Cancer remains a leading cause of death worldwide, with an estimated 10 million cases and a total economic medical treatment burden of 1.16 trillion US dollars in 2020 only (Ferlay et al., 2021, Sung et al., 2021). The constant threat of cancer and the apparent insufficiency of current therapies prompt the search for new alternative treatments to combat this disease. Herbal products contain many constituents that act on various targets in the human body to induce pharmacodynamic responses (Cheon, 2021). According to recent data, the anticancer activity of certain herbal medicines is attributable to the promotion of autophagic cell death, apoptosis, modulation of the tumor microenvironment, and suppression of tumor angiogenesis (Cheon, 2021b; Sun et al., 2020a). Therefore, phytochemicals are a promising addition to anti-cancer drugs currently in clinical use and represent potential adjuvants for future cancer treatments. For instance, medicinal herb-derived shikonin and hypericin induce immunogenic cell death of specific cancer cells resulting in the activation of tumor cell recognition by the host’s immune system (Yin et al., 2017). Studies on the natural compounds reveal the pleiotropic-pharmacological nature of phytocompounds, a so-called concept of “multiple targets, multiple effects, and complex disease” (Yin et al., 2017).

The inhibition of cancer cell growth by bioactive phytochemicals is frequently associated with redox homeostasis disruption. Cell growth is regulated by reactive oxygen species (ROS), and phytochemicals with antioxidant activity may block cell proliferation by scavenging ROS (Aggarwal et al., 2019). Different antioxidants present in foods, including phenolics, flavonoids, carotenoids, etc., are also recognized as potent antioxidants displaying anticancer properties (Ullah et al., 2020). The controlled manipulation of ROS levels with antioxidants may induce selective apoptosis in the cancer cells as the normal cells operate in a different redox environment than the cancer cells and are less sensitive to cellular redox status fluctuations. With this approach, it might be possible to ameliorate the negative impact of oxidative stress on the surrounding non-transformed cells and overcome the resistance of cancerous cells against chemotherapeutics (Barrera et al., 2021). Herbal extracts or isolated phytochemicals can also be used in combination with chemotherapeutic drugs (Seca and Pinto, 2018). Acting together, they were reported to have additive or synergistic therapeutic effects (Fujiki et al., 2018, Liu et al., 2015). Even though sometimes more attention is given to the bioactive phytochemicals on their own, there is evidence that multi-constituent plant extracts are sometimes even more effective than their isolated active components (Caesar and Cech, 2019, Koss-Mikołajczyk et al., 2019b, Koss-Mikołajczyk et al., 2019a; Koss-Mikołajczyk, I. et al., 2019). This phenomenon may be due to the fact that many active phytochemicals lose their activity after they are separated from their original complex mixture, as their activity is high only when multiple constituents are present and these act synergistically to provide the biological effect (Caesar and Cech, 2019). Therefore, in this study, the antioxidant and antitumor properties of the ethanolic extract of Rumex obtusifolius L. (RO) seeds were investigated.

Apart from mentioned pleiotropic activities, plant constituents may occasionally exhibit specific interactions, either inhibitory or stimulatory, with enzymes crucial for cancer progression. For instance, pharmacological strategies that interfere with L-arginine metabolism hold great promise in the treatment of various types of cancers (Engblom et al., 2016, Rath et al., 2014). Earlier reports focused on the expression of arginase and nitric oxide synthase (NOS) in murine or human primary cancer tissue as well as malignant cell lines (Munder, 2009) and emphasized their potential role in the promotion of tumor growth via polyamine synthesis or the down-regulation of nitric oxide-mediated tumor cytotoxicity (Xue et al., 2018). Our previous results have shown that polyamine and nitric oxide (NO) levels are decreased by NG-hydroxy-nor-L-arginine (nor-NOHA) and/or Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME). Moreover, these also attenuate tumor growth, occurrence, and cancer progression in vivo (Avtandilyan et al., 2022, Avtandilyan et al., 2019, Avtandilyan et al., 2018). We also measured several parameters indicatory for the potential side effects of L-NAME and nor-NOHA treatment, including their effects on lipid peroxidation and hyperammonemia, and observed no significant changes.

Polyamines are vital for cell proliferation (Park and Igarashi, 2013), and their abundance allows products of oncogenes to overcome the immune response (Soda, 2011). Over the past decades, NO has emerged as a molecule of interest in carcinogenesis and tumor growth progression (Bonavida, 2015, Choudhari et al., 2013, Vahora et al., 2016). NO has been suggested to modulate different cancer-related events, including angiogenesis, apoptosis, cell cycle, invasion, and metastasis. On the other hand, it is also emerging as a potential anti-oncogenic agent. Strategies for manipulating its production in vivo and for evaluating its therapeutic value are being investigated (Choudhari et al., 2013).

Although nor-NOHA and L-NAME led to a delay in cancer progression, this observation has not been convincing enough for the development of a treatment scheme. Nonetheless, considering the current perspective on developing combined treatment approaches, including phytochemicals to successfully fight different cancers, we aimed to explore a combination of nor-NOHA or L-NAME with multicomponent herb extracts that display promising anticancer or modulating properties. The regulation of the L-arginine metabolic pathway by nor-NOHA and L-NAME and the concomitant targeting of other cellular cancer features with plant extract compounds may be novel and effective anticancer therapies.

Plants within the genus Rumex have a long tradition in folk medicine for treating diabetes, various infectious diseases, diarrhea, jaundice, different inflammatory diseases, etc.(Vasas et al., 2015). Rumex spp. is well-known for its pharmacological effects, including homeostatic, anti-inflammatory, anti-allergic, antimicrobial, antioxidant, neuroprotective, and also anticancer actions (Berillo et al., 2022, Stompor–gorący, 2021, Sun et al., 2020). For instance, the promising antitumor activity of extracts from R. vesicarius aerial parts in rat mammary cancer models has been previously reported (Shahat et al., 2015). Additionally, in a past study, we explored the biological activities of one representative of this genus: R. obtusifolius L. The wild plant is commonly called broad-leaved dock and it is widely distributed worldwide. It belongs to the Polygonaceae family. RO seed alcoholic extracts exhibit particularly high antioxidant activity in chemical tests and have a high total phenolic content (Ginovyan et al., 2020, Ginovyan et al., 2021). Moreover, we found that this herb extract has high antimicrobial and antibiotic modulatory properties as well (Ginovyan et al., 2017, Ginovyan and Trchounian, 2019). Based on these data, it was hypothesized that the RO seed extract may also modulate the anticancer effect of chemotherapeutic agents when used in combination.

Thus, this study aimed to explore the antitumor potential of the R. obtusifolius seed ethanol extract in vitro and in vivo, either individually or in combination with L-arginine/polyamine/nitric oxide pathway inhibitors. The goal was also to elucidate the characteristics of the extract that may contribute to its anticancer activity, explore possible side effects and mechanisms of action, etc. These analyses included elucidating its antioxidant/prooxidant properties by both chemical tests and cellular models, characterizing its phytochemical composition using advanced chromatographic techniques, and assessing its genotoxic and DNA protective properties in cellular models. Moreover, using the 7,12-dimethylbenz[a]anthracene (DMBA) induced breast cancer rat model, we aimed to elucidate the impact of the RO seed extract and chemotherapeutic agent (nor-NOHA and/or L-NAME) combinations on various biological markers, including the biosynthesis of polyamines and nitric oxide, the inhibition of lipid peroxidation, the activity of arginase, the regulation of interleukin-2 (IL-2) levels in the blood and tumor, histopathological and vasculature changes in the tumors, rat mortality rate, the size and number of tumors, etc The cancer preventive properties of RO extract could be of great interest; however, we did not refer to it in the current manuscript because we started treatment after the development of tumors.