Microbiome signatures in prostate cancer.

[pjoshea13]

New study [1]. I don't believe this subject has come up before.

There was a recent thread where one view was that PCa is largely due to lifestyle, while I lean more to the estimate that two-thirds of cases are due to chance.

But didn't Einstein say that "God does not play dice with the prostate"?

Well, if you don't like the chance idea, or the thought that we are to blame because of poor lifestyle choices, we now have the microbiome to ponder.

Much has been written about the gut microbiome, & that we have no free will, because the gut microbiome controls the brain [2]. LOL

But the new study is about the microbiome signatures in PCa cells.

Here is a creepy part for those who are yawning:

"A surprising result from our study is the presence of parasite signatures in prostate tumor samples. However, parasites have been directly or indirectly associated with several different cancers (70–77). For example, Anisakis has been suggested to be a risk factor in colon and stomach cancers (76); Toxoplasma has been shown to induce prostatic inflammation and hyperplasia (75); Blastocystis is found predominantly in colorectal cancer (77); Schistosoma in bladder cancer (72); and Strongyloides has been associated with gastric and other cancers (71,74). In addition, the intestinal nematode Anisakis has been reported previously in the male urinary tract (78). We report finding signatures for these parasites in the prostate tumor samples. We also detected Plasmodium in the prostate tumor samples, which is interesting because it has been reported to activate EBV from latency (73). In addition, Plasmodium has been reported to be a potent mutagen that can indirectly induce chromosomal damage (79), produce reactive oxygen species (80) and inhibit apoptosis (81), potentially facilitating oncogenesis."

The link is to the full text - it's a long paper. Here is the Discussion section:

"In previous studies, we defined the microbial signatures associated with different breast cancer types (12,13), oral cancer and ovarian cancer (14,15). These studies showed that the tumor microbiome is quite diverse compared with normal tissue and that different tumor types have distinguishing microbiome signatures. The microbiome in prostate tumors was detected at significantly higher levels than in control tissue; however, the levels of viruses and microorganisms in the prostate tumor microbiome were still relatively low. Thus, it is unlikely that we are detecting a meaningful replicative infection. We suggest that although the presence of the tumor microbiome may affect the course of the cancer, it is likely that the tumor microenvironment provides a specialized niche in which viruses and microorganisms can persist more readily than in normal tissue. In the present study, we examined the microbiome of prostate cancer compared with prostate tissue from BPH patients and reported a diverse and distinct prostate tumor microbiome compared with that of the controls.

"Our study is not the first to suggest the presence of viruses and bacteria in the prostate. Several studies have documented that viral and bacterial infection of the prostate are risk factors associated with prostate cancer development (1,7,9,18,40–43).

"In this regard, our data suggest a prominent representation of tumor viruses in the prostate cancer samples compared with the BPH controls. It is noteworthy that many of the viral families were detected by PathoChip probes that represent conserved regions found in all members of the virus family. Probes for specific family members were the least represented, possibly suggesting significant strain variation among individual family members. Alternatively, the higher detection by conserved probes may suggest heretofore uncharacterized members of the virus family. Our data neither support nor deny that these viruses have any direct consequence in prostate oncogenesis (44). Previous studies (7,9,18,40,42,45–47) support our findings of viral signatures in prostate cancers, these include the detection of HPV [including HPV18 (11)], HCMV, EBV, JCV and BKV. Also, our detection of the oncogenic papillomavirus HPV18 in prostate cancer has been reported (11). However, reports of the association of viruses with prostate cancer have been controversial, for example, the association of the endogenous retrovirus, xenotropic murine leukemia-related virus in familial prostate cancer patients (43,48). In this regard, we did not detect xenotropic murine leukemia-related virus, but did detect signatures of other endogenous retroviruses, such as the alpharetrovirus RSV, betaretrovirus MMTV and gammaretrovirus MMLV. In agreement, a previous study reported that MMTV-like virus DNA was found in 36% of prostate cancers tested (45). Overall, past studies support our finding of a diverse virome in prostate cancer.

"Considering the bacterial microbiome, several previous studies have reported an abundance of Proteobacteria associated with dysbiosis-related diseases including cancer (49–51). A recent study using ultra-deep pyrosequencing showed the dominance of Actinobacteria in cancerous, pre-cancerous and non-cancerous prostate tissues, Propionibacterium being the most abundant, followed by Corynebacterium (17). Most of the bacterial genera detected in that study were also detected in the present study. An association of Chlamydia trachomatis and P.acnes has correlated with increased risk for prostate cancer development due to their pro-inflammatory host responses (52,53). We detected signatures of both, with medium and low hybridization signal in at least 85% of the prostate cancer cases studied. Another study has suggested that chronic mycoplasma infection may contribute to prostate cancer development in BPH cells (34). In this regard, we detected mycoplasma signatures with high hybridization signal intensity in at least 90% of the prostate cancer samples examined. Thus, there is good agreement between previous studies and our study as to the presence of bacterial signatures in prostate cancer.

"We also detected signatures of Helicobacter in >90% of prostate cancer cases. In this regard, previous studies have suggested that H.pylori infection may contribute to prostate diseases (38,39). One of these studies demonstrated H.pylori DNA in the prostatic tissue of both BPH and a prostate cancer patient (39). Our study detected signals from few Helicobacter probes in the BPH controls, none of which were significantly higher than in the cancers (Supplementary Table S3, available at Carcinogenesis Online). Thus, Helicobacter is likely to be a low-level component of the BPH microbiome as suggested by the PCR validation (Figure 4). In the present study, we found sequences of H.pylori to be integrated at certain locations in the human somatic chromosomes 17, 7 and 11 (17q21.31, 7q21.3, 11q23.2). The integration of the cagA gene sequence in PPP1R9A and NCAM1 gene locations may result in deregulation of their gene expression. Although PPP1R9A gene overexpression seen in prostate cancer (54) provides growth advantage to malignant cells, downregulation of NCAM1 gene has been identified in several human cancers suggesting that it might function as a tumor repressor (55). It was thus interesting to find H.pylori cagA gene integrations in PPP1R9A and NCAM1 genes, which may be a contributing factor to prostate tumorigenesis. Notably, one previous report has suggested Helicobacter DNA integration in a stomach adenocarcinoma (56). Although few studies have been done to examine the integration of bacterial sequences in human cell DNA, such integrations have been reported more frequently in tumors than in controls (56). Our study suggests that there is a marked increase in integration of viral and microbial sequences in prostate tumor DNA; we have reported similar findings for other tumors (14,15).

"The integrated H.pylori DNA that we detected in prostate tumor cells include the sequences of the cagA gene, which encodes the immune-dominant cagA virulence factor (57). CagA is also associated with more severe gastric cancer (57–61). Gastric cancer patients are at least twice as likely to be infected with an H.pylori strain that is cagA positive than one that is cagA negative (59,61). This is significant because cagA is known to activate proto-oncogenes and inactivate tumor suppressor genes (33,62); thus, cagA plays an important role in disease progression in cases of gastric cancer (57,63). Thus, the finding of cagA sequences integrated in prostate cancer cell DNA poses the intriguing possibility that it may function in the establishment or progression of the cancer.

"Among fungi, dermatophytes comprised the largest number of the fungal signatures detected in the prostate cancer samples. This may be because they are commonly detected in cancer patients (64). Similarly, the abundant detection of yeasts in the cancer cases is consistent with studies showing that opportunistic yeast infections are common in cancer cases (65–67). Also, consistent with the present study are previous reports of high incidence of microsporidia, such as Encephalitozoon and Fonsecaea, in cancer (68,69). In particular, chronic chromoblastomycosis, caused by Fonsecaea, has been suggested to promote squamous cell carcinoma (69). As was the case with viral and bacterial signatures, there is substantial agreement between previous studies and our study as to the presence of fungal signatures in prostate cancer.

"A surprising result from our study is the presence of parasite signatures in prostate tumor samples. However, parasites have been directly or indirectly associated with several different cancers (70–77). For example, Anisakis has been suggested to be a risk factor in colon and stomach cancers (76); Toxoplasma has been shown to induce prostatic inflammation and hyperplasia (75); Blastocystis is found predominantly in colorectal cancer (77); Schistosoma in bladder cancer (72); and Strongyloides has been associated with gastric and other cancers (71,74). In addition, the intestinal nematode Anisakis has been reported previously in the male urinary tract (78). We report finding signatures for these parasites in the prostate tumor samples. We also detected Plasmodium in the prostate tumor samples, which is interesting because it has been reported to activate EBV from latency (73). In addition, Plasmodium has been reported to be a potent mutagen that can indirectly induce chromosomal damage (79), produce reactive oxygen species (80) and inhibit apoptosis (81), potentially facilitating oncogenesis.

"Hierarchical cluster analysis showed that the microbiome signatures of the prostate tumors could be grouped into distinct clusters (1, 2a and 2b), suggesting that within prostate tumors different microbiomes are present. Thus, the microbiome may correlate with diagnostic aspects of the disease. Using the limited clinical data that were available for de-identified samples (Gleason grades, Gleason scores and the reported stages of the cancer), we looked for correlative trends between the clinical data and the specific clusters. The sample size is small, and our findings are largely correlative; however, we did find correlations that suggest that specific microbiome signatures may have prognostic and/or diagnostic value. In this regard, we examined the correlations between specific viral and microbial signatures and Gleason score and stages of cancer. We found that certain signatures were significantly higher in prostate cancer with lower Gleason scores (Supplementary Table S5, available at Carcinogenesis Online), where other signatures were higher in prostate cancer with higher Gleason score (see Results; Figure 3C; Supplementary Table S5, available at Carcinogenesis Online). These finding suggest that hybridization intensity of a group of specific viruses and microorganisms can provide significant prognostic and diagnostic value. It is likely that a study of a larger number of samples will clarify and expand the number of distinct clusters and more closely align clinical data to specific clusters and specific signatures.

"As in our previous PathoChip studies of tumor microbiota (14,15), we validated the specific Papillomaviridae and Herpesviridae signatures by sequencing the prostate tumor samples captured by hybridization to selected positive probes of PathoChip screen. Since the tumors are heterogeneous, we pooled samples for the probe-capture sequencing as we only wanted to validate the presence of those signatures in prostate cancer samples. The results validated the PathoChip results. Probably, the most intriguing result from the verification studies was the finding that some captured viral and microbial sequences contained flanking sequences that aligned to human sequences, thus suggesting sites of viral and microbial DNA integration in human chromosomes. We found many examples of viral and microbial integration in the tumor DNA suggesting that tumor cells exhibit greatly increased recombinatorial activity during the development and expansion of the tumor. We show specific hotspots for integration, which may perturb gene expression or miRNA/lncRNA function in ways that potentially modulate or potentiate oncogenesis.

"The controls for the study were derived from patients with BPH since normal prostate samples are very rare. BPH is an inflammatory pathologic condition of the prostate which in some cases could be caused by microbial infections, and may be a precursor to prostate cancer development (3,82). Thus, it is quite possible that the microbiome between BPH and cancerous tissue may be shared and that viral and bacterial integrations may occur during a pre-cancerous BPH condition. Using the same primers used to validate several microbial insertions in the prostate cancer (Figure 5, Supplementary Figure S7, available at Carcinogenesis Online), we also analyzed integration in BPH. These analyses showed similar amplicon from the prostate controls (Supplementary Figure S9, available at Carcinogenesis Online), which, when sequenced, confirmed these integrations in BPH and the cancer. Overall, the BPH and prostate tumor microbiomes may overlap; however, our data show that there is clearly more diverse microbiome in tumor.

"Observing similar HPV18 and KSHV insertions in the BPH samples as in the cancers were not surprising, as inflammatory prostate of BPH patients were not devoid of those viral detections, although significantly lower than in the cancers (Supplementary Table S3, available at Carcinogenesis Online). However, we did perform quantitative RT-PCR on the affected genes to see whether the gene expression were different in the cancers compared with the controls (Supplementary Figure S10, available at Carcinogenesis Online). The host genes, in which microbial insertions were detected, are already known to be associated with oncogenesis (83–86), and the differential expression of those genes that we detected in the prostate cancer samples and in the controls (Supplementary Figure S10, available at Carcinogenesis Online) were also previously reported (83–86). This may or may not be directly related to microbial genomic insertions within those genes.

"In conclusion, we have identified diverse microbiome signatures associated with prostate cancer samples. Many of the viruses and microorganisms we detected have previously been associated with prostate cancer or other cancers. Our observation of integrations of viruses and bacteria into both BPH and prostate cancer cells is the first demonstration of the diversity of viruses and microorganism that can integrate. The prevalence of integrations, especially in the cancer cells, suggests that these cells may have heightened recombinatorial activity. In several cases, the integrations of viral (HPV18, KSHV) and bacterial (Helicobacter) sequences potentially result in gene expression perturbations, which could influence the initiation or progression of the cancer. Finally, the hierarchical clustering analysis of the prostate tumor microbiome suggests that microbiome signatures may correlate with clinical data, suggesting that the signatures may provide biomarkers for diagnostic and prognostic purposes."

-Patrick

[1] academic.oup.com/carcin/adv...

[2] the-scientist.com/news-opin...

[cesanon]

Perhaps some material portion of this can be explained as being vectored through the inflammation mechanism?

[FCoffey]

Thanks, very interesting. Do you have access to the full paper? The link is limited to the abstract, thanks for including the details.

Many alternative medicine practitioners have been talking about this for years. I've read and met clinicians who believe that PCa has a fungal component, a bacterial component, and/or a microbiome component. Like most alternative docs, they are smeared and harshly criticized for not limiting their thoughts and practice to topics covered in randomized clinical trials. Like far too many cases, when conventional science looks into the issue, there is often some evidence to support the clinical observations.

[pjoshea13 in reply to FCoffey]

When I receive the journal notification, the link was to the full text - I don't know why - & that's what I pasted into the post. But I can no longer access it. Just as well that I copied the Discussion while I had it. Glad that you found it as interesting as I did.

-Patrick

[NPfisherman]

This raises the question in my mind since HPV was shown in the prostate cancer samples, that with the new vaccine for HPV, will we see a decrease in prostate cancer rates overall in the next 30 years? ... My sons both received the vaccine...

Fish

[sedgley]

Interestingly the Care Oncology protocol includes Mebendazole which is an anti parasitic - as one of the 4 cornerstones of the trial......

[helvi in reply to sedgley]

Logo of ecancerms

Ecancermedicalscience. 2014; 8: 443.

Published online 2014 Jul 10. doi: 10.3332/ecancer.2014.443

PMCID: PMC4096024

PMID: 25075217

Repurposing Drugs in Oncology (ReDO)—mebendazole as an anti-cancer agent

Pan Pantziarka,1,2 Gauthier Bouche,1 Lydie Meheus,1 Vidula Sukhatme,3 and Vikas P Sukhatme3,4

Author information Article notes Copyright and License information Disclaimer

This article has been cited by other articles in PMC.

Abstract

Mebendazole, a well-known anti-helminthic drug in wide clinical use, has anti-cancer properties that have been elucidated in a broad range of pre-clinical studies across a number of different cancer types. Significantly, there are also two case reports of anti-cancer activity in humans. The data are summarised and discussed in relation to suggested mechanisms of action. Based on the evidence presented, it is proposed that mebendazole would synergise with a range of other drugs, including existing chemotherapeutics, and that further exploration of the potential of mebendazole as an anti-cancer therapeutic is warranted. A number of possible combinations with other drugs are discussed in the Appendix.

Introduction

Mebendazole (MBZ) is a broad-spectrum benzimidazole anti-helminthic drug, in the same class as albendazole, flubendazole, oxfendazole, and others. It is commonly prescribed to treat a range of parasitical worm infections, including threadworm, tapeworms, roundworms, and other nematode and trematode infections in humans and domestic animals. MBZ is available as a generic drug; common trade names have included Vermox (Janssen Pharamceutica) and Ovex (McNeil Products Ltd) in the US and Europe. It is generally available over the counter in European countries, but the last US manufacturer, Teva Pharmaceuticals, ceased production at the end of 2011, although the drug retains US Food and Drug Administration (FDA) approval. It is available in the US from compounding pharmacies, for example, Pavillion Compounding Pharmacy in Atlanta.

Dosage

For human use, the most common formulation of MBZ is as 100 mg chewable tablets. The dosage varies according to the type of helminthic infection being treated. Pinworms are treated with a single 100 mg treatment, whereas roundworms or hookworms are treated with 100 mg twice a day for three days. MBZ, along with albendazole, is also used on a long-term basis for the treatment of human cystic and alveolar echinococcosis (also known as hydatid disease). According to the guidelines published by the World Health Organisation (whqlib-doc.who.int/bulletin..., long-term treatment of cystic echinococcosis using MBZ is at a dosage of 40–50 mg/kg/day for at least 3–6 months. For alveolar echinococcosis, the dose is 40–50 mg/kg/day, with treatment for at least two years, and possibly longer for patients with inoperable disease. Indeed, there are documented cases of treatment periods of ten or more years [1, 2].

Go to:

Toxicity

MBZ has low toxicity, though patients may suffer from transient symptoms, such as abdominal pain and diarrhoea in cases of massive infection and excretion of parasites. Hypersensitivity reactions, such as rash, urticaria, and angioedema, have been observed on rare occasions. MBZ is contraindicated during pregnancy. Caution is also recommended in treating infants below the age of 2, primarily due to a lack of data in such cases [3].

In the case of long-term administration of MBZ for echinococcosis, the evidence is that, in general, the treatment is well tolerated, but the specific treatment for some patients has to be discontinued. For example, in one open-labelled observational study, the patients treated with MBZ for alveolar echinococcosis (average: 24 months) experienced few adverse reactions, and in only three patients (of 17), the treatment was changed to albendazole due to intolerable side effects (reversible alopecia, psychological disturbance, and drop in performance) [4].

Go to:

Pharmacokinetics

First-pass metabolism of MBZ ensures that only about 20% of the oral dose reaches systemic circulation, with maximum plasma concentration reached 2–4 hours post-administration. Dosing with a high-fat meal is known to modestly increase bioavailability [5]. Chronic dosing of MBZ increases plasma concentration by a factor of between two and three compared to single dose [3, 6]. In one series of patients treated with chronic MBZ at a dose of 40 mg/kg/day for hydatid disease, the mean peak plasma level was 137.4 ng/ml [0.47 μM] after a single dose of 10 mg /kg; however, there was high inter-patient variability (99.4–500 ng/ml [0.34–1.69 μM]). For patients not on chronic treatment, an initial treatment of MBZ at the same dose produced a mean peak plasma level of 69.5 ng/ml [0.24 μM], (17.5–116.2 ng/ml [0.06–0.39 μM]) [6].

The poor bioavailability has long been recognised, and strategies to improve this remain actively researched, these strategies have included alternative formulations with vegetable oils [7–9], altering the crystalline structure of MBZ [10] and investigations into PEGylation [11].

Albendazole and MBZ interact with cimetidine, which inhibits metabolism and has been documented to increase MBZ plasma levels, (maximum serum levels rose to 82.3 ± 41.8 ng/ml [0.28 ± 0.14 μM ] from 55.7 ± 30.2 ng/ml [0.19 ± 0.10 μM], on 1.5 g of MBZ following chronic dosing of cimetidine at 400 mg three times a day for 30 days) [12]. This may be an important interaction with clinical relevance in that it suggests a strategy to increase bioavailability should that be required to increase the anti-cancer effect. Given that cimetidine may also have some anti-cancer activity [13], it also suggests that an investigation into possible synergies with MBZ over and above the effect on bioavailability would be an interesting avenue to explore.

High intra- and inter-patient variability may be an important factor in assessing response to MBZ as a possible anti-cancer therapeutic. However, it is clear that plasma levels achieved by chronic and high-dosing schedules are in the range necessary for clinical activity based on the pre-clinical evidence assessed in the following section.

Go to:

Pre-clinical evidence in cancer—in vitro and in vivo

In 2002, Mukhopadhyay and colleagues showed that MBZ induced a dose- and time-dependant apoptotic response in a range of lung cancer cell lines [14, 15], with an IC50 of ~0.16 μM. Cells were arrested in the G2-M phase before undergoing apoptosis. Just as importantly, MBZ had no effect on normal HUVECs or WI38 fibroblasts, even at a concentration of 1 μM. Overall, the in vitro results showed that MBZ inhibited lung cancer cell growth 5-fold compared to controls. Additionally, the authors confirmed the growth inhibitory effects of MBZ against breast, ovary, colon carcinomas, and osteosarcoma, producing IC50s that varied from 0.1 to 0.8 μM.

To test the in vivo response to the MBZ treatment, nu/nu mice were inoculated with subcutaneous injections of H460 non-small cell lung cancer cells [14]. Animals with established tumours (~3 mm diameter) were treated with 1 mg orally of MBZ every other day. Treated animals showed a dose-dependent arrest in tumour growth. The experiment was also repeated with C3H mice and the K1735 mouse cell line, and MBZ inhibited tumour growth in this syngeneic mouse model also. Mice treated with MBZ showed no side effects. Finally, the investigators also assessed whether MBZ might inhibit the formation of lung metastases and injected A549 cells into the tail vein of mice. In untreated controls, approximately 300 metastatic colonies appeared in the lungs by 21 days. Mice treated with 1 mg of MBZ every other day showed a mean colony count 80% lower than controls. Treatment with the established anti-microtubule agent paclitaxel showed no such reduction in colony formation.

Further pre-clinical evidence of MBZ anti-cancer activity was shown in adrenocortical cancer in 2008 [16], both in vitro and in vivo. H295R, SW-13 and WI-38 (normal fibroblast) cells lines were treated with different concentrations of MBZ in vitro, and the two cancer cells lines showed dose-dependent growth arrest, with IC50 of 0.23 μM for H295R and 0.27 μM for SW-13 cells, with no effect on the normal fibroblast cells. Tumour spheroid inhibition was tested against a dose of 1 μM of MBZ, which completely disaggregated the tumour spheroids and killed all cancer cells in about 20 days.

In vivo treatment of athymic nude mouse models of adrenocortical cancer showed that treatment with 1 mg and 2 mg MBZ significantly inhibited tumour growth in both implanted adrenocortical cancers. While there was little difference between the response of the primary tumours to 1 mg and 2 mg doses, the latter dose inhibited the formation of metastases from 50% of controls to 75%. No side effects were noted in the treated animals. Of note, a dose of 1 mg/day in a mouse weighing 20 gm corresponds to a human dose of approximately 500 mg daily for a 70 kg person, if extrapolated by surface area.

In 2008, the in vitro activity of MBZ against chemoresistant melanoma cell lines was assessed by Doudican et al [17]. A screening of 2000 small molecules against melanoma cells lines picked out ten compounds that had inhibitory action against the M-14 and SK-Mel-19 chemoresistant melanoma cells lines, but were non-toxic to normal melanocytes. Of these ten compounds, four were benzimidazoles —mebendazole, albendazole, fenbendazole, and oxybendazole—and of these four compounds, MBZ was selected for more detailed analysis based on its relative lack of toxicity and well-characterised pharmacokinetics. MBZ was shown to induce dose-dependent apoptosis in both cell lines with an average IC50 of 0.32 μM, while the equivalent for the non-cancerous melanocyte cell line was IC50 of 1.9 μM. MBZ also had the greatest inhibitory effect against the melanoma cells of the four benzimidazoles tested.

Subsequently, MBZ was shown to inhibit human melanoma xenograft growth in athymic female nude mice fed 1 mg or 2 mg oral MBZ every other day [18]. Tumour growth was reduced by 83% for the 1 mg dose and 77% for the 2 mg in comparison to controls. This was comparable to the growth inhibitory activity of 100 mg/kg of temozolomide (TMZ) by an intraperitoneal injection for 5 days, used as a positive control as it represents a well-characterised option for melanoma treatment. These results showed that oral MBZ produced equivalent responses to high-dose TMZ, but with no observed side effects.

MBZ activity in glioblastoma multiforme (GBM) was discovered serendipitously in 2011 by investigators, who observed that GBM xenografts were failing after mice models were fed albendazole to fight a spate of pin worm infections [19]. Further investigation showed that both albendazole- and MBZ-induced apoptosis in two GBM cells lines in vitro and in vivo. The in vitro IC50 of MBZ was 0.24 μM in the GL261 mouse glioma line, and 0.1 μM in the 060919 human GBM. In vivo results showed that oral MBZ treatment significantly extended mean survival up to 63% in syngeneic and xenograft orthotopic mouse glioma models.

Screening of compounds for activity against colon cancer cell lines also identified MBZ as a candidate molecule in work by Nygren and colleagues [20]. The authors set out to screen 1600 existing drugs for activity against two well-established colon cancer cell lines (HCT 116 and RKO) and found 64 candidate drugs, including a cluster of benzimidazoles (albendazole, mebendazole, oxybendazole and fenbendazole). Of these, further analysis was performed on MBZ and albendazole because, in the words of the authors, ‘they are registered pharmaceuticals for clinical use in humans, thus easily accessible for clinical testing’.

Diagnosis-specific activity was assessed using the NCI 60 z score data, which showed a high level of activity against leukaemia, colon cancer, CNS and melanoma panels of cell lines, with lesser activity in breast, ovarian, renal and NSCLC lines. It should be noted that the leukaemia panel had the highest level of sensitivity to MBZ, a finding that has not been further investigated to date. In the colon cancer panel, 80% of cells lines were sensitive to MBZ. Detailed in vitro treatment against five colon cancer cell lines (HCT 116, RKO, HT29, HT-8 and SW626), showed that all displayed IC50 of <5 μM, whereas the drug was largely inactive in the non-malignant cell lines.

Some work on in vitro efficacy against a chemoresistant breast cancer cell line (SKBr-3) was performed by Coyne and colleagues in 2013 [21]. A range of benzimidazoles, including MBZ and albendazole, were tested and found to cause significant growth arrest and apoptosis, with flubendazole and MBZ showing the greatest level of cytotoxic activity. MBZ reduced cell survival by 63.1% at a dose of 0.5 μM.

Finally, Schmit showed that a range of benzimidazoles, including MBZ, possess anti-neoplastic activity against the DS 17 canine osteosarcoma cell line in vitro [22]. Canine osteosarcoma is an excellent animal model of the human disease. The results obtained showed that MBZ induced cell cycle arrest and apoptosis at MBZ doses that are clinically achievable with oral dosing.

Go to:

Human data in cancer

No clinical trials of MBZ as a cancer treatment have been completed to date. However, there are two well-documented case reports in the literature in favour of re-purposing MBZ as an anti-cancer therapy.

In 2011, a case of long-term tumour control in metastatic adrenocortical cancer was published [23]. Adrenocortical cancer is a relatively rare malignancy with few treatment options in the case of non-resectable disease. The patient had experienced disease progression despite multiple chemotherapeutic protocols and several rounds of surgery. After all other treatment options had been exhausted, the patient discovered the pre-clinical evidence of MBZ action against adrenocortical cancer via Pubmed and forwarded the information to the clinicians, who agreed to use it based on this evidence and the relatively low toxicity of treatment. Monotherapy commenced with MBZ at a typical anti-helminthic dose of 100 mg twice a day. The patient experienced some regression in metastatic lesions, and overall the disease remained stable for 19 months of MBZ monotherapy, tolerating the treatment without side effects, and his quality of life returned to his baseline prior to his initial surgery. However, 24 months after the commencement of oral MBZ a scan showed disease progression, and everolimus was added to the MBZ but without additional benefit in disease control.

A case of metastatic colon cancer treated with MBZ was described by Peter Nygren and Rolf Larsson in 2013 [24]. Here, a 74-year-old patient suffering from progressive metastatic colon cancer had been treated first with capecitabine, oxaliplatin, and bevacizumab, and then by capecitabine and irinotecan in the face of disease progression, and who had no standard treatment options available was started on an oral dose of MBZ of 100 mg twice a day. MBZ was selected based on the author’s previous pre-clinical work with MBZ [20]. After six weeks of monotherapy, radiological evaluation showed near complete remission of metastatic lesions in the lungs and lymph nodes and a good partial remission in the liver. However, the patient experienced elevated liver enzymes (AST and ALT), so MBZ was temporarily stopped and then started at half the dose, with the patient reporting no ill effects. Liver enzymes normalised and a subsequent round of CT scans confirmed the initial disease response. After ceasing treatment for approximately three months, the patient developed brain metastases that were treated with radiotherapy, following by evidence of disease in the lymph nodes. MBZ treatment was not recommenced following the discovery of the brain metastases or in subsequent disease progression. A further five patients have been treated, with one experiencing a minor remission [Private communication from Peter Nygren].

Go to:

Clinical trials

There are currently two clinical trials of MBZ in cancer, both for brain tumours.

One is a Phase I open label study, at John Hopkins Hospital, of MBZ in newly diagnosed high-grade glioma patients receiving temozolomide (clinicaltrials.gov/ct2/show.... Patients recruited to the trial are treated on a 28 day cycle of 500 mg MBZ tablets three times a day. The primary end point is to determine the maximum tolerated dose of MBZ with temozolomide. A secondary end point is to determine if MBZ with current standard of care can slow tumour progression. Study completion is scheduled for November 2014 (at the time of writing in January 2014).

The other clinical trial is at Cohen Children’s Medical Centre of New York in paediatric patients with low-grade gliomas (clinicaltrials.gov/ct2/show.... This is a Phase I and II pilot study of MBZ in combination with vincristine, carboplatin, and temozolomide. The study design is non-randomised and open label, with comparison between standard of care and standard of care plus MBZ arms. The MBZ dose is 100 mg twice a day over the 70 weeks of treatment. The primary objective of the Phase I part of the trial is to determine if the standard dose of MBZ 100 mg twice daily is ‘well-tolerated’ when used in combination with the current three-drug regimen. At the end of Phase I potential subjects will be offered the chance to receive the three-drug regimen + MBZ for 70 weeks, or else to enrol as part of the control group receiving the three-drug regimen alone. For the Phase II portion of the study, the outcome variables of interest are progression-free and overall survival. Study completion is scheduled for December 2017 (as of December 2013).

Go to:

Mechanism of action

The anti-parasitic action of MBZ is due to its action as a microtubule-disrupting agent acting to prevent the polymerisation of tubulin in the gut of helminths, causing the parasites to die [25]. Tubulin is vital to cell division and is therefore a cancer target for several widely used chemotherapy drugs, including paclitaxel, colchicine, and vincristine. MBZ, as with the other benzimidazoles, binds to the colchicine-binding domain of tubulin [26].The inhibition of tubulin polymerisation by MBZ has been confirmed in vitro in a glioblastoma model [19] and in a melanoma model [17]. The latter work suggested that the apoptotic response to microtubule disruption is mediated by Bcl-2 phosphorylation. Subsequent work on melanoma confirmed this result, and also showed that MBZ decreased the levels of X-linked inhibitor of apoptosis (XIAP) [18], but to date this has not been confirmed in non-melanoma cell lines.

While there are rare reports of reversible alopecia, urticaria, rash, gastro-intestinal upset, leukopenia, and neutropenia in some patients treated with high-dose MBZ, all adverse effects associated with other microtubule disruption agents, there do not appear to be any reports of peripheral neuropathy, which is commonly considered a classic adverse effect of microtubule disrupting agents, including the taxanes and the vinca alkaloids [27]. While this may suggest that the action of MBZ is independent of microtubule disruption, it may also be related to the fact that MBZ acts via the colchicine-binding domain, and that like colchicine, there is little effect in terms of neuropathic pain [28]. Of course, it is also possible that the anticancer activity of MBZ is mediated by additional molecular targets yet to be elucidated.

MBZ appears to be effective through p53-dependent and independent pathways. For example, in lung cancer cell lines, it was found that MBZ treatment caused post-translational p53 stabilization and the downstream expression of p21 and MDM2 [14]. In p53-null lung cancer cells exposure to MBZ caused cytochrome c accumulation, activation of caspase-9 and caspase-8, and cleavage of PARP and procaspase-3. This independence of p53 status is also evident in the analysis of melanoma cells, where wild-type and mutant p53 cell lines were sensitive to MBZ [17].

There has been conflicting evidence regarding the effect that MBZ has on tumour neo-vascularisation, with some reports finding evidence that it has an anti-angiogenic effect and others finding none.

In the earliest work on the anti-cancer activity of MBZ, Mukhopadhyay and colleagues reported an anti-angiogenic effect on human lung cancer xenograft models [14]. However, in vivo analysis of adrenocortical cancer models failed to detect any anti-angiogenic activity compared to controls [16]. Some support for an anti-angiogenic action comes from an in silico study, which indicated that MBZ inhibits the action of VEGFR-2 by binding to it, a finding validated in vitro using a human umbilical vein endothelial cell (HUVEC) based angiogenesis functional assay [29]. Of note, the related drug albendazole has shown anti-angiogenic properties in an ovarian cancer model and in drug-resistant cell lines [30, 31], suggesting that an anti-angiogenic action may be common across a number of benzimidazoles.

To date, the effect of MBZ or other benzimidazole on the immune response in cancer has not been investigated, though there is some evidence that albendazole synergised to stimulate the cellular immune response in mice treated for alveolar echinococcosis with the immunotherapeutic agent liposomal muramyl tripeptide phosphatidylethanolamine (L-MTP-PE) used in the treatment of osteosarcoma [32]. There is also increasing evidence that existing microtubule disrupting agents used at low or metronomic doses, including the taxanes and vinca alkaloids, exert a positive immunomodulatory action that may help to reverse the immunosuppressive effect of cancer [33–36]. We can speculate, mechanistically, that some of this immunomodulatory action is related to microtubule dynamics. Therefore, there may be a similar effect with MBZ and other benzimidazoles, and this may also be a factor in the anti-cancer effects of these drugs.

Go to:

Our take

Next steps

Based on the evidence summarised in Table 1, it is our contention that human clinical trials of mebendazole in a range of cancer types is warranted. The known pharmacokinetics, relatively low toxicity (even with extended high-dosing protocols), low cost, and strong pre-clinical evidence make this an ideal candidate for re-purposing. Currently, there are only two early phase clinical trials getting under way, both in glioma. In addition to these, the evidence suggests that candidate cancer types to take to human trial include:

melanoma,

non-small cell lung cancer,

adrenocortical cancer, and

colon cancer.

Table 1.

A summary of pre-clinical evidence by cancer type.

Cancer TypeIn VitroIn VivoCase Report/Trial

Adrenocortical[16][16][23]

Breast[14, 21, 20]

Colon[14, 20][14][24]

Glioma[19][19]NCT01729260, NCT01837862

Leukaemia[20]

Lung[15][15]

Melanoma[17, 20][18]

Osteosarcoma[14, 22]

Ovary[14, 20]

Additional cancer types, which should be further investigated in animal studies include:

breast cancer,

leukaemia, and

osteosarcoma.

As with other anti-cancer agents, it is most likely that MBZ will be more effective in combination with other drugs or treatment modalities. It should be noted that the first two clinical trials are using MBZ with current standard of care treatment in glioma, which in this case means a combination protocol with other drugs, principally temozolomide. Given the primary putative mechanism of action—microtubule disruption—there are a number of additional agents that warrant investigation for synergy with MBZ, some of which are listed in the Appendix.

Other options

Finally, improved efficacy may also be possible through improvements in the bioavailability of MBZ. As touched on previously, there is evidence that the combination of MBZ with cimetidine increases plasma levels of MBZ [12], potentially improving the therapeutic effect. An alternative means of increasing the bioavailability is through the liposomal encapsulation of MBZ. While this approach has not been explored in an oncological context, some work in this area have been done to enhance the anti-parasitic action of MBZ and other benzimidazole anti-helminthics, including one paper that explored the combined effect of a liposomal benzimidazole (albendazole) and cimetidine and reported a very significant increase in therapeutic effect (including a 75–94% reduction in biomass of the hydatid cysts and a significant increase in survival time) in an animal model [37]. It is possible that a similar approach could yield improvements in the anti-cancer effect of MBZ.

New protocols

Adding MBZ to the existing standard of care protocols, as the first two clinical trials have done, provides an opportunity to test whether there are incremental improvements in outcomes compared to the standard of care alone. However, we should also seek opportunities to create new protocols that combine MBZ with other repurposed drugs with similar low toxicity and potential anti-cancer activity. The intention is to create novel treatment options that are multi-targeted and which present minimal risk of toxicity. Of necessity, given our current state of knowledge, the combinations proposed in the supplementary material are speculative and informed primarily by mechanistic considerations and pre-clinical data. Additional pre-clinical studies are required, but given the urgency of unmet patient needs and the low toxicity of the proposed combinations, it may be argued that small patient trials or even off-label usage may also be warranted.

Go to:

Conclusion

The evidence for an anti-cancer effect of mebendazole treatment comes from in vitro, in vivo, in silico, and human data. Mechanistically, the microtubule action is well characterised in the laboratory and provides a similar rationale to some of the major classical chemotherapeutic drug classes, such as the taxanes and vinca alkaloids. With well-established pharmacokinetics and an excellent toxicity profile, this low-cost agent is a strong candidate for drug repurposing as an oncological treatment, both in combination with the existing standard treatments and alongside other candidate repurposing agents in a number of specific cancer types. We have outlined a number of these multi-drug combinations in the hope that clinicians can act upon this information to initiate clinical trials as a matter of some urgency.

​

Table A1.

Proposed drug combinations with MBZ for specific indications.

DiseaseTargetsDrug Combination

Malignant MelanomaMicrotubule disruption, inhibition of autophagy, anti-angiogenic and immunomodulationHydroxychloroquine (NCT00962845) Diclofenac or Celecoxib [24] Oral cyclophosphamide [39]

NSCLCMicrotubule disruption, AMPK/mTOR, Hedgehog signalling, COX-2 inhibitionMetformin (NCT01997775) Itraconazole [40] Diclofenac or Celecoxib (NCT00520845)

Adrenocortical CarcinomaMicrotubule disruption, anti-angiogenic, Hedgehog signallingItraconazole Oral cyclophosphamide [41]

Glioblastoma MultiformeInhibition of autophagy, microtubule disruption, Hedgehog pathway inhibition, anti-angiogenicHydroxychloroquine (NCT00224978) Itraconazole

Colorectal CarcinomaMicrotubule disruption, AMPK/mTOR, immunomodulation, anti-histamine, COX-2Metformin (NCT01941953) Cimetidine [42] Diclofenac Oral vinorelbine [43]

Osteosarcoma/ Soft-tissue SarcomaMicrotubule disruption, AMPK/mTOR, IGF-I, Hedgehog pathway inhibition, tumour vascularity, anti-angiogenicMetformin Itraconazole Losartan Oral cyclophosphamide [44]

Acute Myeloid LeukaemiaMicrotubule disruption, induction of apoptosisAlbendazole or oral vinorelbine [45] Diclofenac

Breast Cancer (ER+ invasive ductal carcinoma)Microtubule disruption, AMPK/mTOR, anti-angiogenicMetformin (NCT01929811) Oral cyclophosphamide and/or oral vinorelbine (NCT00954135)

Ovarian CarcinomaOvarian CarcinomaMetformin (NCT02050009) Itraconazole Diclofenac (NCT01124435)

Open in a separate window

Note that references to clinical trials or published papers are indicative of trials or case reports where the drug (or analogue) has been used for the specific indication.

Go to:

Acknowledgments

Peter Nygren, Gregory Riggins and Gary Gallia.

Go to:

Appendix. 

Introduction

The following drugs warrant further investigation in combination with mebendazole (MBZ), both in pre-clinical studies and potentially in clinical trials. These combinations listed in Table A1 have been selected on the basis of existing pre-clinical and clinical experience in each of the indications. In some cases, these combinations replicate existing protocols currently being tested in clinical trials, but substitute known and repurposed drugs for the newer and/or more toxic agents currently being investigated. All these proposed combinations are expected to display relatively low toxicity and use low cost and generally available agents.

Higher-priority agents

The agents listed below have a high degree of clinical evidence of efficacy and are currently either in clinical use in oncology or are currently being investigated in clinical trials. They have been selected as potential agents to be used in combination with MBZ. Note that these drugs are not listed in order of priority.

Metformin: There is pre-clinical evidence that metformin potentiates the action of existing microtubule disrupting drugs in a range of cancer types, including endometrial cancers and paediatric sarcomas [1–3]. Given the low toxicity of metformin and its potential as an anti-cancer agent, the combination with MBZ should be explored, both in animal models and potentially in small clinical trials.

Metronomic chemotherapy: While there is intense interest in the area of metronomic chemotherapy using taxanes or vinca alkaloids, progress has been restricted because of a lack of oral formulations of many of these drugs, with the exception of oral vinorelbine. Where existing microtubule targeting drugs without oral formulations are used in metronomic settings, it is normally as a weekly infusion in combination with daily dosing of oral cyclophosphamide or capecitabine. A number of clinical trials using oral vinorelbine have reported both low toxicity and evidence of clinical benefit in advanced cancers [4, 5]. MBZ also offers the possibility of exploring daily oral dosing of a microtubule disrupting agent in combination with low dose oral chemotherapy drugs and other agents used in such protocols (e.g. celecoxib or other anti-inflammatory). It is theorised that one of the principal methods of action of metronomic chemotherapy is through inhibition of neo-angiogenesis, and that escape from angiogenic control is associated with treatment failure. The addition of MBZ with existing metronomic protocols may increase the anti-angiogenic effect of treatment and prolong the therapeutic benefit.

Taxanes or Vinca Alkaloids: Combinations of microtubule targeting agents, for example, paclitaxel or docetaxel and vinorelbine, act synergistically, and there are numerous trials exploring multiple combinations of different microtubule agents [6]. Pre-clinical evidence shows that the benzimidazole flubendazole synergises with vincristine and vinblastine in vitro and in vivo [7]. Given that MBZ has such low toxicity in comparison to many existing microtubule agents, combination therapy of MBZ with taxanes or vinca alkaloid drugs would seem a promising avenue to explore in human trials. One prospect is to combine MBZ with oral vinorelbine, offering the prospect of dual oral microtubule disrupting drugs, with low toxicity, either in standard dosing of vinorelbine or at metronomic dosing of both agents.

Albendazole or other benzimidazole: There is evidence that the different benzimidazoles vary in their molecular targets and that combining them may improve efficacy and reduce the risks of acquired resistance. While this approach has not been explored in a cancer setting, there is pre-clinical and clinical evidence that the combination of MBZ and albendazole is a more effective treatment in certain hard to treat parasitic conditions [8, 9]. There is also some in vitro and in vivo evidence where albendazole exerts an anti-angiogenic action by down-regulating vascular endothelial growth factor (VEGF), an effect mediated through inhibition of tumoural hypoxia inducible factor (HIF-1α) [10]. As HIF-1α is implicated in multi-drug resistance

[ITCandy]

Perhaps this ties into the perceived benefit of taking the Dukoral vaccine.

[George71 in reply to ITCandy]

I was thinking the same thing about the Dukoral vaccine- and what sedgley said about Mebendazole too.

Also many of the drugs being investigated for re purposing showing promising results in PCa are antibacterial -- anti-parasitic anti fungal and mold.

Doxycycline kills CS cells along with vitamin C.

[George71]

"Tapeworm drug fights prostate cancer"

sciencedaily.com/releases/2...

[Kuanyin]

Patrick, what this study shows is that there is a Witches' Brew composed of bacteria, viruses and parasites nesting in the prostate, colon, ad nauseam, in our bodies. It seems that H. pylori might be a place to start since certain antibiotics are effective against this organism, however, killing parasites is another matter. While drugs designed to kill parasites do kill the parasite, they do not their eggs. Also, after this brew gets tied into our DNA through viral insertion, the problem becomes much more complex. Let's not LOL too soon. Finally, as I recall, aren't you currently taking an antibiotic: how is that working?

[George71]

Gut flora is 80% of the immune system -- made from hundreds of strains of billions of microbes --- they are a complete living "organ" inside of our body essential to life.

1md.org/lp/is-this-epidemic...

[Advo__cate]

Interestingly enough, my husband was found to have a parasite at the time of his PCa diagnosis. The integrative doctors found it through an extensive stool test. He was put on an antibiotic and then was treated with billions of probiotics and all things anti-inflammatory to heal his gut. He also had an overgrowth of candida, trouble with sinuses, skin rash, etc. I don’t think H-pylori was found to be a problem for him. Since this time late 2017 he has not had sinus issues, skin rashes, candida, etc. He does continue with all things that heal the gut. I have noticed his eosinophils run a little high so I’m considering getting him tested for the parasite once again.

The human microbiome has been an area of interest to me for numerous years.

Thanks for posting.

[yamobedeh]

Thanks for this. Although this is not my field by any stretch, this has really piqued my interest. Eyes only glazed momentarily, until I read further into the inflammation and microbial links. I had a DNA-based test to confirm ureaplasma infection just when I was diagnosed with PCa two years ago. Also been dealing with a recurrent herpes infection, courtesy of the ex-wife. In the 2-3 years prior to my PCa Dx, I was dealing with BPH issues. I suppose it will be a few years before this field generates potential treatments. Thanks again.

[jerigroves]

So interesting. Hyperthermia kills viruses, so it makes sense that it was super-effective for my husband, Guy. His PSA post-hyperthermia is 0.6, down from 34.95 prior to hyperthermia.

[j-o-h-n]

Patrick, I bet when you were working you got paid by the word....

Good Luck, Good Health and Good Humor.

j-o-h-n Tuesday 02/26/2019 11:02 AM EST

[j-o-h-n]

Speaking of Parasites: My ex-wife is my ex-wife.

Good Luck, Good Health and Good Humor.

j-o-h-n Tuesday 02/26/2019 11:12 AM EST

[monte1111]

At least yamobedeh's ex-wife gave him something. Thanks to all of the knowledgeable persons who contributed to this post. I knew I had an alien inside me. Now it appears there are thousands, just waiting to eat me from the inside out. Enjoy.

[Advo__cate in reply to monte1111]

30 - 400 trillion ...of various gut microbes, is the estimation.

[Captain_Dave]

Didn't Hippocrates say that all disease starts in the gut?

[softwaremom00]

Cool post. Thanks for sharing. Also thanks for the jokes.. you guys often make me smile! I will have to email this post to my husband.

[AlanLawrenson]

Very interesting. I was DX in 2012. Had h.pylori infection about 5 years previously. Had PBT in Seoul, SK in 2013. Been good since. Had h.pylori infection again a year ago. Tri-drug Nexium therapy almost killed me, but finally killed the bug.

Spent the day researching PCa stem cells. I think I deserve a glass of Chardonnay!

[helvi]

"The CDC estimates

that nearly one out of four American adults has been exposed to Toxoplasma

gondii>" (CDC, 2000a)

"Stealth Parasites: The Under Appreciated Burden of Parasitic Zoonoses in North America

Authors

Authors and affiliations

J. L. GauthierAnuj GuptaPeter Hotez