International Journal of Radiation Biology, May 2013; 89(5): 346–355
© 2013 Informa UK, Ltd.
ISSN 0955-3002 print / ISSN 1362-3095 online
DOI: 10.3109/09553002.2013.762136
Inhibitory effects of Rhenium-188-labeled Herceptin on prostate cancer
cell growth: A possible radioimmunotherapy to prostate carcinoma
Hsin-Yi Wang1,2, Wan-Yu Lin2, Mei-Chih Chen1, Teh Lin9, Chih-Hao Chao3, Fu-Ning Hsu1, Eugene Lin1,4,
Chih-Yang Huang5,11, Tsai-Yueh Luo6 & Ho Lin1,7,8,10
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1Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan, 2Department of Nuclear Medicine,
Taichung Veterans General Hospital, Taichung, Taiwan, 3Department of Internal Medicine, Chang Bing Show Chwan Memorial
Hospital, Changhwa, Taiwan, 4Department of Urology, Chang Bing Show Chwan Memorial Hospital, Changhwa, Taiwan,
5Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan, 6Institute of Nuclear Energy Research,
Taoyuan, Taiwan, 7Graduate Institute of Rehabilitation Science, China Medical University, Taichung, Taiwan,
8Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan, 9Department of Radiation Oncology,
Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA, 10Department of Urology, University of Texas Southwestern Medical
Center, Dallas, Texas, USA, and 11Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan
Introduction
Abstract
Purpose: Herceptin is widely used in treating Her2-overexpressing
breast cancer. However, the application of Herceptin in prostate
cancer is still controversial. Our previous results have indicated
the relevance of Her2 in the transition of the androgen requirement in prostate cancer cells. In this study, the effects of radioimmunotherapy against Her2 in prostate cancer were investigated.
Materials and methods: DU145, an androgen receptor-negative
prostate cancer cell line, was used in vitro and in vivo to evaluate
the effects of Herceptin labeled with a beta emitter, Rhenium188 (Re-188). Its effects on cell growth, extent of apoptosis, the
bio-distribution of Re-188 labeled Herceptin (Re-H), and protein
levels were determined.
Results: Treatments with Re-188 and Re-H reduced the proliferation of DU145 cells in dose- and time-dependent manners
compared to the Herceptin-treated group. Growth inhibition
and apoptosis were induced after Re-H treatment; growth inhibition was more distinct in cells with high Her2/p-Her2 levels.
Our in vivo xenograft studies revealed that Re-H treatment
significantly retarded tumor growth and altered the levels of
apoptosis-related proteins. The bio-distribution of Re-H in mice
demonstrated a tissue-specific pattern. Importantly, the levels
of p35 protein, which is related to cancer cell survival and invasion, dramatically decreased after Re-H treatment.
Conclusions: Our data demonstrate that Re-188-labeled
Herceptin effectively inhibited the growth of DU145 cells
compared to the Herceptin- and Re-188-treated cohorts.
This implies that targeting Her2 by both radio- and immunotherapy might be a potential strategy for treating patients
with androgen-independent prostate cancer.
Prostate cancer is the most common malignancy in
males (Jemal et al. 2007). Hormone therapy is the primary
treatment for locally advanced and metastatic prostate cancers (Henry and O’Mahony 1999). However, these tumors
tend to convert to a metastatic hormone-refractory state
which is currently incurable (Stavridi et al. 2010). Human
epidermal growth factor receptor 2 (Her2) is a transmembrane receptor tyrosine kinase normally involved in cell
growth, differentiation, and transformation (Dougall et al.
1994). The Her2 expression rate is approximately 9–30%
in primary prostate cancers; it increases after hormone
treatment to 59–68% in localized tumors and 78–80% in castrated metastatic tumors (Signoretti et al. 2000, Osman
et al. 2001, Shi et al. 2001, Morris et al. 2002, Calvo et al.
2003, Hernes et al. 2004). It has been reported that PTEN
(phosphatase and tensin homolog) stratification with high
levels of Her2/3 expression is important to the prognosis
of prostate cancer patients (Ahmad et al. 2011). Importantly, the elevated expression of Her2 is associated with
exposure to hormone therapy and the androgen independence of prostate cancer (Shi et al. 2001).
Herceptin (trastuzumab), a humanized anti-Her2
monoclonal antibody, is widely used for the treatment of
patients with Her2-overexpressing breast cancer (Albanell
et al. 2003). Although applied in prostate cancer, the
therapeutic effect of Herceptin is not as encouraging as
that in breast cancer (Ziada et al. 2004). In xenograft models,
Herceptin demonstrates growth inhibition in androgendependent tumors but not in androgen-independent
tumors (Agus et al. 1999). Several lines of evidence indicate
Keywords: Herceptin, Rhenium-188, prostate cancer, Her2, Cdk5
Correspondence: Prof. Ho Lin, PhD, Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan. Tel: + 886 4228 40416 ext. 310.
Fax: + 886 4228 74740. E-mail: hlin@dragon.nchu.edu.tw
(Received 9 July 2012; revised 2 November 2012; accepted 14 December 2012)
346
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Rhenium-188-labeled Herceptin and prostate cancer 347
that combination therapy of Herceptin with other drugs
(such as docetaxel or gefitinib) would be beneficial to treat
prostate cancer (Formento et al. 2005, Legrier et al. 2007).
Radio-immunotherapy refers to the treatment of a disease
by using radiopharmaceuticals which are composed of an
antibody labeled with a therapeutic radionuclide (Sharkey
and Goldenberg 2011). The antibody targets the disease
site through its specificity for a disease-associated antigen
and the radionuclide delivers a lethal dose of therapeutic
radiation to the disease site. Radiolabeling of Herceptin
has been used to develop a therapeutic radiopharmaceutical for Her2-overexpressing tumors (Rasaneh et al. 2010).
Rhenium-188 (Re-188), a β-emitting radionuclide, is widely
utilized in radio-immunotherapy due to its optimal radiation energy (maximum of 2.12 MeV for β ), suitable halflife (16.9 hours), and convenient production (Chen et al.
2009, Luo et al. 2009). Apart from its application to breast
cancer and nasopharyngeal carcinoma, the relevance of
Re-188-labeled Herceptin in treating prostate cancer has
not yet been studied.
Cyclin-dependent kinase 5 (Cdk5) is primarily found in
bovine brain and responsible for nervous system development (Lee et al. 1996, Dhavan and Tsai 2001). Cdk5 is regulated by p35 protein, not cyclins, is expressed in neurons
and contributes to neuronal differentiation (Tsai et al.
1994). In recent years, Cdk5 overactivation in the brain has
been associated with neurodegenerative diseases, such as
Alzheimer’s disease (Patrick et al. 1999). Our previous studies in a neurodegenerative model also indicated that Cdk5
can be overactivated by an upstream kinase (Lin et al.
2007a). With regard to cancer cells, Cdk5 and p35 may control the growth of thyroid cancer cells under Her2 regulation (Lin et al. 2007b). Hyperactivation of Cdk5 also
determines the fate of prostate cancer cells after drug treatment (Lin et al. 2004). Our recent findings have indicated
that p35/Cdk5 activation regulates protein stability of the
androgen receptor and the tumor growth of prostate cancer
cells (Hsu et al. 2011b). Moreover, we have demonstrated
that Her2 is required for stability of the androgen receptor
during the transition of androgen requirement of prostate
cancer cells (Hsu et al. 2011a). In addition, Strock et al.
(2006) found that Cdk5 controls the motility and metastatic
potential of prostate cancer cells. These findings suggest
that the protein levels of p35/Cdk5 and Cdk5 kinase activity
in cancer cells might be correlated to their functions. Therefore, Cdk5 and p35 might be potential indicators in our
study.
To the best of our knowledge, the inhibitory effect of
Re-188-labeled Herceptin in prostate cancer is not known.
The primary aim of this study was to determine these effect
by treating Her2-positive/androgen receptor-negative DU145
prostate cancer cell lines in vitro and in vivo with Re-188labeled Herceptin and compare them to cohorts treated with
Herceptin alone and Re-188 alone. The results indicated that
Re-188 labeling enhanced the effectiveness of Herceptin
treatment on the growth inhibition and apoptosis of Her2positive prostate cancer cells. Therefore, Re-188-labeled
Herceptin is a potential therapeutic for the treatment of
Her2-positive/androgen receptor-negative prostate cancer.
Materials and methods
Cell culture
Five human prostate cancer cell lines, DU145 (BCRC-60348),
LNCaP (BCRC-60088), PC3 (BCRC-60122), CWR22Rv1
(22Rv1, BCRC-60545), and BT-474 (BCRC-60359) were purchased from Food Industry Research and Development
Institute, Taiwan. LNCaP, CWR22Rv1, and PC3 cells were
cultured in RPMI-1640 (Sigma, St Louis, MO, USA), and
DU145 cells were cultured in DMEM (Sigma) supplemented
with 10% heat-inactivated fetal bovine serum (FBS,
Invitrogen, Carlsbad, CA, USA), 1% penicillin and streptomycin (Sigma, St Louis, MO, USA). HPEpiC is a normal prostate
epithelial cell line cultured in a specific commercial conditioned medium (purchased from ScienCell Research
Laboratories, Carlsbad, CA, USA). All cell lines were maintained in a humidified incubator at 37°C in an atmosphere
containing 5% CO2.
Preparation of Re-188 labeled Herceptin
Generation and labeling of Rhenium-188 (Re-188) onto
Herceptin was performed in the Institute of Nuclear
Energy Research (INER), Taoyuan, Taiwan (Luo et al. 2009).
The anti-Her2/neu antibody, Herceptin (trastuzumab), was
purchased from Genentech (San Francisco, CA, USA).
Evaluation of cell proliferation inhibition induced by
Re-188-labeled Herceptin
Doses of Herceptin (H) and Rhenium-188 (Re-188)
comparable to those used for Re-188-labeled Herceptin
(Re-H) were administered to DU145 cells. DU145 cells were
cultured in 24-well plates (20,000 cells/well) 8 h before
administering the therapeutic agents. Re-H: The radioactivity
of Re-H was measured by a dose calibrator (Atomlab 100,
Biodex). Seven serial doses (1, 5, 10, 20, 40, 80, and 160 μCi)
of Re-H were administered in triplicate. H: Seven serial
concentrations of Herceptin that matched the Herceptin
concentrations in Re-H (0.3125, 0.625, 1.25, 2.5, 5, 10 and
20 mg/ml) were administered in triplicate. Re-188: Seven
serial doses of Re-188 (1, 5, 10, 20, 40, 80, and 160 μCi)
were administered in triplicate. Control: Each cell line was
treated with PBS (phosphate buffered saline) solution instead
of the therapeutic agent in triplicate and used as control.
Three hours later, the supernatants were removed. The wells
were washed gently with PBS to remove non-binding agents.
The culture medium was then added into each well, and the
plates were incubated at 37°C in an atmosphere containing
5% CO2. The cells were cultured for 4 and 6 days before
evaluating proliferation by MTT assay (Sigma, St Louis, MO,
USA) as described previously (Hsu et al. 2011b).
LNCaP, DU145, and PC3 cells, expressing different levels of
Her2, were cultured as mentioned above and treated in triplicate with comparable doses of Re-H and Re-188 (75, 150, and
300 μCi) as well as with a comparable volume of PBS as the
control for 3 h. Three hours after drug administration, the cells
were washed with PBS, the appropriate culture medium was
added, and the cells were then cultured for 3 days. Cell proliferation was evaluated by MTT assay 3 days after treatment
and presented as the percentage of the PBS-treated group.
348 H.-Y. Wang et al.
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TUNEL assay
Cell apoptosis was characterized using a TUNEL assay
with an In Situ Cell Death Detection Kit from Roche Applied
Science (Mannheim, Germany) according to the manufacturer’s instructions. Briefly, cells were grown on cover
glasses and fixed with 4% paraformaldehyde in PBS for 1 h at
room temperature. After washing three times with PBS, the
cells were permeabilized with 0.1% Triton X-100 (Sigma, St
Louis, MO, USA) in 0.1% sodium citrate for 10 min on ice.
The cells were then stained for the TUNEL assay, and green
fluorescent apoptotic bodies were visualized under a fluorescence microscope (Zeiss Axioplan2 fluorescence microscope, Germany). DAPI was used for counterstaining. The
experiments were performed in duplicate and repeated
twice (Lin et al. 2007a).
Evaluation of tumor growth inhibition induced by
Re-188 labeled Herceptin
Male severe combined immunodeficiency (SCID) mice, 4–
6 weeks old, were maintained in a pathogen-free
environment. The mice were inoculated subcutaneously in
the left thigh with 1 × 107 DU145 cells mixed with Matrigel
(BD Biosciences, Bedford, MA, USA) and used for experiments after the tumor size was larger than 20 mm3. The
mice were assigned randomly into four groups. Three
therapeutic groups were treated via tail vein with Re-188labeled Herceptin (10 μCi/g body weight, number of mice
[n] = 8), Re-188 (10 μCi/g body weight, n = 9), and Herceptin
(2.5 μg/g body weight, Herceptin concentration comparable
to Re-H, n = 8). The mice (n = 8) in the control group
were treated with 0.05 ml of normal saline via tail vein. The
body weight and two perpendicular diameters of the tumor
were measured twice per week with vernier calipers. Tumor
volumes were calculated as mentioned previously
(π/6 × larger diameter × smaller diameter2). Tumor growth
inhibition (TGI) was calculated as the ratio of the tumor
volume in the treated group to that of the control group
at a given time × 100. The mice were ethically killed when
their tumors reached a volume of 2,000 mm3. The animal
use protocol has been reviewed and approved by the Institutional Animal Care and Use Committee in National Chung
Hsing University, 96–91.
Bio-distribution of Re-188 labeled Herceptin
Gamma scintigraphy was used for the sequential, noninvasive evaluation of the in vivo tissue distribution of Re188-labeled Herceptin in tumor-bearing mice. Six mice
with a DU-145 xenograft on the left thigh were administered 11.1 MBq of Re-H via the tail vein and sedated
for scintigraphic evaluation 17 and 24 h later. The static
images were acquired with a gamma scintillation camera
equipped with a pinhole collimator (E-cam, Siemens,
Knoxville, TN, USA) for 5,000 counts. The matrix size was
128 × 128 with zoom 2. Photopeak was at 155 keV with
10% width. The images were evaluated by an experienced
nuclear medicine physician. Evaluation by tissue counting:
The six mice bearing DU-145 tumors on the left thigh
used for scintigraphic evaluation were sacrificed 24 h
after being injected with 11.1 MBq of Re-H , immediately
following completion of scintigraphic acquisition. The main
organs, such as the liver, lung, spleen, kidney, testis, muscle,
bone, blood, and tumor, were weighed and counted by
gamma counter. The injected dose of Re-188-labeled
Herceptin was counted as well. The bio-distribution of
Re-188-labeled Herceptin is expressed as the percentage
of the injected dose per gram of tissue (% injected dose/g).
Western blotting
Protein expression in cell lysates was analyzed by Western
blotting (30 μg/lane) as described previously (Hsu et al.
2011b). Antibodies against Her2, phospho-Her2, Cdk5, p35,
PARP, Bax, Bcl2, tubulin (Santa Cruz Biotechnology, Santa
Cruz, CA, USA), and actin (Chemicon, Temecula, CA, USA)
were used. Peroxidase-conjugated anti-mouse or anti-rabbit
antibodies (Jackson ImmunoResearch Laboratory, West
Grove, PA, USA) were used as the secondary antibody.
ECL detection reagent (Perkin Elmer, Shelton, CT, USA) was
used to visualize the immunoreactive proteins on PVDF
membranes (Perkin Elmer, Shelton, CT, USA) after transfer
using a Trans-Blot SD (Bio-Rad, Hercules, CA, USA).
Statistics
The data are presented as the means 士 standard deviation.
For the apoptosis study, Student’s t-test was used to evaluate
the differences between the tests and controls. Results with
P values less than 0.05 were considered statistically
significant.
Results
Her2 levels in different cell lines
The protein levels of Her2 and phospho-Her2 in different
prostate cell lines were surveyed; BT-474, a breast cancer
cell line, was used as a positive control (Figure 1A). The
normal prostate epithelial cell line, HPEpiC, showed low
Her2/phospho-Her2 expression; BT-474 cell line showed
high Her2/phospho-Her2 expression; DU145, an androgenindependent prostate cancer cell line without androgen
receptor (AR) expression, showed high Her2/phospho-Her2
expression; LNCaP, an androgen-dependent prostate
cancer cell line with AR expression, showed high Her2/
phospho-Her2 expression; CWR22Rv1, an androgenindependent prostate cancer cell line with AR expression,
showed moderate Her2/phospho-Her2 expression; and
PC3, an androgen-independent prostate cancer cell line
without AR expression, showed low Her2/phosphoHer2 expression. Previous studies from our lab indicate
that Her2 is important to the growth of prostate cancer cells
as the cells transition from an androgen-dependent to an
androgen-independent state (Hsu et al. 2011a). Because
DU145 is an androgen refractory cell line and has high
Her2 expression, and a high phospho-Her2/Her2 ratio, it was
suitable for this study.
Inhibitory effects of Re-188-labeled Herceptin on
DU145 proliferation
Serial doses of Re-188-labeled Herceptin (Re-H), Rhenium188 (Re-188), and Herceptin (H) were used to treat DU145
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Rhenium-188-labeled Herceptin and prostate cancer 349
Figure 1. The effects of Herceptin (H), Re-188, and Re-188-labeled Herceptin (Re-H) on the proliferation of Her2-expressing prostate cancer
cell lines. (A) Protein levels of Her2 and phospho-Her2 (Y1221/1222) in HPEpiC cells (a normal prostate epithelial cell line), four prostate
cancer cell lines (DU145, PC3, LNCaP, CWR22Rv1), and BT-474 (a breast cancer cell line) were evaluated by Western blotting. Tubulin served as
an internal control. DU145 cells cultured in 24-well plates (20,000 cells/well) were treated with Re-H (1, 5, 10, 20, 40, 80, and 160 μCi), H (0.3125,
0.625, 1.25, 2.5, 5, 10 and 20 mg/ml), Re-188 (1, 5, 10, 20, 40, 80, and 160 μCi), and the control in triplicate. Three hours later, the supernatant
was removed. The cells were gently washed with PBS to remove non-binding agents. The appropriate medium was then added into each
well, and the cells were incubated at 37°C for additional four days (B) and 6 days (C) before evaluating proliferation with the MTT assay.
LNCaP (D), DU145 (E), and PC3 (F) were treated with Re-188 or Re-H (75, 150, and 300 μCi) as well as the control for 3 hours, and the cells were
incubated for three additional days before measuring proliferation as
described above. The data are presented as the percentages of viable cells to
the control group. + and + +, p < 0.05 and p < 0.01 versus H group; ∗ and ∗∗, p < 0.05 and p < 0.01 vs. the group of concentration = 0; #, p < 0.05
versus Re-188 group.
cells for 3 h. Unbound drugs were then washed out, and the
cells were cultured in fresh medium for an additional 4 or
6 days. Proliferation of the DU145 cells was evaluated by
MTT assay. A significant inhibition of cell proliferation
induced by Re-H after culturing for 4 days was observed at
160 μCi, while there was no significant inhibition after treatment with Re-188 or H (Figure 1B). The Re-H-triggered
growth inhibition was more significant after 6 days of
treatment and was dose-dependent (Figure 1C). Under
these conditions, the Re-188 group showed significant
inhibition after high dosage treatments; however, the inhibition triggered by Re-188 was still less than that of the Re-H
treatments (at a dose of 160 μCi, the survival fraction was
83.9% vs. 70.9% on the 4th day and 22.8% vs. 19.9% on the
6th day, respectively). The H group demonstrated no effect
on cell proliferation after the six-day treatment (Figure 1C).
Analysis of the effect of Re-188-labeled Herceptin on
different cell lines
To differentiate the potential inhibitory effects of Re-H and
Re-188 treatments on different prostate cancer cell lines,
DU145, LNCaP, PC3, and their control counterparts
were analyzed. The doses of Re-H and Re-188 administered
were 0, 75, 150, and 300 μCi. Herceptin treatment was not
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Rhenium-188-labeled Herceptin and prostate cancer 350
included because the data mentioned earlier demonstrated
no correlation between the H group and cell proliferation
(Figure 1B and 1C), in accordance with previous investigations (Agus et al. 1999). The experimental procedure was
similar to those in Figures 1B and 1C, except that the cells
were now incubated for 3 additional days after treatment.
The data are presented as the percentage of the control group
and indicates that both Re-H and Re-188 showed dosedependent inhibition of proliferation in the three cell lines
(Figure 1D–1F). Additionally, the differences in growth
inhibition between the Re-H and Re-188 treatments were
greater in the DU145 and LNCaP cells (p < 0.01) than in the
PC3 cells (p < 0.05) at 300 μCi, which implies the possible
correlation between Her2/phospho-Her2 expression and
drug response in prostate cancer cells.
Re-188-labeled Herceptin induces apoptosis in DU145 cells
Because the growth inhibition induced by Re-H was
significant (Figure 1), we investigated the role of apoptosis
during Re-H treatment. TUNEL staining and measurement
of apoptosis-related proteins were performed to evaluate the
effects of the four DU145 groups on apoptosis. Re-H
(160 μCi), Re (160 μCi), H (20 mg/ml), and Control (c) were
treated in culture medium for 3 h and then replaced by fresh
medium and cultured for an additional 3 days; the cells were
observed under a fluorescent microscope, and a green signal
indicated apoptotic cells. The cell lysates were collected and
analyzed by Western blotting. As seen in Figure 2A, Re-H
treatment significantly increased apoptosis when compared
to the control group and the Re and H treatment groups
(Figure 2A). Apoptosis-related proteins, including cleaved
PARP and Bax, increased after Re-H treatment, while the
(A)
Control
Re-H
anti-apoptotic protein Bcl-2 dramatically decreased (Figure 2B).
This is the first demonstration indicating that Re-188-labeled
Herceptin can directly cause apoptosis of cancer cells.
Effects of Re-188-labeled Herceptin on tumor growth
in xenografts
Tumor growth in the xenograft model in the four treatment
groups (Re-H, Re-188, H and C) was determined as described
in Materials and methods. The tumor growth curves are
shown in Figure 3A. A significant decrease in tumor growth
was observed 13 days after Re-H injection when compared to
the Re-188 and H groups, and the Re-188 and H groups
showed a moderate but not significant decrease in tumor
growth compared to the control group (Figure 3A) while
the average body weights among four groups of animals
showed no significant different (data not shown). Tumor
growth inhibition (TGI) calculated as the ratio of tumor volume (treatments/control) at a given time is shown in Table I.
In contrast, the levels of apoptosis-related proteins in tumor
samples from individual animals of the four groups were
also monitored. The data indicate that Re-H treatment can
increase the protein levels of cleaved PARP and Bax, and
decrease Bcl-2 levels in tumors when compared to other
groups (Figure 3B). Bax protein levels slightly increased after
Re-188 treatment. Thus, our protein expression analysis suggests that Re-H treatment might cause apoptosis in tumor
xenografts. Taken together, in vivo treatment of Re-H could
effectively retard tumor growth, potentially through apoptosis.
Bio-distribution of Re-188-labeled Herceptin in mice
Since the in vivo treatments have systemic effects, biodistribution of the isotope in the whole body is important
Re
H
TUNEL
DAPI
(B)
cleaved PARP
Bax
Bcl-2
Actin
Figure 2. Re-188-labeled Herceptin induces apoptosis in DU145 cells. DU145 cells were treated with Re-188-labeled Herceptin (Re-H, 160 μCi), Re188 (160 μCi), Herceptin (H, 20 mg/ml), or Control (c) for 3 hours. TUNEL staining (A) and Western blotting (B) were performed after three
days of incubation. Apoptosis-related proteins were then detected. The green-fluorescent signal represents cells that underwent apoptosis.
DAPI was used as a nuclear stain. Actin served as the internal control. This Figure is reproduced in color in the online version of International
Journal of Radiation Biology.
350 H.-Y. Wang et al.
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Tumor size (cm3)
(A)
(B)
Mice ID:
C
H
Re
ReH
1 2 3 5 6 7
1 2 3 4 5 6
1 2 3 4 6 7 8
1 2 3 6
cleaved PARP
Bax
Bcl-2
Actin
Figure 3. Tumor growth of the DU145 xenografts in SCID mice was inhibited exclusively by Re-188-labeled Herceptin treatment. SCID mice
were treated with Herceptin (H, 2.5 μg/g body weight), Re-188 (10 μCi/g body weight), Re-188-labeled Herceptin (Re-H, 10 μCi/g body weight),
and normal saline, used as the control (Ctrl), on day 34. (A) The tumor volume was recorded twice a week by calculating the tumor diameter. ∗ and
∗∗, p < 0.05 and p < 0.01 vs. the control group. (B) Apoptosis-related proteins in tumor samples from individual animals of four groups (indicated
by numbers) were detected by Western blotting. Actin served as the internal control.
to understand potential side-effects. Figure 4A showed the
posture of the mouse while scintigraphic images were
recorded. The site of the tumor xenograft is indicated by an
arrow. The scintigraphic images of Re-H distribution 17 h
and 24 h after injection were shown in Figure 4B and 4C,
respectively. Radioactivity at the tumor site on the left thigh
was significantly higher, revealing the specific binding of
Re-H at the tumors (indicated by the arrows). In addition, the
spleen and liver were determined to be the dose-limiting
organs as observed by the accumulation of the tracer, indicated by the arrow heads. After image acquisition, the mice
were sacrificed for isotope counting of individual organs. The
bio-distribution of Re-H 24 h after intravenous injection is
Table I. Tumor growth inhibition (TGI) induced by the treatments
of Herceptin (H), Re-188 (Re), and Re-188 labeled Herceptin (Re-H)
in xenografted model. The TGI was calculated as the ratio of relative
volume of the xenograft in the treated to control group at a given time.
Days
23
27
30
34
37
41
43
48
51
55
H
100.0%
76.7%
82.7%
140.1%
103.9%
120.2%
83.5%
85.9%
90.3%
96.7%
Re
100.0%
93.7%
77.7%
100.6%
70.6%
91.9%
61.2%
64.4%
70.1%
87.4%
H, Herceptin; Re, Re-188; Re-H, Re-188 Herceptin.
Re-H
100.0%
103.5%
92.5%
131.2%
95.4%
110.4%
74.6%
55.1%
37.8%
36.6%
shown in Figure 4D. The relative high tumor uptake of isotope indicated specific binding at the tumor site. However,
the dose-limiting organs, spleen and liver, were highly
radioactive, as shown in Figure 4B and 4C, respectively. In
addition, higher activities in kidney and urine resulted from
the excretion of isotope via the urinary system.
Effects of Re-188-labeled Herceptin on p35/Cdk5 proteins
Cdk5 has been reported to be involved in the response of
cells to radiation (Bolin et al. 2012). Previous studies from
our laboratory have indicated that Cdk5 is important to
the survival of cancer cells (Lin et al. 2004, 2007b, Hsu
et al. 2011b). Therefore, we wanted to test whether Cdk5
mediated the decrease in the survival of prostate cancer cells
after Re-H treatment. We monitored the protein levels of
Cdk5 and its regulator, p35, both in vitro in DU145 cells
and in vivo in tumor xenografts. Our analysis revealed a
gradual decrease in the levels of Cdk5 and p35 proteins with
increasing doses of Re-H (Low: 80 μCi and High: 160 μCi for
3 h) (Figure 5A). In tumor xenografts, although the levels of
Cdk5 protein in tumors were not significantly different
between control and treated groups, the levels of p35 protein
in the Re-H-treated group dramatically decreased. Notably,
the level of p35 protein in the Re-188-treated group also
slightly decreased when compared to the control and H
groups (Figure 5B). The data suggests that activation of
p35/Cdk5 might mediate growth inhibition as a result of
Re-188-labeled Herceptin treatment in prostate cancer cells.
352 H.-Y. Wang et al.
(A)
(B)
14
12
Bio-distribution (%)
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(D)
(C)
10
8
6
4
2
0
Figure 4. Immuno-specific binding of Re-188-labeled Herceptin to DU145 xenografts was determined by scintigraphy. (A) The image shows the
posture of SCID mice during acquisition and the site of the DU145 xenograft on the left thigh. Scintigraphic images with a gamma scintillation
camera equipped with pinhole collimator (E-cam, Siemens) for 5,000 counts, matrix size of 128 × 128 with zoom 2, and a photopeak at 155 keV with
10% width were acquired 17 h (B) and 24 h (C) after 11.1 MBq of Re-188 Herceptin was injected via the tail vein. The arrows indicate the significant
accumulation of radioactivity at the site of tumor xenograft on the left thigh, which confirmed the specific targeting of Re-188 Herceptin to the
Her2-expressing tumor. The arrowheads indicate high tracer uptake at the dose-limiting organs, namely, the spleen and liver. (D) The biodistribution of Re-188-labeled Herceptin in SCID mice with DU145 xenografts was evaluated at 24 h after injection. The organs and tumors
were weighed and counted by gamma counter. The injected dose of Re-188-labeled Herceptin was counted as well. The values of bio-distribution
of Re-188-labeled Herceptin were expressed as the percentage of the injected dose per gram of tissue (% injected dose/g). This Figure is reproduced
in color in the online version of International Journal of Radiation Biology.
Discussion
Her2, a tyrosine kinase receptor, plays an essential role in the
survival of several types of cells. Overexpression or mutation
of Her2 can transform cells into cancer cells (Neve et al.
2001). In prostate carcinomas, elevated Her2 expression
has been noted, especially in hormone-refractory prostate
cancers (59–80% vs. 9–30%) (Shi et al. 2001). Refractory prostate carcinomas are also frequently metastatic with a poor
prognosis. Therefore, novel treatments for prostate cancer
such as targeted therapy are being developed. Here,
we report Re-188-labeled Herceptin treatment as a potential
alternative strategy for treating patients with refractory
prostate cancer with Her2-targeted radiotherapy.
DU145, an androgen receptor-negative prostate cancer
cell line with high Her2 expression (Figure 1A), was
treated with different doses of radioactive Herceptin to
determine the effective doses. Effective growth inhibition
is observed at high doses of treatment or after a long
incubation time (Figure 1B and 1C). In addition to direct
effects, this may be a result of the indirect cytotoxic effects
of radiation inducing the production of free radicals
(Chen et al. 2009, Luo et al. 2009). Since the first effect of
radiation is to damage DNA and arrest cell proliferation,
only cells with irreparable damage would die. This explains
why the survival difference between the treated and control
cells becomes more significant after longer incubation
time even though the duration of the treatment is identical
(Figure 1B and 1C). A previous report indicates that
Herceptin is more effective in androgen-dependent than
androgen-independent cells (Agus et al. 1999). Here, we
found that Herceptin treatment showed no significant
growth inhibition on DU145 cells in contrast to the effects
of Re-188-labeled Herceptin (Figure 1B and 1C). In addition, the growth of DU145 cells in vitro was also affected
by Re-188 treatment (Figure 1C). This might be due to the
limited reaction environment in vitro; thus, beta rays
emitted from the free Re-188 could damage adjacent
cells without specifically binding to them. Our in vivo
tumor studies support this hypothesis because the growth
Rhenium-188-labeled Herceptin and prostate cancer 353
(A)
p35
Cdk5
β-actin
Int J Radiat Biol Downloaded from informahealthcare.com by Taipei Veterans General Hospital on 05/01/13
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(B)
C
H
1
2
3
5
1
2
3
4
6
1
7
Re
5 6
7
2 3 4
8 9
5 6
7 8
ReH
1 2 3 6
Cdk5
β-actin
p35
β-actin
Figure 5. Cdk5 and p35 proteins in DU145 cells were altered by treatment with Re-188-labeled Herceptin both in vitro and in vivo. (A) DU-145
cells were treated for 3 hours as follows: control, low dose (80 μCi) of Re-188-labeled Herceptin (low), and high dose (160 μCi) of Re-188-labeled
Herceptin (High). The cells were washed off the drugs and were incubated for three additional days before analyzing the protein levels by Western
blotting. (B) The protein samples from the same experiment in Figure 3 were detected by Western blotting. Actin served as the internal control.
inhibitions of Re-188 and Herceptin treatments were comparable, but significantly less than the decreased proliferation observed with Re-188-labelled Herceptin treatment
(Figure 3).
To further elucidate the correlation between Herceptinmediated Her2 targeting and growth inhibition, Re-188 and
Re-188-labeled Herceptin were administered to three prostate cancer cell lines (DU145, PC3, and LNCaP) exhibiting
different Her2 expression levels. Expectedly, the growth differences of LNCaP and DU145 cells (with high levels of Her2
expression) between treatments of Re-188-labeled Herceptin and Re-188 were more significant when compared to the
treatments in PC3 cells (with low Her2 expression) when
exposed to radiations below 300 μCi (Figures 1D–1F). In
addition to Her2 expression levels, the levels of other ErbB
members, such as epidermal growth factor receptor (EGFR),
might also affect the efficiency of Her2 targeting because
they are dimerization partners. EGFR itself can also be
a treatment target in PC3, LNCaP, and DU145 (Sgambato
et al. 2004, Telliez et al. 2007). Therefore, EGFR expression
levels and the protein interaction between EGFR and Her2
in different cells affect the efficiency of Herceptin inhibition.
This may explain why we did not observe a Her2 dosedependent effect for growth inhibition in different cell lines.
In addition to Her2 expression levels, androgen dependency might also play a role in growth inhibition by Herceptintargeted treatment. A previous report indicates that Her2 may
modulate AR function through its DNA binding and protein
stability (Mellinghoff et al. 2004). Our previous findings also
suggest that Her2 is important in stabilizing AR during the
transition from androgen dependence to independence in
prostate cancer cells (Hsu et al. 2011a). Therefore, prostate
cancer cells with androgen dependency, such as LNCaP,
were more susceptible to Herceptin, whereas androgenindependent cells such as DU145 and PC3 were not. The high
growth inhibition of LNCaP after Re-188-labeled Herceptin
treatment might result from the high Herceptin susceptibility
to androgen dependency and the fact that high Her2
expression increases the conjugation of Herceptin with
lethal radioisotopes. For the androgen-independent cell line
DU145, after treatment with Herceptin labeled with Re-188,
there was an increase in growth inhibition although Herceptin alone was ineffective (Figure 1B and 1C). Radio-induced
growth inhibition was also noted in the PC3 cell line, which is
another androgen-independent prostate cancer cell line
thought to be unsusceptible to Herceptin (Figure 1F). These
data imply that Re-188-labeled Herceptin might be a potential treatment for androgen-independent prostate cancer.
Int J Radiat Biol Downloaded from informahealthcare.com by Taipei Veterans General Hospital on 05/01/13
For personal use only.
354 H.-Y. Wang et al.
Previous studies have demonstrated that the major contribution of radiation-induced tumor shrinkage is apoptosis
(Stephens et al. 1991, Meyn 1997). TUNEL staining and
analysis of apoptosis-related proteins showed significant
apoptosis of DU145 cells after Re-188-labeled Herceptin
treatment compared to the control group (Figure 2). In addition, in vivo results also indicated that the apoptosis-related
proteins were significantly affected by Re-188-labeled Herceptin while Herceptin and Re-188 treatments alone did not
show similar effects (Figure 3). Taken together with previous
findings, our data suggests that the decrease of tumor size
might be induced by β radiation emitted from Re-188 conjugated with Herceptin.
We also explored the molecular mechanisms of Re188-labeled Herceptin treatment to elucidate its inhibitory
effects. Several lines of evidence have recently demonstrated
that Cdk5 and its regulator, p35, play important roles in cancer biology (Lin et al. 2007b, Eggers et al. 2011, Demelash
et al. 2012). Our report indicates that hyper-activation of
Cdk5 is important to determine the fate of prostate cancer
cells (Lin et al. 2004). Subsequently, Cdk5 was found to
have a significant role in controlling cell motility and metastasis in prostate cancer (Strock et al. 2006). Our recent study
further illustrated that Cdk5 may regulate the androgen
receptor through phosphorylation (Hsu et al. 2011b). In this
study, we found that Re-188-labeled Herceptin treatment
significantly decreased the levels of Cdk5 and its activator,
p35, in DU145 cells. In addition, the inhibitory effects of
Re-188-labeled Herceptin on p35 protein levels became
more significant in tumor xenografts. Indeed, knock-down of
Cdk5 or p35 resulted in a significant decrease of DU145 cell
growth (data not shown). These data suggest that the decrease
of p35/Cdk5 protein and its activity might mediate decreased
prostate cancer cell proliferation upon treatment with Re188-labeled Herceptin.
In conclusion, the inhibitory effects in Her2-expressing
and androgen-independent prostate cancer cells suggest
that Re-188-labeled Herceptin may be therapeutically beneficial for Her2-expressing prostate cancer patients in the
future.
Acknowledgements
The authors thank Dr S.L. Hsu and Ms M.C. Liu (Taichung
Veterans General Hospital, Taiwan); Dr H.C. Chen, Dr T.H.
Lee, Dr Y.M. Liou, Dr J.W. Chen and Dr C.M. Cheng (National
Chung Hsing University, Taiwan) for technical support.
Declaration of interest
The authors report no conflicts of interest. The authors alone
are responsible for the content and writing of the paper.
The work was supported in part by grants from the
Taichung Veterans General Hospital Research Program
(TCVGH-986703B), Taichung Veterans General Hospital/
National Chung Hsing University Joint Research Program
(TCVGH-NCHU-1017607 to H. Lin), Taiwan National
Science Council (NSC97-2320-B-005-002-MY3 to H. Lin),
and Taiwan Ministry of Education under the ATU plan.
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