Multiple mechanisms contribute to cancer drug resistance, including increased expression of adenosine triphosphate (ATP)-binding cassette (ABC) transporter-mediated drug extrusion [e.g., P-glycoprotein (P-gp/ABCB1)], breast cancer resistance protein (BCRP/ABCG2), multidrug resistance protein 1 (MRP1/ABCC1), impaired DNA repair, growth factor receptor (GFR) dysregulation, inhibition of downstream cell death pathways, activation of survival signaling pathways, altered drug metabolism, gene amplification, transfer and expression of miRNAs, pre-existing or acquired tumor cell heterogeneity—especially cancer stem-like cells (CSCs)—fibrotic responses, hypoxia, and immune activation, as well as dysregulation of interconnected inflammation, apoptosis, and oxidative stress pathways (Fakhri et al., 2023). MBZ utilizes several mechanisms of action to counteract cancer initiation, progression, metastasis, and resistance. These include microtubule disruption, induction of apoptosis, inhibition of angiogenesis, induction of autophagy, stimulation of antitumor immune responses, and regulation of multiple signaling pathways.

Mitogen-activated protein kinases (MAPKs) could be classified into three prominent protein families, including the p38 proteins, ERK, and stress-activated protein kinases/c-Jun N-terminal kinase (SAPK/JNK). Furthermore, it should be noted that Ras, Raf, and MAP–ERK kinase (MEK) are proteins with proximal positioning in the ERK pathway (Fan and Chambers, 2001; Kim and Choi, 2010). Studies have demonstrated that ERK plays a crucial role in cell survival, activation, and differentiation. Andersson et al. have shown that MBZ passed immune-modulating activity through the ERK signaling pathway (Andersson et al., 2020). From the mechanistic insight, MBZ makes a specific interaction with the tubulin structure. It also generally binds to the colchicine-binding domain of tubulin; however, such an effect has also been reported with other benzimidazoles. Furthermore, the inhibition of the dual specificity tyrosine-phosphorylation-regulated kinase 1B (DYRK1b) enzyme by marginal concentrations of MBZ may trigger the activation of ERK. Consistently, MBZ strongly inhibits BRAF8 in wild-type RAF cells, leading to inconsistent ERK activation. Additionally, ataxia telangiectasia-mutated (ATM) kinase inhibition effectively decreases the apoptosis induced by MBZ in the zebrafish retina (Gao et al., 2013; Nygren et al., 2013).

The involvement of Hedgehog (Hh) signaling pathways in several types of cancer renders them attractive targets for novel anticancer agents. The Hh signaling pathway initiates through the interaction between Hh ligands and the patched 1 (PTCH1) receptor. In the absence of a bound ligand, PTCH1 impedes the initiation of Smoothened (SMO), a frizzled class receptor within the primary cilium, which plays a crucial role in the transmission of diverse chemical and mechanical signals. In the presence of a ligand, PTCH1 undergoes dissociation from the cilium. It instigates the activation of downstream effectors, including the transcription factor glioma-associated oncogene (GLI), in a process orchestrated by the activation of SMO. Genetic mutations in the gene encoding PTCH1 or aberrations in the Hh signaling pathways are frequently observed in numerous types of cancer (Yauch et al., 2008; Guerini et al., 2019).

It has been demonstrated through empirical observation that the activation of the Hh signaling pathway is evident in many gliomas, melanomas, lung cancers, ovarian cancers, adrenal cancers, and colon cancers, all of which respond to MBZ. Inhibition of primary cilia formation, attenuation of downstream Hedgehog pathway effectors’ expression, as well as reduction in the proliferation and survival of human medulloblastoma cells, were accomplished through the administration of MBZ. In addition, MBZ inhibited the activation of SMO mutant proteins (Larsen et al., 2015; Jain et al., 2020; Staedtke et al., 2020).

By regulating effective signaling pathways in cancer (e.g., ERK and Hh), MBZ could potentially exhibit anticancer effects.

Rapid innate immune responses to infection depend on pattern recognition receptors produced by cells of the innate immune system. These receptors identify molecules associated with pathogenic agents. Because they are located in repetitive patterns of pattern recognition receptors on the surface of professional phagocytes such as dendritic cells, macrophages, and neutrophils, they receive pathogens and deliver them to lysosomes for destruction (Murray, 2017). Macrophages are divided into two subtypes, M1 and M2. M1 macrophages exhibit anti-tumor effects due to their phagocytic and antigen-presenting activity and the production of Th-1 activating cytokines. On the other hand, M2 macrophages promote cancer cell proliferation by stimulating matrix remodeling, angiogenesis, and immune tolerance (Aras and Zaidi, 2017). The regulatory function of MBZ has been observed to regulate the transcription of specific cytokine genes - notably tumor necrosis factor (TNF), interleukin 6 (IL-6), and 8 (IL-8) - that are associated with the M1 phenotype, together with the expression of surface markers CD80 and CD86, and T cell chemokines. In contrast, no alterations in the expression of M2 markers were observed. Additionally, exposure to MBZ resulted in the production of interleukin-1 (IL-1), while the administration of other benzimidazoles had no discernible impact on the release of IL-1. In line with this, it can be inferred that MBZ can intensify the immune-stimulatory and anticancer properties of anti-CD3/IL2-activated peripheral blood mononuclear cells (PBMCs), thereby suggesting its potential contributions to the anticancer mechanisms (Rubin et al., 2018).

Additionally, another effect of MBZ in inducing the antitumor response is through the inhibition of DYRK1B, a mediator of immune-modulating activity. Accordingly, DYRK1B inhibition could provide a limited role in the development of MBZ-induced immune reactions. Therefore, the inhibitory effects of MBZ on DYRK1B are implicated as a contributing aspect of the mechanism of action that prompts the polarization of M2 towards M1 macrophages, ultimately eliciting anticancer responses (Blom et al., 2019).

MMPs are the most critical gelatinases and are produced and secreted by several cell lines, including fibroblasts, leukocytes, and tumor cells. The expression of metalloproteinases is influenced by a multitude of factors, including cytokines, growth factors, oncogenes, intercellular interactions, and extracellular matrix components (Mondal et al., 2020). MBZ significantly reduced the activity of MMP-2 in different concentrations, while it was ineffective on the activity of MMP-9 (Pinto et al., 2015).

Several studies have provided evidence of the combined application of MBZ and radiation in reducing cell growth and angiogenesis, while increasing apoptosis in cancer cells (Guerini et al., 2019). A recent investigation has revealed that MBZ has the potential to modify DNA damage response proteins, thereby increasing the radiation sensitivity of cancer cells. In their study, co-treatment with MBZ and radiation increased γH2AXl as a marker of DNA damage in cancer cells. Overall, there is suggestive evidence to indicate that the simultaneous administration of MBZ and radiation induces a synergistic antagonism of the DNA damage response. Further investigations are warranted to substantiate the assertion that MBZ and radiation impede the development of microtubules, ultimately resulting in DNA impairment within neoplastic cells (Skibinski et al., 2018). Figure 1 presents the anticancer potential of MBZ.

The management of triple-negative breast cancer (TNBC) poses a challenge due to the lack of effective drugs. Accordingly, in an in vitro study, Zhang et al. showed that 0.7 µM of MBZ effectively increased the sensitivity of TNBC cells to radiation therapy. For this purpose, cells of TNBC were administered a singular dose of MBZ for 24 h before exposure to escalated levels of ionizing radiation. The results showed that MBZ at all tested radiation doses on MDA-MB-231 and SUM159PT cells leads to a significant increase in radiation sensitivity (Zhang et al., 2019).

In 2013, Coyne and colleagues evaluated the in vitro effectiveness of MBZ on the cell line resistant to breast cancer (SKBr-3). Based on the results, MBZ at a concentration of 0.5 μM decreased the survival of cancer cells by 63.1% (Coyne et al., 2013). Additionally, the combination of MBZ and methotrexate was evaluated to reduce the survival of breast cancer cells. Their results showed that the combination therapy of MBZ at doses of 1.5–100 µM and methotrexate (0.5–100 µM) exhibited synergistic effects against breast cancer cell lines (MDA-MB-231 and MCF-7) in an in vitro study. Upon administering MBZ, the mean reduction in cell viability for MCF-7 and MDA-MB-231 cells was found to be 81.3% and 66.8%, respectively (Alam et al., 2018).

Chronic inflammation of the intestinal epithelium is a trigger for colorectal cancer (CRC). Typically, the dysregulation of prostaglandin-endoperoxide synthase 2 (PTGS2 or COX2) and the increased concentrations of prostaglandin E2 (PGE2) represent pathophysiological agents that facilitate the processes of inflammation and tumorigenesis (Guerini et al., 2019). In addition, VEGF/VEGFR2 pathways are activated during colon tumor progression. Based on previous reports, an in vitro study on platelets from people with colon cancer has shown that MBZ exhibits anti-CRC clinical activity through tubulin disruption and VEGFR2-mediated anti-angiogenic potential (Holler et al., 2012).

Williamson et al. confirmed that MBZ slowed down the growth of colon cancer xenografts and might be used as a way to prevent cancer in people who have a high chance of getting it. In a study conducted in vivo, it was observed that administering 35 mg/kg MBZ in combination with sulindac to ApcMin/+ mice resulted in a significant reduction in the occurrence of polyps by 90% compared to the group that received no treatment. Conversely, the use of sulindac alone did not prove to be an efficacious measure in preventing colon adenoma formation in ApcMin/+ mice. It may even lead to tumor formation in the colon (Williamson et al., 2016). In mice bearing colon cancer, MBZ significantly reduced the tumor volume (1,177 ± 1,109 mm³; P ≤ 0.001) and tumor weight (2.30 ± 1.97 g; P ≤ 0.0001) compared to the negative control group (weight 12.45 ± 2.0 g; volume 7,346 ± 1077 mm³). Additionally, MBZ increases mean survival time (MST) and the percentage of life span (ILS) in the animal study (51.2% ± 37% vs. 93%, respectively). This study suggests that MBZ strongly and selectively inhibits proliferation and induces apoptosis in colon cancer cells.

Gastric cancer ranks as the fourth most prevalent form of cancer and the second principal cause of cancer-related mortality worldwide (Jemal et al., 2011). Because gastric cancer is somewhat resistant to current clinical treatments, there is still no proven evidence-based treatment for such malignancy. The therapeutic approach for individuals diagnosed with gastric cancer involves the utilization of a treatment regimen consisting of 5-fluorouracil (5-FU)/cisplatin.; however, the toxicity of 5-FU, which leads to myelosuppression and gastrointestinal toxicity, may be a genuine and common issue for numerous cancer patients (Al-Batran et al., 2004).

Accordingly, in an in vitro study, Pinto et al. detailed the impacts of 5-FU combined with 0.15–20 μM of MBZ on a human ascites cell line derived from a gastric cancer tumor. MBZ is orally available and warrants further consideration as an anticancer agent due to its favorable pharmacokinetics, potential to enhance efficacy, and the significant safety profile of other drugs. Their finding demonstrated that MBZ/5-FU inhibited the proliferation of the malignant ascites cell line (i.e., AGP-01) derived from a primary intestinal-type gastric cancer. Additionally, their study showed the disruption of microtubule formation in AGP-01 cells treated with MBZ (Pinto et al., 2015).

Non-small cell lung cancer (NSCLC) is the most frequent type of lung cancer that, despite early diagnosis, has a poor chance of treatment. Due to systemic metastases, chemotherapy is prescribed in more than 75% of patients with NSCLC. However, chemotherapy for such patients does not lead to improvement, even with high-rate treatment (Sasaki et al., 2002). Therefore, targeting molecular mediators sensitive to chemotherapy could be a promising strategy for finding a cure for certain types of cancer. Accordingly, tubulin, the major protein component of microtubules, is of great importance.

Sasaki et al. showed that MBZ exhibits a moderate level of spindle inhibition activity, yet it showcases a robust ability to combat tumors effectively. According to the results of their study, 0.1 µM of MBZ prevented the growth of human NSCLC in vitro. In this way, treatment of A549 and H460 human NSCLC cell lines with MBZ significantly suppressed cell proliferation. Moreover, MBZ induced depolymerization of tubulin and inhibited the formation of the normal spindle in NSCLC cells, resulting in mitotic arrest and subsequently apoptosis (Sasaki et al., 2002). Overall, another in vitro study demonstrated that a 0.165 µM dose of MBZ inhibited lung cancer cell growth 5-fold compared to control groups (Mukhopadhyay et al., 2002).

In vivo studies showed that MBZ in doses of 25–50 mg/kg (oral) could suppress the Hh pathway in the DAOY human medulloblastoma cell line (Larsen et al., 2015). Additionally, Bai et al. demonstrated that MBZ could act as an angiogenesis inhibitor (50 mg/kg/day, administered orally in food) in the D425 human medulloblastoma cell line and murine parental or SMO-D477G mutated medulloblastoma (Bai et al., 2011). Another study showed that MBZ, as a single drug and even through synergistic effects with cisplatin, could result in the induction of apoptosis to increase CAL27 and inhibit SCC15 cells (IC₅₀ values of 1.28 and 2.64 µM, respectively) (Zhang et al., 2017). De Witt et al. examined the viability suppression of GL261 murine glioma cells exposed to MBZ. Based on the results of their in vitro study, MBZ led to the depolymerization of microtubules (132 nM) followed by the induction of cell division arrest (192 nM) (De Witt et al., 2017). Additionally, Markowitz et al. showed that radiosensitization with MBZ (EC₅₀ value of 35 nM) in murine GL261 glioma cells could lead to widespread anticancer effects in vitro (Markowitz et al., 2017).

XIAP is one of the most critical regulatory proteins for apoptosis, as it prevents cell death by inhibiting caspases 3, 7, and 9. Several investigations have demonstrated that enhanced expression of XIAP is associated with advanced disease progression stages in the context of melanoma (Daoud et al., 2022). Doudican et al. assessed the impact of MBZ on human melanoma cell lines, specifically M-14 and A-375. The research findings indicate that the utilization of MBZ resulted in a reduction of XIAP levels. This decrease in XIAP levels demonstrated a reciprocal relationship with the elevation of apoptotic markers, specifically PARP and caspase-9. They also established an M-14 xenograft model to evaluate the anticancer effect of 1 and 2 mg/kg doses of MBZ. MBZ inhibited tumor growth at the two doses tested without toxicity (83% and 77% inhibition, respectively) (Hiscutt et al., 2010; Doudican et al., 2013) (Table 1).