2.2.1 EGFR mutation analysis
The EGFR mutation status was tested by polymerase chain reaction (PCR) and
sequencing. No mutation was found in exon 18, 19, 20 and 21 of NTUB1 or T24 cells.
2.2.2. Clonogenic assay
For erlotinib, the radiosensitizing activity is not significant. (Fig. 2-1 and Fig. 2-2)
Figure 2-1. Clonogenic assay of erlotinib +/- RT in T24 cells
Figure 2-2. Clonogenic assay of erlotinib +/- RT in NTUB1 cells
For trastuzumab, the radiosensitizing activity is not significant, either. (Fig. 2-3 and Fig. 2-4)
Figure 2-3. Clonogenic assay of trastuzumab +/- RT in T24 cells
Figure 2-4. Clonogenic assay of trastuzumab +/- RT in NTUB1 cells
For lapatinib, there was no effect of lapatinib in both cell lines. (Fig. 2-5 and Fig. 2-6)
Figure 2-5. Clonogenic assay of lapatinib +/- RT in T24 cells
Figure 2-6. Clonogenic assay of lapatinib +/- RT in NTUB1 cells
CHAPTER THREE: RADIOSENSITIZING EFFECT OF AFATINIB IN A MURINE BLADDER CARCINOMA MODEL
3.1 Rationale and Approach
As the mechanism of EGFR-mediated radioprotection may differ in various time
sequence, we will test the radiosensitivity after afatinib pretreatment of bladder cancer cells
in (a) immediate early phase (1-4 h) by immunofluorescence detection of γH2AX foci, (b)
early phase (4-24 h) by flow cytometry analysis, and (c) post double-strand break (DSB)
repair phase (>24 h) by clonogenic assay according to a model of EGFR-mediated
radioprotection (D. J. Chen & Nirodi, 2007). The efficacy of afatinib will also be validated in
mouse xenografts. The advantage of murine bladder cancer model is that the in vivo
experiments can be performed in immunocompetent animals. The tumor graft in mice will be
validated by animal imaging and immunohistochemistry.
3.2 Materials and Methods
Murine bladder tumor cell line
The murine (C3H/HeN) bladder tumor cell line, MBT-2, was obtained from the Japanese
Collection of Research Bioresources (Okayama, Japan). Cells were cultured in RPMI-1640
supplemented with 10% fetal bovine serum and 50 U/ml penicillin/streptomycin. Cells were
cultured at 37 °C in a humidified atmosphere of 5% CO2 and 95% air.
Reagents
Afatinib was purchased from Selleck Chemicals (Houston, TX, USA). For in vitro studies,
stock solutions of afatinib were prepared in dimethyl sulfoxide (DMSO) and diluted in
culture medium containing 10% fetal bovine serum. For in vivo studies, afatinib was
suspended in a vehicle (0.5% methylcellulose [wt/vol] and 0.4% Tween 80 [vol/vol] in sterile
water) for oral administration to C3H/HeN mice bearing xenograft tumors.
Irradiation of cells
MBT-2 cells in culture flasks were irradiated with different doses of radiation, using a 6-MV
photon linear accelerator. The distance from the radiation source to the bottom of the flask
was set at 100 cm.
Colony formation assay
Cells (500/well) were seeded in six-well plates and treated with different doses of radiation
following 30-min pretreatment with various doses of afatinib (200–1000 nM) or DMSO
vehicle. Cells were then cultured for an additional 7 days, after which the number of colonies
(clusters of more than 50 cells) was counted in each well using an inverted phase-contrast
microscope at 100X magnification and photographed. The effect on colony number was
analyzed using CompuSyn software (ComboSyn, Inc., Paramus, NJ, USA).
Western blot analysis
Aliquots of cell lysates containing 90 μg of protein were separated by SDS–PAGE (6–15%
polyacrylamide) and then transferred onto polyvinylidene difluoride membranes and
immunoblotted with various antibodies. Bound antibodies were detected using appropriate
peroxidase-coupled secondary antibodies followed by enhanced chemiluminescence (ECL,
Boehringer Mannheim, Mannheim, Germany). Antibodies to the phospho-HER2 and
phospho-EGFR were obtained from Epitomics, Inc. (Burlingame, CA, USA), EGFR and
HER2 from GeneTex, Inc. (Irvine, CA, USA), poly(ADP-ribose) polymerase (PARP) and
cleaved PARP from Cell Signaling Technology (Danvers, MA, USA), beta-actin from Santa
Cruz Biotechnology (Santa Cruz, CA) and histone variant H2AX, phospho-H2AX (Ser139)
and clone JBW301 from Millipore Corporation (Billerica, MA, USA).
Cell cycle phase analysis
The distribution of cells among the phases of the cell cycle was determined by quantifying
the cellular content of propidium iodide-stained DNA. Cells (106/ml) were treated as
indicated, harvested by centrifugation, stained with propidium iodide (PBS containing 0.5%
Tween 20, 15 μg/ml propidium iodide and 5 μg/ml DNase-free RNase), and analysed using a
Becton Dickinson FACScan flow cytometer equipped with Cell Quest software (Becton
Dickinson Immunocytometry Systems, San Jose, CA, USA).
γH2AX immunofluorescence microscopy
Cells were plated on polylysine-coated coverslips, allowed to attach overnight and exposed to
ionizing irradiation of 2.5 Gy either alone or combined with 100 nM afatinib. After treatment,
cells were incubated for 30 min, washed three times with ice-cold phosphate-buffered saline
(PBS), fixed in 4% formaldehyde/PBS for 30 min, permeabilized in 0.5% Triton X-100 in
PBS for 1 h, blocked in 5% bovine serum albumin for 1 h at room temperature, incubated
with the antibody (fluorescein isothiocyanate [FITC] conjugated anti-phospho-Histone γH2AX [Ser139; 1:1500; Millipore, Billerica, MA, USA]) for 2 h at room temperature in the
dark, washed with PBS and mounted in Vectashield mounting medium containing
diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). γ-H2AX foci were examined
using a Zeiss Axio Imager A1 fluorescence microscope. In each sample, the number of
γ-H2AX foci per nucleus was counted by focicounter under high power field and the average of
150 nuclei was calculated. The average number of γ-H2AX foci per nucleus represents the
amount of double strand breaks.
In vivo studies
Female C3H/HeN mice (6 weeks of age) were obtained from the National Laboratory Animal
Center and used for ectopic (subcutaneous) xenograft implantation. Body weights were
measured weekly. Mice from each group were sacrificed on day 8. The tumor was fixed in
10% neutral buffered formalin and processed for histopathological and immunohistochemical
evaluations. Tumor volumes were measured with a set of calipers and calculated using a
standard formula: width2 × length/2. All experimental procedures using these mice were
performed in accordance with protocols approved by the National Taiwan University
Institutional Animal Care and Use Committee.
Ectopic tumor model
Ectopic tumors were established by subcutaneous injection of MBT-2 cells (2 × 106) into the
right hind leg of mice. As the tumors became established (mean starting tumor volume = 162
mm3), the mice were randomized into 4 groups to receive the following treatments: (1)
methylcellulose/Tween 80 vehicle; (2) afatinib (10 mg/kg/day of body weight) on day 1–7;
(3) methylcellulose/Tween 80 vehicle plus 15 Gy of radiotherapy on day 4; (4) afatinib plus
radiotherapy. Small animal positron emission tomography/computed tomography (PET/CT)
scans with [18F]-2-fluoro-2-deoxy-D-glucose (FDG) were performed on the 8th day of
treatment. The mice were intravenously injected with 14 MBq (378 Ci) of FDG in saline via
the tail vein.
Irradiation of mice
Mice were immobilized using a customized harness. With the body shielded, the thigh tumor
was irradiated with a half-beam rectangular field. A 6-MV photon beam from a linear
accelerator was used to irradiate the thigh tumor with 15 Gy on day 4.
Histological evaluation
After fixation, tumor tissues were embedded in paraffin blocks and sectioned (5 μm). Tumor
cells were detected in representative stained sections. The expressions of phospho-EGFR
(Cell Signaling Technology, Inc., Danvers, MA, USA) and phospho-HER2 (Abcam PLC,
Cambridge, UK) were evaluated after immunohistochemical staining using specific
antibodies.
Statistical analysis
The tumor volume data satisfied the assumptions of normality and homogeneity of variance
for parametric analysis; thus, group means on day 21 for the ectopic tumor models were
compared with a one-way analysis of variance (ANOVA) followed by Fisher’s least
significant difference method for multiple comparisons. Differences were considered
significant at p < 0.05.
3.3 Results (Tsai et al., 2013)
3.3.1 Radiosensitization of MBT-2 cells by afatinib
Clonogenic cell survival decreased dose-dependently either with irradiation (2.5–10 Gy) or
afatinib treatment (200–1000 nM (Fig. 3-1A). To determine if the interaction between
afatinib and radiation was synergistic, combination index (CI) values were calculated from
the dose–response data. In MBT-2 cells treated with highest doses of irradiation and afatinib,
CI values of < 1 were achieved and indicative of synergism (Fig. 3-1B).
Figure 3-1. Radiosensitization of MBT-2 cells by afatinib. (A) Quantitative results of the
clonogenic assays after combination treatment with afatinib and irradiation. Cells were
cultured at a density of 500 cells per well in six-well plates and pretreated with different
doses of afatinib (200–1000 nM for 30 min and then irradiated with different doses (2.5–10
Gy). After 7 days, the cells were fixed, stained and photographed (100X). The images were
used to count the number of colonies containing more than 50 cells in each well. The number
of MBT-2 colonies at each dose level is expressed as a percentage of those in the
corresponding control group. Lines, mean (n = 3); Bars, S.D. (B) CI for each dose level of
irradiation and afatinib were calculated and plotted as a function of the MBT-2 cell fraction
affected (Fa). CI values < 1 indicate synergism.
3.3.2 Radiation activates EGFR/HER2 and Akt protein expressions in a time-dependent
manner
It has been reported that receptor tyrosine kinases, such as Erb-B family proteins, are
activated by irradiation. Besides, activation of the PI3K/Akt pathway is associated with
radioresistance. We found in Western blotting assays that levels of both HER2 and EGFR
proteins increased time-dependently, starting at 2 h and 6 h after irradiation in MBT-2 cells
with 2.5 Gy and 10 Gy, respectively (Fig. 3-2). Similarly, the increased expression of
phospho-Akt was induced in a time-dependent manner.
Figure 3-2. Radiation activates EGFR and PI3K/Akt pathways at both low and high doses.
MBT-2 cells were treated with irradiation (2.5 Gy and 10 Gy). Cell lysates were prepared for
Western blotting of the phosphorylation forms of EGFR, HER2 and Akt, and the effects of
irradiation can be seen to unfold in a time-dependent manner.
3.3.3 Afatinib inhibits radiation-induced EGFR/HER2 and Akt protein expressions in
MBT-2 cells
Since radiation induces Erb-B family protein expression, we investigated whether the dual
EGFR/HER2 inhibitor, afatinib, can suppress induced expression of these proteins. In MBT-2
cells that received irradiation (either alone or in combination with afatinib), the increased
expression of HER2 and EGFR proteins as well as expression of activated phospho-Akt were
inhibited by afatinib at 6 h (Fig. 3-3).
Figure 3-3. Afatinib inhibits the radiation-activated EGFR phosphorylation and PI3K/Akt
pathways. MBT-2 cells were pretreated with afatinib (100 nM and 200 nM for 24 h and then
irradiated (2.5 Gy and 10 Gy). After 6 h, cell lysates were prepared for Western blotting of phosphorylated forms of EGFR, HER2 and Akt, with β-actin as a loading control.
3.3.4 Afatinib combined with irradiation increases the apoptosis of MBT-2 cells
Our analysis of the cell cycle distribution of MBT-2 cells at 6 h after irradiation (10 Gy) with
or without pre-treatment of afatinib (100 nM, 30 min) revealed that the combination
significantly increased the sub-G1 population (p < 0.05), indicating apoptotic cell death (Fig.
3-4A). Radiation alone failed to cause a statistically significant increase in the sub-G1
population, but it did insignificantly increase the proportion of cells in G2/M phase arrest and
insignificantly decrease the proportion of cells in S phase. Afatinib alone, however, did not
cause any significant change in the cell cycle phases. Moreover, Western blot analysis of
cleaved PARP revealed that pretreatment with afatinib strongly increases the expression of
this apoptotic marker in response to irradiation (Fig. 3-4B).
Figure 3-4. Afatinib enhances radiation-induced apoptosis in MBT-2 cells. (A) MBT-2 cells
were pretreated with afatinib (200 nM for 30 min and then with radiation (RT; 10 Gy). The
cell cycle distributions were assessed 6 h after afatinib alone, RT alone and in combination.
Columns, mean (n = 3); Bars, S.D. ∗, p < 0.05. (B) MBT-2 cells were pretreated with afatinib
(50 nM and 100 nM for 30 min and then with RT (2.5 Gy and 10 Gy). After 6 h, cell lysates
were prepared for Western blotting to detect the apoptotic marker PARP (cleavage form).
3.3.5 Afatinib combined with irradiation increased DNA damage of MBT-2 cells
Fig. 3-5A and 3-5B show the result of immunofluorescence staining of γ-H2AX, a marker of
DNA double-strand breaks. While sham-irradiated cells exhibited a minimal number of γ-H2AX foci (0 ± 0.05/cell), radiation alone induced immediate increases in γ-γ-H2AX foci (13.0
± 0.28/cell) that were evident at 30 min as a result of cellular DNA damage. In contrast, treatment with afatinib had no effect on γ-H2AX foci (0 ± 0.03/cell). However, in cells
pretreated with afatinib prior to irradiation, the number of γ-H2AX foci was significantly
increased over that observed after irradiation alone (20.0 ± 0.46/cell versus 13.0 ± 0.28/cell, p
< 0.001). Western blot assay revealed dose-dependent changes in γ-H2AX levels in MBT-2
cells pre-treated with afatinib (50 or 100 nM for 30 min followed by irradiation (2.5 or 10
Gy; Fig. 3-5C).
Figure 3-5. Afatinib enhances radiation-induced DNA damage of MBT-2 cells. (A)
Micrographs (1000X) of γ-H2AX foci, a marker of DNA double-strand breaks, of MBT-2
cells at 30 min after pretreatment with afatinib (100 nM for 30 min and then with radiation (RT; 2.5 Gy) show the 4’,6-diamidino-2-phenylindole (DAPI) staining for cells, FITC for
γ-H2AX (green foci) and the merged images. (B) The number of γ-γ-H2AX foci counted in 150
cells per group. Data presented are the mean number of foci per cell in each group. Columns,
mean; Bars, S.D. ∗, p < 0.05. (C) MBT-2 cells were pretreated with afatinib (50 and 100 nM
for 30 min and then RT (2.5 Gy and 10 Gy). After 30 min, cell lysates were prepared for
Western blotting to detect phospho-γ-H2AX (p-H2AX) and H2AX (loading control).
3.3.6 The combination of afatinib and radiotherapy exhibits an enhanced ability to
control the growth of ectopic MBT-2 xenograft tumors
Daily oral treatment of mice with afatinib (10 mg/kg for 7 days) in combination with
radiotherapy on day 4 suppressed the growth of xenograft tumors to a greater extent than
radiotherapy alone (Fig. 3-6A). Afatinib itself did not satisfactorily control growth. Treatment
with afatinib enhanced radiation-induced suppression of MBT-2 tumor growth by 64%.
One day after the treatment (day 8), thigh tumors were imaged by micro-PET/CT with
18F-FDG. Tumor viability was decreased after combined afatinib and radiotherapy, when
compared to either modality alone or sham treatment (Fig. 3-6B). The treatment with afatinib
alone failed to reduce metabolic tumor volume, but radiotherapy to thigh tumors (15 Gy) by
itself partially reduced tumor size. Importantly, co-treatment with afatinib at 10 mg/kg
significantly improved this radiotherapeutic effect.
The expressions of HER2 and EGFR were assessed immunohistochemically in MBT-2
tumors harvested at 8 days after initiation of treatments. Radiotherapy itself increased the
expressions of both HER2 and EGFR (Fig. 3-6C). Moreover, combined treatment with
radiotherapy and afatinib suppressed radiation-activated expression of HER2 and EGFR in
tumor tissues.
Figure 3-6. Combined afatinib and irradiation (RT) enhances tumor suppressive activity in
ectopic murine bladder tumor model. (A) C3H/HeN mice bearing subcutaneous MBT-2
tumors were randomized into 4 groups (n = 5 in each group) to receive RT alone (15 Gy on
day 4), oral afatinib (10 mg/kg/day from day 1–7) alone, combined afatinib and RT or control
treatment (sham). Data presented are the mean tumor volume for each group measured on the
indicated days. Points, mean; bars, S.D. (B) Mice bearing ectopic MBT-2 tumors were
randomized as in (A) to receive afatinib (10 mg/kg/day) alone, RT alone, combined RT and
afatinib or sham treatment. Mice were scanned by positron emission tomography/computed
tomography to determine tumor metabolism on day 8. Images of representative mice from
each group are shown. Arrows indicate viable right thigh tumors. The standardized uptake
value (SUV) and volume of each tumor were shown on top of the panel. (C) Mice bearing
ectopic tumors were sacrificed on day 8. Microscopic images (200X) of tumor tissue
sectioned and immunohistochemically stained with antibodies against HER2 (left panel) and
EGFR (right panel) from a representative mouse in each group are shown.
CHAPTER FOUR: RADIOSENSITIZING EFFECT OF AFATINIB IN HUMAN BLADDER CANCER MODELS
4.1 Rationale and Approach
Erlotinib (Maemondo et al., 2010) is a first-generation EGFR tyrosine kinase inhibitors
and have well-established efficacy in non-small cell lung cancer patients with EGFR
activating mutations. Afatinib, on the other hand, is a second-generation EGFR tyrosine
kinase inhibitor and found to be of benefit to patients with advanced lung adenocarcinoma
who failed previous gefetinib or erlotinib (Miller et al., 2012). First RTK array was done to
detect the change of signaling pathways after irradiation with or without drugs. Then the
experimental design is similar to murine bladder cancer model and includes: (a) clonogenic
assay, (b) flow cytometry analysis, (c) immunofluorescence detection of γH2AX foci and (d)
animal study. The advantage of human bladder cancer model is that the result may benefit
patients clinically. Finally the mechanism was explored.
4.2 Materials and Methods
Cell lines
The human bladder urothelial carcinoma cell line, T24, was purchased from the American
Type Culture Collection / Bioresource Collection and Research Center (Hsinchu, Taiwan) in
2011. The cells were authenticated in BCRC by short-tandem repeat (STR)-PCR profiling.
They were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal
bovine serum. Another human bladder carcinoma cell line, NTUB1, was established from
human bladder cancer tissue and kindly provided by Dr. Hong-Jeng Yu (Yu, Tsai, Hsieh, &
Chiu, 1992). It was not authenticated. NTUB1 cells were cultured in RPMI-1640 medium
with 10% fetal bovine serum. Both cell lines were incubated at 37°C in a humidified
atmosphere of 5% CO2 and 95% air. We sequenced the cDNA of both cell lines, and none of
the common EGFR mutations was found.
Reagents
Afatinib and erlotinib were purchased from Selleck Chemicals (Houston, TX). For in vitro
experiments, afatinib and erlotinib stock solutions were prepared in DMSO and 50%
acetonitrile, respectively. Both compounds were diluted in culture medium before dosing. For
in vivo experiments, afatinib and erlotinib were suspended in a vehicle (0.5% methylcellulose
[wt/vol] and 0.4% Tween 80 [vol/vol] in sterile water) for oral administration to ICR nude
mice (BioLASCO, Ilan, Taiwan) bearing tumor xenografts.
Irradiation of cells and animals
T24 and NTUB1 cells cultured in flasks were irradiated with various doses of ionizing
radiation, using a 6-MV photon beam from a Siemens Primus linear accelerator (Siemens
Oncology Medical Systems, Inc., Concord, CA). Mice were immobilized using a customized
harness. With the body shielded, the thigh tumor was irradiated with a half-beam rectangular
field. The distance from the radiation source to the bottom of the flask or the thigh tumor of
nude mice was set at 100 cm.
RTK signaling antibody array
The PathScan® RTK signaling antibody array kit from Cell Signaling Technology (Danvers,
MA) contained 39 antibodies against phosphorylated forms of receptor tyrosine kinases or
key signaling proteins. T24 cells were first treated with 100 nM afatinib or erlotinib for 30
min, and then with 10 Gy of radiation. After 24 h of incubation, the cells were processed for
RTK array analysis according to the manufacturer's instructions. The membrane was
developed with LumiGLO® and Peroxide reagent (Cell Signaling Technologies), and RTK
spots were visualized using a UVP imaging system and densitometrically quantified with
ImageProPlus software. Each kinase array dot was manually selected, and an average
intensity for each kinase was calculated. For comparison of different stimulation conditions,
sets were normalized to allow equal intensities of positive controls.
Clonogenic assays
T24 or NTUB1 human bladder cancer cells (1×103 per well) were cultured in 6-well plates,
treated with different doses of radiation following 1-h pretreatment with afatinib or erlotinib
on day 1, re-treated with the drugs on day 2 and day 3 using the same concentrations,
incubated for 7 days, and stained with 0.5% crystal violet (Sigma-Aldrich; St. Louis, MO) in
10% methanol for 30 min at room temperature. Colonies with more than 50 cells were
counted. At each drug concentration, the surviving fraction was determined by dividing the
total number of colonies after irradiation by the number of colonies without irradiation. Each
point on the survival curve represents the mean surviving fraction from 3 independent
experiments.
Cell-cycle analysis
Cell cycle stages were analyzed using a BD FACSCan Flow Cytometer (Becton Dickinson;
Franklin Lakes, NJ). In brief, T24 or NTUB1 bladder cancer cells were pretreated for 30 min
with vehicle, 200 nM afatinib, or 200 nM erlotinib, irradiated (2.5 Gy), incubated 24 h, fixed in 70% ethanol, and stained with a solution containing 50 μg/mL propidium iodide and 0.1
mg/mL RNAase (both from Sigma-Aldrich) in the dark for 30 min. Ten thousand events were
examined for each determination. The relative proportions of cells in different cell cycle
phases were determined using WinMDI software.
Determination of apoptosis with fluorescence microscopy
Apoptotic cells were detected using the annexin V/FITC apoptosis detection kit (AVK050,
Strong Biotech, Taipei, Taiwan) according to the manufacturer’s instructions. The annexin
V-positive cells were examined using a Zeiss Axio Imager A1 fluorescence microscope.
Representative images from different treatment groups were taken into account and at least 50
cells were calculated in every group. The portion of annexin V-positive cells was calculated as
the ratio of positively stained cells divided by the total cell numbers.
Western blotting and immunoprecipitation
Aliquots of T24 and NTUB1 bladder cancer cell lysates containing 50 μg of protein were
separated by SDS-PAGE (8–15% polyacrylamide), and the separated proteins were
transferred to polyvinylidene difluoride (PVDF) membranes and immunoblotted with various
antibodies. For immunoprecipitation experiments, we used the Catch and Release v2.0
Reversible Immunoprecipitation System (Millipore) according to the manufacturer's instructions. The immunoprecipitates (50 μg) of cells were eluted, resolved by 8%
SDS-PAGE, electrotransferred to PVDF membranes and incubated with primary antibodies. For
whole-cell preparations, tumor tissue from individual animals was homogenized with a motor
driven pestle and then lysed in 0.2 ml of RIPA lysis buffer/20 mg tissue. The homogenate was
then centrifuged (13,000 g) for 10 min and the supernatant was used as whole-cell extract.
Bound antibodies were detected using appropriate peroxidase-coupled secondary antibodies
Bound antibodies were detected using appropriate peroxidase-coupled secondary antibodies