SN-38-induced apoptosis in gastric cancer

A

B

Control
Control

SN-38
20 nM

SN-38

SN-38

250 nM

250 nM

Fig. 3. Inﬂuence of SN-38 to MKN1 cells.
A: Effect of 20 nM or 250 nM SN-38 on the cell cycle progression of MKN1 cells after 48 h incubation determined by ﬂow cytometry.
B: Fluorescence microscopy dyeing, Hoechst 33342 after 250 nM SN-38 treatment for 48 h.

PI3K inhibitor reduced the expression levelof pAkt and induced apoptosis in MKN1 cells

Sub-G0G1 population

45

Through SN-38 treatment, pAkt expression in
MKN45 cells was reduced but not in MKN1 cells
while MKN45 cells have a higher sensitivity to
SN-38 than MKN1 cells. To elucidate that thePI3K-Akt pathway is involved in SN-38-induced
apoptosis, we examined whether LY294002 could
affect SN-38-induced apoptosis in MKN1 cells.
LY294002 is a speciﬁc PI3K inhibitor that has been
widely used to study the role of PI3K in various
biological responses.
The expression level of pAkt in MKN1 cells
reduced in an SN-38 dose-dependent manner after

40
35

(%)

MKN45
MKN1

*

*

30
25
20
15
10
5
0

0 nM

20 nM

SN-38

250 nM

Fig. 4. Sub-G0G1 population in MKN45 and MKN1
cells treated with 0, 20 or 250 nM SN-38 for 48 h.
*P < 0.05 using Studentʼs t-test.

11

S. Yoshioka et al.

MKN45
SN-38

0

5

10

MKN1

20

100

250

0

5

10

20

100

250

(nM)

pAKT
PTEN
β-actinFig. 5. Western blotting of pAkt and PTEN expression after 0, 5, 10, 20, 100 or 250 nM SN-38 treatment for 48 h.

pretreatment with 50 µM LY294002 (Fig. 6). We
also examined PTEN expression using Western
blotting, but LY294002 had no inﬂuence on PTEN
expression (Fig. 6).
Moreover, by cell cycle analysis, the apoptotic
sub-G1 fraction appeared by ﬂow cytometry (Fig.
7A). By morphological examination, apoptotic
cells were increased by treatment with SN-38 andLY294002 (Fig. 7B). The apoptotic sub-G1 fraction of the population of MKN1 cells was 35.9%
48 h after treatment with 250nM SN-38 and 50 µM
LY294002 (Fig. 8). Exposure to 50 µM LY294002
increased SN-38-induced apoptosis in MKN1 cells
to as much as that in MKN45 cells after treatment
with only SN-38 (Fig. 8). These results indicatedthat the PI3K-Akt pathway plays a signiﬁcant rolein SN-38-induced apoptosis in human gastric cancer cells.
LY294002
SN-38

–
–

+
–

+

+

20

250 (nM)

Discussion
The MKN45 cells were more sensitive to SN-38
than the MKN1 cells. The difference of sensitivity mainly comes from the percentage of apoptotic
cells. The pattern of tumor response after in vivo
topotecan treatment was reported to correlate withthe ability of the drug to induce apoptosis but notwith its in vitro antiproliferative activity (Caserini
et al., 1997). Apoptosis is a major mode of cell
death in response to chemotherapeutic drug treatment (Hickman, 1992; Kerr et al., 1994), andresistance to apoptosis induction has been proposed as a critical mechanism in drug resistance(Kataoka et al., 1994; Chen et al., 1996; Seimiya
et al., 1997). However, the mechanisms by which
chemotherapeutic agents induce apoptosis are notfully understood. The sensitivity of cells to chemotherapeutic drug-induced apoptosis appears to
depend on the balance between proapoptotic andantiapoptotic signals. Therefore, it is possible thata chemotherapeutic drug may induce apoptosis not
only by increasing the proapoptotic signals but also
by decreasing the antiapoptotic ones.
The PI3K-Akt signaling pathway is known totransmit survival signals and to protect a varietyof cells from apoptosis. Akt plays a critical role incontrolling the balance between cell survival andapoptosis (Balendran et al., 1999). Phosphorylation of Akt is promoted by phosphatidylinositides
converted by PI3K products. Previous reports have

pAKT
PTEN
β-actinFig. 6. Western blot of pAkt and PTEN expression.
MKN1 cells were treated with 0, 20 or 250 nM SN-38
for 48 h in the presence of 50 µM LY294002.

12

SN-38-induced apoptosis in gastric cancer

B

A

DMSO

LY294002 50 µM
LY294002 50 µM

LY294002 50 µM+
SN-38 20 nM

LY294002 50 µM+
SN-38 250 nM

Fig. 7. Inﬂuence of LY294002 to MKN1cells.
A: Cell cycle analysis by ﬂow cytometry.
B: Mor phologica l detection of apoptosis by
Hoechst 33342 staining.

LY294002 50 µM + SN-38 250 nM
Cell line/LY294002

shown that Akt delivers antiapoptotic survival
signals by phosphorylating Bad and activating caspase-9 (Cardone et al., 1998; Brunet et al., 1999).
In addition, inhibition of Akt signaling can induce
apoptosis in some human cancer cell lines (Pageet al., 2000; Yuan et al., 2000). It has also beenreported that Akt plays an important role in cell
survival when cells are exposed to different apoptotic stimuli, such as growth factor withdrawal,
ultraviolet radiation, matrix detachment, cell cycle
discordance and DNA damage (Hemmings, 1997;
Kulik et al., 1997; Kulik and Weber, 1998; Ng etal., 2000; Brognard et al., 2001). Moreover, inactivation of PI3K using a specific inhibitor has
led to dephosphorylation of Akt at Ser473, consequently causing translocation of Akt to nuclei,

Sub-G0G1 population

45
40

(%)

MKN45/ 0 M
MKN 1/ 0 M
MKN 1/50 M

*

*

35
30
25

*

20
15
10
5
0

20 nM

250 nM

SN-38
Fig. 8. Sub-G0G1 population in MKN45 and MKN1
cells treated with 0, 20 or 250 nM SN-38 in the presenceor absence of LY294002 for 48 h. *P < 0.05 using Studentʼs t-test.

13

S. Yoshioka et al.
from PIP 3, which is a primary activator of Akt(Maehata and Dixon, 1998). Loss of PTEN resultsin high basal activity of Akt in a variety of tumors,while introduction of PTEN suppresses the activity.
It is reported (Saga et al., 2002) that overexpressionof PTEN increased sensitivity to SN-38 in ovariancancer cells. However, in our study, the expressionof PTEN was not correlated with SN-38-induced
apoptosis; in human gastric cancer cells, SN-38
might suppress the PI3K-Akt signaling via a PTEN
independent pathway. Rokudai et al. (2002) reported that suppression of PDK1, but not of PI3K,
was one of the mechanisms of chemotherapeutic
drug-mediated Akt inactivation, because VP-16
indirectly suppressed PDK1 kinase activity in 293T
cells. However, the mechanisms of SN-38-mediated Akt inactivation remain unclear. Overexpression of constitutive active Akt using a transfected
active form of akt cDNA overcame chemotherapeutic drug-induced apoptosis. Akt inactivation might
be a sign of chemotherapeutic drug sensitivity ofcancer cells, because it was found only in chemosensitive cells (MKN45).
Nuclear factor-κB (NF-κB) is a ubiquitously
expressed transcription factor involved in a wide
spectrum of cellular functions like cell cycle control, stress adaptation, inﬂammation and control ofapoptosis (Pahl, 1999). NF-κB is constitutively activated in various types of tumors, including gastriccancer (Sasaki et al., 2001). Several investigators
have reported that inhibition of the NF-κB pathway
by adenoviral delivery of an IκBα superrepressor,
small interfering RNA, against the p65 subunit of
NF-κB or TNFα increased SN-38-induced apoptosis (Camp et al., 2004; Guo et al., 2004; Valente etal., 2003). Besides the PI3K-Akt pathway, the NFκB pathway might also facilitate an antiapoptotic
signal to inhibit SN-38-induced apoptosis.
In conclusion, we found that SN-38 induced
apoptosis by decreasing the PI3K-Akt survival signaling, the antiapoptotic signals in human gastriccancer cells. A selective small molecule inhibitorof Akt, the Akt/protein kinase B signaling inhibitor-2 (API-2) was recently discovered by screening
the National Cancer Institute Diversity Set (Yang etal., 2004). Such an Akt inhibitor might be a useful

where it is believed to regulate the transcription ofgenes mediating cell survival or apoptosis (Hemmings, 1997). In a previous study, TRAIL and Fas
induced apoptosis by inhibition of the PI3K-Akt
pathway in human gastric cancer cells (Nam et al.,
2003; Osaki et al., 2004). Our study showed that
apoptosis was induced in cells where pAkt expression was reduced (MKN45), but not in cells wherepAkt expression showed no change (MKN1) by
SN-38. It has been reported that exposure to camptothecin induced a dose-dependent trapping in theG2/M phase affects and dose-dependent delay in
the S phase. Treatment of MKN1 cells with 48 h
20 nM SN-38 induced G2/M phase arrest, and 48
h 250 nM SN-38 treatment induced S phase delay.
Meanwhile in MKN45 cells, 48 h 20 nM SN-38
treatment induced G2/M phase arrest tendency
and 48 h 250 nM SN-38 treatment induced sub-G1
peak. We speculate that low dose SN-38 treatment
below 20 nM induces the G2/M arrest in MKN45
cells and 20 nM SN-38 treatment begins to induce
S phase cells. Moreover, in MKN45 cells, 250 nM
SN-38 treatment induces apoptosis by reducing
pAkt expression but MKN1 cells showed resistancefor apoptosis by no change in pAkt expression.
Therefore, inactivation of pAkt using the PI3K
inhibitor, LY294002, finally induced apoptosis
in MKN1 cells as well as in only SN-38-treated
MKN45 cells. It was also reported (Nakashio etal., 2000) that constitutively active Akt results ina reduction of the cytotoxic effect of topotecan
in lung carcinoma cells. The PI3K-Akt pathway
might thus play a signiﬁcant role in SN-38-induced
apoptosis in human gastric cancer cells.
The tumor suppression gene PTEN is a natural biological inhibitor of Akt cell survival signals.
PTEN expression sensitizes cells to death receptormediated apoptosis induced by a variety of stimuli,
such as tumor necrosis factor (Mayo et al., 2002),
TRAIL (Thakkar et al., 2001) and chemotherapeutic agents (Tanaka et al., 2000). Akt is abnormally
activated in several human tumors by loss of PTEN
(Di Cristofano and Pandolfi, 2000; Simpson andParsons, 2001). Recent studies have documented
that PTEN appears to negatively regulate PI3K-Akt
survival signaling by removing the D-3 phosphate14SN-38-induced apoptosis in gastric cancer

anti-tumor agent in combination with a chemotherapeutic agent, such as CPT-11.

9 Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y,
et al. Akt phosphorylation of BAD couples survival
signals to the cell-intrinsic death machinery. Cell
1997;91:231–241.
10 Di Cristofano A, Pandolﬁ PP. The multiple roles ofPTEN in tumor suppression. Cell 2000;100:387–390.
11 Editorial Board of the Cancer Statistics in Japan,
The. Cancer statistics in Japan 2003. Tokyo: Foundation for Promotion of Cancer Research; 2003.
12 Falk SJ, Smith PJ. DNA damaging and cell cycle
effects of the topoisomerase I poison camptothecin in irradiated human cells. Int J Radiat Biol
1992;61:749–757.
13 Franke TF, Kaplan DR, Cantley LC. PI3K: downstream AKTion blocks apoptosis. Cell 1997;88:435–
437.
14 Guo J, Verma UN, Gaynor RB, Frenkel EP, Becerra
CR. Enhanced chemosensitivity to irinotecan by
RNA interference-mediated down-regulation of thenuclear factor-kappaB p65 subunit. Clin Cancer
Res 2004;10:3333–3341.
15 Hemmings BA. Akt signaling: linking membrane events to life and death decisions. Science
1997;275:628–630.
16 Hickman JA. Apoptosis induced by anticancer
drugs. Cancer Metastasis Rev 1992;11:121–139.
17 Kataoka S, Naito M, Tomida A, Tsuruo T. Resistance to antitumor agentinduced apoptosis in a mutant of human myeloid leukemia U937 cells. Exp
Cell Res 1994;215:199–205.
18 Kawato Y, Aonuma M, Hirota Y, Kuga H, Sato
K. Intracelular roles of SN-38, a metabolite of thecamptothecin derivative CPT-11, in the antitumor
effect of CPT-11. Cancer Res 1991;51:4187–4191.
19 Kerr JF, Winterford CM, Harmon BV. Apoptosis.
Its signiﬁcance in cancer and cancer therapy. Cancer 1994;73:2013–2026.
20 Kulik G, Klippel A, Weber MJ. Antiapoptotic signalling by the insulin-like growth factor I receptor,
phosphatidylinositol 3-kinase, and Akt. Mol Cell
Biol 1997;17:1595–1606.
21 Kulik G, Weber MJ. Akt-dependent and -independent survival signaling pathways utilized by insulinlike growth factor I. Mol Cell Biol 1998;18:6711–
6718.
22 Le Good JA, Ziegler WH, Parekh DB, Alessi DR,
Cohen P, Parker PJ. Protein kinase C isotypes controlled by phosphoinositide 3-kinase through theprotein kinase PDK1. Science 1998;281:2042–2045.
23 Maehama T, Dixon JE. The tumor suppressor,
PTEN/MMAC1, dephosphorylates the lipid second
messenger, phosphatidylinositol 3,4,5-trisphosphate.
J Biol Chem 1998;273:13375–13378.

Acknowledgments: We thank Dr. Mitsuhiko Osaki (Division of Molecular Genetics and Biofunction, Tottori
University) for his excellent technical advice.
This work was supported in part by a Grant-in-Aid
for Scientiﬁc Research from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan (Grant
number: 16790779).
References
1 Alessi DR, James SR, Downes CP, Holmes AB,
Gaffney PR, Reese CB, et al. Characterization of a3-phosphoinositide-dependent protein kinase which
phosphorylates and activates protein kinase Balpha.
Curr Biol 1997;7:261–269.
2 Balendran A, Casamayor A, Deak M, Paterson A,
Gaffney P, Currie R, et al. PDK1 acquires PDK2
activity in the presence of a synthetic peptide derived from the carboxyl terminus of PRK2. Curr
Biol 1999;9:393–404.
3 Brognard J, Clark AS, Ni Y, Dennis PA. Akt/protein
kinase B is constitutively active in non-small celllung cancer cells and promotes cellular survival andresistance to chemotherapy and radiation. Cancer
Res 2001;61:3986–3997.
4 Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P,
Hu LS, et al. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription
factor. Cell 1999;96:857–868.
5 Camp ER, Li J, Minnich DJ, Brank A, Moldawer
LL, MacKay SL, et al. Inducible nuclear factor-kappaB activation contributes to chemotherapy resistance in gastric cancer. J Am Coll Surg. 2004;199:
249–258.
6 Cardone MH, Roy N, Stennicke HR, Salvesen GS,
Franke TF, Stanbridge E, et al. Regulation of cell
death protease caspase-9 by phosphorylation. Science -1998;282:1318–1321.
7 Caserini C, Pratesi G, Tortoreto M, Bedogne B,
Carenini N, Supino R, et al. Apoptosis as a determinant of tumor sensitivity to topotecan in humanovarian tumors: preclinical in vitro/in vivo studies.
Clin Cancer Res 1997;3:955–961.
8 Chen Z, Naito M, Mashima T, Tsuruo T. Activation of actin-cleavable interleukin 1beta-converting
enzyme (ICE) family protease CPP-32 during chemotherapeutic agent-induced apoptosis in ovariancarcinoma cells. Cancer Res 1996;56:5224–5229.

15

S. Yoshioka et al.
35 Seimiya H, Mashima T, Toho M, Tsuruo T. c-Jun
N H2-ter m inal k inase-mediated activation ofinterleukin-1beta-converting enzyme/CED-3-likeprotease during anticancer drug-induced apoptosis.
J Biol Chem 1997;272:4631–4636.
36 Simpson L, Parsons R. PTEN: life as a tumor suppressor. Exp Cell Res 2001;264:29–41.
37 Stephens L, Anderson K, Stokoe D, ErdjumentBromage H, Painter GF, Holmes A et al. Protein
kinase B kinases that mediate phosphatidylinositol
3,4,5-trisphosphate-dependent activation of protein
kinase B. Science 1998;279:710–714.
38 Tanaka M, Koul D, Davies MA, Liebert M, Steck
PA, Grossman HB. MMAC1/PTEN inhibits cell
growth and induces chemosensitivity to doxorubicin in human bladder cancer cells. Oncogene
2000;19:5406–5412.
39 Thakkar H, Chen X, Tyan F, Gim S, Robinson H,
Lee C, et al. Pro-survival function of Akt/protein
kinase B in prostate cancer cells. Relationship with
TRAIL resistance. J Biol Chem 2001;276:38361–
38369.
40 Valente P, Arzani D, Cesario A, Margaritora S,
Carbone E, Russo P. TNF increases camptothecininduced apoptosis by inhibition of NF-kappaB. Eur
J Cancer. 2003;39:1468–1477.
41 Yang L, Dan HC, Sun M, Liu Q, Sun XM, Feldman
RI, et al. Akt/protein kinase B signaling inhibitor-2,
a selective small molecule inhibitor of Akt signaling
with antitumor activity in cancer cells overexpressing Akt. Cancer Res 2004;64:4394–4399.
42 Yoshida A, Ueda T, Wano Y, Nakamura T. DNA
damage and cell killing by camptothecin and its
derivative in human leukemia HL-60 cells. Jpn J
Cancer Res 1993;84:566–573.
43 Yuan ZQ, Sun M, Feldman RI, Wang G, Ma X,
Jiang C, et al. Frequent activation of AKT2 and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase/Akt pathway in human ovariancancer. Oncogene 2000;19:2324–2330,.
44 Xu Y, Villalona-Calero MA. Irinotecan: mechanism of tumor resistance and novel strategies formodulating its activity. Ann Oncol 2002;13:1841–1851.

24 Mayo MW, Madrid LV, Westerheide SD, Jones DR,
Yuan XJ, Baldwin AS Jr, et al. PTEN blocks tumor
necrosis factorinduced NF-kappa B-dependent transcription by inhibiting the transactivation potentialof the p65 subunit. J Biol Chem 2002;277:11116–
11125.
25 Nakashio A, Fujita N, Rokudai S, Sato S, Tsuruo
T. Prevention of phosphatidylinositol 3'-kinaseAkt survival signaling pathway during topotecaninduced apoptosis. Cancer Res 2000;60:5303–5309.
26 Nam SY, Jung GA, Hur GC, Chung HY, Kim WH,
Seol DW, et al. Upregulation of FLIP(S) by Akt, apossible inhibition mechanism of TRAIL-induced
apoptosis in human gastric cancers. Cancer Sci
2003;94:1066–1073.
27 Ng SSW, Tsao MS, Chow S, Hedley DW. Inhibition of phosphatidylinositide 3-kinase enhances
gemcitabine-induced apoptosis in human pancreaticcancer cells. Cancer Res 2000;60:5451–5455.
28 Osaki M, Kase S, Adachi K, Takeda A, Hashimoto
K, Ito H. Inhibition of the PI3K-Akt signaling pathway enhances the sensitivity of Fas-mediated apoptosis in human gastric carcinoma cell line, MKN45.
J Cancer Res Clin Oncol 2004;130:8–14.
29 Page C, Huang M, Jin X, Cho K, Lilja J, Reynolds
RK, et al. Elevated phosphorylation of AKT and
Stat3 in prostate, breast, and cervical cancer cells.
Int J Oncol 2000;17:23–28.
30 Pahl HL. Activators and target genes of Rel/ NFkappaB transcription factors. Oncogene 1999;18:
6853–6866.
31 Rokudai S, Fujita S, Kitahara O, Nakamura Y, Tsuruo T. Involvement of FKHR-dependent TRADD
expression in chemotherapeutic drug-induced apoptosis. Mol Cell Biol 2002;22:8695–8708.
32 Romashkova JA, Makarov SS. NF-kappaB is atarget of AKT in anti-apoptotic PDGF signalling.
Nature 1999;401:86–90
33 Saga Y, Mizukami H, Suzuki M, Kohno T, Urabe M,
Ozawa K, et al. Overexpression of PTEN increases
sensitivity to SN-38, an active metabolite of the
topoisomerase I inhibitor irinotecan, in ovarian cancer cells. Clin Cancer Res 2002;8:1248–1252.
34 Sasaki N, Morisaki T, Hashizume K, Yao T, Tsuneyoshi M, Noshiro H, et al. Nuclear factor- kappaB
p65 (RelA) transcription factor is constitutively
activated in human gastric carcinoma tissue. Clin
Cancer Res 2001;7:4136–4142.

Received November 12, 2004; accepted December 20, 2004
Corresponding author: Masahide Ikeguchi, MD

16