Its effectiveness as an antiparasitic drug results from differences in the structures of tubulin in mammalian cells or in lower organisms, which lead to its greater binding to tubulin and, as a result, greater inhibition of polymerization in the parasites. Fenbendazole acts on Helminthes primarily by binding to tubulin and disrupting the tubulin microtubule equilibrium. (Spagnuolo et al., 2010) In addition, fenbendazole’s restricted intestinal absorption causes tissue levels of the medication and its active metabolites to be lower than those in the gut, where the targeted parasites are exposed. (Prichard et al., 1981)
Fenbendazole may have antitumor effects since many commonly used anticancer medications block microtubule depolymerization or formation (paclitaxel, docetaxel, or vincristine), respectively, to induce their antineoplastic effects. Some evidence from the literature lends credence to this theory. (Spagnuolo et al., 2009) Gao et al. reported that a fenbendazole-containing diet combined with high supplemental doses of vitamins inhibited the growth of a human lymphoma xenografted into SCID mice; however, it was not clear whether this was due to the drug’s direct impact on tumor cells or the drug’s stimulation of host immune responses. Fenbendazole was shown to lessen the engraftment of brain tumors in nude mice, according to a study by Bai et al. High dosages of fenbendazole, albendazole, and mebendazole were shown to limit the development of paclitaxel-resistant tumors, according to Chung et al. in a conference presentation. (Chung et al., 2010)
Microtubules are one of the most effective anticancer therapeutic targets due to their significance in cell division, motility, intracellular trafficking, and their involvement in modifying cellular shape in response to the environment. In the therapy of cancer, a variety of substances that alter microtubule dynamics have been used. Microtubules are among the top cancer targets that have been found so far, especially in light of the relative effectiveness of mitotic agents in treating cancer. (Mukhtar et al., 2014)
Agents that target microtubules may be generally divided into two kinds. Microtubule-destabilizing substances, which prevent microtubule polymerization, make up the first class. The vinca alkaloids (vinblastine, vincristine, vinorelbine, vindesine, and vinflunine), estramustine, colchicine, and combretastatin are only a few of the substances in this family of anti-mitotic medications that are now being utilized in cancer therapy or are being studied in this setting. Agents that stabilize microtubules make up the second class. These medications include discodermolide, paclitaxel, docetaxel, and epothilones. Metaphase arrest and apoptosis induction are the results of altering tubulin and microtubule dynamics with both medicines in dividing cells. (Todd et al., 2018)
A broad-spectrum benzimidazole anthelmintic, fenbendazole (methyl N-(6-phenylsulfanyl-1H-benzimidazol-2-yl) carbamate) is authorized for use in a variety of animal species7. Repurposing animal pharmaceuticals for human use may significantly cut down on the time and expense needed to generate new medications. Most species tolerate fenbendazole quite well, and it is recognized to have a large safety margin. In test animals, it is very safe and poisonous in very little amounts. (Hinz, 1978) In this work, we demonstrate that fenbendazole (FZ) has a significant anticancer impact, shown by both in vitro and in vivo tests, but only modest microtubule depolymerizing activity towards human cancer cells. Our findings suggest that FZ achieves its anticancer effects through interfering with microtubule dynamics, activating p53, and altering the expression of genes involved in several cellular processes. Due to the downregulation of essential glycolytic enzymes and GLUT transporters during FZ therapy, glucose absorption in cancer cells was also decreased.
Since several genes and proteins that affect different cell signaling pathways are involved in carcinogenesis, single-target medications have a restricted therapeutic window and run the risk of drug resistance. Therefore, it is believed that drugs with many cellular targets would not only be more effective but also be able to avoid the development of resistance.
Overall, the research shows that FZ has a pleiotropic impact on cancer cells, resulting in cell death. Consequently, FZ may have a future in medicine as a treatment. (Pao et al., 2005)
Studies were carried out by Yale University policies, those of the National Institutes of Health, the Association for the Assessment and Accreditation of Laboratory Animal Care, and those outlined in the Guide for the Care and Use of Laboratory Animals. Protocols for in vivo experiments were reviewed and approved by the Yale Institutional Animal Care and Use Committee. (National Research Council et al., 1988) Female BALB/cRw mice, 2.5–3 months old, with the right flank shaved, were bred in our specific–pathogen-free production colony and tumors were created by intradermally injecting 2 105 EMT6 tumor cells, suspended in 0.05 ml of growth media, into the skin. Vernier calipers were used to measure the tumors’ three orthogonal dimensions three times each week, and the formula for a hemiellipsoid—the geometric form that most closely resembles their shape—was used to compute the tumors’ volumes. Mice were divided into treatment and control groups according to the average tumor volume, which was 100 mm3, around two weeks after injection.
Fenbendazole was given intravenously after being dissolved in sterile, pyrogen-free physiological saline. The target mice were sedated with 100 mg/kg of ketamine and 10 mg/kg of xylazine before the tumors were locally treated with 10 Gy of 250 kV x-rays supplied at 15 mA, 2 mm Al filtration, and a dose rate of 6.4 Gy per minute from a Siemens Stabilipan (Malvern, PA, USA), as previously reported. The radiation to vital normal tissues was less than 0.5 Gy and did not significantly harm the mice since their torso and limbs were insulated. (Duan et al., 2012)
Once a tumor reached a volume of 1000 mm3, it was monitored thrice weekly. To assess tumor growth across groups receiving various treatments, the amount of time it took for each tumor to increase from its volume at randomization to four times that volume was calculated for each tumor individually. (Kim et al., 2012) The growth curves on the figure are plotted using a logarithmic Y axis to allow for rigorous comparisons of volumes in the various groups over the full range of tumor growth. Points are geometric means+/-SEM of the volumes of the individual tumors within the group. The tumors were measured over a range of volumes from 1 mm3 to 1000 mm3.
At each measurement, mice were also weighed and examined for appearance (such as fur condition, eye appearance), behavior (such as changes in grooming, spontaneous movement, or response to handling, or breathing rate and pattern), and weight. If pre-specified toxicity levels were reached, animals were euthanized. Mice were put to sleep, and their necropsies were performed to examine the tumor’s local infiltration and metastatic dissemination. The lungs, which were removed, preserved in Bouin’s solution, rinsed in 95 percent ethanol, and then kept in ethanol (the most typical location of metastases for this tumor line). A blinded observer tallied the tumors on the lung surfaces using a dissecting microscope. (Rockwell et al., 2012)
Experiments With Tumors In Vivo
The trials are shown in figure 1 compared the development of EMT6 tumors that had not been treated to tumors that had been treated with 10 Gy of x-rays, three daily i.p. injections of fenbendazole, or fenbendazole + x-rays. The development of the fenbendazole-only-treated tumors was identical to that of the untreated tumors. As anticipated, the tumors’ ability to develop was significantly inhibited by 10 Gy of radiation treatment. Three fenbendazole treatments were given one day before, two hours before, and one day after irradiation did not affect the development curve for tumors that had received radiation.
Figure 1: Effect of three intravenous Fenbendazole injections on the development and response to radiation of EMT6 tumours in BALB/cRw mice. At a mean tumour volume of 100 mm3, tumor-bearing mice were randomly assigned to one of four treatment options: 10 Gy of x-rays were administered at the time shown by the large open arrow, three daily i.p. injections of 50 mg/kg/day fenbendazole, three daily injections at the times shown by the three dark arrows, or Fenbendazole plus x-rays. Points represent geometric means; SEM; 7-8 mice per group.
The mouse was killed and necropsied after each tumor reached a volume of 1000 mm3. There were no mice with metastases to the lymph nodes or local invasion of the body wall. Neither for unirradiated mice (unpaired t-test, p=0.54) nor for mice that had undergone local tumor irradiation (p=0.44), there were no significant changes in the number of lung metastases in Fenbendazole-treated and non-drug-treated animals.
By estimating the time required for each tumor to increase from its volume at the time of randomization for treatment to four times that volume and comparing these periods for different groups, tumor growth after various therapies was systematically compared. These investigations showed that fenbendazole did not affect the development of radiation-treated or non-radiated tumors. The data in the table also come from a second experiment that looked at how the same three injections of fenbendazole affected the growth of unirradiated tumors. This confirms the result from the first experiment (growth curve not shown). It also includes the time to four-fold volume data from our previous study that looked at how long a continuous therapeutic diet containing fenbendazole had an impact on the development of both irradiated and unirradiated EMT6 None of these studies found any appreciable influence of fenbendazole on the development or response to radiotherapy of EMT6 tumors. (Rockwell et al., 2012)
Table 1: Fenbendazole’s effects on the development of EMT6 tumours in BALB/c mice. In these three tests, tumours were administered intravenously, and the volume of each tumour was measured three times weekly from the moment it became palpable until it reached a volume of 1000 mm3. Mice were divided into groups and given treatment when tumours had a mean volume of less than 100 mm3. It was calculated how long it would take for each tumour to increase from its volume at stratification to four times that volume. Fenbendazole did not affect tumour development in either irradiated or unirradiated tumours, whether it was consumed orally three times a day or administered intravenously. Fenbendazole did not affect the animals’ health or the quantity of spontaneous lung metastases discovered after necropsy in any of these studies.
Treatment Days to four-fold volume (mean ± SD)
Control Fenbendazole 10 Gy Fenbendazole + 10 Gy
Fenbendazole given as three daily injections, 50 mg/kg/day, i.p. 10.4±3.0 12.4±2.6 18.3±5.1 17.5±7.5
Growth curves shown in figure 1
Fenbendazole given as three daily injections 50 mg/kg/day, i.p. 11.4±3.0 11.5±1.0 ND ND
Growth curves not shown
Fenbendazole in diet, 150 ppm, continually during tumor growth 12.7±0.1 12.1±1.2 17.5±3.7 20.4±2.4
At each tumor measurement during the period of the experiment shown in figure 1, the mice’s appearance, behavior, and weights were observed. The mice in the fenbendazole-treated and non-drug-treated groups behaved and looked the same. Throughout the trial, there were no discernible variations in the weights of the mice in the four groups, according to ANOVA analyses. The results of the other studies shown in the table were similar.
Major cytoskeleton components, microtubules are crucial for several cellular functions, including mitosis, cell polarity, intracellular trafficking, and maintaining cell shape and structure. Multiple tumor types are being treated therapeutically using microtubule-targeted medicines (MTAs), although these agents’ efficacy is often greatly hampered by drug resistance mechanisms. Although interactions with tubulin and disruption of microtubule activity are the main mechanisms behind the cytotoxicity of MTAs, variations in their tubulin-binding properties modify their modes of action, significantly affecting the effectiveness or toxicity profile of each drug. It may be very important to use newer drugs with enhanced effectiveness, tolerability, and the capacity to at least partly overcome resistance.
According to the available evidence, FZ is a mild microtubule targeting drug that causes mitotic arrest and cancer cell death. FZ is a very mild microtubule targeting drug, yet it has a special capacity to activate p53 to a very high level.
We have shown the action of FZ as a proteasomal interfering agent in prior work. In response to FZ, the current research found that cyclin B1 levels were early elevated and stabilized, suggesting a progression of cell death as opposed to mitotic exit and polyploidy. Interestingly, the FDA-approved proteasomal inhibitor bortezomib has likewise been shown to cause lymphoma cell death via inducing mitosis. Therefore, the relevance of FZ as a potential anti-cancer drug is increased by proteasomal inhibition linked to a mitosis-selective treatment strategy. (Shen et al., 2008)
It is well known that benzimidazole compounds may disrupt the host’s ability to use carbohydrates for energy. They prevent the absorption of glucose and the production of ATP, which eventually results in the parasite’s demise. For a very long time, it has been known that normal and cancer cells have different energy metabolisms. In many malignant cell lines, glucose consumption is often greater than normal. Therefore, the inhibition of glucose absorption has been used as a therapeutic strategy. The fact that FZ could successfully prevent glucose absorption in NSCLC cells suggests that inhibiting glucose uptake by cancer cells contributes to the FZ-induced cancer cell death. (Aft et al., 2002)
Certain substances, such as dichloroacetic acid, and 3-bromopyruvate, have been shown to increase oxidative phosphorylation and decrease glycolysis, which has been linked to the death of tumor cells. Since cancer cells, in contrast to healthy cells, depend heavily on enhanced glycolysis to produce ATP, disruption of this process might have lethal effects on only these cells. Additionally, it has been proposed that free tubulin controls mitochondrial activity in cancer cells but not in primordial cells that have not transformed, altering the potential of the mitochondrial membrane. As a result, drugs that target microtubules and have the power to alter the glucose metabolic pathways may be very effective against cancer. Hexokinase (HK II) and Glut-4 transporter expression were downregulated after exposure to FZ, which may impact p53 activation and microtubule dynamics. Key glycolytic enzyme hexokinase II works with GLUT transporters to enhance glycolysis and suppresses mitochondria-induced apoptosis52. Therefore, targeting this important enzyme is being researched as a potential way to stop cancer cell proliferation successfully. Proline oxidase, SCO2, TIGAR, and glutaminase 2 (GLS2) are other p53 target genes reported to be activated by FZ therapy and are all involved in controlling cellular metabolism. As a result, FZ-induced tubulin depolymerization and p53 activation may also influence the glycolytic pathway and glucose absorption. (Liu et al., 2009)
Our research demonstrates that FZ-induced preferential elimination of cancer cells occurs both in vitro and in vivo via a combination of microtubule disruption, p53 stability, and interference with glucose metabolism.
In conclusion, our studies did not find any evidence that fenbendazole merits further investigation as a potential agent for use in cancer therapy, even though its mechanisms of action overlap with those of the hypoxia-selective nitroheterocyclic cytotoxins and radiosensitizers, the taxanes, and the vinca alkaloids. Related compounds, however, may very well turn out to be effective anti-cancer medications. It may be worthwhile to investigate other antihelminthic created in the past to determine whether this class of agents includes compounds that might be useful in cancer therapy, either because of their significant differential effects on hypoxic cells or through their effect on other cells. This is important because the US Food and Drug Administration is currently interested in examining the possibility of “repurposing” previously approved drugs with well-defined characteristics and good toxicology data for new uses. Agents with higher intestinal absorption (and consequently higher host toxicity) that were not pursued use as antiparasitic agents may offer new anticancer agents or new lead compounds useful in creating novel anticancer drugs.
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