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THE USE OF PROTEOLYTIC ENZYMES IN INHIBITING CANCER AS ANTI-ANGIOGENESIS AGENTS: MECHANISMS OF ACTION
A.A. Ausaj, W.B. Abed, H.S. Al-Shmgani*
Department of Biology, College of Education for Pure Sciences/Ibn Al-Haitham, University of Baghdad, Baghdad, Iraq. * Corresponding author: [email protected]
Abstract. The potential role of proteolytic enzymes in impeding the growth of cancer by the anti-angiogenesis process has gained attention recently. This review explores proteolytic enzymes that have been studied for their capacity to obstruct the development of new blood vessels that are essential for the growth of tumors. These enzymes include matrix metalloproteinase and serine proteases. The mechanisms by which these enzymes inhibit angiogenesis including degradation of extracellular matrix proteins and inhibition of signaling pathways involved in blood vessel formation are discussed. Also, proteolytic enzymes' possible therapeutic applications as anti-angiogenesis drugs in the treatment of cancer are highlighted.
Keywords: proteolytic enzymes, anti-angiogenesis, tumor microenvironment, extracellular matrix, proteolytic therapeutic applications.
List of Abbreviations
MMPs - matrix metalloproteinase ECM - extracellular matrix TME - microenvironment IFN-y - interferon-gamma NK - natural killer
VEGF - vascular endothelial growth factor FGF - fibroblast growth factor PI3K - phosphoinositide-3-kinase MAPK - protein kinase
Introduction
Traditional medicine has long employed plants for their medicinal benefits, and numerous substances produced from plants have been shown to have anti-cancer activities (Jasim et al., 2023). These substances, referred to as phy-tochemicals have been demonstrated to stop tumors from spreading and to stop the formation of cancer cells. Consequently, there is a growing interest in investigating the possibility of using plant extracts as innovative anti-cancer agents.
The angiogenesis process is the formation of new blood vessels from old ones. There are various mechanisms of action for angiogenesis agents, one approach is the use of plant extracts (Al-Shmgani et al., 2017). Plants contain prote-olytic enzymes that can degrade proteins and promote the synthesis of new proteins. These plant-derived proteases have unique features
such as stability, substrate specificity, and a wide pH range for enzymatic activity, making them suitable for biomedical applications. They have been used in the treatment of pathological conditions, including antitumor, antibacterial, and antifungal activities, as well as wound healing and digestive disorders (Fields, 2019; Ali & Mutlak, 2024). Another approach is the use of proteolytic enzymes, such as matrix metallo-proteinases (MMPs), which directly regulate angiogenesis. Matrix metalloproteinase inhibitors have been developed to selectively inhibit MMP activity and target tumor angiogenesis. These inhibitors can bind to protease secondary binding sites and block the active site or by inhibition of proMMP. The research investigated the inhibition of these inhibitors showing promise in tumor angiogenesis treatment (Balakireva et al., 2019).
Uncontrolled cell growth and invasion are the characteristics of cancer cells. Surgery, chemotherapy and therapy are the main cancer therapies that have frequently negative side effects (Anand, 2022). As a result, there has been growing interest in investigating alternative therapeutic approaches, including the use of proteolytic enzymes. This review aims to provide an in-depth analysis of the mechanism of action of proteolytic enzymes in inhibiting cancer through their anti-angiogenic properties and elucidate their mechanisms of action. The ad-
ministration of proteolytic enzymes from either plant or animal sources is a type of alternative medicine that is primarily used to lessen the effects of cancer therapy.
1. Proteolytic enzymes and cancer
1.1. Definition and types
Proteolytic enzymes, also known as proteases or proteinases, are naturally occurring substances that play crucial roles in different physiological processes, including immune response modulation, inflammation regulation and tissue remodeling (Al-Ibadi 2019; Ashij et al., 2023). They are capable of breaking down proteins into smaller peptides or amino acids. They are classified into various types based on their mechanism of action, including serine proteases (e.g. trypsin), cysteine proteases (e.g. ca-thepsins) and metalloproteases (e.g. matrix metalloproteases) (Vizovisek, 2018). Also based on their origin can be divided into animal, plant and microorganisms' proteases (Razzaq et al., 2019). Another type of classification is based on the sequence of amino acids used to build proteolytic enzymes; they can be categorized using structure-based evolutionary relationships (De Lencastre, 2016). Additionally, one of the first uses of specialized proteins for medicinal purposes is to find out which plants are used to treat wounds, identify fungal, bacterial and viral illnesses and resolve integrative system issues (Rosenberg, 2014). It has been documented that certain plant protein enzymes like bromelain and papain can be used either as commercial form or as punishment for surgical wounds, or in animal experiments as wound healing agents (Hirche et al., 2017)
1.2. Proteolytic enzymes' role in cancer progression
Proteolytic enzymes play a critical role in cancer progression by facilitating tumor invasion, metastasis, and angiogenesis. They degrade the extracellular matrix (ECM) components surrounding tumors, enabling tumor cells to migrate and invade adjacent tissues (Weijers et al., 2013; Al-Badri et al., 2023).
By now, it was established that the neighboring microenvironments are a factor influencing
cancer cell development. The microenvironment (TME) contributes to the development, invasion or metastasis (Hanahan et al., 2012).
Extracellular matrix (ECM) can be recognized by cancer cells by their receptors and then modified them by degrading or depositing (Ka-ramanos, 2019). Apart from the adhesive function to hold and a barrier function used in partitioning tissues or organs, ECM acts as signal-emitting molecules, a reservoir of cytokines and growth factors plus also a working scaffold for migrating cells (Hussain et al., 2017). Metastasis is the main complication of malignant cancers, changes in epithelial cells transformed develop proliferation capacity, arrest adhesion in cells and basement membrane breakdown.
Invasion of cancer cells process disrupts normal structure of the tissue and destroys the ability to perform their functions. The significant mechanism used by cancer cells to disrupt microenvironment signaling is by proteolytic enzyme process, by which these enzymes destroy ECM in tissue (Piperigkou et al., 2021). Limited processing can produce bioactive fragments from ECM proteinases. It also breaks down soluble elements or metabolizes cancer cells' signaling processes. Cytokine, growth factor, TM receptors, as well as ECM bindings and adhesion are also shed by proteinases. Pro-teolytic processes are irreversible transformations and therefore efficient in regulating the activity of molecules (Ashij et al., 2021). It has long been regarded that one of the major pro-teinases to destroy EMC in invasion under this situation is MT1-MMP (Wolf et al., 2013; Itoh et al., 2025; Gifford & Itoh, 2019).
1.3. Direct effects of proteolytic enzymes on tumor cells
Proteolytic enzymes can directly affect tumor cells by inducing apoptosis (programmed cell death) or inhibiting their proliferation. For instance, bromelain, derived from pineapple stems, has been shown to induce apoptosis in various cancer cell lines by activating caspases and disrupting mitochondrial function additionally to revealing inhibitors activity for NF-kB by arresting G2/M point in cell cycle in different cancer cell lines (Dhandayuthapani et al.,
2012). Similarly, papain from papaya latex has demonstrated anti-proliferative effects on breast cancer cells through cell cycle arrest and inhibition of key signaling pathways (Mahrous et al., 2023). Also, bromelain shows apoptotic activity in tumor cells through increased p53 expression and upregulation of Bax expression in mitochondria along with increased cyto-chrome c released (Tysnes et al., 2001).
Anti-angiogenic effects of bromelain have been demonstrated in cancer cell lines and animal models, where it showed the ability to suppress the response to FGF2 stimulation and down regulated MMP-9 expression (Wallace, 2002; Juhasz et al., 2008; Karlsenet et al., 2011). Moreover, when hepatocellular carcinoma cells were treated with bromelain, their levels of COX-2 and VEGF angiogenic bi-omarkers dropped (Manosroi et al., 2012).
Papain another proteolytic enzyme had a potent anti-angiogenic action that was similarly observed with bromelain and ficin, interference with important signaling pathways such as AKT, MEK, ERK1/2 and p38-MAPK signaling is most likely the cause of this impact (Mohr & Desser, 2013).
1.4. Modulation of tumor microenvironment
Proteolytic enzymes can also modulate the
tumor microenvironment by influencing angio-genesis (formation of new blood vessels) and immune response regulation. Matrix metallo-proteinases, a family of proteolytic enzymes, are involved in extracellular matrix degradation during tumor invasion and metastasis. Targeting MMPs using inhibitors or natural proteo-lytic enzyme extracts has shown potential for inhibiting tumor growth and metastasis (Egeblad & Werb, 2002). Since proteolytic events are irreversible, they are useful for modifying how molecules operate (Itoh, 2023).
1.5. Immune system activation
Proteolytic enzymes can enhance immune
system activation, leading to improved anti-tumor responses and stimulating some cytokines production, that play crucial roles in immune cell activation and tumor cell recognition such as interferon-gamma (IFN-y) and IL-2. Addi-
tionally, proteolytic enzymes can increase natural killer (NK) cell activity and promote the maturation of dendritic cells, thereby enhancing antigen presentation and adaptive immune responses (Farhadi et al., 2018). By promoting the expression of proteolytic enzymes, cyto-kines improve the invasion of cancer cells and the angiogenesis of tumors (Kenig et al., 2010; Sevenich et al., 2014).
Proteases influence the production of cyto-kines through several signaling pathways and modify their activity through proteolytic processing after translation (Savickas et al., 2020). Proteases preferentially cleave cytokines which can change the specificity of the receptor or activate or inactivate cytokine activity. Therefore, proteolytic activation is necessary for some cy-tokines to operate (Breznik et al., 2018). Also, proteases have the ability to deactivate cyto-kines and change how they bind to receptors, which can hinder the progression of cancer (Sevenich et al., 2014).
2. Anti-angiogenic properties of proteolytic enzymes
2.1. Inhibition of endothelial cell proliferation
It has been demonstrated that proteolytic enzymes prevent endothelial cell growth, a key process involved in angiogenesis. Targeting particular signaling pathways that promote the development of endothelial cells, these enzymes can suppress the growth of blood vessels within tumors. Phosphorylation levels of Akt, MEK1/2 and SAPK/JNK indicated that endothelial cells were less susceptible to VEGF activation, migration and development of tubes in cells when treated with papain a proteolytic enzyme (Mohr & Desser, 2013).
2.2. Disruption of extracellular matrix remodeling
Proteolytic enzymes have the ability to alter the ECM's remodeling, that is necessary for an-giogenesis. They cause the vascular network to become unstable, which suppress the development of new blood vessels by degrading ECM elements including collagen and fibronectin (Mongiat et al., 2016). ECM contains a vast collection of biochemical and structural compo-
nents which are categorized into proteoglycans, proteins and glycoproteins, each of these contains a variety of component subclasses as well as distinct biochemical and physical characteristics like laminin, fibronectin and nidogen also contribute to the formation of the matrix network (Vakonakis & Campbell, 2007).
2.3. Inhibition of angiogenic factors
Proteolytic enzymes have the ability to block the effect of angiogenic agents, such as fibro-blast growth factor (FGF) and vascular endothelial growth factor (VEGF) (Qaddoori & Al-Shmgani, 2023). These enzymes can directly degrade these factors or indirectly regulate their expression, thereby reducing their pro-angio-genic effects. Proteolytic activation also targets angiogenesis-inducing growth factors indirectly. Plasminogen activators, for instance, control bone angiogenesis by stimulating transforming growth factor (Vazquez et al., 1999).
3. Mechanisms of action
3.1. Regulation of cell signaling pathways
Proteolytic enzymes can modulate various
cell signaling pathways involved in angiogene-sis, phosphoinositide-3-kinase (PI3K) / Akt pathway and mitogen activated protein kinase (MAPK) pathway are among them. By interfering with these pathways, proteolytic enzymes can suppress endothelial proliferation and migration. The antiapoptotic impact of VEGF on ECs was eliminated by transfection with negative Akt mutant or pharmacological suppression of PI3K (Chin et al., 1999).
3.2. Modulation of angiogenic factor activity
Proteolytic enzymes can directly cleave and inactivate angiogenic factors like VEGF and bFGF. Additionally, and by cleaving their receptors, they can control how these factors are expressed or binding proteins, thereby reducing their availability for promoting angiogenesis, moreover, proteases alter the characteristics and availability of growth factors (Boeringer et al., 2019).
4. Clinical applications and challenges
4.1. Potential therapeutic applications
The anti-angiogenic properties of proteolytic enzymes have shown promise in preclinical
studies as possible medicinal ingredients for the treatment of cancer. These enzymes could be used alone or in combination with other anticancer therapies to inhibit tumor growth and metastasis (Raju & Kei, 2022). Given the established correlation between cancer and the inflammatory cascade, studies investigated the impact of proteolytic enzymes on key molecules and events in this process. For instance, when immune cells were stimulated in an overproduction state by proinflammatory cytokines, bromelain reduced IL-ip, IL-6 and TNF- a secretion, this resulted in a modulation of the inflammatory response through prostaglandin reduction and activation of a cascade reaction that activates neutrophils and macrophages as well as an acceleration of the healing process (Alves Nobre et al., 2024). Proteases from outside the tumor cell, which may be attached to the cell surface or produced from the tumor cell itself, aid in local proteolysis. Researches indicate that by breaking down extracellular matrix, proteases of tumor cells may also contribute to local proteolysis. Several histological analyses of tumor tissue reveal the role of stromal cells in the development of malignancy, proteases show promise as diagnostic markers for oral squamous cell carcinoma. The complex pathophysiology of oral carcinoma includes dysregulation of proteolytic systems including various matrix metalloproteases (Sarode et al., 2024). A researcher by Lu and his co-workers suggested that gastric cancer tissues have decreased Cys-tatin SA protein level and selectively inhibit the development of gastric cell lines and increases sensitivity to oxaliplatin via PI3K/AKT signaling pathway so that CST2 functions as a tumor suppressor gene that increases susceptibility to oxaliplatin (Lu et al., 2024).
Researchers have shown that applying some proteolytic plant enzymes such as bromelain and papain topically to the skin can hasten the healing of wounds and promote the creation of new tissue (Ajlia et al., 2010; Rosenberg et al., 2014). pineapple extract has been proven to contain saponin and bromelain. Both human epidermoid carcinoma-A431 and melanoma-A375 cell lines showed a decrease in proliferation when bromelain was added in different
concentrations, and its ability for anchorage-independent growth was inhibited. Furthermore, inhibition of AKt-regulated nuclear factor-kap-paB activation by suppression of inhibitory-KappaBa phosphorylation and concurrent decrease in cyclooxygenase-2 (Bhui et al., 2012). Our previous studies demonstrated that brome-lain congregated with gold nanoparticles showed an interesting protective effect on different organ tissues in mice (Abeed et al., 2023; Abeed & Al-Shmgani, 2024). Furthermore, numerous products derived from antibody cleavage by proteases are offered as kits from commercial companies. In some circumstances, it might be better to generate the Fab fragments in sufficient numbers and high quality using recombinant expression in cell lines. For instance, due to the increased quality of the protein sample, it was discovered that the Fab fragments generated by recombinant expression were more suitable for the crystallization investigations (Zhao et al., 2009). Proteases are vast in diversity and specificity and are valuable tools for creating medicines agents, treating burns and wounds, broad spectrum antibiotics are used with subtilisin or clostridial collagenase (Chiplonkar et al. , 1985). Furthermore, alkaline proteases with fibrinolytic activity have been used as thrombolytic agents, and preparation with high electrolytic activity from B. subtilis 316M was immobilized on a bandage for the therapeutic application in the treatment of burns and purulent wounds, furanches and deep abscess (Kim et al., 2013). A higher metabolism of amino acids in the tumor microenvironment, which is necessary for cancer to develop, multiply, and endure, makes several enzymes that target these compounds appealing therapeutic options. To address the elevated ar-ginine and tryptophan presence in the tumor microenvironment, respectively, PEGylated argi-nine deaminase has been licensed while
PEGylated kynureninase has been studied; thus, PEGylated kynureninase inhibits tumor growth by breaks down the l-tryptophan which is non-toxic and easily to removed (Triplett et al., 2018). Proteases that are appealing have been proposed as treatments against CoV-2 in an in silico analysis. The research investigated the ability of fungal proteases bound to various SARS-CoV-2 proteins. According to the hypothesis, these proteases bind to inactivate virions making them appealing candidates for further research into COVID-19 treatment (Christopher et al., 2021).
4.2. Challenges and limitations
Despite their potential benefits, several challenges need to be addressed before proteolytic enzyme-based therapies can be widely implemented in clinical settings. These include issues related to enzyme stability, delivery methods, dosage optimization, and potential off-target effects, patient immune system reaction against the enzyme (De la Fuente, 2021).
Conclusion
Proteolytic enzymes offer a promising avenue for cancer treatment because of the way they directly affect tumor cells, alteration of the tumor's surrounding environment, and immune system activation. Proteolytic enzymes have emerged as promising anti-angiogenic agents in inhibiting cancer progression. While more investigation is required to completely comprehend their modes of action and maximize their therapeutic potential, existing studies provide valuable insights into their efficacy. Incorporating proteolytic enzyme-based therapies into conventional cancer treatments may lead to improved outcomes with reduced side effects.
Conflicts of interest: the authors declare no conflicts of interest.
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