ArticlePDF Available

Effects of fenbendazole and vitamin E succinate on the growth and survival of prostate cancer cells

Authors:

Abstract and Figures

We describe antitumor activities of vitamin E succinate (VES), an anti-oxidant and fenbendazole (FBZ), a commonly used veterinary anthelmintic. We used VES and FBZ, at low concentrations, singly and in combination, to test their inhibitory effects on proliferation of human and mouse prostate cancer cells in vitro. Administered alone, FBZ inhibited proliferation faster than VES in both mouse and human prostate cancer cell lines and a synergistic effect between both was also observed. Apoptosis was the likely mechanism for the observed effect. These drugs may deserve to be tested for their efficacy in the control of prostate cancer using in vivo models.
Content may be subject to copyright.
Journal of Cancer Research and Experimental Oncology Vol. 3(9), pp. 115-121, December 2011
Available online http://www.academicjournals.org/JCREO
DOI: 10.5897/JCREO11.047
ISSN 2141-2243 ©2011 Academic Journals
Full Length Research Paper
Effects of fenbendazole and vitamin E succinate on the
growth and survival of prostate cancer cells
Ari N. Aycock-Williams1*, Linda K. Pham2, Mengmeng Liang2, Helty A. Adisetiyo2, Lauren A.
Geary2, Michael B. Cohen3, Donald B. Casebolt1,2 and Pradip Roy-Burman2
1Department of Animal Resources, University of Southern California, Los Angeles, CA, USA.
2Department of Pathology, University of Southern California, Los Angeles, CA, USA.
3Department of Pathology, University of Iowa Carver College of Medicine, Iowa City, IA, USA.
Accepted 23 November, 2011
We describe antitumor activities of vitamin E succinate (VES), an anti-oxidant and fenbendazole (FBZ),
a commonly used veterinary anthelmintic. We used VES and FBZ, at low concentrations, singly and in
combination, to test their inhibitory effects on proliferation of human and mouse prostate cancer cells
in vitro. Administered alone, FBZ inhibited proliferation faster than VES in both mouse and human
prostate cancer cell lines and a synergistic effect between both was also observed. Apoptosis was the
likely mechanism for the observed effect. These drugs may deserve to be tested for their efficacy in the
control of prostate cancer using in vivo models.
Key words: Prostate cancer, fenbendazole, vitamin E succinate.
INTRODUCTION
Although various treatments are available for the early
stages of prostate cancer (PCa), the options are,
however, quite limited for advanced or recurrent disease
because of ultimate resistance of these tumors to
chemotherapy and endocrine manipulation (Ismail and
Gomella, 1997; Sullivan et al., 1998). For this reason,
finding novel and alternative methods of treating PCa
have received much attention (Agarwal, 2000; Deutsch et
al., 2004; Gronberg, 2003; Kline et al., 2004; Vashchenko
and Abrahamsson, 2005). Benzimidazoles are a class of
drug that are traditionally used for treating parasitic
infections in humans and animals and are remarkably
safe and efficacious (Lacey, 1988). A primary mechanism
of action relates to the binding of benzimidazoles to
*Corresponding author: E-mail: ariaycockw@gmail.com or
aycock@med.usc.edu. Tel: (734)330-8898. Fax: (323) 442-
2322.
Abbreviations: CRPC, castration resistant prostate
cancer; FBZ, fenbendazole; PCa, prostate cancer;
TUNEL, terminal deoxynucleotidyl transferase mediated
dutp nick end labeling; VES, vitamin E succinate.
nematode β–tubulin that inhibits microtubule
polymerization and thereby interfering with cell division
(Lacey, 1988; Laclette et al., 1980; Sasaki et al., 2002).
Several other cancer chemotherapeutics are also known
to either stabilize microtubulin (paclitaxel and docetaxel)
or destabilize microtubulin (vinblastine and vincristine),
but because of high toxicity their therapeutic efficacy is
limited (Mukhopadhyay et al., 2002). While parasites are
the primary targets of benzimidazoles because of their
affinity for non-mammalian microtubulin and rapidly
dividing cells (Lacey and Gill, 1994), Mebendazole, a
derivative of the Benzimidazoles, has been shown to
selectively induce apoptosis in adrenocortical carcinoma
in vivo and in vitro (Martarelli et al., 2008), and in
melanoma cells in vitro (Doudican N et al., 2008). A
Phase I clinical trial reported by Morris et al., 2001
showed Albendazole, another Benzimidazole, had a high
maximum tolerated dose when given to 36 patients with
malignant tumors, 16% of which showed a decrease in
tumor markers (Morris et al., 2001) .
Fenbendazole is a benzimidazole drug commonly used
for treating pinworm outbreaks in laboratory rodents
(Coghlan et al., 1993). It has a wide margin of safety with
an oral LD50 in mice of >10,000 mg/kg (O'Neil, 2001).
This drug was shown to have a synergistic inhibition of
116 J. Cancer Res. Exp. Oncol.
lymphoma growth in SCID mice when combined in the
diet with supplemental vitamins (Gao et al., 2008).
However, a specific vitamin was not identified.
There is evidence to suggest that vitamin E (RRR-α-
tocopherol) may be beneficial in preventing or delaying
PCa growth (Basu and Imrhan, 2005; Fleshner, 2002;
Gronberg, 2003; Malafa et al., 2006). Vitamin E succinate
(VES), the most potent derivative of vitamin E for anti-
tumor activity (Basu and Imrhan, 2005), has been shown
to induce apoptosis in PCa in vitro and in vivo through
caspase-4 activation (Malafa et al., 2006). An in vitro
study showed that when human PCa PC-3 cells were
treated in culture with 20 µM of VES, there was a growth
inhibition of 40% after 3 days of treatment (Zu and Ip,
2003). Especially when administered in the diet, VES has
displayed chemopreventive effects (Basu and Imrhan,
2005). In a study performed on the LADY (12T-10)
transgenic mouse strain which harbors expression of
SV40 large T antigen but not small t antigen, a diet with a
combination of antioxidants lycopene, selenium, and VES
was fed from the time of weaning. In these mice, PCa
incidence decreased by 4 times that of controls
Venkateswaran et al., 2004. Supporting data from these
animal models have led to consideration in clinical
medicine. An ongoing human clinical trial, the selenium
and vitamin E cancer prevention trial (SELECT) that is
projected to end in 2013, will likely be informative on
whether vitamin E, in conjunction with selenium, is a
chemopreventive agent (Klein et al., 2003; Lieberman,
2003; Ni and Yeh, 2007).
Several spontaneous mouse models of prostate cancer
have been developed based on genetic alterations that
are prominent in human prostate cancer to capture the
natural course of initiation and progression of this cancer
(Mimeault and Batra, 2011; Roy-Burman et al., 2004).
One such model, the conditional Pten deletion mouse
model with a Luciferase reporter (cPten-/- L) (Wang et al.,
2003; Liao et al, 2007), leads to development of prostatic
adenocarcinoma. After castration, tumor burden begins to
decrease rapidly due to the dependence of the primary
tumor on androgen presence. However, the residual
tumors undergo recurrent growth to give rise to castration
resistant prostate cancer (CRPC).
The E8 cell line is a neoplastic epithelial cell line
derived from the primary prostate tumor of the cPten-/-L
mouse which maintains sensitivity to androgen for growth
(our unpublished data). The previously described cE1
carcinoma cell line (Liao et al., 2010) was derived from a
CRPC tumor of the cPten-/-L model. This cell line can
grow in the absence of androgen but still remains
sensitive to androgen stimulation. The PC-3 cell line, a
human prostate cancer cell line of bone metastatic origin,
was obtained from ATCC (Manassas, VA).
In this study we used malignant epithelial cell lines
(Liao et al, 2010) derived from both primary and CRPC
tumors of this model along with a human prostate cancer
cell line to determine the effect of FBZ and VES, singly
and in combination, on their in vitro growth potential.
MATERIALS AND METHODS
E8 and cE1 cells were cultured in DMEM media supplemented with
10% FBS, 1% penicillin/streptomycin, 25 µg/ml bovine pituitary
epithelium, 5 µ g/ml Insulin, and 6 ng/ml recombinant human
epithelial growth factor. PC-3 cells were cultured in DMEM media
supplemented with 1% Penicillin and Streptomycin, 10% FBS, 1x
Non-essential amino acid, and 1x essential amino acid. All cells
were grown until 70% confluent.
To evaluate the inhibitory effect of FBZ (Santa Cruz
Biotechnology, Santa Cruz, CA) and VES (supelco analytical,
bellfonte, PA) on the growth of E8, cE1, and PC-3, the cells (2 ×
104) were seeded in 12-well plates in the maintenance media for 12
h. A dose response curve was performed on E8 cells to determine
the activity of each agent. FBZ was diluted serially from 2:1, 1:1,
1:10, and 1:100, from a starting concentration of 450 ng/ml to 2.25
ng/ml. VES was serially diluted in the same manner but with a
starting c oncentration of 50 µg/ml diluted down to 0.025µg/ml.
Intermediate c oncentrations were also tested that included 56, 140,
168, and 200 ng/ml for FBZ. For VES, intermediate concentrations
included 30 and 40 µg/ml. The vehicle control media contained
ethanol in equal volume. The cultures were monitored for up to 4
days. Once it was determined that FBZ had a stronger effect than
VES on proliferation, an experiment was performed on the PC-3
cell line to see whether these drugs had a synergistic effect on
proliferation. Synergy was measured by cell counts that were
smaller in number when the two drugs were used together at the
optimal dosage than when used alone. The VES concentration of
25 µg/ml was chosen since it was within the IC50 of the drug and
was the highest dose with which growth inhibition was not observed
in the E8 c ells for the first four days. A 12 well plate was treated
with dosage c ombinations of VES+FBZ with a corresponding
vehicle control well. The dosage of 25 µg/ml VES and 14 ng/ml of
FBZ were determined to be the optimal dosages for which growth
inhibition occurred without acute detachment and cell floatation,
and these dosages were used on the other cell lines.
Each cell line was seeded onto six-well plates where three wells
were treated with the vehicle c ontrol and three were treated with 25
µg/ml of VES and 14 ng/ml FBZ. The culture medium was changed
every 2 days and the cell proliferation rate was determined at time
points 1,3,5, and 7 days by cell counting (Beckman Coulter Cell
Counter, Brea, CA).This growth analysis was repeated in
independent triplicates on all 3 cell lines.
Cellular apoptosis in E8, cE1, and PC-3 cells was measured
using APO-BRDUTM Terminal deoxynucleotidyl transferase
mediated dUTP Nick End Labeling (TUNEL) kit (Phoenix Flow
Systems, San Diego, CA). After signs of cell death were observed
(4 days for E8 and cE1, 3 days for PC-3), cells were collected, and
TUNEL was performed and quantified by flow cytometric analysis
on an LSRII machine (SORP, BD biosciences) following the
instructions of the manufacturer.
For the pilot in-vivo experiment, normal mice were euthanized
with an overdose of Isoflurane anesthesia followed by cervical
dislocation at 11 months of age. Their prostates were dissected out
into the separate lobes, paraffinized, and cut into sections using a
mictrotome, (Mikron Instruments Inc, Germany). Sections were
stained with hematoxylin and eosin. Immunohistochemical analysis
was performed on s lides of parallel paraffin s ections of
paraformaldehyde-fixed tissue using a modified Avidin Biotin
Complex technique, as described previously (Zhou et al., 2006).
Antigen retrieval was accomplished by boiling the s lides in 10
mmol/L of citric acid buffer (pH 6.0) for 15 min. Antibodies to
cytokeratin 8 (CK8; 1:100 TROMA-1 antibody; Developmental
Studies Hybridoma Bank, University of Iowa, Iowa City, Iowa),
androgen r eceptor ( ARsc-815 1:200; Santa Cruz Biotechnologies,
Santa Cruz, CA), and vimentin (R28 1:50; c ell signaling technology,
danvers, MA) were incubated overnight at C. Sections were
incubated with biotinylated secondary antibody for 30 min at room
temperature. Apoptosis was detected with TUNEL assay using the
In Situ death detection kit from Roche according to manufacturer’s
directions. All sections were then detected with the ABC elite kit
(Vector Laboratories Inc, Germany) and 3, 3-diaminobenzidine
(DAB Sigma, Dako North America, Carpinteria, CA) as substrate.
All slides were dehydrated through graded alcohols to xylenes and
mounted with coverslips. Animal studies were approved by the
University of Southern California Institution Animal Care and use
committee and housed according to federal guidelines.
Statistical analysis
Statistical analysis was performed with excel 2007 (microsoft,
Redmond, MA). The results of the cell proliferation assay were
evaluated as the mean ± SD of at least three different experiments
performed in triplicate. For both the cell proliferation assay and the
TUNEL assay, differences between control cell numbers were
compared with that of treated cells and were analyzed by
independent t-test, P values of < 0.01 were considered st atistically
significant. Flow cytometric data was collected on BD FACS Diva
software version 6.0 (San Jose, CA) and analyzed on Flow Jo
software version 9.3 (Treestar, Ashland, OR).
RESULTS AND DISCUSSION
Growth assays for all three cell lines were performed
(Figure 1A to D). After testing E8 cell line singly with FBZ
then VES, we determined that neither FBZ at 22.5 ng/ml
nor VES at 25 µg/ml had any significant inhibitory effect
on proliferation, at least during the four days of exposure
(Figure 1A). However, when we used a lower
concentration of FBZ (14 ng/ml) together with VES (25
µg/ml), beginning at the third day, a synergistic inhibitory
effect on proliferation was observed that became robust
in the subsequent days (Figure 1B). A similar pattern of
inhibition was seen with the cE1 (Figure 1C) and PC-3
cells (Figure 1D). All cells were seeded at 2 × 104 cells/ml
and proliferation reached to 3.5 × 106 for control E8 cells,
3.91 × 104 for treated E8 cells, 2.37 × 106 for control cE1
cells, 1.2 × 105 for treated cE1 cells, and 3.7 × 105 for
the control PC-3 cells, 733 for treated PC-3 cells by day
7. The treatment of each cell line yielded a highly
significant (P<0.01) level of cell inhibition at 5 to 7 days.
Representative results from the APO-BRDUTM terminal
deoxynucleotidyl transferase mediated dUTP Nick end
labeling (TUNEL) assay for each cell line are shown
(Figure 1E to G). The cells treated with ethanol as the
vehicle control were represented by the white and
farthest left peak. There was some overlap with cells from
the FBZ+VES treated group which indicated a minimal
amount of apoptosis was occurring in these cells.
However, as determined by the TUNEL assay and flow
cytometric quantification analysis, an average of 42.6
±5.91% of treated E8 cells underwent apoptosis by day 4,
in contrast to only 1.05 ± 1.22% of vehicle control cells.
An average of 22.4 ± 9.42% treated cE1 cells underwent
Aycock-Williams et al. 117
apoptosis by day 4 compared to 2.25 ± 1.71% of vehicle
control cells. For PC-3, apoptosis occurred in 93 ± 2.9%
of treated cells in contrast to 27.2 ± 9.89% labeled control
cells. These results of increased apoptosis induced by
the combination of FBZ and VES were all statistically
significant (P<0.01), indicating that the observed
inhibitory effect on proliferation might be related to
induction of apoptosis by the agents at the concentrations
used.
FBZ+VES significantly inhibited growth of PCa cells
and induced apoptosis in vitro. To our knowledge, this is
the first time these drugs have been tested together in
PCa cell lines. In a previous study, a concentration of 50
µg/ml of VES was shown to be effective on PC-3 cells
(Malafa et al., 2006). For FBZ, we tested a range of
concentrations based on a dosage used for Mebendazole
treatment of melanoma cells in culture (Doudican et al.,
2008). A dosage of 25 µg/ml of VES and 14 ng/ml of FBZ
was optimal for the treatment of prostate cancer cell lines
we tested. Higher concentrations of FBZ produced a
rapid rate of cell death during the initial 48 h period and
lower concentrations did not cause any change in cell
growth compared to controls. The agents were combined
at the specified dosages because alone neither produced
results that were as striking as when they were together.
This synergistic ability of inhibition is very interesting,
although the mechanisms involved is unclear. One
hypothesis is that vitamin E activates multiple pro-
apoptotic pathways such as targeting NF-κΒ (Ni and Yeh,
2007), and caspase-4 (Malafa et al., 2006), and if used
with an agent that inhibits cell division such as one of the
benzimidazoles, can be antagonistic to tumor growth.
These agents are worth further exploration since they are
both relatively safe and easy to administer.
We determined that apoptosis occurred in all three cell
lines as a result of the combination treatment. PC-3 had
the shortest treatment time and the most cell death when
compared to E8 and cE1. This may be as a result of
human cells being more sensitive to this combination of
drugs versus mice having a higher tolerance. Between
the two mouse cells lines tested, the more androgen
independent cE1 cells derived from CRPC appears to be
more resistant to the effects of FBZ+VES than the
primary tumor E8 cells from the same model. Further
studies will be needed to understand which mediators
may be responsible for drug resistance specific to these
cell types. Because all three cell lines had a generally
similar response to the treatment in combination, it can
be concluded that both mouse androgen dependent as
well as human and mouse CRPC cells are susceptible to
FBZ +VES. Thus, these drugs may potentially have
efficacy independent of the hormonal environment.
As a preliminary study, a small group of normal mice
were fed with either FBZ, VES, or a VES+FBZ
combination administered in the feed for 206 days at
which point they were humanely euthanized. Mice fed a
normal rodent diet that was not supplemented were used
118 J. Cancer Res. Exp. Oncol.
Figure 1. Effect of VES+FBZ on the proliferation and apoptosis of human and mouse prostate cancer cell lines. (A) Growth inhibitory
properties of dosages of VES or FBZ on the E8 mouse primary prostate cancer c ell line. (B) Inhibitory effect of combination of VES and FBZ
at low concentrations on the proliferation of E8 c ells. Similar treatment of a mouse prostate cancer c ell line, cE1 derived from a recurrent
cancer (C), and a human prostate cancer cell line PC-3 derived from a bone metastasis (D). W ells were treated with 25 µg/mL VES and
14ng/mL FBZ and an equal volume of Ethanol as the vehicle c ontrol. Representative TUNEL ass ays c onducted for E8 c ells tr eated for 4
days (E), cE1 cells treated for 4 days (F), and PC-3 cells treated f or 3 days (G) with the same concentrations of VES+FBZ. These graphs
are merged to show the vehicle control cells in r elation to that of the experimental groups of c ells. The white peaks represent the population
of live cells (vehicle control) and the gray peaks represent the c ell fraction undergoing apoptosis (FBZ+VES treated). Asterisk (*) denotes
statistical significance at a P value <0.01.
as age-matched controls. While serum markers of organ
failure would have been ideal indicators of toxicity, we
focused on how these drugs affect the function and
anatomy of the prostate on histopathology. General
physical changes such as weight loss, hair coat,
behavior, ability to urinate and defecate normally,
respiration, and activity were monitored by a veterinarian
throughout the study for signs of toxicity and illness but
Aycock-Williams et al. 119
H and E
Tunel
Vimentin
Figure 2. Illustration of comparative histological analysis of the prostate tissue sections of normal mice kept on the FBZ + VES diet (first
row) and control diet not containing FBZ or VES (second row). Representative staining for Hematoxylin and Eosin (H and E), Cytokeratin 8
(CK8), androgen r eceptor (AR), terminal deoxynucleotidyl tr ansferase mediated dUTP Nick end labeling (TUNEL), and vimentin on serial
sections of the dorsolateral prostatic lobe are shown. Arrows, areas enlarged in the insets (400X). Bar, 100 µm.
no abnormalities were observed. Body weight was
measured monthly and significantly increased (P<0.001)
by the end of the study indicating the diet was palatable,
non-toxic, and the mice maintained a positive energy
balance. Upon necropsy, grossly, the dorsolateral,
anterior, and ventral prostatic lobes were morphologically
normal. Histopathology analysis was performed on their
prostates and normal glandular structures were observed
in all groups (Figures 2 and 3). No significant observable
differences in architecture were seen between the groups
and controls except there was more secretory material
present in the lumen of the glands from the FBZ+VES
group. We do not believe this is due to the drugs since
the single treatments did not produce the same result.
This is more likely to be a physiological process that is
occurring such as an increase in prostatic fluid at the time
of death. Expression of androgen receptor and
cytokeratin 8 was present and did not differ between
cells. Stromal proliferation as marked by Vimentin
staining was minimal and also did not differ between
groups. The amount of cells in each group had a minimal
population of cells undergoing apoptosis and also did not
differ. The similarities between groups may indicate these
drugs are non-toxic even when given at 150 ppm of FBZ
and 2000 IU of VES orally for prolonged time periods.
While the preliminary data showed these drugs are
relatively benign further in vivo studies will be necessary
to establish the validity of these observations at higher
concentrations. Additionally, a large study in which
animal models that develop spontaneous tumors are fed
120 J. Cancer Res. Exp. Oncol.
H and E
Tunel
Vimentin
Figure 3. Illustration of comparative histological analysis of the prostate tissue sections of normal mice kept on the VES alone
supplemented diet (first row) and FBZ alone supplemented diet (second row). Representative staining for hematoxylin and eosin (H and
E), Cytokeratin 8 (CK8), androgen receptor (AR), terminal deoxynucleotidyl transferase mediated dUTP Nick end labeling (TUNEL), and
vimentin on serial sections of the dorsolateral prostatic lobe are shown. Arrows, areas enlarged in the insets (400X). Bar, 100 µm.
these agents in the diet at the highest non-toxic
concentrations would be worthwhile for exploration of
these drugs’ efficacy in vivo.
In summary, combination therapy with VES and FBZ
deserves further investigation as a possible treatment
modality for prostate cancer.
ACKNOWLEDEGMENTS
This study was supported by NIH grant No. RO1
CA59705 (to P. Roy-Burman). The authors would like to
thank the animal care and technical staff at the University
of Southern California for the continuous feeding and
monitoring of the animals. Much appreciation also goes
to Lora Barsky of the Flow Cytometry Core and to Dr.
Carrie Schultz at Land O’Lakes Purina Mills for her
guidance with the diets.
REFERENCES
Agarwal R (2000). Cell signaling and regulators of cell cycle as
molecular targets for prostate cancer prevention by dietary agents.
Biochem. Pharmacol., 60: 1051-1059.
Basu A, Imrhan V (2005). Vitamin E and prostate cancer: Is vitamin E
succinate a superior chemopreventive agent? Nutr. Rev., 63: 247-
251.
Coghlan LG, Lee DR, Psencik B, Weiss D (1993). P ractical and
effective eradication of pinworms (Syphacia muris) in rats by use of
fenbendazole. Lab. Anim. Sci., 43: 481-487.
Deutsch E, Maggiorella L, Eschwege P, B ourhis J, Soria JC,
Abdulkarim B (2004). Environmental, genetic, and molecular f eatures
of prostate cancer. Lancet Oncol., 5: 303-313.
Doudican N, Rodriguez A, Osman I, Orlow SJ (2008). Mebendazole
induces apoptosis via Bcl-2 inactivation in chemoresistant melanoma
cells. Mol. Cancer Res., 6: 1308-1315.
Fleshner NE (2002). Vitamin E and prostate cancer. Urol. Clin. North
Am., 29: 107-113, ix.
Gao P, Dang CV, Watson J (2008). Unexpected antitumorigenic effect
of fenbendazole when combined with supplementary vitamins. J. Am.
Assoc. Lab. Anim. Sci., 47: 37-40.
Gronberg H (2003). Prostate cancer epidemiology. Lancet, 361: 859-
864.
Ismail M, Gomella LG (1997). C urrent treatment of advanced prostate
cancer. Tech. Urol., 3: 16-24.
Klein EA, Thompson IM, Lippman SM, Goodman PJ, Albanes D, Taylor
PR, Coltman C (2003). SELECT: The selenium and vitamin E c ancer
prevention trial. Urol. Oncol., 21: 59-65.
Kline K, Yu W , Sanders BG (2004). Vitamin E and breast cancer. J.
Nutr., 134: 3458S-3462S.
Lacey E (1988). The role of the cytoskeletal protein, tubulin, in the mode
of action and mechanism of drug resistance to benzimidazoles. Int. J.
Parasitol., 18: 885-936.
Lacey E, Gill JH (1994). Biochemistry of benzimidazole resistance. Acta
Trop., 56: 245-262.
Laclette JP, Guerra G, Zetina C (1980). Inhibition of tubulin
polymerization by mebendazole. Biochem. Biophys. Res. Commun.,
92: 417-423.
Liao CP, Liang M, Cohen MB, Flesken-Nikitin A, J eong JH, N ikitin AY,
Roy-Burman P (2010) .Mouse prostate cancer cell lines established
from primary and post-castration recurrent tumors. Horm. Cancer, 1:
44-54.
Liao CP, Zhong C, Saribekyan G, Bading J, Park R, Conti PS, Moats R,
Berns A, Shi W , Zhou Z, Nikitin AY, Roy-Burman P (2007). Mouse
models of prostate adenocarcinoma with the c apacity to monitor
spontaneous carcinogenesis by bioluminescence or fluorescence.
Cancer Res., 67: 7525-7533.
Lieberman R ( 2003). Evolving strategies for prostate cancer
chemoprevention trials. World J. Urol., 21: 3-8.
Malafa MP, Fokum FD, Andoh J, Neitzel LT, Bandyopadhyay S, Zhan
R, Iiizumi M, Furuta E, Horvath E, Watabe K (2006). Vitamin E
succinate suppresses prostate tumor growth by inducing apoptosis.
Int. J. Cancer, 118: 2441-2447.
Martarelli D, Pompei P, Baldi C, Mazzoni G (2008). Mebendazole
inhibits growth of human adrenocortical c arcinoma cell lines
implanted in nude mice. Cancer Chemother. Pharmacol., 61: 809-
817.
Mimeault M, Batra SK (2011). Animal models relevant to human
prostate carcinogenesis underlining the critical implication of prostatic
stem/progenitor cells. Biochim. Biophys. Acta, 1816: 25-37.
Morris DL, Jourdan JL, Pourgholami MH (2001). Pilot study of
albendazole in patients with advanced malignancy. Effect on serum
tumor markers/high incidence of neutropenia. Oncol., 61: 42-46.
Mukhopadhyay T, Sasaki J, Ramesh R, Roth JA (2002). Mebendazole
elicits a potent antitumor effect on human c ancer cell lines both in
vitro and in vivo. Clin. Cancer Res., 8: 2963-2969
Aycock-Williams et al. 121
Ni J, Yeh S (2007). The roles of alpha-vitamin E and its analogues in
prostate cancer. Vit. Horm., 76: 493-518.
O'Neil M (2001). The Merck Index. Whitehouse Station: Merck and Co.
Inc.
Roy-Burman P, Wu H, Powell WC, Hagenkord J, Cohen MB (2004).
Genetically defined mouse models that mimic natural aspects of
human prostate cancer development. Endocr. Relat. Cancer, 11:
225-254.
Sasaki J, Ramesh R, Chada S, Gomyo Y, Roth JA, Mukhopadhyay T
(2002). The anthelmintic drug mebendazole induces mitotic arrest
and apoptosis by depolymerizing tubulin in non-small cell lung cancer
cells. Mol. Cancer Ther., 1: 1201-1209.
Sullivan GF, A menta PS, Villanueva JD, Alvarez CJ, Yang JM, Hait WN
(1998). The expression of drug resistance gene products during the
progression of human prostate cancer. Clin. Cancer Res., 4: 1393-
1403.
Vashchenko N, Abrahamsson PA (2005). Neuroendocrine
differentiation in prostate cancer: Implications for new treatment
modalities. Eur. Urol., 47: 147-155.
Venkateswaran V, Fleshner NE, Sugar LM, Klotz LH (2004).
Antioxidants Block Prostate Cancer in Lady Transgenic Mice.
Cancer Res., 64: 5831-5896.
Wang S, Gao J, Lei Q, Rozengurt N, Pritchard C, Jiao J, Thomas GV, Li
G, Roy-Burman P, Nelson PS, Liu X, W u H (2003). Prostate-specific
deletion of the murine Pten tumor suppressor gene leads to
metastatic prostate cancer. Cancer Cell, 4: 209-221.
Zhou Z, Flesken-Nikitin A, Corney DC, Wang W, Goodrich DW, Roy-
Burman P, Nikitin A Y (2006). Synergy of p53 and Rb deficiency in a
conditional mouse model f or metastatic prostate c ancer. Cancer
Res., 66: 7889-7898.
Zu K, Ip C (2003). Synergy between selenium and vitamin E in
apoptosis induction is associated with activation of distinctive initiator
caspases in human prostate cancer cells. Cancer Res., 63: 6988-
6995.
... Furthermore, there was a synergy of efficacy between FEN and VES. 19 Han et al demonstrated that FEN elicited anticancer effects against HL-60 cells by producing reactive oxygen species (ROS). 20 Dogra et al showed that FEN inhibited proteasomal function, activated the mitochondrial pathway, and caused apoptosis in human lung cancer cell lines. ...
Article
Full-text available
Purpose: We aimed to develop a nanocarrier formulation incorporating fenbendazole (FEN) and rapamycin (RAPA) with strong efficacy against A549 cancer cells. As FEN and RAPA are poorly soluble in water, it is difficult to apply them clinically in vivo. Therefore, we attempted to resolve this problem by encapsulating these drugs in polymeric micelles. Methods: We evaluated drug synergy using the combination index (CI) values of various molar ratios of FEN and RAPA. We formed and tested micelles composed of different polymers. Moreover, we conducted cytotoxicity, stability, release, pharmacokinetic, and biodistribution studies to investigate the antitumor effects of FEN/RAPA-loaded mPEG-b-PCL micelles. Results: We selected mPEG-b-PCL-containing FEN and RAPA at a molar ratio of 1:2 because these particles were consistent in size and had high encapsulation efficiency (EE, %) and drug loading (DL, %) capacity. The in vitro cytotoxicity was assessed for various FEN, RAPA, and combined FEN/RAPA formulations. After long-term exposures, both the solutions and the micelles had similar efficacy against A549 cancer cells. The in vivo pharmacokinetic study revealed that FEN/RAPA-loaded mPEG-b-PCL micelles had a relatively higher area under the plasma concentration-time curve from 0 to 2 h (AUC0-2 h) and 0 to 8 h (AUC0-8 h) and plasma concentration at time zero (Co) than that of the FEN/RAPA solution. The in vivo biodistribution assay revealed that the IV injection of FEN/RAPA-loaded mPEG-b-PCL micelles resulted in lower pulmonary FEN concentration than the IV injection of the FEN/RAPA solution. Conclusion: When FEN and RAPA had a 1:2 molar ratio, they showed synergism. Additionally, using data from in vitro cytotoxicity, synergism between a 1:2 molar ratio of FEN and RAPA was observed in the micelle formulation. The FEN/RAPA-loaded mPEG-b-PCL micelle had enhanced bioavailability than the FEN/RAPA solution.
Conference Paper
To screen the small molecule inhibitors blocking the interaction of PD-1 and PD-L1, the plasmids encoding GST-PD-L1 (AA26-236) and His-Trx-PD-1 (AA21-163) were constructed, and the fusion proteins were expressed in E. coli BL21-CodonPlus (DE3)-RIL cells and purified. GST pull-down experiment indicated that PD-1 directly interacts with PD-L1. The screening model of small molecule inhibitors blocking PD-1/PD-L1 interaction using competitive ELISA assay was established, and two small molecule chemicals named S2468 (Fenbendazole) and S2084 (Duloxetine HCl) were screened with inhibition rate 0.49 and 0.52. While S2485 (Mitoxantrone Hydrochloride) and S1957 (Sulfamethizole) were also screened with inhibition rate 1.8 and 1.6, which could increase the binding between PD-1 and PD-L1.
Article
Full-text available
The clinical course of prostate cancer is grouped into two broad phases. The first phase, which is the growth of the androgen-dependent cancer (AD-Ca) responds well to androgen depletion treatment while the second phase, that could be termed as androgen depletion-independent cancer (ADI-Ca) does not. We used two separate prostate tumors, one AD-Ca and one ADI-Ca from the conditional Pten deletion mouse model to generate from each a pair of cell lines. The AD-Ca cell lines (E2 and E4) and the ADI-Ca cell lines (cE1 and cE2) display bi-allelic deletion at the Pten gene locus, an event which is specific for the prostate epithelium for this mouse model, and a fairly similar level of expression of the androgen receptor (AR). The ADI-Ca cell lines (cE series) grow well in the absence of androgen, display increased AR transcription under androgen-deprived environment, and retain the sensitivity to increased proliferation when androgen is supplemented. The AD-Ca cell lines (E series) grow slowly in the absence of androgen, and, unlike cE cells, do not show increased AR expression when maintained in the absence of androgen. The detection of epithelial cell markers, such as CK8, CK14, CK18 and E-cadherin in the cE series is conforming with the polygonal epithelial morphology of these cells in culture. The E cells also present mostly polygonal-shaped morphology with a small percent of cells with fibroblastoid morphology, and produce little or very low levels of cytokeratins, but increased levels of vimentin, Twist and Slug, the markers known to be associated with epithelial-mesenchymal transition. Each of the cell lines, when inoculated subcutaneously into male or female NOD.SCID mice induced tumors within eight weeks with 100% incidence. Histopathological examinations of the tumor sections, however, led to noticeable biological differences. The cE series engenders adenocarcinomas, particularly in male hosts, and the E series induces sarcomatoid carcinomas (positively stained for CK8 and AR as well as vimentin expression) in either male or female hosts. These new cell lines are promising models for the elucidation of the androgen metabolism and their role in prostate cancer.
Article
Full-text available
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 x 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.
Article
Full-text available
Oxyurid parasites are common contaminants of laboratory rodents, and despite many described treatments, no method has assumed preeminence. Limitations in drug efficacy and the general inability to control for exposure to infective eggs are the primary contributors to treatment failure. In addition, some effective drugs must be eliminated from consideration because of narrow safety margins, other toxic aspects, or concerns related to particular uses of the experimental animals. As an alternative to currently described treatments or surgical derivation, we conducted an efficacy study against Syphacia muris in rats with a new fenbendazole-based protocol. Fenbendazole is a highly efficacious broad-spectrum anthelmintic with adulticidal, larvicidal, and ovicidal actions. Its pharmacokinetic behavior, ovicidal activity, and exceptionally wide safety margin in rats and mice make it an attractive choice for pinworm treatment. We used a 150-ppm medicated feed formulation to reach a targeted dose of 8.0 to 12.0 mg/kg/day in three separate studies designed to assess drug intake and efficacy under different housing conditions and in breeding and nonbreeding populations of ACI rats. In all cases, drug was given on alternating weeks, and nonbreeding populations were medicated for a cumulative period of 14 days. The same schedule was used for breeding populations, but the treatment was repeated after a 2-week rest period to ensure sufficient exposure for newly weaned animals. The results of our study indicate that our described treatment, in combination with environmental control measures against pinworm eggs, is capable of eliminating S. muris.
Article
We have found that mebendazole (MZ), a derivative of benzimidazole, induces a dose- and time-dependent apoptotic response in human lung cancer cell lines. In this study, MZ arrested cells at the G(2)-M phase before the onset of apoptosis, as detected by using fluorescence-activated cell sorter analysis. MZ treatment also resulted in mitochondrial cytochrome c release, followed by apoptotic cell death. Additionally, MZ appeared to be a potent inhibitor of tumor cell growth with little toxicity to normal WI38 and human umbilical vein endothelial cells. When administered p.o. to nu/nu mice, MZ strongly inhibited the growth of human tumor xenografts and significantly reduced the number and size of tumors in an experimental model of lung metastasis. In assessing angiogenesis, we found significantly reduced vessel densities in MZ-treated mice compared with those in control mice. These results suggest that MZ is effective in the treatment of cancer and other angiogenesis-dependent diseases.
Article
Epidemiological investigations may allow conclusions on the etiology of prostate cancer. Such data from autopsy studies and morbidity as well as mortality statistics are based on local or national cancer registries and informations obtained from death certificates. Clear cut evidence of certain environmental factors to be involved in prostate cancer development derives from the situation in Japan: (1) high incidence of latent carcinoma despite a 10-fold lower morbidity of manifest carcinoma compared to the western hemisphere; (2) increased grade of malignancy of latent carcinoma and approximating morbidity and mortality rates to the population of the country of immigration. A comparable situation is seen in the Jewish population. In Europe an absolute increase of morbidity is encountered: Cancer Registries of Birmingham, Hamburg, Sweden, Halle (DDR). According to the UICC following incidence rates can be calculated n per 100,000 population per year): USA - 33.9; Sweden - 26.5; England - 17.4; GFR - 16.5; Japanese immigrants - 12.6; Japan - 3.8, China - 0.9. German mortality data (Cancer Atlas of the GFR), which are based on death certificates, show a steady increase between 1955 and 1975 without conclusive differences for the various 'Bundeslander'. On a world-wide basis neither studies on endocrinology nor on the socioeconomic status or sexual life, infectious as well as genetic factors have provided a valuable clue for the etiology of prostatic carcinoma.
Article
Recent development of animal models relevant to human prostate cancer (PC) etiopathogenesis has provided important information on the specific functions provided by key gene products altered during disease initiation and progression to locally invasive, metastatic and hormone-refractory stages. Especially, the characterization of transgenic mouse models has indicated that the inactivation of distinct tumor suppressor proteins such as phosphatase tensin homolog deleted on chromosome 10 (PTEN), Nkx3.1, p27(KIP1), p53 and retinoblastoma (pRb) may cooperate for the malignant transformation of prostatic stem/progenitor cells into PC stem/progenitor cells and tumor development and metastases. Moreover, the sustained activation of diverse oncogenic signaling elements, including epidermal growth factor receptor (EGFR), sonic hedgehog, Wnt/β-catenin, c-Myc, Akt and nuclear factor-kappaB (NF-κB) also may contribute to the acquisition of more aggressive and hormone-refractory phenotypes by PC stem/progenitor cells and their progenies during disease progression. Importantly, it has also been shown that an enrichment of PC stem/progenitor cells expressing stem cell-like markers may occur after androgen deprivation therapy and docetaxel treatment in the transgenic mouse models of PC suggesting the critical implication of these immature PC cells in treatment resistance, tumor re-growth and disease recurrence. Of clinical interest, the molecular targeting of distinct gene products altered in PC cells by using different dietary compounds has also been shown to counteract PC initiation and progression in animal models supporting their potential use as chemopreventive or chemotherapeutic agents for eradicating the total tumor cell mass, improving current anti-hormonal and chemotherapies and preventing disease relapse.
Article
Most metastatic melanoma patients fail to respond to available therapy, underscoring the need for novel approaches to identify new effective treatments. In this study, we screened 2,000 compounds from the Spectrum Library at a concentration of 1 micromol/L using two chemoresistant melanoma cell lines (M-14 and SK-Mel-19) and a spontaneously immortalized, nontumorigenic melanocyte cell line (melan-a). We identified 10 compounds that inhibited the growth of the melanoma cells yet were largely nontoxic to melanocytes. Strikingly, 4 of the 10 compounds (mebendazole, albendazole, fenbendazole, and oxybendazole) are benzimidazoles, a class of structurally related, tubulin-disrupting drugs. Mebendazole was prioritized to further characterize its mechanism of melanoma growth inhibition based on its favorable pharmacokinetic profile. Our data reveal that mebendazole inhibits melanoma growth with an average IC(50) of 0.32 micromol/L and preferentially induces apoptosis in melanoma cells compared with melanocytes. The intrinsic apoptotic response is mediated through phosphorylation of Bcl-2, which occurs rapidly after treatment with mebendazole in melanoma cells but not in melanocytes. Phosphorylation of Bcl-2 in melanoma cells prevents its interaction with proapoptotic Bax, thereby promoting apoptosis. We further show that mebendazole-resistant melanocytes can be sensitized through reduction of Bcl-2 protein levels, showing the essential role of Bcl-2 in the cellular response to mebendazole-mediated tubulin disruption. Our results suggest that this screening approach is useful for identifying agents that show promise in the treatment of even chemoresistant melanoma and identifies mebendazole as a potent, melanoma-specific cytotoxic agent.
Article
The interaction of Mebendazole (methyl-5-benzoyl benzimidazole-2-carbamate), a new antihelminthic drug, with tubulin was studied. Ultramicroscopic and turbidimetric evidence shows an inhibitory effect of Mebendazole on the “in vitro” polymerization of tubulin. Scatchard plot analysis shows a single binding site for Mebendazole per tubulin dimer. This site has an affinity constant of 2.8 × 105 M−1. Competition experiments demonstrate that this binding site is the same as for Colchicine, even when both compounds are not chemically related. Mebendazole is proposed as a useful tool for the study of tubulin assembly.
Article
Heavy reliance on the benzimidazole (BZ) anthelmintics since their introduction in the 1960's for the control of gastrointestinal parasites of livestock has led to widespread BZ resistance in target parasite species. The BZs exert their primary action by binding to tubulin, the major protein component of microtubules. This review discusses the biochemistry of the interaction between the BZs and tubulin from mammalian and BZ-resistant and -susceptible parasite sources, exploring aspects of the selective toxicity of these drugs and examining the mechanism of BZ resistance. Although tubulin is a highly conserved protein present in both the host and the parasite, the BZs demonstrate relatively low mammalian toxicity. The selectivity of these drugs can be explained by the much higher affinity of the BZs for tubulin from the parasite at 37 degrees C compared to their affinity for tubulin from the host. This difference in affinity reflects the considerably slower rate of BZ dissociation from parasite tubulin. BZ-resistance in parasitic nematodes is characterised by a loss of high affinity BZ-parasite tubulin interactions and a corresponding increase in lower affinity interactions, although there are still significant differences between BZ-resistant parasite tubulin and tubulin from the host. These differences suggest the potential for the design of new generation BZs active against 'BZ-resistant' parasites.