This is truly incredible. Had to share! This is truly one of the most promising things I've ever seen, in my cancer research. This man, Joe Tippens, was told his body was riddled with cancer - in his lungs, neck, liver, pancreas and bones. He was told he would die in 3 months.
I read on this great Rex Research page of research about Fenbendazole, that it can be very effective as an anti-fungal. Well, it has been theorized that cancer is actually a FUNGUS (you can read more about cancer being theorized as being a fungus, on THIS PAGE). So, to me, it makes sense that it could work in some cancers.
From RexResearch:
"...when tested against Cryptococcus neoformans (an encapsulated fungal organism that can cause disease such as meningoencephalitis in immunocompromised hosts), it has been shown that Fenbendazole was more active than Mebendazole or other drugs against this opportunistic fungus."
I also can't help if it might have some kind of effect on the pancreas, or pancreatic enzymes (since the first 3 letters in the medication are "pan"). If you haven't already read about the Trophoblastic theory of cancer, please check it out on my page about Dr. Nick Gonzalez - it is fascinating, and if you listen to Dr. Gonzalez' research on Dr. Beard's work, you'll see, it makes a lot of sense!
Please watch the video below, and then visit Joe's site, here: https://www.mycancerstory.rocks/single-post/2016/08/22/Shake-up-your-life-how-to-change-your-own-perspective
September 2025 update: it appears that Joe's site it's currently inaccessible, but there is an archived copy on the wayback machine!
Update 1-9-19
I just watched another video interview with Joe Tippens and learned even more. It was a huge relief for me to hear that Fenbendazole is actually inexpensive to make, and you can get it in Canada and Mexico for less than $5. So, even if Big Pharma tries to jack up the price to over $500 (which according to Joe, they already tried to do once), I can take that $500 to go across the border to get it myself in a neighboring country!
Here's the video:
This is the de-wormer he took (please be sure to read his webpage also.... again, he took CBD and Vitamin E oil in addition to this, but seems to feel that this was the main thing that helped).
Panacur C Canine Dewormer (fenbendazole), 1 gram
https://www.hopkinsmedicine.org/news/publications/doorways_to_discovery/files/sebindoc/o/i/E20FF6E7CB00F25F1ED9927C2946478F.pdf
Research on goats, quail and rats
Comparative studies on the effect of fenbendazole on the liver and liver microsomal enzymes in goats, quail and rats.
Department of Physiology and Pharmacology, School of Veterinary Medicine, Tuskegee University, AL 36088.
Abstract
To compare the effect of fenbendazole on the liver and liver microsomal mono-oxygenases of goats, quail and rats, an oral dose of 25 mg/kg was administered to the animals daily for 9 consecutive days. On the tenth day, blood samples and livers were collected from both the control and the treated animals for preparation of serum and microsomes respectively. Determination of the activities of sorbitol dehydrogenase (SDH, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum samples showed that there was no significant increase in the activities of these enzymes in the treated animals as compared to their corresponding controls, suggesting no liver damage. Similarly, no significant difference in the amount of microsomal cytochrome P-450 was found between the control and the treated animals of the same species. Compared to their respective controls, the activities of microsomal benzphetamine N-demethylase and aniline hydroxylase were almost unchanged in the treated goats and rats. The results indicate that fenbendazole is not liver toxic to goats, quail or rats at a dose rate of 25 mg/kg.
In a 150 lb human, this would equate to 1,700mg per day versus the 222mg per day per my protocol (a 120 lb woman would equate to 1,350mg vs 222mg recommended here.)
(just a note, it looks like, as per joe's protocol of 222 mg per day, that equates to about 1.85 mg per pound.. at least from the math i did. do your own to be sure!I am buying this for my 16 pound cat, and as per joe's protocol i think i am going to give him 29.6 mg daily). I believe the 5 pack they sell on amazon would be equivalent to 30 grams, or 30,000 mg. So if I just estimate my cat needs 30 mg (close enough to 29.6 mg) that means one 5-pack order should be over a 2-year supply. I could be wrong, but that is what I calculated).
3. Malignancy in the liver is a wholly different subject matter than effects of Fenben on liver function, and the two should not be confused. Many people are having success with tumors in the liver without effecting the liver enzymes.
4. Of course anyone with a preexisting condition in the liver should be careful and have their blood checked more often than most.
MOST IMPORTANTLY:
5. As hundreds of people have now taken this drug for extended periods of time, we have learned a lot that I can now pass on.
A. "Many people, to my surprise and without my knowledge, have chosen to self dose at much higher levels than I originally recommended, not only in mg/day but also in number of consecutive days. I love it that people can be so brave and bold as to experiment with their own bodies, but it shows you that when people are given "no hope" by traditional methods, their risk tolerance somehow takes a huge leap of faith. And these people that self dosed at much higher levels have shown amazing results, while not reporting any additional or unusual side effects"
B. Most people are now jumpstarting the process with 7 days a week for the first 2-4 weeks, while a few people have jumpstarted with 7 days a week long term. None of those people have reported any new or unusual or added side-effects.
C. Many people are self administering at higher than 222mg per day, and to my knowledge, none of those people have reported any new or unusual or added side effects.
D. There have been a few people report elevated liver enzymes, so everyone needs to decide for themselves their dosage increases over the original recommendation, and everyone needs to decide for themselves the level of increased blood work for peace of mind.
With my usual disclaimer of "I am not a scientist or doctor", I believe it is time for me to state the following:
Many people have successfully jumpstarted for 3 weeks at 7 days a week, and many people have successfully increased the daily intake from 222mg to 2x, 3x and 4d that level. While I am not recommending any specific "higher dosage", I think the evidence from many people indicates each individual should make their own decision on higher dosages and consider what is best for them. At the end of the day, I believe it is what each individual can tolerate, but I believe most people can probably tolerate more than 222mg 3 days a week.
Formula | multiply the mass value by 1000 |
- Article
- Open Access
- Published:
Fenbendazole acts as a moderate microtubule destabilizing agent and causes cancer cell death by modulating multiple cellular pathways
Scientific Reports volume 8, Article number: 11926 (2018) Cite this article
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Abstract
Drugs that are already clinically approved or experimentally tested for conditions other than cancer, but are found to possess previously unrecognized cytotoxicity towards malignant cells, may serve as fitting anti-cancer candidates. Methyl N-(6-phenylsulfanyl-1H benzimidazol-2-yl) carbamate [Fenbendazole, FZ], a benzimidazole compound, is a safe and inexpensive anthelmintic drug possessing an efficient anti-proliferative activity. In our earlier work, we reported a potent growth-inhibitory activity of FZ caused partially by impairment of proteasomal function. Here, we show that FZ demonstrates moderate affinity for mammalian tubulin and exerts cytotoxicity to human cancer cells at micromolar concentrations. Simultaneously, it caused mitochondrial translocation of p53 and effectively inhibited glucose uptake, expression of GLUT transporters as well as hexokinase (HK II) - a key glycolytic enzyme that most cancer cells thrive on. It blocked the growth of human xenografts in nu/nu mice model when mice were fed with the drug orally. The results, in conjunction with our earlier data, suggest that FZ is a new microtubule interfering agent that displays anti-neoplastic activity and may be evaluated as a potential therapeutic agent because of its effect on multiple cellular pathways leading to effective elimination of cancer cells.
Introduction
The importance of microtubules in cell division, motility, intracellular trafficking and their role in modulating cellular shape according to the environment has made them one of the most successful targets of anticancer therapy. Agents that perturb the microtubule dynamics have been widely used in cancer treatment1,2,3,4. Considering the relative success of mitotic agents in the treatment of cancer, microtubules may be termed as one of the best cancer targets identified till now5.
Microtubule targeting agents can be broadly classified into two major classes. The first class consists of microtubule-destabilizing agents, which inhibit microtubule polymerization. This class of anti-mitotic drugs includes several compounds such as the vinca alkaloids (vinblastine, vincristine, vinorelbine, vindesine, vinflunine), estramustine, colchicine and combretastatins, that are being used clinically or are under clinical investigation for cancer treatment. The second class is comprised of microtubule-stabilizing agents. These agents include paclitaxel, docetaxel, epothilones, and discodermolide6. The consequence of disrupting tubulin and microtubule dynamics with both these classes of drugs in dividing cells is metaphase arrest and induction of apoptosis.
Fenbendazole (methyl N-(6-phenylsulfanyl-1H-benzimidazol-2-yl) carbamate) is a broad-spectrum benzimidazole anthelminthic approved for use in numerous animal species7. Repurposing of veterinary drugs showing promising results for human use can result in considerable time and cost reduction required to develop new drugs. Fenbendazole is known to have a high safety margin and most species tolerate it very well. It has very low degree of toxicity and high degree of safety in experimental animals8,9,10,11,12. In this study, we show that fenbendazole (FZ) exhibits a moderate microtubule depolymerizing activity towards human cancer cells, but possesses a potent antitumor effect as evident from in vitro and in vivo experiments. Our results indicate that FZ exerts its antitumor effect through the disruption of microtubule dynamics, p53 activation and the modulation of genes involved in multiple cellular pathways. FZ treatment also resulted in reduced glucose uptake in cancer cells due to down regulation of GLUT transporters and key glycolytic enzymes.
Since the process of tumorigenesis involves a number of genes and proteins altering various cell signaling pathways, single-target drugs show limited efficacy and may lead to drug resistance13,14,15. Agents having multiple cellular targets, therefore, are expected to have improved efficacy besides the ability to circumvent the likelihood of developing resistance.
Overall, the present work demonstrates a pleiotropic effect of FZ on cancer cells leading to cell death. Thus, FZ may have a potential therapeutic application.
Results
FZ destabilizes tubulin network in human NSCLC cells
Benzimidazole carbamates have been reported to inhibit tubulin polymerization and disrupt microtubule function in parasite cells16,17. Results from in vitro studies using enriched extracts of helminthic and mammalian tubulin have suggested that tubulin is the primary molecular target of the benzimidazoles18. Therefore, to examine the effect of FZ on mammalian microtubule network organization, human non small cell lung carcinoma (NSCLC) A549 cells were treated with 1 uM FZ for 24 h and processed for immunofluorescence using α tubulin antibody. Colchicine was used as a positive control. Results showed that FZ treatment caused a partial alteration of the microtubule network (Fig. 1a). The microtubule cage around the nucleus appeared to have lost its intactness when compared with the control mock treated cells. However, this modification in the organization was not as marked as in case of colchicine treatment, which showed complete depolymerization of microtubules into tubulin subunits. This data suggests that FZ causes distorted microtubule framework of the cells.
FZ treatment alters tubulin network of human cancer cells. (a) A549 cells were treated with 1 uM FZ or 50 ng/ml colchicine for 24 h. Following treatment, the cells were processed for immunofluorescence using anti α-tubulin primary and FITC conjugated secondary antibodies. (Nuclei were counter stained with propidium iodide) (b) bovine tubulin (1.8 mg/mL) was incubated with DMSO (control), FZ (10 uM) or colchicine (100 nM) and the effect on polymerization was monitored spectrophotometrically by measuring turbidity at 340 nm as described under “Methods.” (c) Cells were treated with FZ, nocodazole, taxol or colchicine for 24 h and then lysed and fractionated into soluble (S) and polymerized (P) extracts. The extracts were separated with SDS-PAGE, transferred onto PVDF membranes and probed with both anti-α-tubulin and anti-β-actin antibodies. A representative immunoblot analysis in A549 cells is shown. (d) Intensity of each band of the immunoblot was measured by the NIH ImageJ program, and the ratios of soluble and polymerized tubulin and β-actin in each treatment were calculated. (e) Cells were treated with different MTAs as indicated for 24 h and western blotting was then performed using Ac-α-tubulin (6–11B-1) specific and β-actin antibodies. (Full-length uncropped blots are included in Supplementary Fig. S6).
The effect of FZ on tubulin polymerization was further evaluated by an in vitro assay. Purified bovine tubulin was incubated with FZ, and tubulin polymerization was recorded over time. The results showed mild inhibition of tubulin polymerization by FZ in vitro which was not as pronounced as in case of colchicine treatment. (Fig. 1b)
Next, the effect of FZ on tubulin polymerization was compared with that of other microtubule destabilizing agents like nocodazole and colchicine. Polymerized and soluble fractions were prepared after 24 h drug treatment and western blot was performed using α-tubulin and β-actin antibodies (Fig. 1c). Tubulin bands of polymerized and soluble fractions were quantified after normalization with their respective β-actin bands which served as an internal control (Fig. 1d). There was a modest decrease in polymeric tubulin in FZ treated cells as compared to control untreated cells, whereas polymerized form of tubulin was nearly absent in colchicine treated cells. The result confirms the relatively mild tubulin depolymerising activity of FZ as compared to other known microtubule disrupting agents like nocodazole and colchicine.
A major limiting factor of taxanes and vinca alkaloids is their dose-limiting toxicity and susceptibility to multidrug drug-resistance (MDR) occurring commonly due to the high expression of p-glycoprotein (p-gp; MDR1)19,20. Overexpression of β-tubulin isoforms and mutations are also known to confer resistance to taxanes21. Unlike taxanes and vinca alkaloids, agents targeting the colchicine-binding site are advantageous in that they show minimal multidrug resistance in addition to their ability to overcome the effect of β-tubulin isoforms’ overexpression22,23,24. However, the major drawback of colchicine and its derivatives is their acute toxicity to humans22,25. Therefore, a microtubule inhibitor that binds to the colchicine-binding site but has low toxicity can be highly efficacious26,27. The result of a fluorescence based competitive colchicine binding assay suggests that FZ may bind to the tubulin at the colchicine binding site (Fig. S1).
Tubulin acetylation has been associated with the stability of microtubules. Therefore, to examine the acetylation status of tubulin following treatment, human NSCLC cells were treated with different microtubule targeting agents for 24 h and the cell extracts were subjected to western blot analysis using Ac-α-tubulin specific antibody (6–11B-1). As shown in Fig. 1e, while nocodazole, colchicine and vincristine resulted in a marked reduction of acetylated tubulin, FZ did not alter the amount of acetylated tubulin as compared with control mock treated cells. This result further confirmed the relatively mild effect of FZ on mammalian tubulin as compared with other known microtubule depolymerizing agents.
FZ is not a P-gp substrate or inhibitor
Development of drug resistance is a major concern in cancer treatment. Multidrug resistance (MDR) caused by the overexpression of the MDR-1 gene that encodes P-glycoprotein (P-gp) is a critical mechanism of drug resistance which results in a cross-resistance to multiple classes of drugs28,29. A large number of commonly used chemotherapy drugs like taxanes and vinca alkaloids are P-gp substrates30. However, efforts to inhibit P-gp have not shown encouraging results due to unavoidable side effects31,32. Therefore, discovery and development of novel anti-proliferative compounds that are not substrates of P-gp is an effective approach to overcome drug resistance.
To test whether FZ is a substrate or inhibitor of P-gp, we investigated cancer cell growth inhibition by FZ in the presence of P-gp inhibitor verapamil. The results showed that inhibition of P-gp by verapamil did not enhance the inhibitory effect of FZ on cancer cell proliferation (Fig. 2c). The fluorescent dye rhodamine 123 (Rho123) is a well-known reference P-gp substrate frequently used to determine the P-gp inhibitory potential of drugs33. No significant difference in Rho123 accumulation was observed between control untreated and FZ treated cells, implying the absence of any interaction of FZ with P-gp. (Fig. 2a,b) In the presence of verapamil, the treated and untreated cells showed comparable levels of Rho123 accumulation affirming that FZ is not a substrate or inhibitor of P-gp.
P-gp inhibition has no effect on FZ mediated cell death. (a) A549 cells were left untreated or treated with 10 uM FZ or 10 uM Verapamil for 6 h. Rh123 was then added and fluorescence images were acquired after washings with PBS as described under “Methods”. (b) The cells were treated as before and the fluorescence was measured at Ex507/Em529 using a Tecan multimode plate reader. (c) A549 and H460 cells were treated with 1 uM FZ in the absence or presence of 10 uM verapamil for 24 h. Cell proliferation was then measured by MTT assay.
FZ treatment results in early G2/M block accompanied by cell death
Since inhibition of tubulin polymerization blocks cell-cycle progression and may induce mitotic catastrophe, the effect of fenbendazole on the cell cycle progression was examined. A549 cells were synchronized by serum starvation for 48 h and treated with 1 uM FZ for different time intervals. During cell division, cyclin B1 binds to cyclin-dependent kinase 1 (CDK1) allowing the transition from G2 to mitosis. Thereafter, progression from metaphase to anaphase requires ubiquitination and proteasome mediated degradation of cyclin B1 induced by anaphase-promoting complex (APC)34. Our results show an early elevation of cyclin B1/CDK1 levels in FZ treated cells (8 h as compared to 16 h in case of control untreated cells) (Fig. 3a). In addition, histone pH3 (Ser10), an indicator of mitotic progression, was used to evaluate whether FZ induced cell cycle arrest specifically in the mitosis phase. As seen in Fig. 3b, p-histone H3 (Ser10) was found to be up-regulated at 12 and 24 h post FZ treatment in A549 cells. This data confirms that FZ causes cell cycle arrest in the mitotic phase in human NSCLC cells. Cells in different phases of cell cycle and those undergoing apoptosis were quantified by flow cytometry. The number of apoptotic cells increased in a time dependent manner with simultaneous decrease in cyclin B1 levels, and ~30% cells had undergone apoptosis after 32 h of FZ treatment (Fig. 4a). The cyclin B1 levels went down further at 40 and 48 h of treatment, corresponding to a further increase in the percentage of apoptotic cells. Thus, a decrease in cyclin B1 levels, with a concomitant increase in apoptotic cell death suggests that A549 cells apparently underwent mitotic exit followed by cell death in response to FZ treatment.
FZ causes early elevation of cyclin B1 levels and induces mitotic arrest. a(i) & a(ii) A549 cells were synchronized by serum starvation for 48 h and then left untreated or treated with 1 uM FZ or 50 nM colchicine for the indicated time intervals. The cell extracts were then processed for western immunoblotting using cyclin B1, cdk1 and β-actin antibodies. b(i) & b(ii) A549 cells were treated with 1 uM FZ for the indicated time intervals and the extracts were then processed for western blotting using pH3 and β-actin antibodies. The bands were quantitated using ImageJ software. (Full-length uncropped blots are included in Supplementary Fig. S6).
Mitotic arrest followed by cell death in response to FZ in human NSCLC cells. (a and b) Cells were treated after synchronization as before and FACS analysis was done to detect the fraction of populations in different phases of cell cycle, as well as apoptotic cells. (c) H460 cells were treated with 1 uM FZ for 24 h and cell morphology was observed under phase contrast microscope (i & ii) or after Hoechst 33342 staining under fluorescence microscope (iii & iv). TdT staining was done to detect apoptotic nuclei (v & vi).
While a significant apoptotic population (sub-G1 phase) was observed after 32 h FZ treatment, in contrast, colchicine treated cells underwent polyploidy at the same time point (Fig. 4b). FZ enforced cells to accumulate in mitosis, leading to a dramatic increase in apoptosis without evidence of progression to a G1-like status. Figure 4c shows apoptotic cells following 24 h FZ treatment. Overall, this data suggests that FZ causes cell-cycle arrest and mitotic cell death, consistent with its effects as a microtubule inhibitor. In our earlier work, we reported the absence of any effect of broad spectrum caspase inhibitor Z-VAD-FMK on FZ induced cell death35. The present data suggesting mitotic cell death caused by FZ offers a possible explanation for this result since mitotic cell death can be caspase independent and is known to remain unaffected by the caspase inhibitor Z-VAD-FMK36,37.
Tumour cell lines with wild-type p53 show enhanced sensitivity to FZ induced apoptosis
Treatment of H460 and A549 human NSCLC cell lines with 1 uM FZ significantly reduced cell growth as determined by MTT assay (Fig. 5a). Tumour cell lines having wild-type (WT) p53 appeared to be highly sensitive to FZ action as compared to p53 mutant or null cells (Fig. 5b), indicating enhanced apoptosis inducing activity of FZ in the presence of WT p53. Consistent with this data, p53 null H1299 cells exposed to FZ following transient transfection with WT p53 construct showed enhanced apoptotic cell death (Fig. 5c). Remarkably, FZ showed less toxicity towards primary epithelial cells cultured from rat lung tissue as compared to a lung cancer cell line (Fig. 5d). Wild-type p53 target genes were induced upon FZ treatment (Fig. 6a), suggesting that FZ induced WT p53 protein is transcriptionally active. However, FZ could also induce p21 independent of p53 (Fig. 6b,c), which may be via its effect on the proteasomal pathway as reported earlier35. Dose dependent FZ treatment led to increased nuclear accumulation of WT p53 in these cells which correlated well with enhanced apoptotic activity (Fig. 6d). Tubulin acetylation has been reported to enhance stress-mediated translocation of p53 to the nucleus where it is transcriptionally active38,39. Hence, enhanced p53 nuclear accumulation is concurrent with earlier data showing steady levels of acetylated tubulin in FZ treated cells. Altogether, evidence supports a proactive role of transcriptionally active p53 in augmenting cell death following mild microtubule disruption by FZ.
FZ mediated inhibition of cancer cells in vitro is affected by p53 status. (a) Human H460 or A549 cells were plated onto 96-well tissue culture plates. Cells were left untreated or treated with different doses of FZ for 48 h. Cell viability was measured by MTT assay. (b) H460, A549, H522 or H1299 cells were treated with 1 uM FZ for 48 h and cell viability was measured by MTT assay. (c) H1299 cells were transfected with p53 expression construct or with vector alone. After 16 h of transfection, cells were left untreated or treated with FZ for 24 h following which cell viability was measured by MTT assay. (d) H460 cells or primary lung epithelial cells derived from rat lung tissue were treated with 1 uM FZ for different time points as indicated. Cell viability was determined by MTT assay. The significance level was set at p < 0.05. (*p < 0.05)
Apoptosis and p53 induction in human NSCLC cell lines following FZ treatment. (a) Western blot was performed for p53 target genes after 24 h of FZ treatment in H460 cells. (b) H1299 cells were transiently transfected with p53 expression construct and left untreated or treated with FZ for 24 h. Western blot was then done for p53 and p21. (c) Quantitation of “b” using Image J software. (Full-length uncropped blots are included in Supplementary Fig. S6) (d) immunostaining for p53 in H460 cells after treatment with increasing concentrations of FZ for 24 h.
FZ induces p53 mitochondrial translocation
We earlier reported decreased mitochondrial membrane potential, release of cytochrome c and PARP cleavage in cells undergoing apoptosis in response to FZ treatment. It was shown that FZ treatment resulted in accumulation of p53 protein mainly due to inhibition of proteasomal degradation, and an accumulation of the ubiquitylated form of p53 was also observed35. We, therefore, looked at the mitochondrial levels of p53 when NSCLC cells were exposed to the drug. For this p53 null H1299 cells were transfected with GFP-p53 construct and its subcellular distribution was examined following treatment with different microtubule targeting agents for 24 h (Fig. 7a). Western blot for p53 showed an increased level of p53 protein in the mitochondrial fraction following FZ treatment (Fig. 7b,c). Additionally, the p53 translocated to mitochondria in response to FZ treatment appeared to be monoubiquitinated, indicating that the increased pool of monoubiquitinated p53 in FZ treated cells is translocated to mitochondria resulting in the mitochondrial cell death pathway in these cells40 (Fig. S3). Correspondingly, mitochondrial membrane depolarization was also observed in FZ treated cells using JC1 voltage sensitive dye (Fig. 7d).
FZ treatment results in increased p53 translocation to mitochondria. (a) H1299 cells were transiently transfected with GFP-p53 expression construct and treated as before. They were then stained with red mitotracker dye (Molecular Probes) and fluorescent images were acquired using a Nikon fluorescence microscope. (b) H460 cells were treated with 1 uM FZ, 500 nM nocodazole or 100 nM taxol for 24 h. After treatment, mitochondria were isolated using a mitochondria isolation kit from Sigma. Whole cell lysates (WC), cytosolic (C) and mitochondrial (M) fractions were then resolved on SDS-PAGE and subjected to western blot analysis using anti p53, β-actin and COX IV antibodies. (c) The band intensities from “a” were quantified using Image J software and normalized with β-actin (WC and C) or COXIV (M) levels. (Full-length uncropped blots are included in Supplementary Fig. S6) (d) H460 cells were either left untreated or treated with 1 uM FZ for 24 h and then processed for JC-1 staining (i, control; ii, FZ).
Inhibition of glucose uptake by FZ sensitizes cancer cells to undergo apoptosis
In studies with parasites, the anthelmintic effects of the benzimidazoles have been related to inhibition of glucose uptake with resultant alterations in glucose metabolism41. We tested the effect of FZ on glucose uptake in human cancer cells. H460 and A549 cells were treated with 1 uM FZ for 4 h and uptake of fluorescent glucose analogue 2-NBDG was observed. FZ treatment resulted in inhibition in glucose uptake in both the cell lines (Fig. 8a). Similar results were obtained when a glucose oxidation assay was performed using culture supernatants from cells treated with increasing concentrations of FZ (Fig. 8b). Expectedly, FZ treatment also resulted in reduced lactate levels (Fig. 8c). Hence, FZ induced cell death appeared to be related to inhibition of glucose uptake.
FZ alters glucose uptake and impairs enzymatic activity of HKII in NSCLC cells. (a) A549 or H460 cells were treated with 1 uM FZ for 4 h and uptake of the fluorescent glucose derivative 2-NBDG was examined thereafter by fluorescence microscopy as described. Representative images of cells from three independent experiments are shown. (b) Human NSCLC H460 cells were exposed to increasing doses of FZ for 24 h. Culture supernatants were then used to assess glucose consumption by glucose oxidation assay using GO assay kit from Sigma. (c) H460 cells were left untreated or treated with 1 uM FZ and the lactate levels in culture supernatants were assessed after the indicated time points using Lactate Assay Kit from BioVision. (d) Human H460 cells were exposed to 1 uM FZ for 24 or 48 h as indicated, total RNA was isolated and RT-PCR was performed using primers specific for the indicated genes. (e) (i) H460 cells were left untreated or treated with 1 uM FZ for 24 h and the cell extracts were then processed for a spectophotometric assay for SDH (A630nm). (ii) A549 cells were left untreated or treated with 1 uM FZ for 20 h following which they were processed for histochemical assay to assess SDH activity. Cells were then observed under a microscope and images were acquired at 40X magnification. (f) H460 cells were left untreated or treated with 1 uM FZ for 24 h. HK enzymatic activity was then determined spectrophotometrically as described under “Materials and Methods”. (g) H460 and A549 cell lysates were incubated with DMSO or FZ for 15 min prior to initiation of reaction. HK enzymatic activity was then determined spectrophotometrically as described under “Materials and Methods”. (h) Purified HKII from S. cerevisae was incubated with increasing doses of FZ and HK activity was then measured spectrophotometrically. (*p < 0.05, **p < 0.01, ***p < 0.005).
Since an increase in soluble tubulin, and both p53 induction and NFκB downregulation [reported earlier35], have been linked with glucose metabolism, the expression of genes involved in the process was examined. An increase was observed in the mRNA levels of Glutaminase 2 and Proline oxidase 48 h after FZ treatment while GLUT-4 and Hexokinase II showed considerably reduced expression. p53 inducible pro-apoptotic genes, TIGAR and SCO2 were also induced to an appreciable extent following FZ treatment (Figs 8d and S4).
Hexokinase II (HKII), a key glycolytic enzyme, plays a critical role in glucose retention and metabolism and is highly advantageous for cancer cell survival and proliferation. Therefore, we examined whether FZ impairs the enzymatic function of HKII in cancer cells. An enzymatic assay for HKII activity was performed after treating NSCLC cells with FZ for 24 h (Fig. 8f). The results showed reduced HKII activity in FZ treated cells, suggesting the inhibition of HKII activity to be a causative factor for reduced glucose uptake in FZ treated cells, eventually leading to the activation of apoptotic signals. In-silico models indicated that this action may be due to the ability of FZ to mimic glucose or glucose-6-phosphate (G6P) by stably binding to its pocket in HKII (Fig. S5 and Table S1). The result was further validated by in vitro hexokinase assays (Fig. 8g and h).
Various benzimidazole compounds have been shown to be highly effective as inhibitors (up to 50% reduction of activity) of the helminth-specific enzyme fumarate reductase in vitro. While fumarate reductase converts fumarate to succinate in microbes and lower organisms, succinate dehydrogenase catalyzes the oxidation of succinate to fumarate in mammalian mitochondria. The two enzymes, therefore, act on the reverse directions of the same enzymatic interconversion. Succinate dehydrogenase (SDH) is a mitochondrial tricarboxylic acid (TCA) cycle enzyme and a known tumour suppressor gene42. Succinate, a TCA cycle metabolite, is accumulated due to SDH downregulation and provides cancer cells with a growth advantage leading to tumour progression42. Spectrophotometric assay as well as histochemical staining43 showed enhanced SDH activity in cells following FZ treatment (Fig. 8e i and ii).
Combination effect of FZ with other drugs
Finally, we evaluated the effect of FZ in combination with the microtubule targeting drug taxol, glycolytic inhibitor 2 deoxyglucose (2DG) and dichloroacetate (DCA) - a pyruvate dehydrogenase kinase inhibitor which acts by shifting the metabolism towards glucose oxidation over glycolysis. In order to determine whether FZ could show an additive or synergistic effect with these drugs, cell proliferation assays were performed and the combination effects were analyzed using the combination index (CI) method44. The Fa-CI plot for the combinations showed that the CI values were <1 over the entire range for combination with DCA, CI value was 0.04 at the 50% effective dose, suggesting a strong synergism by FZ and DCA. (Fig. S2-a) Similarly, CI values for the FZ-2DG combination were also less than 1 for the higher doses with a CI of 0.21 at the Fa value 0.5. (Fig. S2-b). CI <1 was also observed at most doses of FZ-Taxol combination (CI-0.52 at 50% inhibition). (Fig. S2-c). Since CI <1 represents synergism as per the CI method, we conclude that FZ shows synergistic effect with DCA, 2DG and over a range of doses with taxol.
FZ effectively inhibits colony formation of human NSCLC cells in culture
The effect of FZ on cancer cells in vitro was examined by the colony forming ability of A549 and H460 cells in culture. Treatment of these cells with 1 uM FZ for 48 h resulted in significant reduction in number of colonies as compared to control untreated cells (Fig. 9a). Further, anchorage independent growth of control and FZ treated H460 cells was evaluated by soft agar assay. Results of the soft agar assay correlated with colony formation data (Fig. 9b,c) suggesting that FZ is a potential antineoplastic agent that kills cancer cells in vitro.
FZ treatment results in reduced tumorigenicity in vitro and in vivo. (a) Colony formation assay for H460 and A549 cells following treatment with 1 uM FZ for 48 h. (b) Soft agar assay following FZ treatment. Cell colonies were counted after staining with 5% crystal violet (c). (d) Tumors were established in nu/nu mice by subcutaneous injection of 5 × 106 A549 cells. After the tumors were 2–4 mm in diameter, the mice were orally fed with FZ dissolved in olive oil (1 mg/mouse) every second day, whereas control animals received olive oil only. Tumor volumes were then calculated by measuring tumor dimensions using Vernier callipers. (e and f) Tumors were excised, photographed and weighed. (g) Tumor vascularity in vivo was quantified between control and FZ treated mice by measuring hemoglobin spectrophotometrically (A590nm). (h) Tumor sections from control untreated and FZ treated mice were processed for TdT staining to identify apoptotic cells (i & iv), sections were immunostained using p53 specific antibody (ii & v), and immunohistochemistry was performed on sections for CD31 (iii & vi). i, ii & iii are sections from control mock treated mice and iv, v & vi are sections from FZ treated mice (*p < 0.05).
In vivo tumour suppression by FZ treatment
The therapeutic activity of FZ in vivo was examined by giving oral doses to nude mice bearing A549 xenografts. Female athymic nu/nu mice were xenografted with A549 cells and mice bearing tumours (2–3 mm) were fed with FZ (1 mg/mouse) orally every second day for 12 days (Fig. 9d). At the end of 12 days, tumours were excised, measured and weighed. FZ administration led to a marked reduction in tumour size and weight (Fig. 9e,f). Further, the tumor vascularity was quantified in control and FZ treated mice by spectrophotometrically measuring hemoglobin content (A590nm). FZ administration led to a reduction in hemoglobin content in tumors signifying reduced tumor vascularity (Fig. 9g). TUNEL staining of tumour sections showed a significant number of apoptotic cells (Fig. 9h i & iv). These results suggest that FZ inhibits tumour cell growth in vivo by inducing apoptosis of tumour cells. When tumour sections were further examined for p53 protein expression, a number of p53 positive tumour cells were visible in FZ treated mice suggesting p53 induced cell death (Fig. 9h ii & v). Moreover, FZ treated A549 tumours showed very few CD31 positive endothelial cells in xenografts (Fig. 9h iii & vi). These data are in good agreement with our in vitro analysis of FZ mediated cell death.
Discussion
Microtubules are major components of the cytoskeleton and play important roles in a variety of cellular processes like intracellular trafficking, maintenance of cell shape and structure, polarity, cell signalling, and mitosis. Microtubule targeting agents (MTAs) are being used clinically for the treatment of multiple tumour types, but their effectiveness is often largely affected by drug resistance mechanisms. Although the principal mechanism of MTAs’ cytotoxicity relates to their interactions with tubulin and disruption of microtubule function, differences in their tubulin binding characteristics alter their modes of action with a significant impact on the efficacy or toxicity profile of each agent. Newer agents with improved efficacy, tolerability, and the ability to even partially overcome resistance could be of great significance.
The present data reveals FZ as a moderate microtubule targeting agent causing mitotic arrest followed by cancer cell death. Despite being a relatively mild microtubule targeting agent, FZ possesses a unique ability to induce p53 to a considerably high level.
In our earlier work we have shown the activity of FZ as a proteasomal interfering agent35. The present study revealed an early elevation and stabilization of cyclin B1 levels in response to FZ, indicating a progression of cells towards cell death rather than mitotic exit and polyploidy. Interestingly, the FDA approved proteasomal inhibitor bortezomib has also been reported to induce mitotic cell death in lymphoma cells45. Therefore, proteasomal inhibition associated with mitosis-selective therapeutic approach adds to the significance of FZ as a potential anti-cancer agent.
Benzimidazole compounds are known to interfere with energy metabolism of the host, particularly, the carbohydrate metabolism. They block the glucose uptake and ATP formation which ultimately leads to the death of the parasite. Disparity in energy metabolism between normal and cancer cells has been well known for a long time and in many malignant cell lines glucose consumption is several folds higher than in normal cells. Deprivation of glucose uptake, therefore, has been exploited as a therapeutic approach46. FZ could effectively inhibit glucose uptake in NSCLC cells suggesting that FZ induced cancer cell death is, in part, facilitated by blocking glucose uptake of cancer cells.
Enhancement of oxidative phosphorylation and/or inhibition of glycolysis by certain agents (e.g., dichloroacetic acid, 3-bromopyruvate) have been reported to result in tumor cell death47,48. Since unlike normal cells, cancer cells mostly thrive on increased glycolysis for generation of ATP, impairment of this pathway could lead to fatal consequences specifically for these cells. Also, it has been postulated that free tubulin regulates mitochondrial function in cancer cells but not in non-transformed primary cells, via alteration of mitochondrial membrane potential49. Therefore, microtubule targeting agents having the ability to affect glucose metabolic pathways can be immensely beneficial as anti-cancer agents. FZ exposure reduced the expression of Glut-4 transporter as well as hexokinase (HK II), which may be linked to p53 activation and alteration of microtubule dynamics50,51. Hexokinase II is a key glycolytic enzyme, which, besides acting to promote glycolysis in co-operation with the GLUT transporters, also acts to suppress mitochondria-induced apoptosis52. Targeting this crucial enzyme is therefore being investigated as a possible strategy to effectively curb cancer cell growth. Similarly, proline oxidase53,54, SCO255,56, TIGAR42,57,58 and glutaminase 2 (GLS2)59 are all p53 target genes involved in the regulation of cellular metabolism which were found to be induced following FZ treatment. Therefore, tubulin depolymerization and p53 induction caused by FZ may further be leading to modulation of glucose uptake as well as glycolytic pathway.
Altogether, our findings show microtubule disruption, p53 stabilization and interference with glucose metabolism as collective underlying mechanisms of FZ induced preferential elimination of cancer cells both in vitro and in vivo.
Materials
Fenbendazole (FZ), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra- zolium bromide (MTT), colchicine, taxol, JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazoleocarbocyanine iodide), Hoechst 33342, propidium iodide, anti β-actin, anti-mouse IgG-fluorescein isothiocyanate (FITC), horseradish peroxidase (HRP) conjugated anti-mouse, anti-rabbit and anti-goat IgGs, TRI reagent, purified HKII from S. cerevisae as well as all the cell culture reagents were purchased from Sigma. Nocodazole was obtained from Calbiochem. 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) and MitoTracker were purchased from Molecular Probes. Anti p53 (Bp53-12 and DO1), anti-p21, anti MDM-2, anti α-tubulin, anti Ac-α-tubulin, anti pH3 (Ser10) and anti-cyclin B1 antibodies were purchased from SantaCruz Biotechnology.
Cell lines
All cell lines were procured from NCCS, Pune except H1299 which was kindly provided by Dr. Bert Vogelstein. The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1X penicillin/streptomycin antibiotics (100 u/ml penicillin and 100 ug/ml streptomycin).
Methods
Immunofluorescence for tubulin
Tubulin organization following FZ treatment was visualized by immunofluorescence using anti α-tubulin antibody. Human NSCLC A549 cells were grown on coverslips and treated with 1 uM FZ or 0.05 ug/ml colchicine for 24 h. Following treatment, cells were rinsed twice with PEM-PEG buffer (80 mM PIPES, 1 mM EGTA, 0.5 mM MgCl2, 4% PEG-8000) and permeabilized with PEM-PEG buffer containing 0.05% Triton X-100. The cells were then rinsed with PEM-PEG buffer and quickly fixed in 3% formaldehyde in PEM with 1% DMSO for 30 min at RT. After washing with PEM-PEG buffer, primary antibody (anti α-tubulin) incubation was carried out overnight at 4 °C. After washing with PBS, cells were incubated with FITC-conjugated secondary antibody for 2 h at 37 °C, washed several times, and visualized using a fluorescence microscope. Nuclei were counter-stained red using propidium iodide.
Fractionation of soluble and polymerized tubulin
Separation of soluble and polymerized tubulin fractions from A549 cells was carried out as described by Legault et al.60. After drug exposure, about 5 × 106 cells in 100 mm petridishes were washed with PBS at 37 °C and harvested in 1 ml of PBS containing 0.4 ug/ml of paclitaxel using a rubber policeman. Cells were then centrifuged and lysed using 250 ul of microtubule stabilizing buffer [20 mM Tris-HCl (pH 6.8), 140 mM NaCl, 1 mM MgCl2, 2 mM EDTA, 0.5% NP40 and 0.4 ug/ml paclitaxel] and centrifuged at 12,000 × g for 10 min at 4 °C. The supernatants containing soluble tubulin were mixed with 2X Laemmli’s sample buffer. Pellets containing the polymerized tubulin were resuspended in 250 ul of water, followed by two freeze/thawing cycles and finally resuspended in Laemmli sample buffer. Samples were analyzed by western blot using anti α-tubulin antibody.
Tubulin polymerization assay
Polymerization of bovine tubulin was measured according to Beyer et al.61. Briefly, bovine tubulin (1.8 mg/mL; Sigma) was added to ice-cold polymerization buffer (PEM: 80 mM PIPES, 0.5 mM EGTA, 2 mM MgCl2, 10% glycerol, and 1 mM GTP) and centrifuged at top speed in a microcentrifuge for 5 minutes at 4 °C. Supernatant (100 μL/well) was immediately added to a 96-well plate, which contained 10 uM FZ or dimethyl sulfoxide control in PEM buffer. After addition of tubulin, the plate was immediately placed in the spectrophotometer (Tecan multimode reader), which was maintained at 37 °C, and the absorbance was measured every 5 minutes for 2.5 hours at 340 nm.
Competitive tubulin binding assay
10 uM FZ was coincubated with 3 μM colchicine in PEM buffer containing 3 μM tubulin at 37 °C for 60 min. After incubation, the fluorescence of tubulin-colchicine complex was measured using a Tecan multimode reader at excitation wavelength of 380 nm and emission wavelength of 435 nm. PEM buffer was used as a blank. The raw fluorescence values were normalized by setting the fluorescence of 3 μM tubulin with 3 μM colchicine to 100%.
Rhodamine 123 accumulation assay
Cells were seeded in a 6 well plate and incubated with 0.5 uM FZ for 6 or 24 h with or without 10 μM verapamil. Following day, Rho 123 (10 μM) was added and the cells were further incubated for 1 h at 37 °C in the dark. Cells were then washed thoroughly three times with ice cold PBS and images were acquired using a fluorescence microscope. Alternatively, fluorescence was measured at Ex507/Em529 on a Tecan Infinite M200 multimode plate reader.
FACS analysis
Cells were synchronized by serum starvation for 48 h. After 48 h, the media were replaced with fresh media containing serum and cells were treated with 1 uM FZ for the indicated time intervals. Following treatment, cells were harvested, washed with PBS and fixed in 70% ethanol overnight at 4 °C. Next day, cells were centrifuged at 1000 rpm for 5 min, the supernatant was carefully aspirated and the pellet was resuspended in PBS. The cells were again centrifuged, the supernatant removed and the pellet was finally resuspended in PBS containing 40 ug/ml PI and 100 ug/ml RNase A. FACS analysis was done on a BD FACS Array.
RT-PCR and qPCR
Cells were treated with FZ as indicated and total RNA was extracted using TRI reagent from Sigma. Concentrations of RNA in different samples were determined using a spectrophotometer. RNA was reverse transcribed using oligo (dT)18 primers and RT-PCR analysis was performed. For qPCR, RealMasterMix SYBR ROX kit from Eppendorf was used. The reactions were set up according to the manufacturer’s instructions in an Eppendorf Mastercycler Realplex real-time PCR machine.
Immunoblotting experiments
After treatment as indicated, the total cell lysates or the sub-cellular fractions were separated through 10% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes. Protein concentration was measured according to the method of Bradford using bovine serum albumin as a standard62. The membranes were successively incubated in blocking buffer [5% skimmed milk in TBST (50 mM Tris, pH 7.5, 0.15 M NaCl, 0.05% Tween-20)], with primary antibody and then with secondary antibody conjugated with horseradish peroxidase. Detection was carried out using enhanced chemiluminiscense reagent from Millipore. All primary antibodies were used in 1:4000 dilutions for immunoblotting.
Glucose uptake assay
Glucose uptake assay was performed using 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]−2-deoxy-D-glucose (2-NBDG; Invitrogen), a fluorescent analogue of 2- deoxyglucose. After overnight culture, the cells were left untreated or treated with 1 uM FZ for 4 h following which they were pre-incubated in glucose free KRB buffer (129 mM NaCl, 5 mM NaHCO3, 4.8 mM KCl, 1.2 mM KH2PO4, 1.0 mM CaCl2, 1.2 mM MgSO4, 10 mM HEPES, 0.1% BSA) pH 7.4 for 15 min at 37 °C. The cells were then incubated in fresh KRB buffer supplemented with 400 μM 2-NBDG and 3.3 mM glucose for 10 min at 37 °C. Cells were observed and imaged under a fluorescence microscope after washing with KRB buffer.
Glucose oxidation assay
Glucose utilization was estimated in cells exposed to FZ using the commercially available glucose assay kit from Sigma as per manufacturer’s instructions. After 24 h of drug exposure, culture supernatants were collected and centrifuged to remove any cellular debris. Assay reagent was mixed with the culture supernatants and incubated at 25 °C for 30 min, after which the absorbance was recorded at 505 nm on a PerkinElmer VictorX3 spectrophotometer.
Assays for succinate dehydrogenase activity
For spectophotometric determination of SDH activity, 20ul buffer B (200 mM Na-phosphate buffer, pH 7.4),10 ul 2.5 mg/ml NBT, 10 ul 1% Triton-X 100 and 10 ul substrate B (100 mM Na succinate, pH 7.4) were added to the enzyme fraction (cell lysates). The mixture was incubated at 37 °C for 30 min and the reaction was stopped by adding 40ul of 10% SDS. Absorbance was then measured at 630 nm.
For histochemical staining, A549 cells were plated onto coverslips and cultured overnight. After treatment, cells were washed with 1 mM malonate in 0.9% NaCl. The cells were then fixed in acetone for 5 min at −20 °C. After fixing, they were coated with CoQ10 (0.2 mg/ml in acetone) and incubated for 1 h at 37 °C in 50 mM succinate and 0.5 mg/ml NBT in 200 mM phosphate buffer (pH 7.6). The staining was observed and images acquired under a bright field microscope.
Enzymatic assay for hexokinase
Enzymatic assay for determination of hexokinase activity of control and FZ treated cells was performed as described by Darrow and Colowick63 by using glucose as substrate. The final concentrations of the reaction mixture were 8.3 mM glycylglycine, 17 mM ATP, 0.0011% cresol red, 14 mM magnesium chloride, 27 mM glucose. For determining HK activity, the reaction mix was added to the cellular extracts and the decrease in A560nm was measured for approximately 5 minutes.
Colony formation assays
H460 and A549 cells were seeded in a 12-well plate in triplicate (500 cells/well) and left untreated or treated with 1 uM FZ for 48 h. After 48 h, the cells were washed with PBS and the media was replaced with fresh media. The cells were allowed to grow for 8 days in order to form colonies and then stained with Coommassie Brilliant Blue. Anchorage-independent growth was assayed by the ability of cells to grow in soft agar. 5 × 103 untreated or FZ treated A549 cells (1 uM, 48 h) were overlayed in a 0.3% agarose solution in DMEM on a layer of 0.6% agarose in a 6-well plate and incubated for 14 d to allow colony formation. Cell colonies were then counted after staining with 5% crystal violet.
Nude mice experiments
Human NSCLC A549 cells were grown in DMEM with 10% FBS. When cells were 70–80% confluent, 3–4 h before harvesting, medium was replaced with fresh medium to remove dead and detached cells. Cells were then trypsinized and collected in complete medium. They were collected by centrifugation at 1500 rpm for 2–5 min and washed twice with PBS. Cell viability was assessed by trypan blue staining and cell number was determined using a hemocytometer. Cells were resuspended in a volume so that 100 µl contained 5.0 × 106 cells. nu/nu mice used were 6 weeks old and they were acclimatization for 3 days before injection. Cells (5.0 × 106) were injected subcutaneously into the right flank of mice. Oral dosing of FZ (1 mg/mouse) was started after 4 weeks when the tumours had reached an average volume of 2–3 mm3. Tumour diameters were measured with digital callipers, and the tumour volume in mm3 was calculated by the formula: Volume = (width) 2 × length/2.
Experiments on animals in this study were carried out after approval from the Institutional Animal Ethics Committee (IAEC- ACTREC, Mumbai, India). All methods were performed in accordance with the institutional guidelines and regulations.
Statistical Analysis
All results are expressed as means ± S.D. unless otherwise mentioned. Student’s t test was used to calculate the significance, accepting p < 0.05 as the level of significance.
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Acknowledgements
We thankfully acknowledge Dr. Rita Mulherkar, Advanced Centre for Treatment Research and Education in Cancer (ACTREC), Navi Mumbai, India, for valuable assistance with the nude mice experiments.
Author information
Nilambra Dogra
Present address: Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Sector-12, Chandigarh, 160012, India
Ashok Kumar
Present address: Centre for Systems Biology and Bioinformatics, Panjab University, Sector-25, Chandigarh, 160014, India
Affiliations
National Centre for Human Genome Studies and Research, Panjab University, Sector-14, Chandigarh, 160014, India
Nilambra Dogra, Ashok Kumar & Tapas Mukhopadhyay
Contributions
N.D. designed and performed the experiments, drafted the manuscript and prepared all figures. A.K. performed the nude mice experiments. T.M. conceived and supervised the study and reviewed and approved the final manuscript.
Corresponding author
Correspondence to Tapas Mukhopadhyay.
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Competing Interests
The authors declare no competing interests.
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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3158014/
Antiparasitic mebendazole shows survival benefit in 2 preclinical models of glioblastoma multiforme
Associated Data
Abstract
Glioblastoma multiforme (GBM) is the most common and aggressive brain cancer, and despite treatment advances, patient prognosis remains poor. During routine animal studies, we serendipitously observed that fenbendazole, a benzimidazole antihelminthic used to treat pinworm infection, inhibited brain tumor engraftment. Subsequent in vitro and in vivo experiments with benzimidazoles identified mebendazole as the more promising drug for GBM therapy. In GBM cell lines, mebendazole displayed cytotoxicity, with half-maximal inhibitory concentrations ranging from 0.1 to 0.3 µM. Mebendazole disrupted microtubule formation in GBM cells, and in vitro activity was correlated with reduced tubulin polymerization. Subsequently, we showed that mebendazole significantly extended mean survival up to 63% in syngeneic and xenograft orthotopic mouse glioma models. Mebendazole has been approved by the US Food and Drug Administration for parasitic infections, has a long track-record of safe human use, and was effective in our animal models with doses documented as safe in humans. Our findings indicate that mebendazole is a possible novel anti-brain tumor therapeutic that could be further tested in clinical trials.
Glioblastoma multiforme (GBM), which has been classified as a grade IV astrocytoma, is a highly aggressive tumor that invades early into surrounding brain tissues, making cure via surgical resection almost impossible. Standard of care currently consists of radiotherapy combined with chemotherapy of DNA-alkylating/methylating temozolomide (TMZ), which has increased the mean duration of patient survival to 15 months.1 Currently, only 10% of patients with GBM, including all post-treatment living conditions, survive 5 years after diagnosis, despite continuous work and improvement of GBM therapy.2,3
There has been no shortage of clinical trials for GBM. More than 600 clinical trials related to GBM have been run or are actively recruiting, according to the US National Institutes of Health's website (http://www.ClinicalTrials.gov). Unfortunately, few if any recent clinical trials show a clear survival benefit for patients with GBM. The present clinical trial system often does not address the difficulties of GBM therapy. Many therapies tried in the clinic were developed on the basis of results for other cancers, do not account for insufficient drug delivery to the brain, and do not address treatment-resistant migrating glioblastoma cells.4
There is a need to broaden the available treatments for GBM by introducing new therapeutic agents. One possible means to expedite initiation of GBM clinical trials is to examine previously established drugs with known track records of safety in humans, regardless of their intended use. However, searching for anticancer activity in compounds that have been “generally regarded as safe” may or may not turn up a promising drug suitable for GBM.
Mebendazole (MBZ), methyl N-[6-(benzoyl)-1H-benzimidazol-2-yl] carbamate, is a drug developed to treat human helminthic infections. It has been approved by the US Food and Drug Administration (FDA) and is available as generic drug for treating roundworm, common hookworm, American hookworm, pinworm, and whipworm. Clinical application of MBZ has been well documented at various doses for the rarer echinococcosis (hydatid disease).5–8 The closely related albendazole (ABZ) has been approved for treating an even wider range of parasites, including neurocysticercosis in the central nervous system (CNS). Both drugs are used to treat CNS parasitic infections and, therefore, have sufficient brain penetration for these indications. Sufficient drug delivery to the tumor remains a major challenge for most glioblastoma therapy.
The mechanism of action for MBZ and other benzimidazoles is to bind to the tubulin subunits in the gut epithelium of the parasite, preventing polymerization of the tubulin, causing ultrastructural changes, and eventually preventing parasite growth.9,10 Tubulin is vital to cell division and is also a cancer target for several chemotherapy drugs, including paclitaxol, cholchicine, and vincristine.
In addition to antiparasitic activity, MBZ and ABZ have shown preclinical anticancer activity in adrenocortical carcinoma, lung cancer, ovarian cancer, and melanoma cells,11–15 but thus far, the benzimidazole family has not been tested against glioblastoma or any other brain tumors.
Fenbendazole, a benzimidazole antihelminthic used routinely in veterinary medicine, was applied as feed supplement in our mouse colony to fight a pinworm infection. We noted problems with brain tumor intake in the xenograft model after the mice were given fenbendazole, and we further investigated this finding. This eventually led us to test the efficacy of the approved human drugs MBZ and ABZ against GBM in vitro and in vivo. We also tested whether MBZ's predicted interaction with tubulin was evident in glioblastoma cells. We found that MBZ worked best to extend survival in 2 different animal models and to explain our findings.
Materials and Methods
Cell Lines and Tissue Culture
Human GBM U87-MG (U87), D54, H80, H247, H392, H397, H502, H566, and the mouse GL261 glioma cell line were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. The human GBM neurosphere line 060919 was grown in NeuroCult NS-A basal medium containing NeuroCult NS-A proliferation supplements (Stem Cell Technologies), 20 ng/mL epidermal growth factor (PeproTech), 10 ng/mL basic fibroblast growth factor (PeproTech), and 4 μg/mL heparin (Stem Cell Technologies). Normal mouse nontumor astrocytes were obtained by mincing a healthy C57BL6 mouse brain and culturing it in DMEM media supplemented with 10% FBS for 2 weeks. Astrocyte populations emerged as adherent cells and were further cultured and used within 1 month. All cells were maintained at 37°C in humidified air containing 5% CO2.
Cells Growth Assay
ABZ, MBZ, thiabendazole, and fenbendazole were purchased from Sigma-Aldrich. TMZ was kindly provided by the Developmental Therapeutics Program of the National Cancer Institute (NCI)/National Institutes of Health. The viable cells were measured with a Cell Counting Kit-8 (Dojindo Laboratories) containing WST-8 tetrazolium salt at 450 nm on a PerkinElmer VICTOR3 plate reader. Half-maximal inhibitory concentrations (IC50s) were determined by incubating cells at a range of concentrations for 72 h and calculated by GraphPad Prism, version 5.0, using the log(inhibitor) versus response function and nonlinear fit.
Luciferase Expression by Lentivirus
The firefly luciferase cDNA from pGL3-basic (Promega) was subcloned in pFUGW and transfected along with CMVΔR8.91 and pMD.G in 293T cells using Lipofectamine 2000 (Invitrogen). Virus was harvested after 48 h and used to infect GL261 and 060919 cells by incubating with 8 μg/mL polybrene (Sigma).
Animal Experiments
Female C57BL/6 mice (age, 5–6 weeks) were purchased from the NCI. The syngeneic cell line GL261 was transfected with firefly luciferase with lentivirus and used for brain tumor implantation. For the implantation procedure, female C57BL/6 mice were anesthetized via intraperitoneal injection of 60 µL of a stock solution containing ketamine hydrochloride (75 mg/kg; 100 mg/mL; ketamine HCl; Abbot Laboratories), xylazine (7.5 mg/kg; 100 mg/mL; Xyla-ject; Phoenix Pharmaceutical), and ethanol (14.25%) in a sterile 0.9% NaCl solution. Using a stereotactic frame, 40, 000 GL261 cells were injected through a burr hole drilled 2 mm lateral to the sagittal suture and 1 mm anterior to the coronal suture at a depth of 3 mm below the dura at a rate of 1 µL/min.
The human GBM 060919 stem-like neurosphere cells were transfected with firefly luciferase and lentivirus, and 150,000 cells were implanted in female athymic nude mice, as described above.
At day 5 after implantation of the tumor cells, drugs were administered by oral gavage. MBZ tablets (TEVA) and ABZ tablets (GlaxoSmithKline) were resuspended in phosphate-buffered saline (PBS) and mixed with 50% of sesame oil (Sigma) to achieve better gastrointestinal absorption of the drug.5,16 Control animals were fed with PBS mixed with 50% sesame oil. TMZ was dissolved fresh for each use in PBS.
Animals were observed daily for any signs of deterioration, neurotoxicity, or movement disorders. They were inspected for signs of pain and distress, as in accordance with the Johns Hopkins Animal Care and Use Guidelines. If the symptoms persisted and resulted in debilitation, the moribund animals were euthanized according to protocol. The brain and other organs were dissected and placed in formalin for additional pathological studies.
Intracranial luciferase activity was determined by a Xenogen instrument with intraperitoneal injection of 2 mg/mouse D-luciferin potassium salt (Gold Biotechnology). Fifteen minutes after the injection, the animals were scanned for 1 min at a distance of 20 cm.
Tubulin Polymerization Assay
The tubulin polymerization assay was performed as described elsewhere.17 In brief, cells were lysed by resuspension in hypertonic buffer (2 mM of ethylene glycol tetraacetic acid, 1 mM of MgCl2, 2 mM of phenylmethylsulfonyl fluoride, 0.5% NP40, 20 mM of Tis-HCl [pH 6.8], and protease inhibitors). After brief vortexing, the samples were centrifuged at 13, 000 g for 10 min at room temperature. The supernatant containing the depolymerized tubulin was transferred to a new tube, and the pellet containing the polymerized tubulin was resuspended in an equal volume of hypertonic buffer. Equal amounts of protein were analyzed by Western blotting. The anti-α-tubulin Western blots were scanned, and the signals were qualified by the program ImageJ 1.42q (National Institutes of Health).
Western Blots
Cells were lysed in buffer as previously described elsewhere.18 Cell lysates were heated for 5 min with LDS Sample Buffer (Invitrogen) supplemented with 100 mM of dithiothreitol before loading onto a 4%–12% NuPAGE Bis-Tris Gel (Invitrogen). After transfer to a polyvinylidene fluoride membrane (Bio-Rad), immunostaining was performed in accordance with standard procedure. The following antibodies were used in this study: mouse anti-α-tubulin (Calbiochem) and anti-actin-HRP (Santa Cruz, C-11). Signals were visualized by the SuperSignal chemiluminescent system (Pierce).
Immunofluorescence Staining
The staining procedure followed the procedure described above.19 The neurosphere glioblastoma cell line 060919 was transferred from neurosphere medium and grown in DMEM medium containing 10% FBS in adherent single cell condition on chamber slides (Nunc). They were treated with 1 μM of MBZ or 10 nM of cholchicine for 24 h and then fixed for 10 min with 4% paraformaldehyde solution and permeated with methanol for 2 min with 3 washes in PBS in between and after. The slides were first blocked by 10% goat serum in PBS for 1 h at room temperature and incubated with mouse anti-α-tubulin antibody and subsequently with Alexa Fluor 594 (Texas Red) goat anti-mouse IgG (Invitrogen) in 10% goat serum in PBS at room temperature. They were then washed 3 times in PBS in between and after. After staining, the slides were covered with mounting medium containing DAPI (Vector Laboratories) and examined on a fluorescence microscope.
Statistical Analysis
The results are presented as a mean value ± standard deviation. Data were analyzed by GraphPad Prism, version 5.0. The P values were determined by a Mantel-Cox test. P values <.05 were accepted as statistically significant.
Results
We first observed the anti–brain tumor activity of benzimidazole family anthelminthics serendipitously when the animal facility started providing fenbendazole-containing food to the mice colony to eliminate pinworm infections. One of our orthotopic brain tumor models stopped forming tumors, whereas before the fenbendazole therapy, we had consistent, reproducible tumor engraftment. This prompted us to investigate commercially available benzimidazoles: fenbendazole, thiabendazole, MBZ, and ABZ, by first using GBM cell lines in vitro. On the basis of superior IC50s levels (Fig. 1A and data not shown) and FDA approval of human use, we decided to focus on MBZ and ABZ for in vivo preclinical evaluation.
Mebendazole (MBZ) inhibited intracranial tumor growth in the syngeneic GL261 mouse model. (A) Inhibition of GL261 mouse glioma cells by MBZ and albendazole (ABZ) showing the half-maximal inhibitory concentration (IC50) of MBZ at 0.24 μM and the IC50 of ABZ at 0.30 μM. (B) Hematoxylin and eosin staining of a coronal section of a C57BL/6 mouse brain implanted with GL261 glioma cells in the frontal lobe. (C) Mice implanted with GL261 cells expressing firefly luciferase were injected with 100 mg/kg luciferin and measured by Xenogen after 20 days of MBZ treatment. Four animals per group were randomly selected and shown. The total photon counts are displayed in the bar graph to the right. (D) Kaplan-Meier survival curve of C57BL/6 mice implanted with GL261 glioma cells and treated with MBZ. MBZ was given orally beginning 5 days after tumor implantation at a daily dose of 50 mg/kg for the first 20 days of treatment then changed to 50 mg/kg for 5 days, with 2 days off, each week. MBZ treatment increased the mean survival to 49 days compared with the 30 days of control (P < .0001). m, Mean survival days; n, number of animals. (E) Kaplan-Meier survival curve of C57BL/6 mice implanted with GL261 glioma cells treated with ABZ. MBZ was given orally beginning 5 days after tumor implantation at either 50 mg/kg or 150 mg/kg, as indicated, every day for the first 20 days of treatment and subsequently changed to 5 days a week. P < .001 for ABZ, 150 mg/kg, versus control; P = .015 for ABZ, 50 mg/kg, versus control; P = .38 (not significant) for ABZ, 150 mg/kg, versus 50 mg/kg
We used a GL261 syngeneic mouse glioma model to test the in vivo efficacy of MBZ and ABZ. The syngeneic GL261 mouse glioma was induced originally by intracranial injection of 3-methylcholantrene into C57BL/6 mice.20 Intracranial GL261 tumors showed rapid growth, diverse cell populations, necrotic regions, and invasive growth pattern, along with hemorrhages, closely resembling human GBM pathology (Fig. 1B). GL261 cells in vitro were susceptible to MBZ and ABZ, with IC50 levels measured at 0.24 and 0.3 μM, respectively (Fig. 1A). In a dose escalation in C57BL6 mice, daily administration of MBZ at 100 mg/kg led to toxicities, such as weight loss, whereas administration at 50 mg/kg did not show adverse effects.
Oral administration of MBZ at 50 mg/kg from day 5 after tumor implantation slowed tumor growth. The GL261 tumors expressing luciferase were visualized via Xenogen scanning using luciferin, showing smaller tumors after 20 days of MBZ treatment, as reflected by the significantly lower luciferase signals versus those of the control group (Fig. 1C). Mean survival was increased from 30 days in the control group to 49 days in the intervention group, a 63.3% increase (Fig. 1D).
ABZ was tested with the GL261 model at 50 mg/kg and 150 mg/kg, also without showing any significant adverse effects. Compared with the same control group in Fig. 1D, 2 doses of ABZ were able to moderately extend the survival to 36 days (20%) and 39 days (30%), respectively (Fig. 1E). This survival increase was less than that with MBZ.
Next, we tested MBZ and ABZ with the 060919 human GBM stem-like neurosphere cell line. The IC50s of both drugs in vitro were determined to be similar (∼0.1 μM). Of note, this cell line showed resistance to TMZ compared to the GL261 line, with an IC50 at 148 μM (Fig. 2A). The 060919 line grown as an intracranial xenograft showed a highly invasive and neo-vascularized growth pattern similar to the morphology of human GBMs, with features such as brain tissue infiltrations, heterogeneic population, hemorrhages, neoplastic giant cells, necrotic/hypoxic tissues, and pseudopalisading cells21 (Fig. 2B). Treating the mice with MBZ prolonged the mean duration of survival to 65 days, compared with 48 days for the control group, whereas ABZ at 150 mg/kg and TMZ at 15 mg/kg failed to extend the duration of survival (Fig. 2C). The measurement of luciferase activity in 060919 tumors confirmed that the tumor growth was inhibited by MBZ treatment (Fig. 2D and E).
Mebendazole (MBZ) improved the survival in the 060919 human glioblastoma multiforme (GBM) xenograft mouse model. (A) The half-maximal inhibitory concentration (IC50) levels of albendazole (ABZ), MBZ, and temozolomide (TMZ) in the 060919 human GBM neurosphere line are shown. (B) Hematoxylin and eosin staining of a coronal section of a nude mouse brain implanted with 060919 cells in the frontal lobe (10x). White arrows point to the invasive tumor cells. (C) Kaplan-Meier survival curve of 060919 xenografts treated with oral MBZ (50 mg/kg), ABZ (150 mg/kg), or TMZ (15 mg/kg) started 5 days after tumor implantation. Mice were treated daily for 20 days, followed by the same daily dose for 5 days per week. MBZ treatment increased the mean survival to 65 days compared with the 48 days of control. M, mean survival in days; n, number of animals. Significance values were as follows: MBZ versus control, P = .0016; ABZ versus control, P = .45; TMZ versus control, P = .30. (D) Mice implanted with 060919 cells expressing firefly luciferase were injected with 100 mg/kg luciferin and measured by Xenogen before and after 20 days of MBZ treatment. Three animals in each group were randomly selected and shown in the picture on the left side. Different color bars were applied on day 0 and day 20 of treatment. (E) The total photon counts of animals displayed in D were shown in the graph.
Benzimidazole family antihelminthics can exert cellular toxicity by binding to tubulin molecules and thereby disrupting their polymerization and microtubule formation in a similar fashion to colchicines.22 To test whether MBZ interferes with the microtubule formation in GBM cells, we performed a tubulin polymerization assay with the 060919 GBM cell line. Cells were incubated with 0.1 or 1 μM of MBZ, 1 μM of colchicines, or 10 nM of paclitaxel (PTX) for 24 h. PTX is known for hyperstabilizing the microtubule structure, thus causing mitotic arrest. After the lysis by hypotonic buffer and centrifugation, polymerized and depolymerized tubulin resided in the pellet and supernatant fractions, respectively. OneμM of MBZ clearly reduced the polymerized tubulin in the pellet as well as the percentage of polymerized tubulin in the total tubulin amount down to 11%, compared with 38% for the control group (Fig. 3A and B). Accordingly, colchicine severely decreased polymerized tubulin down to 1%, and PTX accumulated it up to 80%. Supplementary Material, Figure S1 shows the antitubulin polymerization of MBZ at 0.1 or 0.2 μM after 72 h, and the concentrations and time were comparable to the results of the condition used to determine the IC50 value of MBZ with 060919 cells. After 72 h, MBZ significantly inhibited the polymerization of tubulin at 0.1 μM. The depolymerization of tubulin by MBZ caused serious disruption in microtubule structure, as demonstrated by the immunofluorescent staining of α-tubulin in Fig. 3C, in which 060919 GBM stem cells were cultured adherently on the chamber slides and incubated with 1 μM of MBZ for 24 h. As a positive control, colchicine showed a similar effect.
Mebendazole (MBZ) disrupted microtubule polymerization in 060919 glioblastoma multiforme (GBM) cells. (A) 060919 GBM neurosphere cells were incubated with MBZ, colchicine (Col), or paclitaxol (PTX) at indicated concentrations for 24 h. After lysing cells with hypotonic buffer, the lysates were separated by centrifugation. The pellets (P) containing polymerized tubulin were resuspended in equal amount of lysis buffer and loaded along with the supernatant (S) containing depolymerized tubulin on SDS-PAGE for anti-α-tubulin (αTub) Western blot. The anti-β-Actin (βAct) blot served as a loading control. (B) The signals on the anti-α-tubulin blot were quantified and the percentage of polymerized tubulin (% P) induced by individual treatments was calculated with the formula: % P = P/(P + S) × 100. Mean and standard deviation (SD) are indicated. (C) MBZ disrupted the microtubule structure of 060919 cells. 060919 cells were cultured in adherent fashion with serum-containing media, treated for 24 h with 1 μM of MBZ or 10 nM cholchicine, and stained with anti-αTub antibody, Texas Red secondary antibody, and DAPI.
To evaluate MBZ therapy in combination with TMZ, we first tested the in vitro efficacies of MBZ and TMZ using 10 GBM cell lines. Although the IC50s of MBZ appeared to be in a close range, between 0.11 and 0.31 μM, these cell lines varied widely in response to TMZ with IC50s ranging from 8.7 to 547 μM (Fig. 4A). When used together in vitro, the combination of MBZ and TMZ slowed growth in GBM cells more than either drug alone (Fig. 4B). However, synergy was not observed in vivo. In the GL261 mouse model, TMZ at 15 mg/kg prolonged the mean survival to 41 days, compared with 29 days in the control group, in this set of experiments. With the addition of MBZ to the treatment regimen, the mean survival was extended to 50 days (Fig. 4C). This was a 72.4% improvement over the untreated control group. However, it is not significantly longer than the survival benefit achieved by MBZ alone observed in the previous set of experiments in Fig. 1D. Because the experiments were done under the same conditions, the various studies are shown superimposed in Supplementary Material, Fig. S2 for direct visual comparison.
Mebendazole (MBZ) plus temozolomide (TMZ) extends survival further than TMZ alone in the GL261 mouse model. (A) The half-maximal inhibitory concentration (IC50) levels of MBZ and TMZ with various glioblastoma multiforme (GBM) cell lines and normal mouse astrocytes. (B) Inhibition of GBM cell growth by MBZ, TMZ, or MBZ and TMZ combined. (C) Kaplan–Meier survival curve of C57BL/6 mice implanted with GL261 glioma cells and treated with TMZ (15 mg/kg) or TMZ plus MBZ (50 mg/kg). MBZ and TMZ were administered daily starting 5 days post tumor implantation for 20 days, followed by dosing 5 days per week. The TMZ + MBZ treatment increased the mean survival to 50 days, compared to the 30 days for controls (P < .0001) and 41 days for TMZ alone. The P = .0015 for TMZ plus MBZ versus TMZ. M, mean survival in days; n, number of animals. (D) Mice implanted with GL261 cells expressing firefly luciferase were injected with 100 mg/kg luciferin and measured by Xenogen after 25 days of MBZ treatment. Four animals in the TMZ and TMZ plus MBZ groups were randomly selected and are shown in the top picture, whereas the total photon counts are displayed on the lower graph. The color bars were set as follows: min = 2e + 5; max = 2e + 7.
Discussion
We accidentally found that fenbendazole, a benzimidazole, reduced brain tumor engraftment in nude mice after the mouse colony was treated for pinworms. Fenbendazole was previously reported to interfere with one lymphoma model in 2008,23 after we had already noted problems with fenbendazole disrupting brain tumor engraftment. We pursued this finding by evaluating whether the 2 most widely used human approved benzimidazoles showed efficacy against glioblastoma models.
The benzimidazoles are widely used for veterinary and human applications, with MBZ and ABZ approved for human parasitic treatment. Benzimidazole drugs, such as MBZ and ABZ, have been used to treat CNS infections of human cystic and alveolar echinococcosis since the introduction in the 1970s and have proven to be well tolerated and safe.24 Although MBZ and other benzimidazoles have reported preclinical antitumor activity, this is, to our knowledge, the first study to have related benzimidazole drugs to the treatment of a brain cancer. MBZ showed efficacy in 2 mouse models, with both having histological and phenotypic features similar to human GBM.
Although ABZ and MBZ revealed similar IC50s with both GL261 mouse glioma cells and 060919 human GBM stem-like neurosphere cells, MBZ extended survival most effectively. One explanation may be the different bioavailability of MBZ versus ABZ in our models. The limited solubility of ABZ and MBZ in water and organic solvents greatly affects absorption and behavior in the body.25 Given orally, ABZ and MBZ suffer from poor gastrointestinal absorption in human and rodents, and fatty meals are usually included to increase the absorption.5,16 The absorption of MBZ has been reported to be as low as 5%–10%, and similar low levels have been measured with ABZ in humans.25 We decided to focus on MBZ because of its superior efficacy.
MBZ and ABZ bind to the (+) end of the microtubule, inhibit tubulin polymerization, and prevent the addition of tubulin subunits in parasites.10,26 Microtubules are composed of α- and β-tubulin and are cytoskeleton components that are required for cellular transport, cell division, and maintenance of structural integrity. In addition, disruption of microtubule formation could also impair the migration of GBM cells. It was shown that the migration of neuronal cells is impaired by an α–tubulin mutation and that microtubule inhibitors can potentially block the mobility of glioma cells.27,28 It is also very likely that the migration/invasion feature of GBM cells renders them more resistant to apoptosis and cytotoxic insults.4,29
Figure 3 shows significant reduction of polymerized tubulin in 060919 cells treated with 1 μM of MBZ, as well as the disruption of microtubule structure. Other mechanisms have been proposed for benzimidazole, such as inhibition of VEGF and HIF-1α expression and inactivation of Bcl-2.15,30,31 Despite clear evidence of an anti-tubulin effect from MBZ, other mechanisms contributing to its efficacy in our preclinical models cannot be excluded. In parasites, a specific resistance to benzimidazoles was found to be conferred by changing the β-tubulin sequence at codon 200 as the result of a single-nucleotide polymorphism from TTC200 to TAC200.32,33 The possibility of tubulin mutation-based acquired resistance should be noted if MBZ therapy proceeds to clinical trials.
Antitubulin drugs other than benzimidazoles have been studied for cancer treatments, but many, such as cholchicine, exhibit severe toxicity. MBZ, flubendazole, and a number of benzimidazoles have been shown to interact with tubulin on the similar binding site–like colchicines, but distinct from the binding site of vinca alkaloids.22,34,35 Vinca alkaloids, such as vinblastine and vincrisine, have been approved for treating lymphomas, acute lymphoblastic leukemia, and nephroblastoma but are associated with considerable adverse effects, including bone marrow suppression and nervous system toxicity.
MBZ and other benzimidazoles seem to target cancer cells preferentially over normal cells, and this may suggest a favorable therapeutic index for in vivo applications. In vitro, MBZ displayed a slightly higher IC50 of 0.4 μM in mouse astrocytes, compared with those in various GBM cell lines, ranging 0.11–0.31 μM. In addition, our 2 different GBM animal models showed survival benefit for MBZ with minimal toxicity. Several other studies have shown in vivo success with benzimidazoles against cancers, including non–small cell lung cancer, adrenocortical carcinoma, leukemia, and colorectal cancer.11,12,23,35,36 There is evidence that MBZ's mode of action in lung cancer cells involves prevention of polymerization of tubulin.12 An additional anti-cancer mechanism associated with MBZ is that it induces apoptosis by BCL-2 inactivation in melanoma cells.15
MBZ has not yet been tried in human cancer therapy. A previous pilot clinical study of ABZ in patients with advanced colorectal cancer and hepatocellular carcinoma at 10 mg/kg/day for 28 days demonstrated anti-tumor efficacy, but there were concerns about severe neutropenia in 3 of 10 patients.37 On the basis of our preclinical results in GBM and the problems faced by ABZ, we would favor a clinical investigation with MBZ for GBM.
We used a daily dosing for 2 months of 50 mg/kg of MBZ, which translates to 4.1 mg/kg/day based on body surface area.38 MBZ has been extensively used for human for treating echinococcosis, with multiple reports of minimal adverse effects at 50–70 mg/kg/day for 6–24 months of continuous use.7,8,39 MBZ was also reported safe in children at 100–200 mg/kg doses for 12 weeks.6 These reported safe doses suggest we could escalate to higher doses than those required for efficacy in animals.
In another study of patients with echinococcosis, dosages as high as 200 mg/kg per day for up to 48 weeks were well tolerated, with 8.6 ng/mL (0.029 μM) of MBZ measured in cerebrospinal fluid, compared to 93 ng/mL maximally attained in serum.40 Other studies have shown safe doses up to 200 mg/kg with daily use.6,41,42 With a molecular weight at 295 Da and lipophilic properties, MBZ is capable of passing through the blood-brain barrier.43 In fact, MBZ has effectively treated CNS echinococcosis in numerous clinical settings before, indicating significant CNS penetration of MBZ.44–46
To determine whether combining MBZ with TMZ was beneficial, we chose to use a modest dose of TMZ that had a survival benefit, but not so large as to mask any potential synergy. At 15 mg/kg, TMZ extended the survival rate by 41.4%. The combination of MBZ and TMZ showed a survival benefit of 72.4%, compared with 63.3% for MBZ alone in a separate set of experiments. This difference was not significant for MBZ versus MBZ plus TMZ when comparing these 2 sets of experiments. Also of interest is that TMZ showed efficacy in only 1 of our 2 mouse models, whereas MBZ showed a significant survival difference in both TMZ-susceptible GL261 (IC50 = 6.9 μM) and TMZ-resistant 060919 (IC50 = 149 μM). Of note, patients with TMZ-resistant GBMs make up more than one-half of the patient population.47 A possible next step is to determine whether MBZ has single-agent efficacy in patients for whom initial therapy has failed.
In summary, MBZ offers a highly promising opportunity for clinical application on GBM. This is because it has a long track record of safety, there is evidence of preclinical efficacy, an anti-cancer mechanism has been revealed, cost is relatively low, the drug widely available as a generic drug, there is good CNS penetration, and there is a great need for better GBM therapy.
Monday December 3, 2018
This update is not about me. It is for Shane, Scott and Steven Sturgeon and their Mom.
Above I told the story about a veterinarian who I talked to to tell me of what would ultimately become my very unlikely story. I never mentioned his name because I wasn't sure I was authorized to, given that he was a licensed veterinarian and shouldn't be prescribing medicine to humanoids.
Dr. David Sturgeon literally saved my life. And as witnessed by this blog, he has saved countless other lives. There isn't enough gratitude in this world for me to express the sadness that Dr. Sturgeon passed away recently (non cancer related). And I think there is no BETTER way to go out, then to know that he had such an amazing impact on so many lives.....and will continue to have that impact posthumously. Only an OSU veterinarian can save human lives after he is gone :)
To the boys and to their Mom, there are scores of people who will be forever grateful and beholden. But that doesn't surprise you. You already knew how special he was to so many and you don't need this blog to remind you.
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