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Association between Expression of p70s6k with
Radiotherapy Response in Cervical Cancer
I. Kurnia1*, B. Siregar2,Z. Alatas1, I. Ramli2, T. Kurjana3,A. Andrijono2,
M.D.L. Tobing3, B.S. Hernowo3, T. Kisnanto1,D. Tetriana1,and S. Soetopo3
1Center
for Technology of Radiation Safety and Metrology, National Nuclear Energy Agency,
Jl. Lebak Bulus Raya No.49, Pasar Jum’at, Jakarta 12440, Indonesia
2Cipto Mangun Kusumo Hospital,Jalan Diponegoro No.71, Jakarta10430,Indonesia
3Hasan Sadikin Hospital, Jalan Pasteur No.38, Bandung 40161, Indonesia
ARTICLE INFO
ABSTRACT
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There aremany important prognostic factors in advanced stage
cervical cancer primary treated with radiotherapy. Besides clinical
factor, many biomarkers have been studied in relation with
radiotherapy response.
P70s6k is one of biomarker plays an
important role in cell proliferation. Increased levels of p70s6kalso
have associated with drug resistance of cancer. In the present study,
wedetermined the association between the expressions of p70s6k
before treatment with radiotherapy response in cervical
cancer.Immunohistochemical staining ofp70s6k, MIB-1, p53 was
conducted in microscopic slide from 21 cancer tissue biopsies before
treatment with radiotherapy. After complete the treatment, early
radiotherapy response was observed by pelvic control method. The
resultsshow thatp70s6k is highly expressed(61.9%, 13/21) and low
expressed (38.1%, 8/21) in the cancer cells andno
significantdifference was foundonAgNOR mean, MIB-1 and p53
index in the different degrees of p70s6k expression (p≥0.05).High
expression of p70s6k had association with good radiotherapy
response compared tolower one (p=0.05). In conclusion, degree of
p70s6k expression before treatment has association with
radiotherapy response.
Keywords:
Cervical Cancer
Radiotherapy
P70s6k
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INTRODUCTION
Cervical cancer was the most malignancy
disease case found in Indonesia. Usually, patient
come to the hospital was already at a local advanced
stage. One of the treatment choices in local
advanced staged form of this cancer is radiotherapy.
This treatment and sometime could be combined
with chemotherapy or concurrent chemoradiotherapy [1,2].
There are many important prognostic factors
in advanced stage cervical cancer primarily treated

Corresponding author.
E-mail address:[email protected]
with chemoradiotherapy are clinical pathologic
factors, including stage and tumor histology [3].
Besides clinical pathologic factors, many
biomarkers have been studied in relation to survival
and/or response to chemoradiotherapy especially
markers involved in tumorigenesis and tumor
progression, such as genes associated with
apoptosis, angiogenesis, and cell growth, that have
been investigated extensively. At the moment, the
focus of improving survival rates is mainly on
targeted therapies in combination with standard
chemoradiotherapy [4].
P70s6k is a serine/threonine protein kinase
responsible for the phosphorylation and activation
of 40S ribosomal subunit protein S6 [5,6]. It acts
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downstream of the mammalian target of rapamycin
(mTOR) in Akt pathway. The activation of p70s6k
and its downstream target ribosomal protein S6
mediates
nutrient
and
mitogen-stimulated
translation, which is essential for cell growth and
proliferation [7]. In some in vitro studies, it was
foundthat activity of p70s6k related with prevention
of apoptosis as one of death kind in cancer cell
treatment with chemotherapy and in radiotherapy
[8-11].
AgNOR, MIB-1 and p53 are biomarkers
that related to cell proliferation. Nucleolar organizer
regions (NORs) are chromosomal segments that
carry ribosomal genes. Silver stained NORs are
called AgNORs and when examined using light
microscopy they appear as well-defined black dots
exclusively located within the nucleoli [12]. MIB-1
is monoclonal antibody to detect Ki-67 antigen.
This antigen can bedetected in the nucleus of cells
during the gap1 (G1), synthesis (S), and gap2 (G2)
and mitosis (M) phases of the cell cycle [13]. Thus,
it can be only detected in the nucleus of proliferating
cells in all active phases of the cell-division cycle,
but is absent in non-proliferating cells [14]. The p53
gene, located on chromosome 17, encodes a 53-kDa
protein. Wild-type p53 protein is a transcription
factor that, when activated, acts to arrest cells
temporarily in the G1/G2 phase of the cell cycle
allowing time for the repair of the damaged DNA,
or to cause cells to proceed to apoptosis if the DNA
damage is irrepairable [15,16].
In our previous publication there were
correlation between apoptosis caspase-3 with better
radiotherapy response, and contrary with high level
of Akt expression that related with poor or bad
radiotherapy response, but there were no association
between some proliferation markers like AgNOR,
MIB-1, p53, with radiotherapy response [17,18].
The aim of this study is to investigate association
between p70s6k as biomarker with radiotherapy
response in cervical cancer.
EXPERIMENTAL METHOD
Patients
Twenty one consecutive patients, who
achieved a complete treatment, from a whole series
of 60 cases suffering from non-metastatic localized
cervical carcinoma in local advance stage (IIB-IIIB),
were prospectively conductedfrom July 2010 to
March 2011. All patients were diagnosed and
treated by definitive concurrent chemoradiotherapy
in Cipto Mangunkusumo and Hasan Sadikin
Hospitals. The treatment consists of external
radiation and internal radiation.
External Beam
Radiotherapy (EBRT) with C-60 with total doses 50
Gy in 25 fractions. Internal radiation in 192Ir High
Dose-Rate Intracavitary Brachytherapy (HDRICBT)
using
a
Microselectron(NucletronInternational, Amsterdam,
Netherlands) followedby EBRT in two fractions
(850 cGy/fraction).Cisplatin was administered at
dose of 40 mg/m2 on days 1, 8, 15, 22, and 29 that
given concurrently with the EBRT in 2 hours or less
before the treatment for corresponding day. Process
of diagnosis and treatment followed the Standard
Operational Procedure (SOP) as show in our
previous publication [17].
Before the studies were carried out, the
research proposal was approved by the Research and
Ethical Committee from Faculty of Medicine,
Universityof Indonesia and the patients received
written informed consent.
Radiotherapy Response
Radiation response was evaluated by
radiotherapist and as grouped by Hong Criteria as
follow [19]:
1. NRT response (no gross residual tumor):
complete or nearly complete regression of
pelvic tumor, non specific fibrosis, or
granulation over the cervix (called as good
response).
2. GT response (gross residual tumor): gross
tumor or palpable nodularity on cervix, and/or
palpable in duration on the parametrium (called
as bad response).
AgNOR Staining and Immunohistochemical
AgNORstaining and Immunohisto-chemical
of MIB-1, p53 and p70s6k were performed on
microscopic slide from biopsy cancer tissue sample
before treatment with radiotherapy. The detail
analysis method of MIB-1 and p53 was conducted
with procedures as in our previous publication
[20,21]. P70s6k expression is observed in the
nucleus and cell cytoplasm. The degree of its
expression was estimated blindly of the clinical
patient characteristics and outcome,with microscope
magnification (10x40) semi-quantitatively. The
scored was from negative (−) to slightly positive
(+), moderately (++) and strongly positive (+++)
[22]. The score from negative to slightly positive
was grouped as low expression and moderate to
strong positive was grouped as high expression
Statistical Analysis
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Analysis of variance (ANOVA) Test was
used to analysescomparison between expression of
p70s6k and AgNOR mean, MIB-1 index and p53
index. Fisher Exact Test was used toanalyze
between expressions of p70s6k with radiotherapy
response. All the test use with p≤0.05 as statistical
significant. Minitab 17 was used to data analysis.
RESULTS AND DISCUSSION
Expression of p70s6k was detectable as
brown colour in the nucleus and cytoplasm of
cancer cell as shown in Figure 1a and b.
Itsexpression can be observed in all of
21microscopic slides from cancer tissue slide before
radiotherapy. Itcan be grouped into high expressed
(61.9%, 13/21) and low expressed (38.1%, 8/21).In
Table 1, there was no statistical different of AgNOR
mean, MIB-1 and p53 index in high and low
expression of p70s6k before treatment. Different
with AgNOR mean that tend lower in high
expression of p70s6k, both of MIB-1 and p53 index
is seem higher in high expression of p70s6k than
lower one (Fig.2 a,b,c). After radiotherapy there was
statistical different in proportion of complete and
partial radiotherapy response in high and low
expression of p70s6k (p=0.05) (Figure 3).
Tendency of low AgNOR meaninhigh
expression of p70s6k is related with different
activity site of p70s6k and AgNOR in part of cell
cycle. AgNOR can be detected in G1, S and G2 in
cell cycle and p70s6k expression is focused in
G1only. The number of AgNOR start to be detected
in G1, increased and accumulated in S and
decreased again in G2 [23]. Another report also
found that when compared in cancer tissue before
treatment and after 9 Gy irradiated AgNOR mean is
lower in after 9 Gy irradiated as considered in G2
phase and S phase beforeirradiation[21]. The
similar data was also foundwhenre AgNOR
observed in stimulated mitotic of hepatocyte cell
[20].
As a mitogen-activated Ser/Thr protein
kinase, p70s6k is required for G1 cell cycle
progression and cell proliferation [8].
High expression of p70s6k related with cell
proliferation. In active proliferation cell, amount of
cell inmitotic (cycling cell) is higher than G0 phase.
Its clearlythat higher expression of p70s6k also
show higher of MIB-1 index[24,25]. Higher MIB1 also mean that higher of growing fraction of
cancer cell[21].
Even though without DNA sequencing
analysis, almost expression of p53in cervical
cancerwas in mutant than wild type.It was the main
reason for this current data explaining higher p53
index in high expression of p70s6k than in lower
one. Invitro investigation reported that wild type
p53 can decrease of p70s6k expression by mTOR
pathway[26,27,28].
In this current result,high expression of
p70s6k made the high proliferated cell and also
prevents the process of apoptosis as one of way the
death of cancer cell after receiving exposure
ofioniczing radiation in radiotherapy. High portion
of proliferating cell also made the cells to be more
radiosensitive than radioresistance. A number of
studies have focused on the involvement of p70S6k
in influencing cellular responses to apoptotic
stimuli. Treatment of Swiss 3T3 and RAT-1 cells
with etoposide and staurosporine resulted in
dephosphorylation and decreased activity of
p70S6kin apoptosis preventing[8,9]. It has also
been shown that cisplatin inhibited the
phosphorylation of p70s6k in mouse myoblasts [10].
Rapamycin, an immune-suppressant blocked the
activation of mTOR/p70s6k, thus compromising the
cell's ability to progress through the G1 phase of the
cell cycle [10,11]. On other in vitro study, using
breast cancer cell,inactivated of p70s6k also let the
process of cell death and should sensitize cancer cell
to chemotherapeutic agents[29].
Cell in high proliferationwere highly
radiosensitive and giving good radiotherapy
response, in otherwise lower proliferation
proportion will give the bad radiotherapy response.
Because in this result the observation of
radiotherapy response as early response, we think
that major of death of cancer cell was
almostcausedby irradiation than cisplatin in
concurrent therapy.
This present result seems have different
with some reports explaining relation between
expression of p70s6k with resistance of some cancer
cells after treatment. Highexpression of p70s6k
before treatment related with resistanceor
poorresponse of cancer, especiallyin cancer treated
with chemotherapy [30-32].
In the future, ifthis result will be able to be
confirmed in bigger sample,we suggested the higher
expressed p70s6k patients as one of consideration
parameter receiving earlier treatment priority than
the lower one. It also haspotential using anti p70s6k
as target therapy in the form of adjuvant therapy
after the patients completed the treatment of
radiotherapy in cervical cancer.
CONCLUSION
Expression of p70s6k have important role
in cell cancer proliferation to make the cancer tobe
more radiosensitive or radioresistantand
have
potential to predict the radiotherapy response and
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also as a consideration parameter in selection
patientbefore treatment.
14.
15.
16.
ACKNOWLEDGMENT
This research was supported by a Research
Grant from Center for Technology of Radiation
Safety and Metrology, National Nuclear Energy
Fiscal Year 2011. The authors thank Dr.
MukhSyaifudin, Head of Radiobiology Group,
Division of Nuclear Medicine and Radiation
Biology,Center for Technology of Radiation Safety
and Metrology, National Nuclear EnergyAgency for
reviewing and editing this manuscript.
REFERENCES
T. Didit and M.Rukmini,Clin.Oncol. Jpn.
J.32 (2002)S17.
2. S. Mufyalaand A.H. Wofson, Cervical
Cancer.In: J.J.Lu andL.W. Brady (Eds).
Radiation Oncology.An Evidence Base
Approach. Berlin:Springer(2008)357.
3. M.G. Noordhuis,J.J. Eijsinkand K.A.T.
Hoor,et al.,Clin. Cancer. Res.15(2009)7389.
4. J.M.D. Campo andP.A. Moreno, et al.,
Gynecol.Oncol. 110(2008)S72.
5. P.Yoon,N. Giafisand J. Smith, et al., Mol.
Cancer.Ther. 5(2006)2815.
6. H.B.J. Jefferies, C. Reinhardand S.C. Kozm,
et al., Proc. Natl. Acad. Sci. USA 91(1994)
4441.
7. N. Pullen andG. Thomas
,FEBS.Lett.410(1997)78.
8. D. Rohiniand B.Alakananda,Int. Jornal
Oncology 32 (2008)1133.
9. A.R. TeeandC.G. Proud,Oncogene 19
(2000)3021.
10. A.R. TeeandC.G. Proud, Cell Death Differ.8
(2001) 841.
11. C. Belka,V. Jendrossek and M. Pruschy, et
al.,Int. J.Radiat.Oncol.
Biol.Phys58(2004)542.
12. D.Trere, Micron31(2000)127.
13. M. Matsumoto , K. Komiyama and M.
Okaue, et al., J. Oral. Sci. 41(1999)53.
17.
18.
19.
20.
1.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
I.R. Kill, J. Cell Sci. 109(1996)1253.
P.A. Hall, D. Meek and D.P. Lane,
J.Pathol. 180(1996)1.
W.A.S. E. Deiry,J.W. Harper
andP.M.O.Connor , Cancer
Res.54(1994)1169.
I. Kurnia,S. Budiningsih and S.
Setiawan,et al.,Indonesian Journal of
Nuclear Science and Technology 14(1)
(2013)51. In Indonesian
I. Kurnia, S. Budiningsihand S.
Setiawan,et al., (Submitted To Hayati
Journal of Biosciences,Manucsript Number
998).
A.D.Gonzales,C.L. Granieland A.G.
Ensico, et al.,Annals of
Oncology13(2002)1212.
D. Ploton, M. Menager and P. Jeannesson,
et al., Histochem J.18 (1986)5.
I. Kurnia,Y. Suzuki and S. Budiningsih,et
al., Austral - Asian Journal of Cancer8(2)
(2009)93.
M. Lioret, P.C. Lara and E.Bordon, et
al.,GynecolOncol106 (2007)8.
V. Sirri,R. Pascal and V.D. Hernandes,
Micron 31(2000)121.
E. Gabele, R. Shimon and T. Shigheki, et
al., The Journal Of Biological Chemistry
280(14)(2005)13374.
S. Fikret, K. Rajesh andA. Onikepe, et al.,
Clinical Cancer Research10(2004)8421.
L.E. Horton, B. MartinandB.B. Diane, et al.,
Oncogene21 (2002) 5325.
X. Wan and L.J. Helman, Neoplasia4
(2002) 400.
D.J. Price, J.R. Grove, V. Calvo, et al.,
Science 257(1992) 973.
R. Dar andA.Basu, Int. Journal of Oncology
32 (2008) 1133.
C.C. Benz andC. Yau, Nat. Rev.
Cancer8(11) (2008)875.
G. Xu, W. Zhang and P. Bertram , et al.,
Int. J Oncol.24 (2004)893.
J.A.V.D. Hage, L.D.V.J.Broekand C.
Legrand, Br. J. Cancer 90 (2004)1543.
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If you find difficulties in placing figures or table in the text above, please insert them in this page!
Fig 1. Expression of p70s6k in cytoplasm (a) and nucleus (b) in cervical cancer before treatment with radiotherapy
(originalmagnification 10 x 40).
Table 1.AgNORmean, MIB-1 index and p53 index in different expression of p70s6k
Biomarker
AgNOR Mean
MIB-1 Index
P53 Index
Low Expression p70s6k
6.01±1,23
0,39±0.10
0.34±0.13
High Expression p70s6k
5,33±0,94
0.47±0,13
0.46±0.16
p value
0.17ns
0,16ns
0.11ns
ns:not significant (p>0.05)
Fig 2. AgNOR mean (a), MIB-1 indexs(b) and p53 indexs (c) in different expression of p70s6k in cervical cancer before treatment
with radiotherapy.
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Fig 3. Expression of p70s6k in different radiotherapy response.