Akt–the Mammalian Target of Rapamycin (mTOR) Pathway Inhibition Increases Cervical Cancer Cell Chemosensitivity to Active Form of Irinotecan (SN-38) | Septiani | International Journal of Integrated Health Sciences 103 352 1 PB

Original Aricle
Akt–the Mammalian Target of Rapamycin (mTOR) Pathway Inhibiion Increases
Cervical Cancer Cell Chemosensiivity to Acive Form of Irinotecan (SN-38)
Leri Sepiani,1 Yudi Mulyana Hidayat,1 Yusuf Sulaeman Efendi,1 Tono Djuwantono,1 Dimas Erlangga
Lutimas,2 Ahmad Faried2
Department of Obstetrics and Gynecology, Faculty of Medicine, Universitas Padjadjaran-Dr. Hasan Sadikin
General Hospital
2
Oncology and Stem Cell Working Group, Health Research Unit, Faculty of Medicine, Universitas PadjadjaranDr. Hasan Sadikin General Hospital

1

Abstract

Objecive: To invesigate the molecular pathway of the cytotoxic efect of SN-38
in human cervical cancer cell lines.
Methods: Two human cervical cancer cell lines were treated with various
concentraions of irinotecan for 24–72 hours and the sensiivity was analysed
using the MTT assay. Apoptosis was further observed through microscopic
examinaions. The protein expression was determined using Western blot
analysis.

Results: CaSki cells demonstrated the highest sensiivity to SN-38, whereas
HeLa cells showed the lowest. In cervical cancer cells, SN-38 induced apoptosis
through an intrinsic- and extrinsic-pathways. In addiion, we showed that SN-38
downregulated the phosphorylaion of Akt-mTOR pathways in CaSki cells, but not
in HeLa cells. Interesingly, in HeLa cells, which were more suggesive of a resistant
phenotype, pre-treatment with LY294002 and rapamycin inhibited acivaion of
Akt-mTOR signaling and signiicantly enhanced the sensiivity of HeLa cells to SN38.

Received:
October 31, 2012
Revised:
February 1, 2013
Accepted:
February 24, 2013

Conclusions: Irinotecan exerts its ani-neoplasic efects on cervical cancer
cells by inducing apoptosis through caspase-cascade. Inhibiion of Akt-mTOR,
LY294002 and rapamycin, which is targeted to Akt-mTOR pathways, may sensiize
irinotecan-resistant cervical cancer cells.
Keywords: Akt-mTOR pathways ani-neoplasic drugs, cervix cancer cells,

LY294002, rapamycin
IJIHS. 2013;1(1):13–21

Introducion
Carcinoma of the cervix is the second most
common neoplasm in Indonesian women and
the second most common neoplasm leading
to female cancer mortality.1 Furthermore, it is
also the second most frequently found cancer
among women in reproducive age between 15–
44 years old.2 It consitutes almost 15,000 new
cases and 7,500 deaths per annum.2
Cervical cancer treatment depends on the
Fédéraion Internaionale de Gynécologie et
d’Obstétrique (FIGO) staging. Early stadium of
Correspondence:
Leri Sepiani, Department of Obstetrics and Gynecology,
Faculty of Medicine, Universitas Padjadjaran-Dr. Hasan
Sadikin General Hospital
Jl. Pasteur No. 38, Bandung, Indonesia

e-mail: lsfaried@gmail.com

cervical cancer is generally treated with radical
operaions but up to now there is no ixed
standard therapy for FIGO stadium Ib-IIa of
cervical cancer. Cancer within these stadiums can
be treated with radical operaion, radiotherapy,
surgery-radiotherapy combinaion, surgeryanicancer drug combinaion, or chemotherapy
although these combinaion therapy protocols
may vary. The administraion of the combinaion
therapy for each paient is based on many factors
including age, general condiion, and paient’s
preference to achieve maximum therapy target
with minimum complicaions.
The standard surgery for cervical cancer in
stadium Ib and IIa is radical hysterectomy with
pelvic lymphadenectomy. The beneit of surgical
therapy is the ability to extract primary tumor
and apply staging accurately which leads to the

Internaional Journal of Integrated Health Sciences. 2013;1(1):13–21

13

Akt–the Mammalian Target of Rapamycin (mTOR) Pathways Inhibiion Increases Cervix Cancer Cell
Chemosensiivity to Acive form of Irinotecan (SN-38)

selecion of the most appropriate type of adjuvant
therapy for the paient. For stadium Ib2 and IIa,
neoadjuvant chemotherapy is one of several
adjuvant therapy alternaives applied. The goal
of this neoadjuvant chemotherapy is to eradicate
micrometastasis and reduce primary tumor
volume so the next treatment efeciveness can
be improved both for surgery and radiotherapy.
In addiion to radiotherapy, chemotherapy is
the standard treatment for advanced cervical
cancers. However, the systemic therapy protocol
for this kind of cervical cancer keeps changing. In
the past, chemotherapy was used for recurrent

and persistent cervical cancer, or cervical cancers
with metastasis. Currently, chemotherapy is the
primary treatment for cervical cancers with a
high risk of reccurrency.
The best possible treatment of locally
advanced cervical cancer is a combinaion of
radiaion and cisplain-based chemotherapy.
Cisplain-based chemotherapy as the main
regimen is sill the primary choice for advanced,
metastaic, and recurrent cervical cancers. Even
with this regimen, the response rate of advanced
stadium paients was only 23% with limited
response ime.3 From this data, it is suggested
that convenional treatment methods have
reached a plateau and, therefore, to overcome
this problem, inding a good prognosic factor
and predictor response to chemotherapy might
be useful.
Irinotecan (CPT-11) and its analog have
given promising results on many clinical trials.

SN-38, which is a semi-syntheic derivaive
of camptothecin, is the acive metabolite of
Irinotecan. Like other camptothecin-derivaives,
SN-38 inhibits topoisomerase I (Topo I), a
nuclear enzyme needed for replicaion and
transcripion by unwinding supercoiled DNA.4
SN-38 interferes with Topo I acivity by trapping
Topo I-DNA cleavage complexes, leading to lethal
replicaion-mediated double strand breaks and
ended with cell death.4 Currently, irinotecan
is used as a standard therapy for colon cancer
but not for cervical cancer. Irinotecan speciicity
against cervical cancer needs to be studied.
Chemotherapeuic agents kill cancer cells
through interacion with several disinct intracellular targets, including factors afecing
cells cycles, apoptosis cell death, and survival
pathways. Class I phosphaidylinositol 3-kinase
(PI3K) and its downstream efectors, such as
Akt and mTOR, have emerged as a prominent
proliferaion and survival pathway. Acivaion

of the PI3K/Akt-mTOR pathway has been
implicated in many human cancers and shown to
be associated with chemoresistance.5 We have
previously shown that advanced cervical cancer
14

expressed Akt and mTOR and the expression
of p-mTOR could predict the response to
neoadjuvants and the survival of the paients
treated with cisplain-based neoadjuvant
chemotherapy.6,7 A therapeuic strategy for
selecively modulaing the expression of a gene
of interest is the use of inhibitor agents of Akt
such as LY294002 and inhibitor agents of mTOR
such as rapamycin. We assessed whether the
acive form of irinotecan treatment enhanced
the inhibiion of proliferaion signals, in this case
the Akt-mTOR pathway, which led to increase of
cell death.

Methods
Cell lines and culture condiions
Two human cervical cancer cell lines, the HeLa
cell line which is an adenoma cell carcinoma with
human papilloma virus or HPV-18 (+) and CaSki
cell line which is a squamous cell carcinoma with
HPV-16 (+), were purchased from the American
Type Culture Collecion (ATCC) (Manassas, VA).
HeLa cells were maintained in Eagle’s Minimum
Essenial Medium (EMEM) obtained from Sigma
Chemical Co. (St. Louis, MO), supplemented with
2 mM L-glutamine, 1.0 mM sodium pyruvate, and
10% heat-inacivated fetal bovine serum (FBS)
obtained from Gibco (BRL, Grand Island, NY).
The CaSki cells were maintained in Rosewell Park
Memorial Insitute-1640 (RPMI-1640) medium
(Gibco) supplemented with HEPES (Sigma) and
10% heat-inacivated FBS.
Drugs and cell treatment
Irinotecan was purchased from Wako Pure

Chemicals (Osaka, Japan) and received as sterile
lyophilized powder. A stock soluion of 5 mg/
mL was made in dimethylsulfoxide (DMSO) and
stored at 4 °C. Further diluions were made in
culture media to reach the desired concentraions
when the cells obtained approximately 80%
conluency. The inal concentraion of DMSO did
not exceed 0.08%, a concentraion that does not
alter the growth or survival properies of any cell
type. We used CPT-11 and SN-38 stock of 3 μM
peak plasma concentraion (ppc) with a range of
9.8 ng/mL–5 μM. LY294002 and rapamycin were
purchased from Calbiochem (San Diego, CA). For
LY294002 (25 μM) or rapamycin (100 nm), cells
were pre-incubated with these inhibitors for 6
hours (h) prior to irinotecan treatment.
Drug sensiivity assay
Cell proliferaion analysis was performed on
human cervical cancer cell lines in the presence of
various concentraions of drugs by a colorimetric

Internaional Journal of Integrated Health Sciences. 2013;1(1):13–21

Leri Sepiani, Yudi Mulyana Hidayat, et al.

methyl thiazolyl tetrazolium (MTT) assay. Briely,
exponenially growing cells (2x104 cells/well)
were plated in 96-well plates. Ater an overnight
culture, the medium was subsituted by a fresh
medium with diferent concentraions of drugs.
At the end of various treatments, 10 μL of a cellcouning soluion (WST-8, Dojindo Labs, Tokyo,
Japan) was added. Ater dissolving the crystals
with 1 N HCl-isopropanol, the absorbance was
measured at a wavelength of 450 nm using a
microiter plate reader (Beckton Dickinson,
Franklin Lakes, NJ). A value of 100% was assigned
to untreated control and the concentraion of
drug that reduced the number of viable cells to
50% ater 24 h of exposure (CPI50) was derived
from cell survival plots. All experiments were

performed in triplicate.
Light microscopy examinaion
All cells were cultured in a 6-cm plate and
treated with drugs as described previously.
Morphological changes were examined at the
imes indicated and photographed using a
regular phase contrast microscope.
Western blot analysis
Western blot analysis was applied as described.5
Cells were harvested at the ime indicated and
prepared in a bufer (20 mM Tris–HCl, pH 7.6, 1

mM EDTA, 140 mM NaCl, 1% Nonidet P-40, 1%
aproinin, 1 mM phenylmethylsulfonylluoride,
and 1 mM sodium vanadate). The concentraion
of the sample protein was determined by
bicinchoninic acid (BCA) protein assay (Pierce,
Rockford, IL). Equal amounts of protein (40 μg)
were subjected to a 5–20% Tris-tricine Ready Gel
(Bio-Rad, Tokyo, Japan) and then transferred to
nitrocellulose membrane (Amersham Pharmacia
Biotech, Buckinghamshire, UK). Membrane was
blocked with Tris-bufered saline/0.1% Tween
20 with 5% nonfat dry milk and incubated in
primary anibodies against caspase-3, caspase-8,
caspase-9 and PARP (Santa Cruz); phosphorylated
Akt (p-Akt, Ser473), phosphorylated mTOR
(p-mTOR, ser2448) and phosphorylated p70 S6
Kinase (pS6K1,Thr389) from Cell Signaling at
4 °C overnight. The bands were visualised by a
chemiluminescence system (Amersham). The
expression of beta-acin (Sigma) served as a
loading control. The density of the bands was
quaniied using Quanity One (BioRad).
Staisical analysis
Quanitaive experiments were analyzed using
Student’s t test. The P value was gained from the
two-sided tests and were considered signiicant
when p