CRC has become one of the most common (third) types of cancer and remains one of the leading causes of cancer-related mortality (second) [1]. The development of CRC influenced by non-modifiable risk factors, including age, gender, ethnicity, a personal history of adenomatous polyps, inflammatory bowel disease (IBD), and genetic predisposition, all of which are beyond individual control [2]. Conversely, modifiable factors such as lifestyle choices and personal habits—such as smoking, poor nutrition, excessive alcohol intake, lack of physical activity, and obesity—can be altered to lower the likelihood of developing the disease [3]. Most CRC cases are adenocarcinomas, a type of tumor that originates in the epithelial tissue of the colon or rectum [4]. In essence, epithelial cells undergo a series of genetic or epigenetic alterations that enable high proliferation rates [5]. Various genes involved in key signaling pathways are frequently dysregulated in CRC due to mutations or altered activity [6]. Given their distinct roles in CRC pathogenesis, ING4 and APC were selected as representative tumor suppressors for their roles in regulating the Wnt/β-catenin pathway and their common loss-of-function mutations in CRC [7]. DHX32, an angiogenesis-related gene that promotes vascular endothelial growth factor (VEGF) expression, was included for its association with tumor progression and prognosis [8]. Although P53 is traditionally a tumor suppressor, its gain-of-function mutations in CRC contribute to oncogenic traits such as apoptosis resistance and increased tumorigenicity [9,10]. These genes collectively represent key molecular pathways implicated in CRC development.

The DHX32 gene is associated with angiogenesis and belongs to the group of RNA helicases that promote vascularization. By upregulating and activating VEGF in tumor cells, it facilitates tumor growth and colorectal cancer progression. The DHX32 protein consists of 743 amino acids with a molecular weight of 84 kDa and is widely distributed across human tissues, including bone marrow, thymus, spleen, rectum, and breast. In summary, DHX32 expression is upregulated in colorectal cancer and is significantly linked to its occurrence, progression, and prognosis, suggesting that DHX32 may serve as a novel biomarker for colorectal cancer [11]. ING4 and APC are considered tumor suppressor genes in CRC, with mutations in these genes playing a crucial role in cancer initiation and progression. Their primary function is mainly associated with the wingless-type (Wnt/β-catenin) signaling pathway. Mutated forms of ING4 and APC exhibit reduced expression and regulatory disruption, leading to anti-apoptotic activity through a transcription-independent mechanism. These alterations are also linked to metastasis and cellular invasion [12,13]. Reduced expression of ING4 has been observed across various cancer types, including head and neck squamous cell carcinoma, hepatocellular carcinoma, gastric adenocarcinoma, lung, breast, and colorectal cancers, and is frequently associated with poor clinical outcomes [7]. According to Chen et al., ING4 plays a critical role in suppressing angiogenesis in colorectal cancer [14]. The APC gene produces a protein that regulates β-catenin levels, controlling cell growth, differentiation, and migration through the Wnt/β-catenin signaling pathway. It forms a degradation complex with Axin to ensure β-catenin remains at proper cellular concentrations [7]. The P53 gene, a critical tumor suppressor, has been shown—according to a study by Effati et al.—to induce apoptosis in SW480, HT-29, and Caco-2 CRC cell lines when its P53 expression is elevated [15]. Under normal conditions, P53 plays a pivotal role in preserving cellular integrity. However, when mutated, it not only loses its tumor-suppressive function but also acquires oncogenic properties [9]. These mutations are often triggered by cellular stressors such as DNA damage, oxidative stress, and osmotic shock. Mutant P53 not only loses its tumor-suppressing ability but also gains oncogenic traits, including uncontrolled proliferation, resistance to cell death, invasion, metastasis, and increased tumor-associated inflammation. In colorectal cancer, P53 mutations enhance tumorigenicity, promote drug resistance, and protect cancer cells from chemotherapy-induced apoptosis [16].

To reduce the incidence and mortality of CRC, individuals at moderate risk are recommended to undergo screening via fecal occult blood (FOB) testing and endoscopy. [17] Treatment approaches include surgical intervention, chemotherapy, radiotherapy, and targeted therapies utilizing monoclonal antibodies. Despite these advancements, modern chemotherapy frequently results in drug resistance, reducing the efficacy of anticancer treatments [2,18].

Probiotics are living microorganisms recognized for their potential anti-cancer properties, as they may help inhibit or prevent the growth of cancer cells [19]. While research has provided valuable insights into the role of probiotic bacteria in carcinogenesis, their use in cancer patients raises concerns regarding possible risks, including infections and immune suppression [20]. Recently, postbiotics—a novel category of microbial-based products—have gained considerable attention. Postbiotics offer several advantages over probiotics, as they can exert biological effects across various body surfaces, including the oral cavity, gut, skin, urogenital tract, and nasopharynx [21]. Moreover, their molecular mechanisms and impact on disease outcomes are easier to evaluate [22,23]. Unlike probiotics, postbiotics are bioactive compounds produced during the lifecycle or following the death of probiotic bacteria. They contribute to gut health and immune function by enhancing mucins, antimicrobial proteins, cytokines, and neurotransmitters [24]. While postbiotics hold promise for disease prevention and therapeutic applications, their exact mechanisms remain under investigation. They encompass a diverse range of components, including metabolites, short-chain fatty acids (SCFAs), microbial cell fractions, functional proteins, EPS, cell lysates, teichoic acid, peptidoglycan-derived muropeptides, and pili-type structures [25]. EPS, derived from probiotics, is a crucial functional component of the metabolic products of lactic acid bacteria (LAB). It plays a vital role in immune regulation, inflammation suppression, tumor cell inhibition, cancer prevention and treatment, and antioxidant activity [26].

The progression of cancer, including CRC, is a complex, multi-step process driven by sequential genetic mutations and key signaling pathways such as Nuclear Factor Kappa-light-chain-enhancer of Activated B Cells (NF-κB), Mitogen-Activated Protein Kinase (MAPK), and Wnt/β-catenin, which regulate essential biological functions like cell proliferation, differentiation, angiogenesis, apoptosis, and survival [27]. Mutations affecting the Wnt/β-catenin signaling pathway play a crucial role in CRC progression. Disruptions in Wnt signaling contribute to tumor development by enabling β-catenin accumulation and promoting cancer-related gene expression [28]. EPS has been shown to inhibit β-catenin production, potentially suppressing cancer growth [29].

Nanotechnology has emerged as a promising approach for the diagnosis and treatment of various diseases, particularly cancer [30]. Among its applications, ZnONPs have gained significant attention in anticancer research due to their simple, safe, and cost-effective structure, as well as their selective cytotoxicity against cancer cells. Despite these advantages—and although many studies have investigated the anticancer effects of ZnONPs synthesized via plant-based methods [15,[31], [32], [33], [34]]—the biological synthesis of metal NPs continues to face challenges, including prolonged reduction times and complex downstream processing [35].

To overcome these issues, researchers have focused on using probiotic exopolysaccharides as safe reducing agents [36]. In line with this approach, the present study introduces a novel biosynthetic method for producing ZnONPs using exopolysaccharides extracted from the probiotic bacterium L. plantarum. This microbial synthesis pathway not only ensures high stability and biocompatibility but also harnesses the unique biochemical properties of probiotic compounds to enhance the structural integrity and therapeutic efficacy of the nanoparticles. Consequently, this strategy opens new avenues for the development of probiotic-based nanomedicines in cancer therapy.

ZnONPs have garnered widespread interest due to their selective toxicity against cancer cells, along with their safety, affordability, and ease of synthesis [37]. Beyond their anticancer properties, zinc also plays a vital role in cellular signaling as a neurotransmitter and secondary messenger, contributing to processes such as differentiation, cell cycle regulation, and gene expression [38]. Moreover, zinc ions have been shown to suppress tumor growth by inhibiting angiogenesis [39]. Recent studies further demonstrate that ZnONPs synthesized via microbial routes possess notable antioxidant activity, inducing oxidative stress, promoting cell cycle progression, and facilitating DNA replication and repair—ultimately triggering apoptosis in various cancer cell lines [40,41].

This research investigates the extraction and purification of EPS from L. plantarum for ZnONP biosynthesis. The NPs were characterized and tested for their cytotoxic properties using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Additionally, quantitative polymerase chain reaction (qPCR) analysis assessed the expression levels of tumor suppressors genes ING4 and APC, the angiogenesis-associated DHX32, and the oncogenic mutant P53, providing insights into their biomedical potential.