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Induction of apoptosis in SF21 cell line by conditioned medium of the entomopathogenic fungus Nomuraea rileyi through Sf-caspase-1 signaling pathway.

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Tseng et al.
Archives of Insect Biochemistry and Physiology 68:206–214 (2008)
Induction of Apoptosis in SF21 Cell Line by
Conditioned Medium of the Entomopathogenic
Fungus, Nomuraea rileyi, Through Sf-Caspase-1
Signaling Pathway
Yu-Kai Tseng, Meng-Shiue Wu, and Roger F. Hou*
The apoptosis in SF-21 cell line can be induced by the conditioned medium (CM) of the entomopathogenic fungus, Nomuraea
rileyi, based on changes in morphology and formation of apoptotic bodies in cultured cells, and with the onset of DNA
fragmentation as shown by TUNEL staining and agarose electrophoresis. Moreover, the induction of apoptosis in SF-21 cells
was inhibited by adding the inhibitor of effector caspase, viz. z-DEVD-fmk, to the CM, indicating that Sf-caspase-1 is involved
in this apoptosis. Similarly, the inhibitor of initiator caspase, viz., z-VAD-fmk, inhibited apoptosis. Therefore, both initiator and
effector caspases are possibly involved in the apoptosis of SF-21 cells. In addition, we detected Sf-caspase-1 activity in the
process of apoptosis in SF-21 cells, suggesting that the effector caspase in SF-21 is similar to that found in mammalian cells.
Our results also indicated that the apoptosis found in this line is accomplished through a Sf-caspase-1 signaling pathway.
Arch. Insect Biochem. Physiol. 68:206–214, 2008. © 2008 Wiley-Liss, Inc.
KEYWORDS: apoptosis; SF21; entomopathogenic fungus; Nomuraea rileyi; conditioned medium; caspase
Apoptosis is defined as an active cell death
(Sloviter, 2002). It is highly conserved among invertebrates and vertebrates, and plays a key role in
tissue homeostasis, normal development, and disposition of unwanted cells including those by damaged physically and by virus (Clem et al., 1996;
Bergmann et al., 1998; Nordstrom and Abrams,
2000; Baehrecke, 2002). Apoptotic cells undergo a
chain reaction of characteristic and dramatic distortion in morphology including cytoskeletal rearrangement, cell shrinkage, membrane blebbing,
chromatin condensation, and DNA fragmentation
(Kerr et al. 1972; Lawen, 2003). These alterations are
initiated either indirectly or directly by the proteolytic
activity of caspases, which is a group of enzymes con-
served highly among families of aspartate-specific cysteine proteases (Cryns and Yuan, 1998).
Caspases are known to be involved apoptosis
in insects as well as in mammals; however, the processes by which caspases are activated including
the hierarchy of initiator and effector are indistinct.
To date, caspases have been identified and characterized from Lepidoptera (Ahmad et al., 1997; Liu
and Chejanovsky, 2005); especially, Sf-caspase-1 is
the principal effector caspases of SF-21 cell line
from Spodoptera frugiperda (Ahmad et al., 1997;
LaCount et al., 2000; Manji and Friesen, 2001).
This line is often used for investigations on
apoptosis due to its sensitivity to various lethal factors, such as baculovirus infection, UV irradiation,
and overexpression of relative apoptotic genes
(Bertin et al., 1996; Clem et al., 1996; LaCount
Department of Entomology, National Chung Hsing University, Taichung, Taiwan 402, Republic of China
Contract grant sponsor: National Science Council, Republic of China; Contract grant number: NSC95-2313-B-005-048; NSC96-2628-B-005-007.
*Correspondence to: Roger F. Hou, Department of Entomology, National Chung Hsing University, Taichung, Taiwan 402, ROC. E-mail:
© 2008 Wiley-Liss, Inc.
DOI: 10.1002/arch.20242
Published online in Wiley InterScience (
Archives of Insect Biochemistry and Physiology
August 2008
Apoptosis in SF21 Cells by N. rileyi
and Friesen, 1997; Manji et al., 1997; Vucic et al.,
1997a,b, 1998).
Pro-Sf-caspase-1 with a short domain is activated by an apical caspase through proteolytic
cleavage when apoptotic signaling is triggered. The
cleavage occurs at the caspase-recognition site between the large and small subunits of pro-Sfcaspase-1 (LaCount et al., 2000). The peptide
analogue, z-VAD-fmk, is a broad-spectrum inhibitor of initiator caspase, which is often used to investigate whether apoptosis occurs together with
caspase activation and inhibits pro-Sf-caspase-1 activation in SF21 cells. The other peptide analogue,
z-DEVD-fmk, is specific to suppress the activity of
Sf-caspase-1 by blocking amplification of Sfcaspase-1 (Manji and Friesen, 2001).
In vertebrates, apoptosis occurs in either target
tissues or immune cells when infected with microorganisms including viruses, bacteria, fungi, and
protozoa (Gao and Kwaik, 1999b; Benedict et al.,
2002; James and Green, 2004; Filler and Sheppard,
2006). In insects, apoptosis occurs also as a result
of virus infection (Lee et., 1993). Although gene
regulation of apoptosis in Drosophila was studied
in comparison with that of nematodes and mammals (McCall and Steller 1997; Chen and Abrams,
2000), information on inducing apoptosis of insect cells by fungi is meager. In this article, we report that the conditioned medium (CM) of the
entomopathogenic fungus, Nomuraea rileyi, could
induce active cell death in SF21 cells through Sfcaspase-1 signaling pathway.
Preparation of Fungal Cells and Conditioned Medium
Several stock cultures of entomopathogenic
fungi have been maintained in our laboratory since
1998 (Tang and Hou, 1998). Among them, N. rileyi
SH1 isolate was originally isolated from mummified larvae of the beet armyworm, Spodoptera exigua
and pure cultured on Sabouraud maltose agar fortified with 1% yeast extract (SMAY) medium at
25°C. After sporulation, a concentration of ~1 ×
108 conidia/ml was suspended in a 0.1% Tween
Archives of Insect Biochemistry and Physiology
August 2008
80 sterile aqueous solution, added to a liquid culture medium (20% potato, 2% sucrose, and 0.2%
yeast extract) to generate a 200-ml conidial suspension, and was incubated with shaking at 25°C
for 12 days. Upon reaching medium denseness, the
mycelia were removed from the cultured medium
by filtering through a Whatman filter paper. The
resulting CM was then sterilized by filtration
through 0.45-µm and 0.22-µm filters. The CM with
more than 90% lethal effect was assayed in every
culture by injecting into the 5th-instar larvae of
Galleria mellonella (15 µl/larva). In addition, 1 ml
of sterilized CM was proteolyzed overnight with
10 µl of proteinase K (20 mg/ml) at 37°C. Another
1 ml of sterilized CM was boiled at 100°C for 10
min. The enzyme- and heat-treated CM were examined electrophoretically for their ability to induce apoptosis in SF21 cells.
Cell Culture and Microscopic Examination of
Medium-Treated Cells With TUNEL Staining
The SF21 cells were cultured in Grace’s insect
culture medium (Gibco-BRL, Grand Island, NY)
containing 10% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel) at 28°C. After forming a monolayer, the cells were plated on
12-well culture plates and covered with the sterilized circular coverglasses (10-mm diameter). After
overnight incubation, the original CM was diluted
to 10-fold with cell culture medium and added to
each well. The cultured cells reacted with CM were
examined under a phase-contrast microscope
(Biophot, Nikon, Tokyo, Japan) at 0, 8, 12, or 24
h after treatment. In addition, the cultured cells at
24 h after culturing were treated with CM and examined using TUNEL staining described as below:
A circular coverglass attached with SF21 cells was
fixed with 10% formalin for 25 min, and then
dipped into phosphate buffer solution (PBS) twice
for 5 min. After washing, the coverglass was soaked
with 0.2% Triton X-100 for 5 min and washed
again with PBS for 5 min twice. The cells anchored
on coverglass were stained with DeadEnd colorimetric TUNEL system (Promega, Madison, WI).
Then, the coverglass was dried at room temperature,
Tseng et al.
sealed with Entellan (Merck, Darmstadt, Germany)
and examined under a phase-contrast microscope
(Biophot, Nikon, Tokyo, Japan).
Genomic DNA Extraction and DNA Ladder Examination
A culture of ~1 × 106 cells was plated onto a
3-cm Petri dish and allowed for attachment for
overnight. After incubation with the original, heattreated (100°C), or proteinase K-treated CMs for
24 h, adherent cells were removed and collected
in 2-ml Eppendorf tubes. Cells were centrifuged at
500g for 5 min, and then the supernatants were
removed. The cell pellets were washed with phosphate buffer, and centrifuged again to remove the
supernatants. The resultant pellets were lysed with
1 ml of lysis buffer (0.25% NP-40; 50 mM, TrisHCl, pH 8; 1 mM, EDTA, pH 8), and then 5 µl of
RNase (10 mg/ml) was added. After incubation for
30 min at room temperature, the mixture was
treated with 10 µl of proteinase K (Sigma, St. Louis,
MO) (20 mg/ml) at 37°C overnight. The genomic
DNA was extracted with a phenol–chloroform mixture (1:1) from proteolyzed solution and centrifuged at 10,000g for 5 min; the supernatants were
transferred into a new Eppendorf tube. The procedures of DNA extraction and purification were repeated twice. The purified DNA solution was
precipitated with 1/10 volume of sodium acetate
(3 M, pH 5.2) and double volume of pre-chilled
100% alcohol at –20°C for 30 min, and then was
centrifuged at 10,000g for 10 min. The DNA pellets were washed with pre-chilled 70% alcohol
twice and dissolved in 50 µl of sterilized water. In
this study, 5 µg of extracted genomic DNA was run
electrophoretically with 1× triacetate-EDTA buffer
in 2% agarose gel to examine the presence of DNA
ladder. At least five gels were run in each treatment.
Treatment of SF21 Cells With Peptide Analogues
Irreversible fluoromethyl ketone peptide inhibitors,
z-VAD-fmk, and z-DEVD-fmk (Merck, Darmstadt,
Germany), were dissolved in dimethylsulfoxide solution (DMSO, Sigma, St. Louis, MO) adjusted to
10 mM. A culture of ~1 × 106 cells was plated onto
a 3-cm Petri dish at 28°C for 24 h and then treated
with 10% CM mixing with 200 µM of peptide analogue inhibitors. After culturing at 28°C for 24 h,
the cells were harvested, and their genomic DNA was
extracted and examined electrophoretically for DNA
ladder as described under genomic DNA extraction
and DNA ladder examination (above section).
Caspase Assay
A culture of ~1 × 106 cells was plated onto a 3cm Petri dish and then treated with 10% CM for
different times. The cells were harvested and assayed with a caspase-3 activity assay kit (Calbiochem, La Jolla, CA), which is customarily used to
determine caspase activity in mammalian cells. Accumulation of fluorescence was monitored using
a fluorescence microplate reader (excitation, 355
nm; emission, 535 nm, Twinkle LB970, Berthold
Technologies, Oak Ridge, TN). Relative fluorescence
units (RFU) were averaged from the result of 3
Apoptosis of SF21 Cells Induced by N. rileyi
Conditioned Medium
After the cultured cells were treated with the
fungal CM, changes in cell morphology, especially
membrane blebbing, occurred at 8 h after treatment compared with normal cells (Fig. 1A,B).
Many apoptotic bodies were released from the cells
observed under a microscope at 12 and 18 h after
treatment (Fig. 1C,D), suggesting that N. rileyi CM
was able to induce apoptosis of SF21 cells based
on microscopic observations. Furthermore, the cultured cells stained with the TUNEL system after CM
treatment were stained positively (in dark) compared to those treated with fresh medium (Fig.
2A,B). In addition, to verify the onset of apoptosis,
it is necessary to characterize the presence of DNA
fragmentation in the treated cells. Genomic DNA
extracted from SF21 cells treated with the CM-generated DNA ladders in agarose gel was compared
with fresh medium at 24 h after treatment (Fig.
Archives of Insect Biochemistry and Physiology
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Apoptosis in SF21 Cells by N. rileyi
Fig. 1. SF-21 cells after treatment with Nomuraea rileyi
condition medium for various times. A: 0 h. B: 8 h. C:
12 h. D: 18 h. Arrows show
cell membrane blebbing (B)
and apoptotic bodies released
from lytic cells (C,D).
3). Hence, the genomic DNA fragmentation in SF21
cells occurred after CM treatment.
The cultured cells treated with the fungal fluids, which had been hydrolyzed with proteinase K
or heated at 100°C, did not form DNA ladders,
while those treated with unhydrolyzed CM or
with the CM incubated overnight at 37°C formed
the ladders (Fig. 4). Therefore, the active component(s) in the CM seems to be proteinacious
and heat labile.
Fig. 2. Micrographs showing apoptosis of SF-21 cells
after staining with DeadEnd
colorimetric TUNEL system.
A: SF-21 cells treated with
fresh medium. B: SF-21 cells
treated with 10% Nomuraea
rileyi conditioned medium,
24 h. Arrows show DNA fragmentations as revealed by
staining in dark, indicating
a positive signal of apoptosis.
Archives of Insect Biochemistry and Physiology
August 2008
Tseng et al.
Fig. 3. Electrophoretic profile of SF-21 DNA. M: marker.
Lane 1, DNA of untreated SF-21 cells; lane 2, DNA of SF21 cells treated with 10% Nomuraea rileyi conditioned medium; lane 3, DNA of SF-21 cells treated with 10% PDB+Y
medium. Note the formation of DNA ladder on lane 2.
Involvement of Caspases and Sf-Caspase-1 in
Apoptosis of SF21 Cells
A broad-spectrum inhibitor of initiator caspase,
z-VAD-fmk, was used to treat SF21 cells to determine the involvement of caspases in programmed
cell death induced by CM from fungal cultures.
Those SF21 cells incubated with the fungal CM and
z-VAD-fmk did not form a DNA ladder in agarose
gel (Fig. 5). In addition, we did not see the formation of apoptotic bodies in these cells under microscopic observation before harvesting them for
DNA extraction. Therefore, induction of apoptosis
in SF21 cells by N. rileyi CM was possibly accomplished through a caspase signaling pathway. For-
Fig. 4. Electrophoretic profile of DNA extracted from SF21 cells treated with protein inactivated conditioned medium of Nomuraea rileyi. M: marker. Lane 1, from untreated
conditioned medium; lane 2, from conditioned medium
heated at 100°C, 10 min; lane 3, from conditioned medium incubated with proteinase K at 37°C overnight; lane
4, from conditioned medium incubated at 37°C overnight.
Note the formation of DNA ladders on lanes 1 and 4.
mation of DNA ladders was also inhibited when
SF21cells were incubated with N. rileyi CM and zDEVD-fmk (Fig. 6). Activity of Sf-caspase-1 was elevated predominantly after 8 h treatment with CM
and reached a peak at 12 h (Fig. 7).
Significant features of apoptosis including membrane blebbing, formation of apoptotic bodies, and
DNA fragmentation characterize apoptotic cells
(Kerr et al., 1972; Lawen, 2003). In our study, SF21
cells treated with N. rileyi CM showed membrane
blebbing at 8 h, apoptotic bodies at 18 h, and DNA
ladders at 24 h after incubation, indicating the onArchives of Insect Biochemistry and Physiology
August 2008
Apoptosis in SF21 Cells by N. rileyi
Fig. 5. Electrophoretic profile of DNA extracted from SF21 cells treated with 10% conditioned medium of Nomuraea
rileyi and 200 µM caspase inhibitor, z-VAD-fmk, for 24 h.
M: marker. Lane 1, untreated SF-21 cells; lane 2, SF-21
cells treated with 10% conditioned medium; lane 3, SF21 cells treated with 10% conditioned medium and 200
µM caspase inhibitor, z-VAD-fmk. Note the formation of
DNA ladder on lane 2.
set of apoptotic cells. The apoptosis of SF-21 was
further confirmed by treating the conditioned cells
with TUNEL staining system. This cell line was reported to release apoptotic bodies at 17 h and DNA
ladders at 12 h after baculovirus infection (Lee et
al., 1993). The same line showed cell membrane
blebbing at 3 h and formed apoptotic bodies at 9 h
after UV irradiation for 10 min (Manji and Friesen,
2001). These reports indicate that SF-21 cells generated typical characterization of apoptosis after either
a microbial infection or a physical damage. Similarly,
we found that the CM of the entomopathogenic fungus could also induce apoptosis in SF21 cells.
The entomopathogenic fungi, Beauveria bassiana
and Metarhizium anisopliae, release protease and
Archives of Insect Biochemistry and Physiology
August 2008
Fig. 6. Electrophoretic profile of DNA extracted from SF21 cells treated with 10% conditioned medium of Nomuraea
rileyi and 200 µM caspase inhibitor, z-DEVD-fmk, for 24 h.
M: marker. Lane 1, SF-21 cells treated with 10% conditioned
medium; lane 2, SF-21 cells treated with 10% conditioned
medium and 200 µM caspase inhibitor, z-DEVD-fmk.
Fig. 7. Changes in sf-caspase-1 activity after treatment
of SF-21 cells with 10% conditioned medium of Nomuraea
rileyi. RFU, relative fluorescent unit.
Tseng et al.
toxic metabolites into the hemolymph, resulting
in inhibition and impairment of hemocytic attachment, spreading, and phagocytic activity in insects
(Mazet et al., 1994; Vilcinskas et al., 1997; Griesch
and Vilcinskas, 1998). Our studies show that
proteinacious substances in N. rileyi CM were capable of inhibiting cellular immune response of
the larvae of Galleria mellonella (Y.K. Tseng, Y.W.
Tsai, M.S. Wu, and R.F. Hou, unpublished data).
Similarly, treatments of N. rileyi CM with either
proteinase K or heat inhibited apoptosis against
SF-21 cells. It thus appears that the fungal metabolites released into cultured medium elicit other
cellular reactions in vitro, e.g., apoptosis, in addition
to interference with host immune responses in vivo.
A group of aspartate-specific cysteine proteases,
i.e., caspases, have been found to be engaged in
most apoptosis of mammalian and Drosophila cells
(Fuentes-Prior and Salvesen, 2004). The initiator
caspase is activated, followed by activation of an
effector caspase with a proteolytic cleavage when
cells receive apoptotic signals. The effector caspase
continues to undergo cleavage into protein substrates, leading to programmed cell death (Cohen,
1997; Degterev et al., 2003). Since caspases undergo a proteolytic cleavage on special amino acid
sequences of protein substrates (Cohen, 1997), different peptide analogues have always been designed
and created as substrates for caspases. One of the
peptide analogues, z-VAD-fmk, which is a broadspectrum inhibitor for caspase activation, was used
to determine whether the caspase pathway was involved in apoptosis (Armstrong et al., 1996; Fraser
and Evan, 1996; McCarthy et al., 1997). Apoptosis
of SF21 cells induced by N. rileyi CM was blocked
by treatment with z-VAD-fmk, suggesting that
caspase pathway is involved in this apoptosis. Similarly, the same pathway was reported in SF21 cells
after baculovirus infection and UV irradiation
(Manji and Friesen, 2001). Therefore, involvement
of caspase pathway in apoptosis appears common
in SF21 cells.
An effector caspase, Sf-caspase-1, present in SF21
cells was a counterpart of caspase-3 in mammalian cells with similar amino acid sequences and
protein structure (Ahmad et al., 1997). The peptide analogue, z-DVED-fmk, could inhibit the activity of Sf-caspase-1. Moreover, a peptide analogue,
DVED, is excised proteolytically by caspase-3 and
Sf-caspase-1, and is useful for measuring the activity of Sf-caspase-1 (Zoog et al., 2002). Our results
showed that apoptosis of Sf21 cells by N. rileyi CM
was inhibited when z-DEVD-fmk was added to the
mixture, and the activity of Sf-caspase-1 was elevated in the process of apoptosis of SF21 cells.
Inhibition of caspases was found in apoptosis of
SF21 cells induced by baculovirus infection and UV
irradiation (Manji et al., 1997; Manji and Friesen,
2001). Hence, it is reasonable to speculate that Sfcaspase-1 is involved in apoptosis of SF21 cells
when treated with N. rileyi conditioned medium.
z-IETD-fmk could inhibit activity of apical
caspase in SF21 cells as a result of UV irradiation
(Manji and Friesen, 2001). In this context, it seems
that apical caspase is a counterpart of caspase-8
based on our observations. Therefore, the signaling pathway leading to apoptosis in SF21 cells is
similar to the caspase cascade in mammalian cells.
Although the counterparts of caspase-8 and -9 were
involved in SF21 apoptosis induced by N. rileyi in
this study, whether both extrinsic and intrinsic
pathways discovered in mammals and Drosophila
occur in the process of apoptosis in SF21 cells
treated with the fungal CM remains to be verified.
The authors thank Dr. Ray Cooper (Hong Kong
Polytechnic University) for valuable comments and
critical reading of this manuscript.
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