Flubendazole suppresses VEGF-induced angiogenesis in HUVECs and exerts antitumor effects in PC-3 cells.

Flubendazole, an FDA-approved anthelmintic, has been predicted to show strong VEGFR2 inhibitory activity in silico screening combined with in vitro experimental validation, and it has shown anti-cancer effects on some human cancer cell lines, but little is known about the anti-angiogenesis effects and anti-prostate cancer effects. In this study, we analyzed the binding modes and kinetic analysis of flubendazole with VEGFR2 and first demonstrated that flubendazole suppressed VEGF-stimulated cell proliferation, wound-healing migration, cell invasion and tube formation of HUVEC cells, and decreased the phosphorylation of extracellular signal-regulated kinase and serine/threonine kinase Akt, which are the downstream proteins of VEGFR2 that are important for cell growth. What's more, our results showed that flubendazole decreased PC-3 cell viability and proliferation ability, and suppressed PC-3 cell wound healing migration and invasion across a Matrigel-coated Transwell membrane in a concentration-dependent manner. The antiproliferative effects of flubendazole were due to induction of G2-M phase cell cycle arrest in PC-3 cells with decreasing expression of the Cyclin D1 and induction of cell apoptosis with the number of apoptotic cells increased after flubendazole treatment. These results indicated that flubendazole could exert anti-angiogenic and anticancer effects by inhibiting cell cycle and inducing cell apoptosis.

In vitro metabolic stress induced by nitazoxanide and flubendazole combination in Taenia crassiceps cysticerci.

Taenia crassiceps is often used as experimental model for T. solium cysticercosis studies. Currently cysticercosis antiparasitic treatment is based on albendazole and praziquantel which may present side effects and parasitic resistance. The search for other antiparasitic drugs is necessary. Nitazoxanide (NTZ) and flubendazole (FLB) are broad spectrum antiparasitic drugs that present anti-cysticercosis effect. Metabolic analyses help to determine the impact of these drugs on parasites. The aim of this study was to determine the impact on the production and excretion of organic metabolites in T. crassiceps cysticerci after in vitro exposure to NTZ and FLB, isolated or in combination. T. crassiceps cysticerci were culture in RPMI medium and exposed to 10 µg/mL of NTZ, 10 µg/mL of FLB or 10 µg/mL of NTZ +10 µg/mL of FLB. 24 h after exposure, the parasites were chromatographic analyzed to determine the impact of these drugs on glycolysis, homolactic fermentation, tricarboxylic acid cycle, fatty acids oxidation and proteins catabolism. It was possible to determine that the drugs combination induced greater metabolic impact on cysticerci in comparison to the isolated drugs exposure. The drugs combination induced gluconeogenesis, metabolic acidosis, increase in tricarboxylic acid cycle and in proteins catabolism. While the NTZ isolated exposure induced metabolic acidosis and protein catabolism and the FLB isolate exposure induced gluconeogenesis and protein catabolism. These results show that the combination of drugs with different modes of action increase the antiparasitic effect and may be indicated as alternative cysticercosis treatments.

Flubendazole induces mitochondrial dysfunction and DRP1-mediated mitophagy by targeting EVA1A in breast cancer.

Breast cancer is still one of the most common malignancies worldwide and remains a major clinical challenge. We previously reported that the anthelmintic drug flubendazole induced autophagy and apoptosis via upregulation of eva-1 homolog A (EVA1A) in triple-negative breast cancer (TNBC) and was repurposed as a novel anti-tumor agent. However, the detailed underlying mechanisms remain unclear and need further investigation. Here, we found that flubendazole impairs the permeability of the mitochondrial outer membrane and mitochondrial function in breast cancer. Meanwhile, flubendazole increased dynamin-related protein (DRP1) expression, leading to the accumulation of PTEN induced putative kinase 1 (PINK1) and subsequent mitochondrial translocation of Parkin, thereby promoting excessive mitophagy. The resultant excessive mitophagy contributed to mitochondrial damage and dysfunction induced by flubendazole, thus inhibiting breast cancer cells proliferation and migration. Moreover, we demonstrated that excessive DRP1-mediated mitophagy played a critical role in response to the anti-tumor effects of EVA1A in breast cancer. Taken together, our results provide new insights into the molecular mechanisms in relation to the anti-tumor activities of flubendazole, and may be conducive to its rational use in potential clinical applications.

Hot-melt extrudability of amorphous solid dispersions of flubendazole-copovidone: An exploratory study of the effect of drug loading and the balance of adjuvants on extrudability and dissolution.

The FDA-approved anthelmintic flubendazole has shown potential to be repositioned to treat cancer and dry macular degeneration; however, its poor water solubility limits its use. Amorphous solid dispersions may overcome this challenge, but the balance of excipients may impact the preparation method and drug release. The purpose of this study was to evaluate the influence of adjuvants and drug loading on the development of an amorphous solid dispersion of flubendazole-copovidone by hot-melt extrusion. The drug, copovidone, and adjuvants (magnesium stearate and hydroxypropyl cellulose) mixtures were statistically designed, and the process was performed in a twin-screw extruder. The study showed that flubendazole and copovidone mixtures were highly extrudable, except when drug loading was high (>40%). Furthermore, magnesium stearate positively impacted the extrusion and was more effective than hydroxypropyl cellulose. The extruded materials were evaluated by modulated differential scanning calorimetry and X-ray powder diffraction, obtaining positive amorphization and physical stability results. Pair distribution function analysis indicated the presence of drug-rich domains with medium-range order structure and no evidence of polymer-drug interaction. All extrudates presented faster dissolution (HCl, pH 1.2) than pure flubendazole, and both adjuvants had a notable influence on the dissolution rate. In conclusion, hot-melt extrusion may be a viable option to obtain stable flubendazole:copovidone amorphous dispersions.

Flubendazole Plays an Important Anti-Tumor Role in Different Types of Cancers.

Flubendazole, belonging to benzimidazole, is a broad-spectrum insect repellent and has been repurposed as a promising anticancer drug. In recent years, many studies have shown that flubendazole plays an anti-tumor role in different types of cancers, including breast cancer, melanoma, prostate cancer, colorectal cancer, and lung cancer. Although the anti-tumor mechanism of flubendazole has been studied, it has not been fully understood. In this review, we summarized the recent studies regarding the anti-tumor effects of flubendazole in different types of cancers and analyzed the related mechanisms, in order to provide the theoretical reference for further studies in the future.

Emerging insights on functions of the anthelmintic flubendazole as a repurposed anticancer agent.

While flubendazole has been used as a macrofilaricide in humans and animals for some 40 years, work in vitro and in preclinical models over the last decade has suggested its potential use as an anticancer agent. This article reviews recent studies in a range of tumor types indicating novel functions for flubendazole in its control of processes associated with tumor growth, spread and renewal including ferroptosis, autophagy, cancer stem-like cell killing and suppression of intratumoral myeloid-derived suppressor cell accumulation and programmed cell death protein 1. Flubendazole's potential use in clinical oncology will require further understanding of its mechanistic roles, range of inhibition of cancer types, capacity for adjunctive therapy and possible reformulation for enhanced solubility, bioavailability and potency.

Alterations in Taenia crassiceps cysticerci cytoskeleton induced by nitazoxanide and flubendazole.

Cysticercosis is the presence of Taenia solium larval stage in tissues such as central nervous system, skin, muscles and eye globe. The current treatment is based on albendazole and praziquantel which already present resistance reports. Therefore, the search for alternative treatments is paramount. The aim of this study was to determine the effect of flubendazole and nitazoxanide on cytoskeleton proteins from Taenia crassiceps cysticerci, an experimental model for cysticercosis. Cysticerci were cultured in RPMI supplemented medium containing nitazoxanide and/or flubendazole. 24 h after the exposure the cysticerci were processed for scanning and transmission electron microscopy and for protein analysis of the cytoskeleton. The proteins were detected through 1D electrophoresis and identified through Western Blot. Nitazoxanide exposure increased tubulin and actin quantifications in T. crassiceps cysticerci. While flubendazole alone and the drugs combinations induced an increase in α-tubulin and actin and decreased ß-tubulin quantifications in the parasite. Morphological changes such as swelling and rupture of vesicle, stiff membrane, decrease in movements were observed when the cysticerci were incubated with the different compounds. In conclusion the drugs induced significative impact in the parasite`s cytoskeleton and may be considered as alternative treatments for cysticercosis.

Antimicrobial and anticancer properties of Carica papaya leaves derived di-methyl flubendazole mediated silver nanoparticles.

BACKGROUND: In this study, a biologically active molecule, di-methyl flubendazole isolated from the extract of Carica papaya leaves confirmed by using GC-MS, 1H NMR, and 13C NMR analysis was applied to synthesize silver nanoparticles (AgNPs). The AgNPs with plant sources an alternative therapeutic agent for synthetic compound used in cancer chemotherapy. METHODS: The AgNPs were characterized using UV, FT-IR, XRD, FESEM with EDX and TEM. The antibacterial effects of AgNPs were determined with agar well diffusion method. The MTT assay used to evaluate the inhibitory effect cell lines. The acridine orange and ethidium bromide and DAPI have used cell morphological effects. RESULTS: The AgNPs were mono-crystalline and their size ranged from 7 to 22 nm. AgNPs showed good antibacterial activity against both Gram-positive and Gram-negative bacteria. Studies on the antiproliferative potential of bioinspired AgNPs in cancer cell lines revealed that the antiproliferative effect was much stronger in HepG2 than in MCF-7 and A549 cell lines. Similarly, AgNPs exerted less cytotoxic activity in Vero cells (normal cells). AgNPs-treated cells showed necrosis, apoptotic morphology evidenced by cell shrinkage, membrane blebbing, cell decay, and necrosis. HepG2 cells treated with biosynthesized AgNPs exhibited a G0/G1 phase (52-53.37%) blockage. Compared to the control, AgNP-treated HepG2 cells showed elevated ®-actin levels; however, Bcl-2 was significantly down regulated in AgNP-treated cells, indicating the involvement of Bcl-2 in apoptosis. CONCLUSION: Overall, the fact that di-methyl flubendazole-based silver nanoparticles showed a novel and cost-effective natural antitumor and antibacterial agent.

Flubendazole, FDA-approved anthelmintic, elicits valid antitumor effects by targeting P53 and promoting ferroptosis in castration-resistant prostate cancer.

On account of incurable castration-resistant prostate cancer (CRPC) inevitably developing after treating with androgen deprivation therapy, it is an urgent need to find new therapeutic strategies. Flubendazole is a well-known anti-malarial drug that is recently reported to be a potential anti-tumor agent in various types of human cancer cells. However, whether flubendazole could inhibit the castration-resistant prostate cancer has not been well charified. Thus, the aim of the present study was to characterize the precise mechanism of action of flubendazole on the CRPC. In this study, we investigated the potential effect of flubendazole on cell proliferation, cell cycle and cell death in CRPC cells (PC3 and DU145). We found that flubendazole inhibited cell proliferation, caused cell cycle arrest in G2/M phase and promoted cell death in vitro, and suppressed growth of CRPC tumor in xenograft models. In addition, we reported that flubendazole induced the expression of P53, which partly accounted for the G2/M phase arrest and led to inhibition of the transcription of SLC7A11, and then downregulated the GPX4, which is a major ferroptosis-related gene. Furthermore, flubendazole exhibited synergistic effect with 5-fluorouracil (5-Fu) in chemotherapy of CRPC. This study provides biological evidence that flubendazole is a novel P53 inducer which exerts anti-proliferation and pro-apoptosis effects in CRPC through hindering the cell cycle and activating the ferroptosis, and indicates that a novel utilization of flubendazole in neoadjuvant chemotherapy of CRPC.

In Vivo Treatment with the Combination of Nitazoxanide and Flubendazole Induces Gluconeogenesis and Protein Catabolism in Taenia crassiceps cysticerci.

PURPOSE: Cysticercosis is the presence of Taenia solium larvae in humans or swines tissues. It is a public health problem related to bad hygienic habits and consumption of infected pork. T. crassiceps is a widely used cysticercosis experimental model. The combination of two effective drugs such as nitazoxanide (NTZ) and flubendazole (FBZ) may potentialize their effect. The aim of this study was to use biochemical analysis to determine the metabolic impact of the combination of NTZ and FBZ on cysticerci inoculated intraperitoneally in mice. METHODS: Balb/c mice intraperitoneally infected with T. crassiceps cysticerci received a single oral dose NTZ/FBZ (50 mg/kg). 24 h after the treatment the cysticerci were removed, frozen and analyzed by high performance liquid chromatography regarding the detection of the following metabolic pathways: glycolysis, gluconeogenesis, homolactic fermentation, tricarboxylic acid cycle, proteins catabolism and fatty acids oxidation. RESULTS: The treatment with the drugs combination induced a statistically significant increase in gluconeogenesis and in protein catabolism when compared to the control groups. CONCLUSION: The drugs combination is potentialized and capable of causing greater metabolic stress than the separate treatment with NTZ or FBZ, showing its potential for an alternative cysticercosis treatment.

Flubendazole elicits anti-cancer effects via targeting EVA1A-modulated autophagy and apoptosis in Triple-negative Breast Cancer.

Background: Triple-negative breast cancer (TNBC) is one of the most prevalent neoplastic diseases worldwide, but efficacious treatments for this pathological condition are still challenging. The lack of an effective targeted therapy also leads to a poor prognosis for patients affected by TNBC. In the present study, we repurposed the distinctive inhibitory effects of flubendazole, a traditional anthelmintic drug, towards the putative modulation of proliferation and migration of TNBC in vitro and in vivo. Methods: According to a series of experimental approaches, including immunofluorescence (IF), immunoblotting (IB), siRNA and GFP-mRFP-LC3 plasmid transfection, respectively, we have found that flubendazole is capable of inducing autophagic cell death and apoptosis, thus exerting some anti-proliferative and anti-migration activity in TNBC cells. The therapeutic effects of flubendazole were evaluated by xenograft mouse models, followed by immunohistochemistry (IHC), IF and IB. Changes in the gene expression profiles of flubendazole-treated TNBC cells were analyzed by RNA sequencing (RNA-seq) and validated by IB. The potential binding mode of flubendazole and EVA1A was predicted by molecular docking and demonstrated by site-directed mutagenesis. Results: We have presently found that flubendazole exhibits a considerable anti-proliferative activity in vitro and in vivo. Mechanistically, the induction of autophagic cell death appears to be pivotal for flubendazole-mediated growth inhibition of TNBC cells, whereas blocking autophagy was able to improve the survival rate and migration ability of flubendazole-treated TNBC cells. Specifically, RNA-seq analysis showed that flubendazole treatment could promote the up-regulation of EVA1A. Flubendazole may regulate autophagy and apoptosis by targeting EVA1A, thus affecting the mechanisms of TNBC proliferation and migration. Furthermore, Thr113 may be the key amino acid residues for the binding of flubendazole to EVA1A. Conclusion: Our results provide novel insights towards the putative anti-cancer efficacy of flubendazole. Furthermore, here we show that flubendazole could serve as a potential therapeutic drug in TNBC. Altogether, this study highlights the possibility of this repurposed autophagic inducer for future cancer treatments.

An analysis of mitotic catastrophe induced cell responses in melanoma cells exposed to flubendazole.

Mitotic catastrophe induced by mictotubule-targeting drugs such as benzimidazole carbamates has been demonstrated to be an efficient mechanism for suppression of tumor cells growth and proliferation, with variable resulting endpoints. The present study was designed to explore some of these endpoints; i.e. the apoptosis as well as autophagy and their related signaling in several stabilized cell lines as well as human explant melanoma cells treated with flubendazole (FLU). FLU-induced mitotic catastrophe resulted in mitochondrial and caspase-dependent apoptosis, which occurred at various rates in all treated cells during 96 h of treatment. The process was characterized by enhanced transcriptional activity of TP53 and NF-κB as well as upregulated Noxa expression. Also, inactivation of Bcl-2, BclXL and Mcl-1 proteins by JNK mediated phosphorylation was observed. Although increased autophagic activity took place in treated cells too, no discernible functional linkage with ongoing cell death process was evidenced. Together these results advance our evidence over the effectiveness of FLU cytotoxicity-related killing of melanoma cells while calling for more extensive testing of melanoma samples as a prerequisite of further preclinical evaluation of FLU antineoplastic potential.

[Flubendazole Inhibits the Proliferation of A549 and H460 Cells and Promotes Autophagy].

BACKGROUND: Flubendazole is an anthelmintic and categorized in benzimidazole. Previous evidence indicates its suppression on proliferation of colon cancer and breast cancer cells. Our study aims to explore the effects of flubendazole on non-small cell lung cancer A549 and H460 cell lines and the underlying mechanism. METHODS: CCK-8 assay was used to detect the effect of flubendazole at different concentrations on viability of both cell lines A549 and H460. We used western blot to detect the expression levels of autophagy-related proteins p62 and LC3 after flubendazole treatment. Cells were transfected with tandem fluorescent adenovirus (mRFP-GFP-LC3), and the impact of flubendazole treatment on autophagic flux were analyzed. RESULTS: Cell viability analysis showed a dose-dependent inhibitory effect on proliferation of both A549 and H460, comparing to cells without flubendazole treating (P<0.001). Level of p62 decreased and LC3 II/I ratio increased in cells treated with 2 µmol/L flubendazole for 24 h and 48 h, compared to control groups (P<0.005). Red fluorescence signals increased in mRFP-GFP-LC3 transfected cells after flubendazole treating, suggesting an elevation in autophagic flux. CONCLUSIONS: Flubendazole may inhibit the proliferation of A549 and H460 cells and promote autophagy.

Flubendazole demonstrates valid antitumor effects by inhibiting STAT3 and activating autophagy.

BACKGROUND: Signal transducer and activator of transcription 3 (STAT3) is an oncogene, which upregulates in approximately 70% of human cancers. Autophagy is an evolutionarily conserved process which maintains cellular homeostasis and eliminates damaged cellular components. Moreover, the STAT3 signaling pathway, which may be triggered by cancer cells, has been implicated in the autophagic process. METHODS: In this study, we found that the anthelmintic flubendazole exerts potent antitumor activity in three human colorectal cancer (CRC) cell lines and in the nude mouse model. The inhibition of cell proliferation in vitro by flubendazole was evaluated using a clonogenic assay and the MTT assay. Western blot analysis, flow cytometry analysis, siRNA growth experiment and cytoplasmic and nuclear protein extraction were used to investigate the mechanisms of inhibiting STAT3 signaling and activation of autophagy induced by flubendazole. Additionally, the expression of STAT3 and mTOR was analyzed in paired colorectal cancer and normal tissues collected from clinical patients. RESULTS: Flubendazole blocked the IL6-induced nuclear translocation of STAT3, which led to inhibition of the transcription of STAT3 target genes, such as MCL1, VEGF and BIRC5. In addition, flubendazole also reduced the expression of P-mTOR, P62, BCL2, and upregulated Beclin1 and LC3-I/II, which are major autophagy-related genes. These processes induced potent cell apoptosis in CRC cells. In addition, flubendazole displayed a synergistic effect with the chemotherapeutic agent 5-fluorouracil in the treatment of CRC. CONCLUSIONS: Taken together, these results indicate that flubendazole exerts antitumor activities by blocking STAT3 signaling and inevitably affects the autophagy pathway. Flubendazole maybe a novel anticancer drug and offers a distinctive therapeutic strategy in neoadjuvant chemotherapy of CRC.

In vitro efficacies of solubility-improved mebendazole derivatives against Echinococcus multilocularis.

Recently, we introduced an epoxy group to mebendazole by a reaction with epichlorohydrin and obtained two isoforms, mebendazole C1 (M-C1) and mebendazole C2 (M-C2). The in vitro effects of mebendazole derivatives at different concentrations on Echinococcus multilocularis protoscoleces and metacestodes as well as cytotoxicity in rat hepatoma (RH) cells were examined. The results demonstrated that the solubility of the two derivatives was greatly improved compared to mebendazole. The mortality of protoscoleces in vitro reached to 70-80% after 7 days of exposure to mebendazole or M-C2, and M-C2 showed higher parasiticidal effects than mebendazole (P > 0.05). The parasiticidal effect of M-C1 was low, even at a concentration of 30 µm. The percentage of damaged metacestodes that were treated with mebendazole and M-C2 in vitro at different concentrations were similar, and M-C1 exhibited insignificant effects on metacestodes. Significant morphological changes on protoscoleces and metacestodes were observed after treatment with mebendazole and M-C2. In addition, the introduction of an epoxy group to mebendazole also reduced its cytotoxicity in RH cells. Our results demonstrate that the introduction of an epoxy group not only improved the solubility of mebendazole, but also increased its parasiticidal effects on E. multilocularis and reduced its cytotoxicity in RH cells.

The anthelmintic flubendazole blocks human melanoma growth and metastasis and suppresses programmed cell death protein-1 and myeloid-derived suppressor cell accumulation.

The incidence of melanoma is increasing faster than any other cancer. In recent years, treatment of melanoma and a range of other deadly cancers has involved immunotherapy with programmed cell death protein-1 (PD-1)/PD-1 ligand (PD-L1) checkpoint blockade which has improved survival. However, many patients do not respond or have partial response, survival benefit is in the order of months and all available PD-1/PD-L1 strategies are antibodies requiring intravenous infusion. There are no clinically approved small molecule pharmacologic inhibitors of the PD-1/PD-L1 system. The benzimidazole derivative flubendazole is a widely used anthelmintic available over the counter in Europe. Here we demonstrate the ability of flubendazole to inhibit human melanoma growth and spread in mice. Flubendazole's ability to block tumor growth and spread was comparable to paclitaxel. Anti-tumor effects were observed when flubendazole was delivered systemically not locally. Flubendazole inhibited CD31/PECAM-1 staining indicating suppression of tumor angiogenesis. Most surprisingly, flubendazole inhibited PD-1 levels within the tumors, but not PD-L1. Western blotting and flow cytometry revealed that flubendazole inhibits PD-1 expression in cultured melanoma cells. Flubendazole also reduced myeloid-derived suppressor cell (MDSC) levels in tumor tissue. Further we found that flubendazole inhibited active (phospho-Tyr705) signal transducer and activator of transcription (STAT3), an upstream regulator of PD-1 expression. These findings uncover that flubendazole is a novel small molecule inhibitor of not only melanoma growth and spread but also of PD-1 and MDSC.

Efficiency of flubendazole-loaded mPEG-PCL nanoparticles: A promising formulation against the protoscoleces and cysts of Echinococcus granulosus.

None of the existing drugs can effectively treat the human cystic echinococcosis. This study aimed to improve the efficacy of flubendazole (FLBZ) against the protoscoleces and cysts of Echinococcus granulosus by preparing polymeric FLBZ-loaded methoxy polyethylene glycol-polycaprolactone (mPEG-PCL) nanoparticles. The protoscoleces and microcysts were treated with FLBZ-loaded mPEG-PCL nanoparticles (FLBZ-loaded nanoparticles) and free FLBZ at the final concentrations of 1, 5, and 10 µg/mL for 27 and 14 days, respectively. The chemoprophylactic efficacy of the drugs was evaluated in experimentally infected mice. The nanoparticles were stable for 1 month, with an average size of 101.41 ± 5.14 nm and a zeta potential of -19.13 ± 2.56 mV. The drug-loading and entrapment efficiency of the FLBZ-loaded nanoparticles were calculated to be 3.08 ± 0.15% and 89.16 ± 2.93%, respectively. The incubation of the protoscoleces with the 10 µg/mL nano-formulation for 15 days resulted in 100% mortality, while after incubation with the 10 µg/mL free FLBZ, the viability rate of the protoscoleces was only 44.0% ± 5.22%. Destruction of the microcysts was observed after 7 days' exposure to the FLBZ-loaded nanoparticles at a concentration of 10 µg/mL. The in vivo challenge showed a significant reduction in the weight and number of the cysts (P < 0.05) in the mice treated with the FLBZ-loaded nanoparticles, yielding efficacy rates of 94.64% and 70.21%, correspondingly. Transmission electron microscopy revealed extensive ultrastructural damage to the cysts treated with the FLBZ-loaded nanoparticles. The results indicated that the FLBZ-loaded nanoparticles were more effective than the free FLBZ against the protoscoleces and cysts of E. granulosus both in vitro and in vivo.

Flubendazole and mebendazole impair migration and epithelial to mesenchymal transition in oral cell lines.

Benzimidazole anthelmintics flubendazole and mebendazole are microtubule-targeting drugs that showed considerable anti-cancer activity in different preclinical models. In this study, the effects of flubendazole and mebendazole on proliferation, migration and cadherin switching were studied in a panel of oral cell lines in vitro. Both compounds reduced the viability of the PE/CA-PJ15 and H376 oral squamous carcinoma cells and of the premalignant oral keratinocytes DOK with the IC50 values in the range of 0.19-0.26 µM. Normal oral keratinocytes and normal gingival fibroblasts were less sensitive to the treatment. Flubendazole and mebendazole also reduced the migration of the PE/CA-PJ15 cell in concentrations that had no anti-migratory effects on the normal gingival fibroblasts. Levels of the focal adhesion kinase FAK, Rho-A and Rac1 GTPases and the Rho guanine nucleotide exchange factor GEF-H1 were decreased in both PE/CA-PJ15 cells and gingival fibroblasts following treatment. Both drugs also interfered with cadherin switching in the model of TGF-ß-induced epithelial to mesenchymal transition (EMT) in the DOK cell line. Levels of N-cadherin were reduced in the TGF-ß induced cells co-treated with flubendazol and mebendazole in very low concentration (50 nM). These results suggest direct effects of both benzimidazoles on selected processes of EMT in oral cell lines such as cadherin switching as well as cellular migration.

Flubendazole elicits anti-metastatic effects in triple-negative breast cancer via STAT3 inhibition.

Tumor metastasis remains the cause of 90% of cancer-related deaths. Cancer stem cells (CSC) are thought to be responsible for the aggressive and metastatic nature of triple-negative breast cancers (TNBC), and new therapeutic strategies are being devised to target them. Flubendazole (FLU) is a widely used anthelmintic agent that also exhibits anticancer activity in several cancer types. The aim of this study was to characterize the mechanism of action of FLU on breast cancer stem cell (BCSC)-like properties and metastasis in TNBC. FLU treatment caused a significant induction of apoptosis, accompanied by G2/M phase accumulation, caspase-3/-7 activation and the dysregulation of STAT3 activation in TNBC cells. The latter phenomenon was associated with impairment of cancer stem-like traits, concomitant with a reduction in the CD24low /CD44high , CD24high /CD49fhigh subpopulation, ALDH1 activity and mammosphere formation. The BCSC-enriched populations exhibited enhanced metastasis with higher STAT3 activation, while FLU administration inhibited tumor growth, angiogenesis and lung and liver metastasis, coinciding with decreased MMP-2 and MMP-9 levels in circulating blood. FLU kills not only rapid proliferating tumor cells but also effectively eradicates BCSC-like cells in vitro and in vivo. Our findings warrant further investigation of FLU as a treatment for metastatic TNBC.

Repurposing and Reformulation of the Antiparasitic Agent Flubendazole for Treatment of Cryptococcal Meningoencephalitis, a Neglected Fungal Disease.

Current therapeutic options for cryptococcal meningitis are limited by toxicity, global supply, and emergence of resistance. There is an urgent need to develop additional antifungal agents that are fungicidal within the central nervous system and preferably orally bioavailable. The benzimidazoles have broad-spectrum antiparasitic activity but also have in vitro antifungal activity that includes Cryptococcus neoformans Flubendazole (a benzimidazole) has been reformulated by Janssen Pharmaceutica as an amorphous solid drug nanodispersion to develop an orally bioavailable medicine for the treatment of neglected tropical diseases such as onchocerciasis. We investigated the in vitro activity, the structure-activity-relationships, and both in vitro and in vivo pharmacodynamics of flubendazole for cryptococcal meningitis. Flubendazole has potent in vitro activity against Cryptococcus neoformans, with a modal MIC of 0.125 mg/liter using European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology. Computer models provided an insight into the residues responsible for the binding of flubendazole to cryptococcal ß-tubulin. Rapid fungicidal activity was evident in a hollow-fiber infection model of cryptococcal meningitis. The solid drug nanodispersion was orally bioavailable in mice with higher drug exposure in the cerebrum. The maximal dose of flubendazole (12 mg/kg of body weight/day) orally resulted in an ∼2 log10CFU/g reduction in fungal burden compared with that in vehicle-treated controls. Flubendazole was orally bioavailable in rabbits, but there were no quantifiable drug concentrations in the cerebrospinal fluid (CSF) or cerebrum and no antifungal activity was demonstrated in either CSF or cerebrum. These studies provide evidence for the further study and development of the benzimidazole scaffold for the treatment of cryptococcal meningitis.