Yonago Acta medica 2005;48:7–16

The PI3K-Akt Pathway in SN-38-Induced Apoptosis in
Human Gastric Cancer Cell Lines
Shizue Yoshioka, Shigeru Tatebe, Naruo Tokuyasu, Kenjiro Taniguchi, Yoshimi
Ogami, Osamu Yamamoto, Keigo Ashida, Yoshihito Gomyo, Akira Kondo, Shunichi
Tsujitani and Masahide Ikeguchi
Division of Surgical Oncology, Department of Surgery, School of Medicine, Tottori University
Faculty of Medicine, Yonago 683-8504 Japan
SN-38, an active metabolite of a topoisomerase I inhibitor, CPT-11, exhibits a cytotoxic effect by inducing apoptosis in cancer cells. Phosphatidylinositol-3-OH kinase(PI3K)-Akt signaling is known to protect a variety of cells from apoptosis. The relationship between resistance to SN-38-induced apoptosis and the PI3K-Akt pathway inhuman gastric cancer cells is unknown. Here, we did an investigation using two gastriccancer cell lines, MKN1 and MKN45. Cell viability was determined by sodium 3'-[1(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonicacid hydrate (XTT) assay. Apoptosis was conﬁrmed by ﬂuorescence microscopy using
Hoechst 33342 staining. Expression levels of phospho-Akt (pAkt) were determined byWestern blotting. After being treated with SN-38, the populations of sub-G1 cells wereinduced by ﬂow cytometry in 36.8% of MKN45 cells more frequently than in 13.5% ofMKN1 cells. SN-38 inhibited the expression of pAkt dose-dependently in MKN45 cells,
but not in MKN1 cells. In MKN1 cells, an additional pretreatment with the PI3K inhibitor, LY294002, led to the inhibition of pAkt expression and induced apoptosis. The results suggested that SN-38 induces apoptosis by decreasing PI3K-Akt survival signaling,
the anti-apoptotic signals, in human gastric cancer cells. Akt inhibitor might be a usefulanti-tumor agent in combination with CPT-11.
Key words: apoptosis; gastric cancer; PI3K-Akt pathway; SN-38

Gastric cancer continues to be one of the most common malignancies in Japan. While the development
of diagnostic modalities and surgical techniques has
improved prognosis, the associated mortality is still
the second highest in Japan after lung cancer (Editorial Board of the Cancer Statistics in Japan, 2003).
This mortality is due to lack of effective treatment,
especially in respect to chemotherapy.

New anticancer agents have recently beenused in gastric cancer therapy. Camptothecins arebroad-spectrum anticancer drugs that specificallytarget DNA topoisomerase I (TopoI). The formation of a cleavable drug-TopoI-DNA complex
results in lethal double-strand DNA breakage
and cell death (Xu and Villalona-Calero, 2002).
CPT-11 is a new semisynthetic derivative of camp-Abbreviations: DMSO, dimethyl sulfoxide;ID80, dose giving 80% inhibition; NF-κB, nuclear factor-κB; pAkt,phospho-Akt; PI3K, phosphatidylinositol-3-OH kinase; PIP 2 , phosphatidylinositol-3,4-bisphosphate; PIP3, phosphatidylinositol-3,4,5-trisphosphate; PTEN, phosphatase and tensin homologue deleted on chromosome 10; TopoI,
topoisomerase I; XTT, sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro) benzene sulfonic acid hydrate

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S. Yoshioka et al.
Here, we analyzed SN-38-induced apoptosis
in human gastric cancer cell lines. Our data suggest that in some human gastric cancer cells, SN-38
induces apoptosis, which might be partly explained
by PI3K-Akt pathway inactivation.

tothecin that is ultimately converted into the active
metabolite SN-38 by carboxylesterase, which is atleast 100-fold more cytotoxic than CPT-11 (Rokudaiet al., 2002). It has also been reported that exposure to camptothecin derivatives induces apoptosis(Yoshida et al., 1993) and affects the cell cycle byinducing a dose-dependent delay in the S phasefollowed by dose-dependent trapping in the G2/M
phase (Falk and Smith, 1992). However, the precise
mechanisms of SN-38-induced apoptosis have notbeen elucidated.
The phosphatidylinositol-3-OH kinase (PI3K)Akt signaling pathway is known to transmit survival-promoting signals and to protect a variety ofcells from apoptosis (Franke et al., 1997). After
growth factor stimulation, PI3K is activated andgenerates phospholipid second-messenger molecules, phosphatidylinositol-3,4,5-trisphosphate(PIP3) and phosphatidylinositol-3,4-bisphosphate(PIP 2), which cause a diverse set of cellular responses. One target of PI3K is the serine/threonine
kinase Akt [also known as protein kinase B orRAC-PK (Balendran et al., 1999)]. With the generation of PIP3 and PIP 2 proteins by stimulation
with growth factors and cytokines, Akt is recruited
to the plasma membrane and is then phosphorylated at two key regulatory sites, Thr308 in the activation loop of the catalytic domain and Ser473 in
the COOH-terminal regulatory domain (Alessi etal., 1997). The phosphorylation of Akt at Thr308
is catalyzed by ubiquitously expressed PDK1(Datta et al., 1997; Le et al., 1998; Stephens et al.,1998). The kinase responsible for phosphorylation of Akt at Ser473 was reported to be PDK2
[PDK1 bound to a fragment of PRK2 (Alessi et al.,1997)]. Phosphorylation at both residues is necessary for full activation of Akt and the subsequent
regulation of many cellular processes. Activated
Akt phosphorylates proapoptotic Bcl-2 family
member Bad, caspase family member, caspase-9,
Forkhead family transcription factor, FKHRL1,and IκB kinase, leading to cell survival (Kawato etal., 1991; Cardone et al., 1998; Brunet et al., 1999;
Romashkova and Makarov, 1999). Thus, PI3K-Akt
signaling pathway inactivation might correlate withapoptosis.

Materials and Methods
Cell culture and reagents
MKN1, MKN28, MKN45, MKN74 and KATOIII cells were purchased from the Riken Cell Bankand grown in RPMI-1640 supplemented with 10%
fetal bovine serum, 100 units/mL of penicillin G
sodium, 100 µg/mL of streptomycin sulfate and0.25 µg/mL of amphotericin B equilibrated with
5% CO2 in air at 37˚C. SN-38 was obtained throughDaiichi Pharm (Tokyo, Japan), and LY294002 waspurchased from Sigma (St. Louis, MO). SN-38 andLY294002 were dissolved in dimethyl sulfoxide(DMSO). Dilutions with DMSO were made immediately before use to adjust the DMSO concentrationin the growth medium to 0.1% for all experiments.
Growth inhibition experiments
The effect of the SN-38 on the proliferation ofcells was evaluated using the sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy6-nitro) benzene sulfonic acid hydrate (XTT) assay(Cell Proliferation Kit II, XTT; Roche Molecular
Biochemicals, Mannheim, Germany). The assay
is based on the cleavage of the yellow tetrazolium
salt XTT to form an orange formazan dye by metabolically active cells. Brieﬂy, cells were seeded into
96-well plates at a density of 2 × 103 viable cells perwell in 100 µL medium. After 24 h incubation, adifferent dose of SN-38 (0–500 nM) was added toquadruplicate wells. Then after 72 h incubation, 50µL of the XTT was added to each well (ﬁnal concentration 0.3 mg/mL), followed by further incubation for 4 h in a humidiﬁed atmosphere containing
5% CO2 at 37˚C. The absorbance of the samples
was measured at 490 nm using a microplate reader(Wellreader; Seikagaku Corporation, Tokyo, Japan).

8SN-38-induced apoptosis in gastric cancer

poly vinilidene difluoride Immobilon membrane.
The antibodies used in this study were anti-phospho
Akt (Ser473) polyclonal antibody (#9271, Cell Signaling Technology, Beverly, MA), anti-phosphataseand tensin homologue deleted on chromosome 10(PTEN) polyclonal antibody (#21940, Upstate Cell
Signaling Solutions, Charlottesville, VA) and antiβ-actin monoclonal antibody (AC-15, Sigma).

The dose giving 80% inhibition (ID80) was calculated in terms of the absorbency in treated cells
relative to the absorbency in untreated control cells.
Cell cycle analysis
Cell cycle distribution was analyzed by determining the DNA content and the sub-G1 peak corresponding to the apoptotic cells using ﬂow cytometry. MKN45 (3 × 106) and MKN1 (5 × 105) cells
were seeded on 75 cm2 tissue culture dishes andallowed to grow overnight at 37˚C with 5% CO2.
Cells were exposed to 50 µM LY294002 for 1 h,or not exposed. Cells were next exposed to 20 nM
or 250 nM SN-38 for 48 h, then collected, resuspended in 70% ethanol and stored at –20˚C until
use. After ﬁxation, cells were incubated for 20 minat 37˚C with RNase A (50 µg/mL) and then for 10min at room temperature with propidium iodide (50µg/mL). After samples were ﬁltered through 35µm nylon mesh, stained cells were analyzed withan FACS Calibur Cytometer (Becton Dickinson,Franklin Lakes, NJ).

Results
The effect of SN-38 on cell viability andapoptosis in MKN1 and MKN45 cells
We ﬁrst examined the susceptibility of the two human gastric cancer cell lines, MKN1 and MKN45,
to SN-38 treatment. Figure 1 shows the dosedependent cytotoxic effect of SN-38 against MKN1and MKN45 cells using an XTT cell proliferation
assay kit. The ID80s of MKN45 and MKN1 cells
were 20 nM and 250 nM, respectively. We also examined the susceptibility of three other human gastric cancer cell lines, MKN74, MKN28 and KATOIII cells. ID80s of MKN74, MKN28 and KATO-IIIcells were 15 nM, 200 nM and 30 nM, respectively.
MKN45, MKN74 and KATO-III cells showed highsensitivity to SN-38 (ID80 < 50 nM), and MKN1
and MKN28 cells showed low sensitivity to SN-38(ID 80 > 100 nM). Then we used MKN45 andDetection of apoptotic cells
Apoptosis was detected morphologically. Trypsinized adherent and ﬂoating cells were washed withphosphate buffered saline and pelleted. After ﬁxation with 3.5% formalin solution, apoptotic cells
were assessed morphologically by staining with
Hoechist 33342, using ultraviolet laser microscope(Optiphoto-2, Nikon, Tokyo).

(%)
10080

Cell survivalWestern blot analysis
MKN45 and MKN1 cells were treated with LY294002
and/or SN-38 as in the case for cell cycle analysis. The cells were lysed in a sample buffer [TrisHCl (pH 6.8), SDS, glycerol, bromophenol blue]
containing protease inhibitor (complete, mini;
Roche Molecular Biochemicals). Following sonication, the total protein was quantiﬁed by a BCA
method (BCA Protein Assay Kit; Pierce, Rockford,IL). Each boiling sample (60 µg protein/lane) wasseparated by 10% SDS-PAGE and blotted onto aMKN1MKN4560402000100200300400500 (nM)

SN-38

Fig. 1. XTT assay of MKN1 and MKN45 cells treatedwith SN-38. The rates of MKN1 cells are shown by opencircles, and those of MKN45 cells, by closed circles.

9S. Yoshioka et al.

AB

Control
Control

SN-38
20 nM

SN-38

SN-38

250 nM

250 nM

Fig. 2. Apoptosis of MKN45 cells by SN-38.
A: Flow cytometry of MKN45 cells treated with 20 nM or 250 nM SN-38 for 48 h.
B: Fluorescent microscopy of MKN45 cells treated with 250 nM SN-38 for 48 h, and stained with Hoechst
33342 ﬂuorescent dye.

the apoptotic sub-G1 fractions of the population of
MKN45 and MKN1 cells 48 h after treatment with
250 nM SN-38 were 36.8 and13.5%, respectively.

MKN1 cells as the representative cell lines for thefollowing studies.
Then, we examined the effect on the cell cycle
in MKN1 and MKN45 cells after SN-38 treatment using ﬂow cytometry. An evident apoptotic
sub-G1 fraction was shown in MKN45 cells 48
h after treatment with 250 nM SN-38 (Fig. 2A).
We further confirmed SN-38-induced apoptosisof MKN45 cells morphologically by fluorescencemicroscopy with a Hoechst 33342: 250 nM SN-38
induced apoptosis in MKN45 cells, in which there
were condensed and fragmented nuclei (Fig. 2B).
However, MKN1 cells treated with 250 nM SN-38
suffered delays in the S phase (Fig. 3A) and had afew apoptotic cells (Fig. 3B). As shown in Fig. 4,

pAkt and PTEN expression after treatment
with SN-38 in gastric cancer cell lines
Next, we examined pAkt and PTEN expression
after treatment with SN-38 by Western blotting. InMKN45 cells, the expression levels of pAkt werereduced in a dose-dependent manner by SN-38(Fig. 5). On the other hand, the expression levels ofpAkt were not affected by SN-38 in MKN1 cells.
The expression levels of PTEN were not affected ineither cell line (Fig. 5).

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