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rhabdospheres
derived
from
the
embryonal
rhabdomyosarcoma cell lines.
Material and Methods
Rhabdospheres enriched in cancer stem like cells were
obtained growing ERMS cells in non adherent condition in
stem cell medium. Stem cell markers were evaluated by
FACS analysis and immunoblotting. ERK1/2, myogenic
markers, proteins of DNA repair and bone marrow X-linked
kinase (BMX) expression were evaluated by
immunoblotting analysis. Radiation was delivered using an
x-6 MV photon linear accelerator. Xenografts were
obtained in NOD/SCID mice by subcutaneously injection of
rhabdosphere cells or cells pretreated with U0126 in stem
cell medium.
Results
MEK/ERK inhibitor U0126 dramatically prevented
rhabdosphere formation and down-regulated stem cell
markers CD133, CXCR4 and Nanog expression, but
enhanced ALDH, MAPK phospho-active p38 and
differentiative myogenic markers. By contrast, MAPK p38
inhibition accelerated rhabdosphere formation and
enhanced phospho-active ERK1/2 and Nanog expression.
ERMS cells, chronically treated with U0126 and then xeno-
transplanted in NOD/SCID mice, delayed tumor
development and reduced tumor mass when compared
with tumor induced by rhabdosphere cells. U0126
intraperitoneal administration to mice bearing
rhabdosphere-derived tumors inhibited tumor growth .
The MEK/ERK pathway role in rhabdosphere
radiosensitivity was investigated in vitro. Disassembly of
rhabdospheres was induced by both radiation or U0126,
and further enhanced by combined treatment. In U0126-
treated rhabdospheres, the expression of the stem cell
markers CD133 and CXCR4 decreased and dropped even
more markedly following combined treatment. The
expression of BMX, a negative regulator of apoptosis, also
decreased following combined treatment, which suggests
an increase in radiosensitivity of rhabdosphere cells.
Conclusion
Our results indicate that the MEK/ERK pathway plays a
prominent role in maintaining the stem-like phenotype of
ERMS cells, their survival and their innate radioresistance.
Thus, therapeutic strategies that target cancer stem cells,
which are resistant to traditional cancer therapies, may
benefit from MEK/ERK inhibition combined with
traditional radiotherapy, thereby providing a promising
therapy for embryonal rhabdomyosarcoma.
PO-0981 Disturbance of redox status enhances
radiosensitivity of hepatocellular carcinoma
H. Zhang
1
, C. Sun
1
1
Institute of Modern Physics- Chinese Academy of
Sciences, Department of Heavy Ion Radiation Biology and
Medicine, Lanzhou, China
Purpose or Objective
High constitutive expression of Nrf2 has been found in
many types of cancers, and this high level of Nrf2 also
favors resistance to drugs and radiation. Here we
investigate how isoliquiritigenin (ISL), a natural
antioxidant, inhibits the Nrf2-dependent antioxidant
pathway and enhances the radiosensitivity of HepG2 cells
and HepG2 xenografts.
Material and Methods
Treatment of HepG2 cells with ISL for 6 h, Keap1
and ubiquitination of Nrf2 were measured by RT-PCR and
Western blot. Pretreatment with ISL for 6 h followed by X-
ray irradiation, confocal microscopy was used to visualize
Nrf2 translocation to the nucleus and γ-H2AX foci. To
investigate
the
radiosensitization
effect
of
ISL, apoptosis, clonogenic potential and HepG2 xenografts
were examined.
Results
Treatment of HepG2 cells with ISL for 6 h selectively
enhanced transcription and expression of Keap1. Keap1
effectively induced ubiquitination and degradation of
Nrf2, and inhibited translocation of Nrf2 to the nucleus.
Consequently, expression of Nrf2 downstream genes was
reduced, and the Nrf2-dependent antioxidant system was
suppressed. Endogenous ROS was higher than before ISL
treatment, causing redox imbalance and oxidative stress
in HepG2 cells. Moreover, pretreatment with ISL for 6 h
followed by X-ray irradiation significantly increased γ-
H2AX foci and cell apoptosis, and reduced clonogenic
potential compared with cells irradiated with X-rays
alone. In addition, HepG2 xenografts, ISL, and X-ray
cotreatments induced greater apoptosis and tumor growth
inhibition, when compared with X-ray treatments alone.
Additionally, HepG2 xenografts, in which Nrf2 was
expressed at very low levels due to ectopic expression of
Keap1, showed that ISL-mediated radiosensitization was
Keap1 dependent.
Conclusion
ISL inhibited the Nrf2-antioxidant pathway by increasing
the levels of Keap1 and ultimately inducing oxidative
stress via disturbance of the redox status. The antioxidant
ISL possessed pro-oxidative properties, and enhanced the
radiosensitivity of liver cancer cells, both
in vivo
and
in
vitro
. Taken together, these results demonstrated the
effectiveness of using ISL to decrease radioresistance,
suggesting that ISL could be developed as an adjuvant
radiosensitization drug. Disturbance of redox status could
be a potential target for radiosensitization.
PO-0982 Fused Toes Homolog (FTS) is a potential
target for Notch-mediated radioresistance in cervical
cancer
W.Y. Park
1
, P.D. Subramania
1
, J.R. Yu
2
1
Chungbuk National University Hospital, Dept of
Radiation Oncology, Cheongju, Korea Republic of
2
Konkuk University, Department of Environmental and
Tropical Medicine, Chungju, Korea Republic of
Purpose or Objective
Radiation therapy is one of the major treatment
modalities for cervical cancer. Increasing evidences
suggest that cancer stem cells (CSC) in tumours contribute
to radioresistance and recurrence. Notch pathway plays a
vital role in maintenance of cancer stemness and its
activation leads to disease progression and metastasis. FTS
gene was initially identified as one of six genes deleted in
a mouse mutant called Fused Toes, due to defects in limb
development, and referred as FT1/FTS. However, the
function of FTS has not been elucidated well in human. We
previously reported that FTS plays an essential role in
nuclear phosphorylation of EGFR and repair of DNA
damage, and epithelial-mesenchymal transition. In this
study, we evaluated the role of FTS in Notch signaling and
CSCs.
Material and Methods
A human cervical cancer cell line (ME180) was used.
Silencing of FTS was obtained using siRNA. Western blot
and immunofluorescence was done to analyze the
expression and localization of the proteins.
Results
Protein expression of Notch 1, cleaved Notch1, Notch 3,
γ-secretase complex and its downstream Hes-1was
increased by ionizing radiation and it was reduced by FTS-
silencing. Spheroid formation ability and cancer stem cell
markers Nanog, Oct-4A, Sox2 were reduced by FTS-
silencing. Cell survival was decreased by FTS-silencing.
Conclusion
FTS is involved in the regulation of Notch signaling and CSC
maintenance. FTS can be a target to overcome Notch–
mediated radioresistance in cervical cancer.
PO-0983 Antrodia cinnamomea Regulates DNA Repair
and Enhances Radiosensitivity of Esophageal Cancer
Cells