Repurposing anti-parasite benzimidazole drugs as selective anti-cancer chemotherapeutics

Cancer chemotherapy is generally associated with many severe adverse effects. Many cancer studies are currently focused on repurposing conventional non-toxic anti-parasite drugs for cancer treatment. Since cancer cells and parasites have many features in common, some anti-parasite drugs such as benzimidazoles have been recently found to possess the anti-cancer activity. Benzimidazoles act against cancer by inhibiting tubulin polymerization, inducing cancer cell apoptosis, arresting cell cycle and over-generating reactive oxygen specimen. In this review, we summarize the anticancer features of these drugs in recent investigations, lead to reconsideration of benzimidazoles as a family of anti-cancer chemotherapeutics with non-toxicity or low toxicity to the normal cells and tissues. We particularly highlight the recent progresses using nanoformulations for enhanced cancer therapy and provide our prospects in the future research.


Introduction
Conventional chemotherapeutics are normally non-selective. These therapeutic agents not only kill cancer cells but also damage healthy tissues and cells, causing adverse effects [1] and influencing patients' quality of life [2]. For this reason, there is a great motivation to repurpose conventional anti-pathogen drugs with a long-term safety history for cancer chemotherapy. The benzimidazole family is one of such safe drugs.
Benzimidazoles are heterocyclic organic compounds, consisting of imidazole and benzene (as illustrated in Figure 1) with a high anti-helminth efficacy and a low level of toxicity to healthy cells [3,4]. Apart from the high anti-helminth activity, benzimidazoles have been also found to possess anti-fungal [4], anti-bacterial [5], anti-viral [6] and anti-inflammatory activity [7]. Due to extensive similarities between parasites and cancer cells [8], benzimidazoles have also shown some anti-cancer activity, as confirmed in many studies [9]. Moreover, benzimidazole drugs are reported to enhance the efficacy in combination with other treatments such as conventional chemotherapy [10] and radiation therapy [11]. Interestingly, some benzimidazole drugs induce apoptosis of drug-resistant cancer cell lines [12] [13]. More importantly, benzimidazole drugs kill cancer cells in a selective way [14], i.e. via targeting tubulin polymerization dominantly in rapidly dividing cells such as parasites and cancer cells [15], leaving healthy cells being not much affected.

Figure 1.
Chemical structure of benzimidazole drugs (albendazole and mebendazole) as heterocyclic organic compounds, consisting of imidazole and benzene, as well as other functional groups [126].
Although benzimidazole drugs have shown promise for cancer therapy, their low water solubility presents a significant challenge for their clinical application and therapeutic outcomes [16]. To overcome this limitation, nanoformulating benzimidazole drugs has emerged as a viable approach to increase their solubility and improve their efficacy in cancer therapy.
In this review, we aim to summarize the anti-parasite and anticancer features of benzimidazole drugs and their mechanisms of action. We further highlight the superior potentials of benzimidazoles such as safety and effectiveness on resistant cancer cells along with the limitation to their clinical applications, and then present the recent progresses using their nanoformulations for enhanced cancer therapy in combination with other drugs or treatment modes.

Common features of cancer cells and parasites suit actions of benzimidazole drugs
Parasites and cancer cells have many common features, as summarized in Table 2. In general, they are both resistant to apoptosis and capable of unlimited proliferation in human and livestock [37]. Both are capable of changing the expression of antigens exposed to the immune system of the host, masking the membrane components to survive in adverse conditions and secreting enzymes such as protease for the facilitated invasion to the host tissue [8]. Finally, both parasites and cancer cells are able to guide many innate immune cells such as monocytes to form a proper microenvironment, allowing them to survive and proliferate well, evading other tissues and escaping from the immune system [38]. Considering the safety and efficacy of benzimidazole drugs for parasite treatment for many years, these drugs are also good candidates for being repurposed as anticancer drugs.
A recent study has examined the impact of a wide range of benzimidazole drugs, including flubendazole, parbendazole, oxibendazole, mebendazole, albendazole, and fenbendazole, on tumor cells derived from paraganglioma, pancreatic, and colorectal cancer [72]. Many of these drugs demonstrated IC50 values within the low micromolar or nanomolar range. In silico analysis indicated no interaction between those drugs and P-gp permeability glycoprotein that plays a pivotal role in drug efflux in tumors. The results also confirmed moderate to good oral bioavailability. Significantly, target prediction analysis of benzimidazole drugs revealed some cancer-related molecular targets for fenbendazole and mebendazole with high probability scores.
In efforts to achieve higher efficacy with low drug dosages, many new benzimidazole derivatives have been developed for the treatment of cancers such as colon cancer [73], breast cancer [74], lung cancer [75], chondrosarcoma [76] and leukemia [77]. Many new derivatives are screened and effective against a very broad range of cancers [78,79]. Furthermore, new benzimidazole derivatives have demonstrated high capability for overcoming drug resistance. For instance, a benzimidazole derivative with a pyrrolidine chain significantly reduced the proliferation and migration of sorafenib-resistant hepatocellular carcinoma cells [80]. Similarly, benzoxazole-based zinc and copper complexes showed remarkably increased apoptosis induction in multidrug resistant L5178Y mouse T-lymphoma compared to non-complex ones [81].
Benzimidazole drugs' side effects are rare and mild. Recent studies have further confirmed the selective mode of actions of benzimidazole drugs against cancer cells. For instance, albendazole inhibited ovarian tumor growth but showed no toxicity to HOSE normal ovary cells [71]. Mebendazole did not show any toxicity to HUVEC cells while it was highly toxic to lung cancer cells [70]. Recently, our group has also shown that 4 benzimidazole drugs induced significant anti-cancer efficacy on B16F0 melanoma cells, but no obvious toxicity to HEK293T healthy cells [82]. Such a distinct mode of action comes from targeting tubulin polymerization overexpressed in cancer cells [83]. Furthermore, cancer cells are more susceptible to oxidative stress [84] and benzimidazole drugs are recognized as ROS generators [85,86].

Inhibition of metastatic cancer cells, cancer stem cells, and drug-resistant cancer cells
Interestingly, benzimidazole drugs exhibit anti-metastatic effect through inhibiting cell migration and invasion [65]. As an example, mebendazole suppressed metastatic potential of anaplastic 8505c cells and prevented lung metastasis in advanced thyroid cancer mouse model [87]. Such inhibitions stems from reducing the activity of matrix metalloproteinases 2 (MMP-2) [62] and repression of signal transducer and activator of transcription 3 (STAT3) [88], which is considered as the key regulator of cancer metastasis by transducing the signals from cell surface receptors to the nucleus. Benzimidazole drugs also suppress telomerase reverse transcriptase (TERT) expression [89], whose activation is associated with metastasis [90].
It is well known that most of chemotherapeutics kill bulk cancer cells but not cancer stem cells, where cancer  [91]. Benzimidazole drugs are potent in targeting cancer stem cells and preventing tumor recurrence. For instance, mebendazole significantly suppressed tubulin polymerization in temozolomide-resistant stem-like glioblastoma cells [92]. It is also reported that mebendazole depleted triple-negative breast cancer stem cells [93].
Benzimidazole drugs are also found to prevent the radiation-induced transformation of cancer cells into radiation-resistant cells, and furthermore sensitize some drug-resistant cells. Many studies have reported the susceptibility of taxane-resistant cancer cells to benzimidazole drugs, especially albendazole [13,94]. The superiority of albendazole to taxane drugs comes from targeting tubulin polymerization [95]. Albendazole also demonstrated higher efficacy in reducing the level of Bcl-2 antiapoptotic protein, whose expression is elevated in drug-resistant cells [94]. Flubendazole showed inhibitory activity on vinblastine-resistant leukemia cells in spite of glycoprotein overexpression [12]. Temozolomide-resistant glioblastoma cells also showed susceptibility to mebendazole [92]. Therefore, benzimidazole drugs own great potentials for the treatment of drug-resistant cancer cells. Note that benzimidazole treatment may also result in tubulin mutation and subsequent resistance [92].

Nanoformulation for enhanced anti-cancer effect
In spite of the broad spectrum of applications in cancer therapy, the low water solubility of benzimidazole drugs impedes their clinical applications and therapeutic outcomes [16]. Chemical modification of benzimidazoles generates many new derivatives with higher water solubility [16,96]. More promisingly, nanoformulating benzimidazole drugs that are safely used for many years has emerged as a feasible approach to increase the bioavailability with extra advantages, including: i.
Extending the lifetime of benzimidazole drugs in blood circulation: Kupffer cells are specialized macrophages that play a crucial role in defending against foreign particles, bacteria, and other debris that enter the bloodstream [97]. However, the unique physicochemical properties of nanoparticles present a challenge to these cells due to their small size. Smaller nanoparticles, particularly those with a neutral or slightly negative charge, are less likely to be recognized and phagocytozed by Kupffer cells and cleared by the immune system [98], resulting in a longer circulation time in the bloodstream. This extended circulation time allows nanoparticles to accumulate at the tumor site with greater specificity and in higher quantity. This "stealth effect" of nanoparticles is dependent on several factors, such as their size [99], shape [100], and surface chemistry [101].  [102]. This accumulation can increase the efficacy of anti-cancer drugs while reduce systemic exposure to healthy tissues, thus improving the therapeutic index of drugs [103].

Review Article
iii. Potential stimuli-responsive release: Stimuli-responsive nanoparticles can be designed to release drugs in response to specific stimulators within the tumor microenvironment, allowing for the targeted delivery of drugs to cancer cells while minimizing the exposure of healthy tissues to high drug concentrations [104].
For instance, lipid-coated calcium phosphate (LCP) nanoparticles released albendazole at pH 6-6.5, which is supposed to be similar to that in the tumor microenvironment while keeping the cargo intact at pH 7.4 [105].
iv. Potential target delivery: Nanoparticles can have targeting capability by functionalizing their surface with specific molecules that can selectively bind to cancer cells. Nanoparticles can circulate until they encounter the target cancer cells, where they can bind to the receptors on the cell surface and enter the cells via endocytosis. This allows the nanoparticles to deliver their therapeutic payload directly to the cancer cells, while minimize exposure to healthy tissues and reduce systemic toxicity. Folic acid has widely been used as a targeting moiety for the targeted delivery of benzimidazole drugs to cancer cells due to overexpression of the folate receptors on cancer cells [106]. As an example, the use of folic acid-targeted chitosan nanoparticles for the delivery of mebendazole in the treatment of murine triple-negative breast cancer has been shown to be particularly effective by significantly reducing tumor size and extending the survival time of mice with triple-negative breast cancers [107]. Similarly, utilizing folate-conjugated bovine serum albumin (BSA) for co-delivery of albendazole and nanosilver simultaneously inhibited the energy metabolism of tumor cells, demonstrating superior anti-tumor efficacy compared to other nanoparticles lacking folic acid modification [108].
Of course, these similar nanoformulations can also be explored for cancer therapy. An example is incorporating albendazole into chitosan-coated PLGA nanoparticles (260-480 nm), which elevated albendazole release to 200-fold compared to untreated albendazole, resulting in superior mucoadhesion and cytotoxicity [116]. Some nanoformulations of benzimidazole drugs have also been specifically studied for cancer therapy. As illustrated in Figure 3, albumin nanoparticles in the size of 7-10 nm were promising carriers of albendazole, reducing tumor size at a very low drug dosage while 200 nm albumin nanoparticles were less effective [117]. In addition to confirming improved anti-cancer efficacy of albendazole in nanoparticle forms, these results confirm the importance of the nanoparticle size to the treatment efficiency. The uptake of nanoparticles plays a pivotal role to increasing the treatment efficiency and smaller nanoparticles are more likely to be taken up by tumor cells [117].

Review Article
Cancer Insight | 10.58567/ci02010003 Cancer Insight | 2023 2(1) 31-52 ©2020-2023 Anser Press Pte.Ltd. All rights reserved.   Recently, our group formulated benzimidazole drugs into 50 nm LCP nanoparticles. These drug-loaded LCP nanoparticles increased the solubility in PBS by 100-200% and significantly enhanced the apoptosis-induced anti-cancer efficacy in the treatment of B16F0 melanoma cells via generating more reactive oxygen species (ROS) and inhibiting Bcl-2 expression, as demonstrated in Figure 4. Very obviously, these drug-loaded LCP nanoparticles did not show any obvious toxicity and Bcl-2 inhibition in HEK293T healthy cells [82,105]. More frequently, benzimidazole drugs are combined with other chemotherapeutics, such as paclitaxel [10], trametinib [118], gemcitabine [119] and methoxyestradiol [120], to enhance the anti-cancer treatment efficacy. Flubendazole and albendazole at the low dosage were found to significantly potentiate the anti-proliferative effect of paclitaxel on colon cancers [10]. 2-Methoxyestradiol was the other microtubule-binding agent, exhibiting synergistic anti-cancer effect in combination with albendazole [120]. Mebendazole in combination with the methyl ethyl ketone (MEK) inhibitor, trametinib, demonstrated encouraging results for melanoma treatment by rapidly phosphorylating MEK and extracellular signal-regulated kinases (ERKs), and increasing apoptosis markers such as cleaved caspase-3 and poly (ADP-ribose) polymerase (PARP) [118]. The therapeutic effects of gemcitabine

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was also promoted in combination with mebendazole [119] and parbendazole [121]. Interestingly, fenbendazole showed excellent anti-tumourigenic effect in combination with supplementary vitamins [122]. In addition to vitamins, fenbendazole in combination with rapamycin demonstrated synergistic anti-cancer effect against A549 cancer cells [123]. We also developed lipid-coated calcium phosphate (LCP) nanoparticles combining albendazole (ABZ) and a TOPK inhibitor, OTS964 for synergistic treatment of skin cancer. The dual-targeting capacity of the LCP nanoparticles to the programmed death ligand-1 (PD-L1) and folate receptor overexpressed on the surface of skin cancer cells enabled the combination therapy to completely suppress the skin tumour growth (Figure 5A and B) [124]. Furthermore, such combination treatment induced a certain level of local anti-cancer immunity by recruiting more CD4+ and CD8+ T cells into the tumor tissues (Figure 5C and D).
Moreover, benzimidazole drugs have also sensitized tumor cells to radiation therapy [93]. For example, as shown in Figure 6, mebendazole enhanced survival time and reduced colony formation of malignant meningioma [11], and also prevented radiation-induced conversion of triple-negative cancer cells into drug-resistant breast cancer-initiating cells and elevated the sensitivity of cancer cells to radiation [93]. Similarly, albendazole sensitized small cell lung cancer and metastatic melanoma cells to radiation therapy [125]. Albendazole not only induced DNA damage in those cells, but also arrested cell cycle at G2/M phase where cells are more sensitive to ionizing radiation. Therefore, combination of this antihelminthic drug with radiation therapy led to a synergistic cancer inhibition.

Conclusions and future direction
Initially emerged as anti-helminth drugs, benzimidazole drugs have recently been repurposed as anti-cancer agents largely due to their safety in the long term applications. Benzimidazoles prevent cancer cell growth by inhibiting tubulin polymerization and disrupting mitotic spindles, leading to cell cycle arrest and inhibition of angiogenic factors such as HIF-1α and VEGF. Moreover, these agents disable cell maintenance by elevating ROS generation and reducing ATP, thus suppressing many key enzymes and inducing apoptosis. A higher level of tubulin polymerization in rapidly dividing cells leads to a selective toxic effect of benzimidazoles on cancerous cells, leaving healthy cells intact. The higher sensitivity of cancer cells to ROS augmentation is the other factor for the selective activity of benzimidazoles. Such a selective effect, along with high efficacy in cancer treatment, represents benzimidazoles as a promising anti-cancer chemotherapeutics with minimum adverse side effects.
Benzimidazoles are also able to synergize other therapeutic approaches such as chemotherapy and radiation therapy. Some members of this family have also shown anti-cancer effect on drug-resistant cells and even cancer stem cells. Benzimidazoles not only enhance the efficacy of radiation therapy but also prevent cells from transformation to be resistant to the radiation treatment. On the other hand, benzimidazole treatment may cause tubulin mutation, and thus the combination with other therapeutics may be considered as the better choice.
Despite the extensive potentials of benzimidazole drugs for cancer treatment, their clinical application is still limited due to low water solubility and bioavailability. Nanoformulation of these drugs is promising for improving the solubility and bioavailability in addition to providing the opportunity of enhancing their circulation and tumor accumulation, targeted delivery and stimuli-responsive release. Until now, there are few studies that have investigated nanoformulations of these drugs for cancer therapy. Therefore, nanoformulation of benzimidazoles represents a promising future direction as it is still at the infant stage of research. Moreover, combination with other therapeutic modules, such as chemotherapeutics, radiation sensitizers, immune checkpoint inhibitors as well as target therapy in nanoformulations would synergize the therapeutic outcomes and bloom the repurpose applications of benzimidazole drugs in the fight with cancers in the near future.