Lopes et al., 2017; Reed et al., 2018; Kim et al., 2021; Masuelli et al., 2021; Almatroodi et al., 2020; Khan et al., 2019; Zhang, W. et al., 2022; Zhang, Y. et al., 2023; Wang W. et al., 2023). In this regard, the possibility of combining first-line therapies used in clinics with these products is being studied. Indeed, certain natural compounds and their mixtures are already being administered to patients’ as adjuvants together with antitumor treatments, as they can enhance their effect (Harvie, 2014; Lee et al., 2021; Wang, S. et al., 2024; Farghadani and Naidu, 2022; Shi et al., 2023; Ng et al., 2022; Abadi et al., 2022; Talib et al., 2022; Chen, Z. et al., 2023). Besides, these supplements, as well as supporting nutrition, can help to mitigate adverse effects of treatments, such as loss of appetite, nausea and fatigue, thus improving tolerance to therapy and the patient’s quality of life (Harvie, 2014; Liu et al., 2021; Wang, K. et al., 2022). Green tea, for instance, is one of the most widely consumed beverages in the world that possesses many health benefits and may serve as a preventive element for cancer development. Its polyphenols, especially the epigallocatechin-3-gallate (EGCG), are known to protect against inflammation, inhibit tumor cell growth, modulate the secretion of metalloproteases and reduce the expression of immune checkpoint inhibitors, among others (Masuelli et al., 2021; Almatroodi et al., 2020). Similarly, many spices have shown antitumor effects against cancer: cinnamon induces apoptosis, impedes cell growth and hinders angiogenesis (Peng et al., 2024; Sadeghi et al., 2019); curcumin suppresses proliferation, enhances apoptosis and triggers mitochondrial stress in tumor cells (Abadi et al., 2022); ginger reduces cell viability, boosts cell death and modulates cytokine secretion (Mahomoodally et al., 2021); licorice also induces apoptosis, inhibits angiogenesis and impedes cell migration and invasion (Zhang, Y. et al., 2023); turmeric inhibits cell proliferation, promotes apoptosis and cell cycle arrest, and blocks Epithelial-Mesenchymal Transition (EMT) and invasion (Zhang, Y. et al., 2023; Wang X. et al., 2023). Likewise, some fruits like berries, grapes and many others, have shown beneficial effects against cancer by increasing cell death, causing cell cycle arrest and by reducing inflammation and the presence of Reactive Oxygen Species (ROS) (Masuelli et al., 2021; Esmeeta et al., 2022).

Related to this, Ocoxin is a mixture containing a combination of plant extracts (green tea, licorice, cinnamon), amino acids, minerals and vitamins. Although the compound was originally designed as a supplement to strengthen the immune system and to improve the general wellbeing of patients suffering from cancer and promote their overall health, several studies have reported its antitumor mechanisms. Briefly, Ocoxin showed to reduce tumor growth in vitro by decreasing cell proliferation, blocking cell cycle, augmenting apoptosis, modulating gene expression and altering the secretome of tumor cells while reducing chemoresistance and improving the effect of different chemotherapeutic drugs. Moreover, in vivo studies performed in mice revealed that, apart from reducing tumor volume, the natural supplement modulated the expression of secreted cytokines, diminished the infiltration of macrophages and Cancer Associated Fibroblasts (CAFs) and reduced angiogenesis (Hernandez-Unzueta et al., 2023; Hernandez-Unzueta et al., 2017; Benedicto et al., 2021; Márquez et al., 2016; Hernandez-Unzueta et al., 2019). Furthermore, several clinical trials have indicated the advantages of using Ocoxin together with cancer standard care therapies to alleviate side-effects and to improve the quality of life of patients (Shumsky et al., 2019; Fundora-Ramos et al., 2021; Kaidarova et al., 2019).

As we mentioned, extensive research has been performed on the antitumor effect of Ocoxin against both tumor cells and tumor microenvironment, which has permitted the comprehension of the biological processes in which the compound participates. However, much remains to be discovered about the underlying mechanisms and altered pathways that may be involved in the response to the treatment with Ocoxin. In order to gain a more complete understanding of how the natural mixture acts in the context of cancer, we have chosen to carry out RNA sequencing of the four cancer types mentioned above. This approach will allow us to identify which specific genes are altered in response to the compound and, in turn, help us to infer which biological pathways might be modified. This more detailed analysis will permit us to gain crucial insights into the possible mechanisms of action of Ocoxin in cancer and may provide a solid basis for future clinical studies and therapeutic developments.

Cancer is one of the most worrisome pathologies in the world. Nowadays, due to the prevention schedules, advances in oncological surgery and the identification of new therapeutic alternatives have permitted to treat, prolong and improve the life quality of patients suffering from many cancers. However, despite the progress, many of them experience recurrence and metastasis and although they can be treated with radiotherapy or chemotherapy, the adverse effects arising from those treatments considerably damage their quality of life. Thus, the latest research is focused on the identification of new therapeutic alternatives that not only treat cancer but also extend life expectancy and improve the wellbeing of cancer patients by reducing the undesired effects caused by the treatments. In this scenario, there is growing interest in the application of bioactive compounds of natural origin, such as, EGCG, curcumin, resveratrol, quercetin and many others, to treat different kinds of cancers and to reduce the non-desirable side effects produced by chemotherapy (Naujokat and McKee, 2021; Ding et al., 2020; Rana et al., 2021; Vladu et al., 2022; Maleki Dana et al., 2022).

Ocoxin is a natural mixture composed of plant extracts, amino acids, vitamins and minerals known for its established antioxidant, anti-inflammatory and immunoregulatory properties, which has further demonstrated to exert antitumor effects in several cancer models in vitro and in vivo by impairing tumor development at different levels. On the one hand, Ocoxin has reduced cell viability in vitro by decreasing cell proliferation and inducing apoptosis and cell cycle arrest in many cancers. On the other hand, the compound has increased the cytotoxic effect of first-line chemotherapeutic drugs while reverting the resistance to chemotherapy caused by CAFs (Hernandez-Unzueta et al., 2017; Benedicto et al., 2021; Hernandez-Unzueta et al., 2019). Besides, Ocoxin has been used in clinics and showed an improvement in the quality of life of patients with hepatocellular carcinoma, lung cancer, PRAD, PAAD, cervical cancer and endometrial adenocarcinoma, among others, while increasing survival in some patients (Al-Mahtab et al., 2015; Kaidarova et al., 2019; Ruiz Lorente et al., 2020; Lima-Pérez et al., 2024; García-Perdomo et al., 2021). Therefore, in the present work, we aimed to deepen into the fundamental mechanisms through which Ocoxin exerts its antitumoral activity against cancer, as well as to identify whether there are any commonalities among the various types of cancer that may offer insights into the functionality of the nutritional supplement.

In relation to that, previous works have described the role of natural products acting as multi-targeted elements to alter different signaling pathways by regulating gene expression or targeting proteins and their secretion (Luesch and Paavilainen, 2020; Irshad and Husain, 2021; Nasir et al., 2020). Considering that cancer is a dynamic disease and that tumor cells possess distinct molecular signatures and sensitivity to therapies, the capacity of bioactive compounds to target cancer through several mechanisms at once needs to be explored since those effects might be an interesting approach to personalized therapies. Therefore, once tumor and patient’s characteristics are defined, instead of using established first-line treatments, those could be combined with natural products, thereby increasing the potential for a successful treatment.

According to our results, the mixture altered several genes in each type of cancer. Notably, TNBC and PAAC showed a substantially higher number of significantly altered genes, 3,438 and 1589 respectively, from which 12% were in common. Contrarily, in PRAD and COAD the number of deregulated genes was around 500 in each cancer. Besides, taking into consideration all the results, Ocoxin deregulated the expression of 13 genes in common in the analyzed tumors, which can be classified in three principal independent groups based on four functional protein association networks. The first group is composed of proteins related to the UPR (ADM2, CHAC1, SESN2, TRIB3). The second group gathers proteins involved in two biological processes such as, zinc homeostasis (MT1F, SLC30A1, SLC39A10) and detoxification of ROS (HMOX1, NQO1, TXNRD1). These molecules can also be englobed as part of the nuclear factor erythroid 2-related factor 2 (NRF2) pathway, which is responsible for the defense against oxidative stress and the maintenance of stable conditions. Finally, the third group contains proteins related to the cell cycle (ESPL1, KIF20, RAD54L). In this context, the upregulation of the genes encoding for the proteins of the first and second group that deal with cell homeostasis could have led to the production of misfolded proteins that are not correctly eliminated by the ER-associated degradation (ERAD) (Smith et al., 2011; Travers et al., 2000), causing ER stress. In fact, some natural bioactive compounds are known to induce or reduce ER stress. Actually, Xue et al. reported that morusin, a flavonoid obtained from white mulberry, induces ER stress on human epithelial ovarian cancer cells by upregulating the heat shock protein family A member 5 (HSPA5), the DNA damage inducible transcript 3 (DDIT3 or CHOP), the endoplasmic reticulum to nucleus signaling 1 (ERN1) and the α subunit of the translation initiation factor 2 (eIF2α) driving cells to a paraptosis-like cell death (Xue et al., 2018) and Zhang et al. observed that curcumin boosts ER stress-associated apoptosis on human papillary thyroid carcinoma cells (Zhang, L. et al., 2018).

It is known that cells, in order to restore the proteostasis, activate a mechanism named the UPR, whereby modulation of the gene expression occurs and protein synthesis becomes diminished (Harding et al., 2000; Ron and Walter, 2007). In this scenario, cells need to make a decision: to continue living or to die. This choice is controlled by eIF2α, whose activation reduces the global protein synthesis and, at the same time, induces the activating transcription factor 4 (ATF4) (Harding et al., 2003). These genes are central players of the integrated stress response (ISR), an adaptive response by which cells have the ability to reprogram gene expression in order to provide an optimized response to deal with stressful conditions. Despite the fact that the ISR is mainly a homeostatic pro-survival mechanism, cell response will depend on the nature and intensity of the driving stimuli, which, in case of prolonged and severe signals, will drive cells to death (Pakos-Zebrucka et al., 2016). In fact, this signaling network could be useful to face cancer as it is known to contribute to drug sensitivity and to promote the expression of immunotherapeutic targets (Obiedat et al., 2020; Lines et al., 2023; Tian et al., 2021). Actually, over the last years ISR modulating therapies have attracted significant attention to such an extent that diverse ongoing clinical trials target different levels of this pathway (Lines et al., 2023). During the ISR, various kinases sense different stimuli, such as nutrient deprivation, heme deficiency, DNA damage, oxidative stress or ER stress and converge on the phosphorylation of eIF2α leading to the induction of the ATF4 (Lines et al., 2023; Tian et al., 2021). In this work, the treatment with Ocoxin only upregulated ATF4 in PAAD. However, ATF3, ATF5, PMAIP1, DDIT4 and CHOP, which are downstream genes of ATF4, were upregulated by Ocoxin in at least one of the studied cancers, while TRIB3 was overexpressed in all of them. Furthermore, despite the fact that ATF4 itself was not deregulated by Ocoxin in all the analyzed cancer models, it arises as a central protein in the correlation among the genes altered by the compound (except for KIF20A, RAD54L and ESPL1) in common in COAD, PAAD, PRAD and TNBC. In fact, most of the genes upregulated by Ocoxin, such as ADM2, CHAC1, HMOX1, MTF1, NQO1, SESN2, TRIB3 or TXNRD1, are related to the ER stress either directly or indirectly. On the one hand, ADM2, CHAC1, SESN2 and TRIB3 are stress sensors of UPR controlled by the ATF4 gene under ISR (Crawford et al., 2015; Kovaleva et al., 2016; Garaeva et al., 2016; Kalinin et al., 2023) and on the other hand, HMOX, MTF1, NQO1 and TXNRD1 are involved in ROS detoxification, whose imbalance causes ER stress (Shi et al., 2023; Dunn et al., 2014; Li, H. et al., 2021; Rodríguez-Menéndez et al., 2018; Scuto et al., 2022). Furthermore, MTF1 encodes for a zinc dependent transcription factor that responds to changes produced in the intracellular heavy-metal homeostasis. Briefly, MTF1 regulates cellular processes by binding zinc to metallothioneins (MTs) or through the transcription of metal transporters of the ZRT/IRT-like protein (ZIP) family and the zinc transporter (ZnT) family (Eide, 2006; Lichten et al., 2011; Lichten and Cousins, 2009). While ZIP family carriers import zinc from the extracellular space or subcellular organelles to the cytoplasm, ZnT family components export the metal from the cytoplasm to the extracellular milieu or organelles (Sun, C. et al., 2024; Cotrim et al., 2019). At this point, it needs to be mentioned that Ocoxin contains high levels of zinc, which could have altered the expression of genes that participate in its homeostasis. In particular, those genes deregulated by Ocoxin were SLC30A1 and SLC39A10 (also known as ZNT1 and ZIP10), which enable zinc discharge and acquisition respectively. In this regard, Ocoxin upregulated the expression of SLC30A1, whereas downregulated that of SLC39A10, resulting in an increase of export to the extracellular compartment and a reduction of intracellular zinc accumulation (Fukada and Kambe, 2011; Palmiter and Findley, 1995). Therefore, cancer cells may activate MTF1 in the presence of high concentrations of this metal, causing an underexpression of SLC39A10 and an overexpression of SLC30A1 in order to recover zinc homeostasis. In fact, this metal is an essential component for the correct functioning of the ER since any homeostatic disruption can originate ER stress and subsequent UPR activation, which, as previously described, are related to the ISR pathway (Homma et al., 2013; Nguyen et al., 2013; Kambe et al., 2017). Therefore, we deem that Ocoxin drives tumor cells to a stressful environment through the ISR. Bearing in mind the intertumoral and intratumoral heterogeneity, and given the implication of the ISR in cellular adaptation and tumor development, modulating its activity could be a formidable treatment for many cancers, especially in combination with other therapies. Due to the multiple cellular mechanisms controlled by the ISR, the manipulation of this pathway could reach several populations of cells with distinct genetic signatures within the same tumor, enabling the overcoming of barriers such as treatment resistance or relapse, which represents a major impediment in medical oncology.

Anyway, it is crucial to consider that our results correspond to cells that had been treated with Ocoxin for 48 h, a period that might not be sufficient for the cells to determine their fate. Probably, as described before, if the stress caused by Ocoxin persisted, cells would engage cell death signaling pathways. This is supported by unpublished data where we observed that the mixture led to an increase in the percentage of dead cells after 72 h, thereby confirming that cells ultimately succumb to death after sustained stress conditions. Nevertheless, all the analyzed tumors exhibited either unchanged or downregulated caspases, indicating that cells may be undergoing a caspase-independent form of programmed cell death or that a duration exceeding 48 h is required for the activation of apoptotic caspases. Here, we show that, among other processes, Ocoxin deregulated the expression of multiple genes associated with autophagy, necroptosis and especially ferroptosis, a type of programmed cell death characterized by the accumulation of free iron within cells and by lipid peroxidation. Due to its clinical importance, ferroptosis has gained attention over the last years as an apoptosis-independent programmed cell death. This mechanism is strongly associated with metabolic processes, such as, carbohydrate metabolism, lipid metabolism and amino-acid metabolism (Ye et al., 2024) as well as iron and selenium metabolism (Conrad and Proneth, 2020) among others. In this regard, keeping in mind that cell metabolism is a crucial process to sustain cell growth and self-renewal, it leads to the fact that metabolic activities and the metabolites produced in the tumor could have an influence on ferroptosis (Jiang et al., 2024). Interestingly, Ocoxin caused an overexpression of HMOX1, a critical mediator in ferroptosis that promotes cell death in the presence of oxidative stress and high iron concentrations inside the cells (Chiang et al., 2018). Moreover, the nutritional supplement also deregulated the expression of NQO1, a gene responsible for redox control that is also involved in ferroptosis (Zhang, L. et al., 2018; Wang W. et al., 2023). Actually, both HMOX1 and NQO1 are downstream genes of NRF2, which play a central role in the NRF2 pathway defending cells against ROS. In consonance, Liu et al. (2022) described that Vitamin C, which is one of the components of Ocoxin, sensitized pancreatic cancer cells to erastin-induced ferroptosis by activating the AMPK/NRF2/HMOX1 pathway (Liu, Y. et al., 2022). In line with this, Wang et al. reported that eriodictyol, a flavonoid obtained from certain plants, regulated ferroptosis through the NRF2/HMOX1/NQO1 signaling pathway in ovarian cancer (Wang X. et al., 2023). Thus, the overexpression of HMOX1 and NQO1 observed in cancer cells treated with Ocoxin could have accounted for the subsequent mortality of those cells when exposed to the compound. Besides, it is important to bear in mind that HMOX1 and NQO1 are also two of the genes related to the main pathway deregulated by Ocoxin in all the four analyzed cancers: cell metabolism. Interestingly, both HMOX1 and NQO1 are downstream genes of ATF4 and, therefore, they are implicated in the ISR pathway mentioned before. Wang W. et al. (2023) reported that a flavonoid derived from licorice increased HMOX1 and decreased metabolic glutathione peroxidase 4 (GPX4), which reduces lipid peroxidation, leading to ferroptosis (Wang X. et al., 2023; Koppula et al., 2021; Zhao et al., 2019; Li, S. et al., 2010). Additionally, Yang and colleagues (2021) described that the suppression of the KIF20A/NUAK1/NRF2/GPX4 signaling pathway induces ferroptosis and increases the sensitivity of COAD to chemotherapy. Interestingly, although the expression of NUAK1, NRF2 and GPX4 was not altered by Ocoxin, the supplement downregulated the expression of KIF20A in the four tumors (Yang et al., 2021). Nevertheless, a deregulation of SLC7A11 was detected in PRAD, PAAD and COAD, which is a downstream gene of NRF2 and the ISR (Koppula et al., 2021). This gene encodes for a cysteine/glutamate antitransporter system composed of two subunits (SLC7A11 and SLC3A2) that exchange extracellular cysteine for intracellular glutamate and regulate ferroptosis through the glutathione/glutathione peroxidase 4 (GSH/GPX4) pathway. Here we observed that Ocoxin upregulated SLC7A11 in three cancers along with SLC3A2 in PAAD, prompting an accumulation of cysteine, which is a component of Ocoxin, and a loss of glutamate inside the cell, impeding the function of GPX4 and, therefore, contributing to ferroptosis (Koppula et al., 2021). Correspondingly, Tang et al. described how certain metabolites obtained from plants used in Chinese medicine altered SLC7A11 and GPX4, among others, which reduced cancer development (Tang et al., 2024). Moreover, Ocoxin overexpressed CHAC1, a gene that also participates in the glutathione degrading metabolism, which curiously was connected to HMOX1 and NQO1 through ATF4 in the protein-protein interaction network analysis. According to several authors, CHAC1 is essential for the maintenance of NRF2 activation and is also involved in ferroptosis (Kreß et al., 2023; Kreß et al., 2023). Actually, He et al. reported that CHAC1 plays a role in hindering tumor development and enhancing the efficacy of docetaxel in prostate cancer by triggering ferroptosis and ER stress (He et al., 2021) and additionally, Chen et al. reported that this gene facilitates ferroptosis as well as necroptosis in breast cancer (Chen, M. et al., 2017). Furthermore, this is corroborated by Liu et al., who described the role of CHAC1 inducing ferroptosis through the glutathione metabolism in retinal pigment epithelial cells (Liu et al., 2023). Taking that into consideration, Ocoxin could have decreased intracellular glutathione concentration through the overexpression of CHAC1 and the subsequent glutathione degradation, driving cells to ferroptosis. Thus, we conclude that Ocoxin induces ferroptosis in tumor cells through several pathways, which could be beneficial for patients that do not respond to chemotherapy. According to several authors, apoptosis-resistant cancer cells have showed to be susceptible to ferroptosis inducers (Hangauer et al., 2017; Tsoi et al., 2018; Viswanathan et al., 2017). In this manner, a greater efficacy of current therapies could be achieved leading to the induction of apoptosis while simultaneously targeting resistant cells via ferroptosis. Moreover, this type of cell death has also been associated with the tumor microenvironment by several authors. On the one hand, Costa da Silva et al. reported that iron uptake by M2-like tumor-associated macrophages drives a differentiation to M1-proinflammatory phenotype which helps to reduce tumor growth (Costa da Silva et al., 2017). On the other hand, Wang et al. described that immunotherapy-activated CD8⁺ T cells enhance tumor ferroptosis by inhibiting the expression of molecules implicated in the glutamate–cystine antiporter system through interferon gamma (IFNγ) release, which causes lipid peroxidation and leads tumor to ferroptosis (Wang et al., 2019). In this regard, we previously mentioned that Ocoxin has an antitumor effect in different cells of the tumor stroma. However, additional analyses need to be carried out so as to identify the possible implication of the compound in the induction of ferroptosis through the tumor microenvironment. Nonetheless, the use of Ocoxin as an adjuvant agent could enhance first-line therapies by targeting the different populations of cancer cells and the tumor microenvironment, thereby increasing the susceptibility of therapy-persistent cancer cells to common therapies and preventing tumor recurrence and metastasis.

Nevertheless, those are not the unique metabolic genes targeted by Ocoxin. As previously mentioned, TNBC and PAAC shared a considerable number of altered genes. In particular, considering only those genes involved in metabolic pathways, those two cancers showed 14% of altered genes in common, while at the same time those were not altered in PRAD and COAD. In this regard, it is important to bear in mind that the analyzed PAAD and TNBC cells come from primary tumors, while those of PRAD and COAD were isolated from metastatic sites. A deeper analysis about the specific metabolic pathways where the genes altered by Ocoxin are involved, showed that in the primary tumors (PAAD and TNBC) the compound targeted genes from the carbohydrate, nucleotide and lipid metabolism, protein lysine methyltransferases (PKMTs) methylate histone lysine and the citric acid (TCA) cycle and respiratory electron transport. Specifically, regarding the respiratory electron transport, Ocoxin downregulated the expression of the mitochondrial oxidative phosphorylation (OXPHOS) genes ATP5F1C, ATP5MC3, ATP5F1E, ATP5PB, ATP5PF and ATP5MG, which could have been also driven by the ISR. Briefly, the phosphorylation of eIF2α mediated by the ISR stimulates the transcription of CHOP, ATF4 and ATF5 genes that participate in the activation of the mitochondrial UPR (UPR^mt) (Zhang, G. et al., 2021; Ogilvie et al., 2005; Boyce et al., 2005) Since both, the ISR and the UPR^mt, are two strongly related processes that function together to maintain the mitochondrial metabolism and function, we can assume that Ocoxin altered mitochondrial homeostasis. Interestingly, central players of this pathway, SIRT7 and HSP60 (HSPD1) (Lu et al., 2022), are overexpressed in the primary PAAD and TNBC but not in the metastatic COAD and PRAD. A similar phenomenon was also observed in this study, while the KEGG enrichment analysis indicated that cell cycle is one of the principal pathways altered in the metastatic PRAD and COAD tumors, in PAAD and TNBC the number of genes involved in the cell cycle was not that remarkable. In this context, it is widely described that one of the antitumor mechanisms of Ocoxin is the alteration of the cell cycle (Díaz-Rodríguez et al., 2018; Diaz-Rodriguez et al., 2017; Diaz-Rodriguez et al., 2016; Benedicto et al., 2021; Hernandez-Unzueta et al., 2023; Hernandez-Unzueta et al., 2019; Hernández-García et al., 2015; Pérez-Peña et al., 2019; Márquez et al., 2016). Among the 13 genes commonly altered by the mixture in the four cancer types, three of them, KIF20A, ESPL1 and RAD54L are implicated in the cell cycle progression. Curiously, although KIF20A is downregulated in all tumors, ESPL1 and RAD54L are differentially expressed between primary and metastatic tumors, being upregulated in PAAD and TNBC while downregulated in COAD and PRAD. We previously discussed the relation of KIF20A with ferroptosis; however, this gene mediates the spindle formation during mitosis and its overexpression is related to both, cancer development and a poor overall survival of patients. In fact, KIF20A is overexpressed in all the cancers present in the Gene Expression Profile Interactive Analysis (GEPIA) server (Tang et al., 2017), including COAD, PAAD, PRAD, and TNBC. Interestingly, in this work we demonstrated that Ocoxin downregulated KIF20A in all the analyzed tumors, that is, the compound reverted the expression of this gene. Besides, previous works report that the nutritional mixture also slowed down the cell cycle of tumor cells by causing an arrest on the G2/M phase in breast cancer while in colorectal carcinoma also affected the cell transition from G1 to S phase (Hernández-García et al., 2015; Márquez et al., 2016). Moreover, it is known that the inhibition of KIF20A hinders the growth of several cancer cells (Jin et al., 2023) and Nakamura and colleagues reported that the suppression of KIF20A arrested cells in the G2/M phase in breast cancer, while Zhang et al. reduced the colorectal cancer cell proliferation and migration by drecreasing the cell transition from G1 to S phase through the E2F1 transcription factor in hepatocellular carcinoma (Nakamura et al., 2020; Wang, H. et al., 2020). Considering all this, the reduction of KIF20A expression could be a specific therapeutic strategy to treat cancer, which proves that the understanding of factors such as the genetic profile of the patient and the mechanism of action of the compound might be a step towards more personalized therapies. In this regard, several authors have reported so far that Ocoxin slows down the cell cycle in different phases depending on the tumor type. Taking this into consideration, we can suggest that ESPL1 and RAD54L, which are differentially expressed between the primary TNBC and PAAC and the metastatic PRAD and COAD, are modulating the cell cycle in various ways. While RAD54L belongs to the DEAD-like helicase superfamily and is involved in the homologous recombination and DNA repair, ESPL1, also known as separase, initiates the final separation of sister chromatids during anaphase. ESPL1, which is typically upregulated in tumor tissue due to the rapid proliferation of cancer cells, is controlled by the PTTG1 regulator of sister chromatid separation, also known as securin, which has the ability to inhibit ESPL1 (Sun, Y. et al., 2009; Zhong et al., 2023). Here we observed that, on the one hand, the treatment with Ocoxin upregulated ESPL1 and downregulated PTTG1 in TNBC and PAAD, and on the other hand, ESPL1 was downregulated in PRAD and COAD, but PTTG1 was not altered. Moreover, while most of the cell cycle genes in common in metastatic tumors, such as, MCM2, MCM3, MCM4, MCM5, MCM7, BUB1, BUB1B and AURKB are downregulated, including CDKN2B, which is downregulated in TNBC and CDKN2C in PAAC, both encoded proteins that function as a cell growth regulator that controls cell cycle G1 progression. Nevertheless, most of the cell cycle genes of the metastatic COAD and PRAD tumors were upregulated, including the abovementioned CDKN2B, showing that Ocoxin regulates the G1 phase of the cell cycle through different routes.

To conclude, we can suggest that Ocoxin shows different mechanisms of action depending on the type and stage of the tumor. In fact, the compound demonstrated a greater sensitivity to primary tumors, where it altered more genes than in metastatic tumors. Nevertheless, the main altered pathway was cell metabolism, with a significant emphasis on the process of ferroptosis among the genes modulated by Ocoxin in common in all the studied cancers. Moreover, it must be mentioned that Ocoxin exerted a varied effect in each cancer by acting on distinct pathways, which, considering tumor heterogeneity, might reflect the multi-target action of the compound. Although the primary process modulated by the supplement was cell metabolism, the effect of Ocoxin in metastatic tumors exerted a substantial effect on the cell cycle. Therefore, we can presume that the antitumor action of Ocoxin is, in part, attributable to mechanisms englobed in the UPR, ISR and metabolic alterations leading to cell death, which may also explain its supportive role in chemotherapy. Modulating those pathways could be a crucial strategy to provide more specific treatments in clinical settings and improve oncological therapies, especially in aggressive or treatment-resistant tumors. However, further studies are needed to delve into the antitumoral mechanism of Ocoxin.