PMCID: PMC2687140
PMID: 19049251
Unexpected Antitumorigenic Effect of Fenbendazole when Combined with Supplementary Vitamins
This article has been cited by other articles in PMC.
Abstract
Diet
containing the anthelminthic fenbendazole is used often to treat rodent
pinworm infections because it is easy to use and has few reported
adverse effects on research. However, during fenbendazole treatment at
our institution, an established human lymphoma xenograft model in
C.B-17/Icr-prkdcscid/Crl (SCID) mice failed to grow.
Further investigation revealed that the fenbendazole had been
incorporated into a sterilizable diet supplemented with additional
vitamins to compensate for loss during autoclaving, but the diet had not
been autoclaved. To assess the role of fenbendazole and supplementary
vitamins on tumor suppression, 20 vendor-supplied 4-wk-old SCID mice
were assigned to 4 treatment groups: standard diet, diet plus
fenbendazole, diet plus vitamins, and diet plus both vitamins and
fenbendazole. Diet treatment was initiated 2 wk before subcutaneous
flank implantation with 3 × 107 lymphoma cells. Tumor size
was measured by caliper at 4-d intervals until the largest tumors
reached a calculated volume of 1500 mm3. Neither diet
supplemented with vitamins alone nor fenbendazole alone caused altered
tumor growth as compared with that of controls. However, the group
supplemented with both vitamins and fenbendazole exhibited significant
inhibition of tumor growth. The mechanism for this synergy is unknown
and deserves further investigation. Fenbendazole should be used with
caution during tumor studies because it may interact with other
treatments and confound research results.
Abbreviation: HIF, hypoxia-inducible factor 1α
Pinworms are a common problem in rodent facilities4,16and typically are treated with anthelminthics.17 Fenbendazole incorporated in the diet is used often because it is safe—the oral LD50 for rats and mice is in excess of 10,000 mg/kg5—and labor-efficient, and adverse effects in research rodents have rarely been reported.20
More than 50% of ingested fenbendazole is absorbed and metabolized in
the liver, primarily to the active form, fenbendazole sulfoxide.19
Fenbendazole inhibits microtubule polymerization, and its efficacy as
an anthelminthic results from its greater affinity for helminth tubulin
than mammalian tubulin.9
During an 8-wk facility treatment for Aspiculuris tetraptera pinworms with fenbendazole diet at our institution, human lymphoma xenografts failed to grow in C.B-17/Icr-Prkdcscid/Crl
(SCID) mice. This well-established xenograft model is used to study the
role of mitochondrial genes in tumorigenesis and usually results in 80%
to 100% successful tumor growth within 21 d. However, during the
facility treatment with fenbendazole, no tumors grew in 40 mice during
the 30 d after injection. The mice in this study had not been diagnosed
with pinworms but were part of a facility treatment. Rodents in this
area customarily were fed a commercial irradiated diet (Global 2918,
Harlan Teklad, Madison, WI). However the equivalent treatment diet
containing 150 ppm fenbendazole was available only in a sterilizable
form (2018S, Harlan Teklad) supplemented with vitamins A, D, E, K, and B
(Table 1) to compensate for loss during sterilization. Because the
animal facility was not configured for dietary sterilization, the
sterilizable diet was fed unautoclaved, with the result that mice
received higher-than-normal concentrations of vitamins. Therefore the
observed antitumor effect could have resulted from either the additional
vitamins or the fenbendazole. Therefore a controlled study was
conducted to test whether fenbendazole, supplemented vitamins, or both
in combination affected the growth of this human lymphoma cell line in
SCID mice.
Materials and Methods
Mice were housed in an AAALAC-accredited facility under conditions compliant with the Guide for the Care and Use of Laboratory Animals.12
Procedures were approved by the Johns Hopkins institutional animal care
and use committee. The mice were housed in individually ventilated
cages (Allentown Caging Equipment, Allentown, NJ) on autoclaved corncob
bedding (Bed-O'Cobs, The Andersons, Maumee, OH). Mice received
hyperchlorinated reverse-osmosis–treated water by means of an in-cage
automated watering system (Edstrom Industries, Waterford, WI). Cages
were changed by using chlorine-dioxide-based disinfectant (MB10 tabs,
100-ppm solution, Quip Laboratories, Wilmington, DE) in filtered-air
change stations (Lab Products, Seaford, DE). The colony tested free of a
wide range of viral and parasitic pathogens by sentinel surveillance;
pathogens included Sendai virus, pneumonia virus of mice, mouse
hepatitis virus, mouse minute virus, mouse parvovirus 1 and 2, Theiler
mouse encephalomyelitis virus, reovirus, epizootic diarrhea of infant
mice, lymphocytic choriomeningitis virus, ectromelia virus, murine
adenovirus, murine cytomegalovirus, Mycoplasma pulmonis, fur mites, and pinworms.
Twenty
4-wk-old male SCID mice (Charles River Laboratories, Boston, MA) were
assigned randomly to 4 groups housed 5 animals per cage: control diet
(2018, Harlan Teklad), diet plus fenbendazole (2018 custom-formulated
with 150 ppm fenbendazole, Harlan Teklad), diet plus supplemented
vitamins (2018S, Harlan Teklad), and diet plus both fenbendazole and
supplemented vitamins (2018S plus 150 ppm fenbendazole, Harlan Teklad).
All diets had the same basic composition (18% protein, 5% fat; Table 1),
and none of the diets was autoclaved. The mice were stabilized on their
respective diets for 2 wk after arrival and before tumor cells were
injected.
Table 1.
Vitamin levels in nonsterilizable (regular) and sterilizable (supplemented) diets
| Diet | ||||
| Vitamins | Regular | Supplemented | Units | % Increase |
| A | 15.4 | 30.7 | IU/g | 100 |
| Retinol | 4.65 | 9.31 | mg/kg | 100 |
| D3 | 1.54 | 2.05 | IU/g | 33 |
| Cholecalciferol | 38.39 | 51.18 | g/kg | 33 |
| E | 101 | 126 | mg/kg | 25 |
| K3 | 51 | 102 | mg/kg | 100 |
| B1 | 16.5 | 117.6 | mg/kg | 613 |
| B2 | 14.9 | 27.2 | mg/kg | 83 |
| Available niacin | 41.2 | 87.3 | mg/kg | 112 |
| B6 | 18.5 | 26.8 | mg/kg | 45 |
| Pantothenic acid | 33 | 141.6 | mg/kg | 329 |
| B12 | 0.08 | 0.15 | mg/kg | 88 |
| Available biotin | 0.3 | 0.82 | mg/kg | 173 |
| Folate | 3.34 | 8.41 | mg/kg | 152 |
The data shown are vendor-reported values.
The
day after arrival, blood was collected by puncture of the facial vein
under manual restraint and processed by using an automated analyzer
(Hemavet 950, Drew Scientific Group, Dallas, TX) for complete blood
count. Human Burkitt lymphoma cells (P493-6 B cell line8)
were cultured in RPM1 1640 plus 10% fetal calf serum containing 100 U/ml
penicillin and 100 µg/ml streptomycin. The cells were washed, counted,
and resuspended in PBS. Each mouse was restrained manually and received 3
× 107 lymphoma cells in 100 µL PBS injected subcutaneously
in the flank. Growth of tumors was monitored every 4 d by using
calipers, and tumor volume was calculated by using the formula length ×
width × width × 0.52 mm3. Once the largest tumors reached a calculated volume of 1500 mm3,
the experiment was terminated. Before euthanasia of mice, blood was
collected from the facial vein and analyzed for complete blood count.
The size of tumors in each group at the endpoint was compared with that
of the control group by using the Student t test. Total white
cell, lymphocyte, and neutrophil counts were compared with those of
controls at the beginning and end of the experiment.
Results
Tumor size.
Tumors in the fenbendazole plus vitamin group were significantly smaller (P = 0.009) and delayed in initial growth compared with those of the control group (Figure 1). Tumor growth did not differ between control and fenbendazole-only (P = 0.12) or vitamin-only (P = 0.82) groups. The apparent trend toward increased tumor size (P = 0.12) in the fenbendazole-only group was due to a single outlier.
Growth
in tumor volume (mean ( SD) in mice on 4 different diets. After
subcutaneous injection of lymphoma cells, tumor growth in mice receiving
fenbendazole- or vitamin- supplemental diet did differ from that in
controls. Tumor growth in mice on fenbendazole- plus
vitamin-supplemented diet was significantly (P = 0.009) inhibited
compared with that in controls.
White cell counts.
One
mouse died during the initial blood collection, and 1 blood sample from
the terminal group was lost. Initial complete blood counts (Figure 2)
were typical of SCID mice, demonstrating a low white cell count with a
paucity of lymphocytes. White cell counts did not differ significantly
between test and control groups on arrival. At the end of the study (Figure 2),
all groups demonstrated a leukocyte response consisting primarily of
neutrophils. The total white cell and neutrophil responses in the
fenbendazole plus vitamin group were significantly smaller (P = 0.001 and P = 0.04, respectively) than in the control group. There was a trend (P = 0.06) toward increased total white cell count and a significantly increased lymphocyte count (P = 0.009) in the fenbendazole-only group compared with controls.
Initial
and terminal white cell counts (mean ± SD) in mice on 4 different diet.
Initial counts did not differ among the 4 groups. Terminal total white
cell and neutrophil counts in mice receiving the (p=0.001 and p=0.04
respectively) lower than those in control animals, and terminal
lymphocyte counts in the fenbendazole-only group were significantly (P =
0.009) higher than those in controls.
Discussion
This
study demonstrated that a combination of supplemented vitamins and
fenbendazole in the diet inhibited growth of a human lymphoma cell line
in SCID mice, whereas fenbendazole or vitamins alone had no growth
inhibitory effect. The mechanism for this synergy is as yet unknown.
However, like other anticancer drugs such as taxanes,14 quinolones,3 and vinca alkaloids,15
fenbendazole inhibits microtubule polymerization. In addition,
fenbendazole is a member of a large group of related anthelminthics, the
benzimidazoles, and another member of this group, mebendazole, exerts
antitumor effects by inhibition of tumor-induced neovascularization.11
However, in the present case, fenbendazole likely contributes to the
antitumorigenic effect through its antimicrotubule activity.
Supplemented
vitamins included B, D, K, E, and A. Vitamins E and A both have
antitumor properties by virtue of their antioxidant properties. Vitamin E
causes antitumor and antimetastatic effects in several animal models of
cancer; for example, it suppresses the nuclear transcription factor
NFκB in prostate cell lines.13
NFκB regulates proapoptotic and prometastatic proteins; thus
suppression results in antitumor effects. Higher intake of dietary
folate and vitamin B has been associated with lower incidence of
colorectal cancer in women.22
Recent work suggests that hypoxia-inducible factor 1α (HIF), which
plays a key role in tumorigenesis by facilitating adaptation to hypoxia,
is diminished by microtubule inhibitors,7 and some antioxidants may exert their antitumor effects through reducing HIF rather than by reducing genetic instability.8,10
We hypothesize that the combination of fenbendazole and supplemented
vitamin antioxidants may have exerted a threshold effect, resulting in
reduction of HIF and inhibition of tumorigenesis. Indeed, preliminary
information from our laboratory confirms that fenbendazole inhibits HIF
transcriptional activity in cell culture in an additive manner with
other HIF inhibitors (data not shown).
This study
demonstrated significant inhibition of tumor growth. Results were less
dramatic than the total inhibition initially observed, perhaps due to
inadvertent inclusion of lower vitamin concentrations during the study
than during the initial observation. Vitamins in prepared diets
deteriorate with time, and the study diet containing both vitamins and
fenbendazole was within a week of its expiration date (6 mo after
manufacture) at the time of this study. In contrast, diet used during
the initial observation was newly ordered to treat the pinworm outbreak
and so was likely more recently manufactured, with higher vitamin
concentrations. Unfortunately expiration dates during the initial
observation were not recorded and vitamin concentrations in the diet
were not analyzed independently so this theory cannot be confirmed.
Figure 1 shows a trend (P = 0.12) toward increased
tumor growth in the fenbendazole only treatment group. This trend was
the result of 1 outlier. Nonetheless, fenbendazole may have
tumor-promoting activity in rats at therapeutic dosages (that is,
through inhibition of connexin 32 and induction of cytochrome P450
enzymes 1A1 and 1A2).21
In our experiment, the apparent increase in tumor size in the
fenbendazole group was due to a single large-tumor outlier, and we
believe that this trend is due to experimental noise. Further studies
are needed to determine whether fenbendazole actually exhibits a
tumor-promoting effect in this model.
Although the
exact composition of T and B cells was not analyzed, complete blood
counts confirmed that the numbers of white cells did not differ
initially between the treatment and control groups, ruling out the
possibility of a chance concentration of relatively immunocompetent
‘leaky’ SCID mice1,2
in 1 group. At study termination, the fenbendazole plus vitamin group
had significantly lower total white cell and neutrophil values (P = 0.001 and P
= 0.04, respectively) than did the control group. This observation is
consistent with significantly smaller tumors causing less compression
and necrosis of adjacent tissues, although this supposition was not
confirmed by histopathology. There was also a trend (P = 0.06) toward increased total numbers of white cells and a significantly larger (P = 0.009) lymphocyte response in the fenbendazole-only group. Fenbendazole has had immunomodulatory effects in sheep and mice18 and stimulated proliferation of T and B cells in healthy mice,6 but most studies have shown no effect on selected immune responses.21
It is not possible to draw any conclusions regarding immunomodulation
from the present study because additional analysis of cell types was not
done.
Our study showed that
fenbendazole alone did not significantly affect growth of the P493-6
human lymphoma cell line in SCID mice. Most importantly, our observation
that fenbendazole in combination with supplemented vitamins
significantly inhibited tumor growth has implications for its
use during antitumor studies because it may cause unpredictable
interactions with test substances and thus alter research results.
Acknowledgments
This
study was in part supported by Leukemia Lymphoma Society grant
LLS6175-08, NIH grant CA57341, and by Johns Hopkins Research Animal
Resources.
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