PrPC-induced drug resistance in gastric cancer cellsJie Liang*, Fulin Ge*, Changcun Guo, Guanhong Luo, Xin Wang, Guohong Han, Dexin Zhang, Jianhong Wang, Kai Li, Yanglin Pan, Liping Yao,
Trang 1PrPC-induced drug resistance in gastric cancer cells
Jie Liang*, Fulin Ge*, Changcun Guo, Guanhong Luo, Xin Wang, Guohong Han, Dexin Zhang, Jianhong Wang, Kai Li, Yanglin Pan, Liping Yao, Zhanxin Yin, Xuegang Guo, Kaichun Wu, Jie Ding and Daiming Fan
State Key Laboratory of Cancer Biology and Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi’an, Shaanxi, China
Cellular prion protein (PrPC) is a ubiquitous
glyco-protein that is localized at the cell surface via a
glycosyl-phosphatidylinositol-anchored membrane
pro-tein, whose pathogenic isoform, PrPSc, has attracted
worldwide attention due to its involvement in the
path-ogenesis of bovine spongiform encephalopathy and
Creutzfeldt–Jakob disease in human beings [1] PrPCis
highly conserved in mammalian species, and has
been postulated to be involved in one or more of the
following activities: neurotransmitter metabolism, cell adhesion, signal transduction, antioxidant activity and programmed cell death [2] However, the exact biologi-cal function of normal PrPCis still unknown [3]
In our previous studies, PrPC was identified as an upregulated gene in the adriamycin (ADR)-resistant gastric carcinoma cell line SGC7901/ADR compared
to its parental cell line SGC7901 by subtractive hybrid-ization and RT-PCR [4] PrPCwas further found to be
Keywords
drug resistance; gastric cancer; P-gp;
PI3K/Akt; prion protein
Correspondence
J Ding, State Key Laboratory of Cancer
Biology and Xijing Hospital of Digestive
Diseases, Fourth Military Medical University,
Xi’an 710032, China
Fax: +86 29 82539041
Tel: +86 29 84771504
E-mail: dingjie@fmmu.edu.cn
D Fan, State Key Laboratory of Cancer
Biology and Xijing Hospital of Digestive
Diseases, Fourth Military Medical University,
Xi’an 710032, China
Fax: +86 29 82539041
Tel: +86 29 84775221
E-mail: fandaim@fmmu.edu.cn
*These authors contributed equally to this
work
(Received 1 October 2008, revised 16
November 2008, accepted 24 November
2008)
doi:10.1111/j.1742-4658.2008.06816.x
Cellular prion protein (PrPC), a glycosyl-phosphatidylinositol-anchored membrane protein with unclear physiological function, was previous found
to be upregulated in adriamycin (ADR)-resistant gastric carcinoma cell line SGC7901/ADR compared to its parental cell line SGC7901 Overexpres-sion of PrPC in gastric cancer has certain effects on drug accumulation through upregulation of P-glycoprotein (P-gp), which is suggested to play
an important role in determining the sensitivity of tumor cells to chemo-therapy and is linked to activation of the phosphatidylinositol-3-kinase/Akt (PI3K/Akt) pathway In the present study, we further investigate the role
of the PI3K/Akt pathway in PrPC-induced multidrug-resistance (MDR) in gastric cancer Immunohistochemistry and confocal microscope detection suggest a positive correlation between PrPC and phosphorylated Akt (p-Akt) expression in gastric cancer Using established stable PrPC transfec-tant cell lines, we demonstrated that the level of p-Akt was increased in PrPC-transfected cells Inhibition of PrPC expression by RNA interference resulted in decreased p-Akt expression Inhibition of the PI3K/Akt path-way by one of its specific inhibitors, LY294002, or by Akt small interfering RNA (siRNA) resulted in decreased multidrug resistance of SGC7901 cells, partly through downregulation of P-gp induced by PrPC Taken together, our results suggest that PrPC-induced MDR in gastric cancer is associated with activation of the PI3K/Akt pathway Inhibition of PI3K/Akt by LY2940002 or Akt siRNA leads to inhibition of PrPC-induced drug resis-tance and P-gp upregulation in gastric cancer cells, indicating a possible novel mechanism by which PrPCregulates gastric cancer cell survival
Abbreviations
ADR, adriamycin; IR, immunoreactivity; MDR, multidrug-resistance; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; P-gp, P-glycoprotein; PI3K/Akt, phosphatidylinositol-3-kinase/Akt; PrP C , cellular prion protein; siRNA, small interfering RNA; VCR, vincristine.
Trang 2highly expressed in the gastric cancer cell lines
SGC7901/ADR and SGC7901/VCR Its
overexpres-sion played a certain role in drug accumulation in
gastric cancer cells, which could induce drug resistance
in SGC7901 cells by decreasing accumulation and
increasing release of these drugs (ADR and VCR) [5]
PrPC-mediated multidrug resistance (MDR) was
thought to be related to upregulation of P-glycoprotein
(P-gp) [6] and inhibition of apoptosis [6–9]; these
stud-ies were the first reports that PrPC can induce the
MDR phenotype in gastric cancer cells
Activation of the phosphatidylinositol-3-kinase/Akt
(PI3K/Akt) pathway is a critical step in determining
the sensitivity of tumor cells to chemotherapy [10]
A previous study demonstrated that MDR in gastric
cancer could be reversed by downregulation of Akt1
by Akt1 small interfering RNA (siRNA) [11] Other
studies strongly suggested that the PI3K/Akt
path-way is involved in certain functions of PrP [12,13]
Some differentially expressed genes involved in the
PI3K/Akt pathway were found to be overexpressed
in breast cancer cell line MCF7, together with
17-fold upregulation of PrP [14]
In present study, we hypothesized that the PI3K/Akt
pathway is involved in PrPC-mediated MDR in gastric
cancer To test this hypothesis, the expression of Akt
and PrPCwas examined by immunohistochemistry and
confocal microscope in human gastric cancer cases and
cell lines Expression of total and p-Akt was detected in
cells transfected with PrPC The effects of PI3K/Akt on
PrPC-induced MDR in gastric cancer were then
exam-ined by a drug sensitivity assay that involved adding the
PI3K/Akt-specific inhibitor LY294002 or
co-transfec-tion with Akt siRNA The underlying mechanisms were
further explored by RT-PCR, Western blotting and the
luciferase reporter assay
Results
Co-expression of PrPCand Akt in gastric cancer
Immunohistochemical staining showed that PrPC was
expressed in the cytoplasm of neoplastic cells in 70.6%
(60/85) of gastric cancer tissues Although PrPC also
showed expression in adjacent nontumor gastric tissue,
this was significantly lower than in gastric cancer cells
(P < 0.05) Phosphorylated Akt (p-Akt)
immuno-reactivity was observed in 88.2% (75/85) of gastric
cancer tissues, with positive signals mainly in the
cyto-plasm of neoplastic cells As PrPC and p-Akt
expres-sion coexisted in gastric cancer tissues, we analyzed
their correlation in 60 cases of PrPC-positive gastric
cancer tissues Spearman analysis showed that there
was a significant correlation between PrPC and p-Akt immunoreactivity, with rs = 0.514, P < 0.01 (Fig 1A) The correlation of p-Akt with PrPC in gastric cancer tissues suggests possible co-expression
To test this hypothesis, PrPC and p-Akt were co-expressed in SGC7901/ADR cells The yellow stain-ing in dual-labelstain-ing experiments indicated overlappstain-ing areas of red and green fluorescence, suggesting co-expression of p-Akt with PrPCin the cytoplasm of SGC7901/ADR cells (Fig 1B) These results suggested
a positive correlation between PrPCand p-Akt expres-sion in gastric cancer
Induced activation of p-Akt in PrPC-transfected cells
Our previous work has shown that PrPC expression was detected in several different histological types of human gastric cancer cell lines, and is relatively low in SGC7901 cells and high in AGS To upregulate or downregulate PrPC expression, the PRNP gene was stably transfected into human gastric cancer cells of line SGC7901 or blocked by siRNA in AGS After cell transfection and antibiotic screening for more than
2 months, multiple resistance clones were selected, and expression of PrPCin the cells was confirmed by Wes-tern blotting (Fig 2A) As close correlation of p-Akt with PrPCwas found in gastric cancer tissues, we won-dered whether PrPC could regulate the expression of Akt in transfected cells As shown in Fig 2B, there was higher expression of the phosphorylated form of Akt (Thr308) in SGC7901 transfected with PrPC (SGC7901/PrPC) than in SGC7901 transfected with empty pcDNA3.1B (SGC7901/pcDNA3.1B) or non-transfected SGC7901, which did not affect the expres-sion of total Akt As the siRNA affected translation but not post-translational events, both the total and phosphorylated Akt levels decreased in AGS/PrPC (RNAi) These results indicate that alteration of PrPC expression led to a corresponding change in the PI3K/ Akt pathway in gastric cancer cells lines, and con-firmed that PrPC regulates the expression of p-Akt
in vitro, consistent with the above in vivo findings
PI3K/Akt is involved in PrPC-mediated MDR in gastric cancer
In order to study whether activation of the PI3K/Akt signaling pathway played a role in PrPC-induced MDR
in gastric cancer cells, the PrPC-overexpressing cell line and the corresponding controls were treated with LY294002, a selective inhibitor of PI3K, or by co-transfection with Akt siRNA [13] The in vitro
Trang 3effects of the anticancer drugs on the growth of
SGC7901/PrPC, SGC7901/pcDNA3.1B and SGC7901
were evaluated by the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay As
shown in Table 1, in the absence of LY294002 or Akt
siRNA, the control and parental cells showed higher
sensitivity to ADR and VCR than adding LY294002
or Akt siRNA However, the inhibitory rate in cells
transfected with PrPCwas significantly different to that
in control cells (P < 0.05) When the cells were treated
with a high dose of the inhibitor LY294002 (40 lm) or
co-transfected with Akt siRNA (1 lg) for 24 h, the
relative inhibitory rate in SGC7901/PrPC was
decreased to levels similar to the controls because of
their toxicity in cell death (P > 0.05)
The effects of LY294002 on adriamycin
accumula-tion and retenaccumula-tion in SGC7901/PrPCcells were further
determined by flow cytometric analysis The
adria-mycin-releasing index in gastric cancer SGC7901 cells
was calculated as follows: releasing index =
(accumu-lation value) retention value)/accumulation value
The results showed less adriamycin accumulation and
retention in SGC7901/PrPC cells compared with that
in controls When the cells were treated with
LY294002 or co-transfected with Akt siRNA for 24 h,
the accumulation and retention of adriamycin
increased in both cell lines, but was more significant in
SGC7901/PrPC (P < 0.05) The above results
indi-cated a partly dose-dependent effect, but the effect
more significant at concentrations of 10 lm LY294002
or 0.2 lg Akt siRNA The releasing index also decreased more sharply at 10 lm LY294002 or 0.2 lg Akt siRNA in SGC7901/PrPC The highest concentra-tions of LY294002 (40 lm) or Akt siRNA (1 lg) resulted in similar releasing rates to each other because
of their toxicity effect (P > 0.05) (Fig 3) The results indicated that inhibition of the PI3K/Akt signaling pathway leads to inhibition of PrPC-induced cell drug resistance in gastric cancer cells
PI3K/Akt is involved in the activation of P-gp by PrPCin gastric cancer
To further investigate the underlying mechanism of PI3K/Akt-mediated PrPC-induced gastric cancer MDR, the PrPC-overexpressing cell lines (SGC7901/PrPC) and corresponding controls (SGC7901/pcDNA) were trea-ted with LY294002 or Akt siRNA to screen the down-stream molecules PrPC-transfected gastric cancer cells were treated with LY294002 (10 lm) or Akt siRNA (0.2 lg), and exhibited downregulation of P-gp at both the mRNA and protein levels (Fig 4A) Inhibition of the PI3K/Akt pathway decreased the expression of P-gp induced by PrPCat both the mRNA and protein levels, suggesting transcriptional regulation of P-gp by PI3K/ Akt in PrPC-transfected gastric cancer cells Therefore, the promoter sequence of MDR-1 ()136 to 10) was amplified, and the luciferase reporter assay was performed to investigate the transcriptional regulation
of P-gp by PI3K/Akt The luciferase activity of P-gp
A
B
(c)
Fig 1 Co-expression of PrPCand Akt in
gastric cancer (A) Serial sections of gastric
cancer tissue were stained with antibodies
against PrPC(3F4, Sigma) or p-Akt (Thr 308;
Cell Signaling Technology) (a) Gastric
can-cer tissue stained with anti-p-Akt (Thr 308).
(b) Gastric cancer tissue stained with
anti-PrP C (c) Negative control (original
magnifica-tion, ·200) (B) Co-expression of PrP C with
p-Akt in SGC7901/ADR cells (a) Confocal
microscopic imaging of p-Akt, stained with
monoclonal antibody against p-Akt and
FITC-conjugated goat anti-mouse IgG (green) (b)
Confocal microscopic imaging of PrPC,
stained with polyclonal antibody against PrP
(Santa Cruz Biotechnology) and tetraethyl
rhodamine isothiocyanate-labeled rabbit
anti-goat IgG (red) (c) Co-expression of p-Akt
with PrP C results in yellow staining (d)
Co-expression of p-Akt with PrPC, with
nuclear staining by
4¢,6-diamidino-2-phenylin-dole (DAPI) (blue) (original magnification,
·400).
Trang 4promoter in gastric cancer cells decreased with or
with-out PrPC transfection by adding LY294002 or
co-transfection with Akt siRNA However, SGC7901
transfected with PrPC showed significantly decreased P-gp promoter activity compared with the control cells (SGC7901/pcDNA and SGC7901 cells) after addition
of LY294002 or co-transfection with Akt siRNA (Fig 4B) PI3K/Akt might therefore be involved in transcriptional activation of P-gp in gastric cancer cells transfected with PrPC Inhibition of the PI3K/Akt path-way by its specific inhibitor or Akt siRNA could reverse the PrPC-induced MDR effect, partly through decreas-ing transcriptional activation of P-gp
Discussion
In the present study, we present the first evidence that PI3K/Akt may be involved in the transcriptional acti-vation of P-gp in gastric cancer cells transfected with PrPC Inhibition of the PI3K/Akt pathway by its spe-cific inhibitor or Akt siRNA reverses PrPC-induced MDR in gastric cancer cells, partly by decreasing the transcriptional activation of P-gp Continuing from our previous work, it further reveals the function of PrPCin the MDR of cancers
Over recent years, many investigations have focused
on the physiological and pathological processes of PrPC [3] Recently, PrPC has been reported to play a positive role in MDR of many types of human cancers [15] Levenson et al [16] analyzed cell lines in which MDR had been induced by genetic suppressor ele-ments of cDNA array hybridization, and found that the transcription level of PrPCwas increased Our pre-vious studies had shown that PrPC is ubiquitously expressed in gastric carcinoma cell lines and tissues but
is weakly or not expressed in normal gastric mucosa [6,7,9,13] In vitro and in vivo drug sensitivity assays indicated that PrPC could promote the drug resistance
of a gastric cancer cell line [5] However, little is known about the molecular mechanism involved in this process
A
B
Fig 2 The inducible effect of PrPCon Akt (A) Western blot
analy-sis of the cells transfected with the empty vector or with PrP C
b-actin was used as a loading control (B) Expression of p-Akt and
total Akt were determined in gastric cancer cells by Western blot.
b-actin was used as an internal control Autoradiograms were
quantified by densitometry and p-Akt protein levels were calculated
relative to the amount of b-actin protein.
Table 1 IC50values (lgÆmL)1) for anticancer drugs for gastric cancer cells Survival rates of gastric cancer cells treated with anticancer drugs were evaluated by the MTT assay as described in Experimental procedures Dose–effect curves for the anticancer drugs were plotted
on semi-logarithmic coordinate paper, and IC 50 values were determined Data are means ± SD of three independent experiments.
Cell lines
Adriamycin
SGC7901/PrP C 6.87 ± 0.79 4.12 ± 0.64 2.58 ± 0.34 0.84 ± 0.17 6.87 ± 0.79 4.92 ± 0.74 3.76 ± 0.49 1.39 ± 0.25 SGC7901/pcDNA3.1B 0.43 ± 0.03 0.37 ± 0.05 0.28 ± 0.02 0.20 ± 0.03 0.43 ± 0.03 0.38 ± 0.06 0.30 ± 0.03 0.24 ± 0.04 SGC7901 0.31 ± 0.03 0.29 ± 0.04 0.25 ± 0.03 0.19 ± 0.02 0.31 ± 0.03 0.32 ± 0.05 0.26 ± 0.02 0.22 ± 0.04 Vincristine
SGC7901/PrP C 7.38 ± 0.78 5.21 ± 0.56 2.69 ± 0.38 0.34 ± 0.21 7.38 ± 0.78 5.83 ± 0.59 3.16 ± 0.43 0.83 ± 0.35 SGC7901/pcDNA3.1B 0.24 ± 0.02 0.20 ± 0.03 0.17 ± 0.02 0.09 ± 0.01 0.24 ± 0.02 0.22 ± 0.06 0.18 ± 0.04 0.12 ± 0.03 SGC7901 0.14 ± 0.02 0.12 ± 0.04 0.10 ± 0.03 0.08 ± 0.02 0.14 ± 0.02 0.13 ± 0.05 0.11 ± 0.04 0.09 ± 0.02
Trang 5P > 0.05
P < 0.05
P < 0.05
P > 0.05
0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0
P > 0.05
P < 0.05
P < 0.05
P > 0.05
SGC7901/PrP SGC7901/pcDNA
25 20 30
15 10 5
Fluorescence intensity 0
0 µ M 10 µ M 20 µ M
LY294002
40 µ M
0 µg 0.2 µg 0.4 µg
siRNA of Akt
siRNA of Akt
1 µg
0 µ M 10 µ M 20 µ M
LY294002
40 µ M
25
20
15
10
5
0
P < 0.05
P < 0.05
0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0
P > 0.05
P > 0.05
P < 0.05
P < 0.05
SGC7901/PrP SGC7901/pcDNA
SGC7901/PrP SGC7901/pcDNA
SGC7901/PrP SGC7901/pcDNA
Fig 3 Fluorescence intensity and releasing index of adriamycin in gastric cancer cells Gastric cancer cells were treated with or without LY294002 (10, 20 or 40 lM) or co-transfected with Akt siRNA (0.2, 0.4 or 1 lg) for 24 h Adriamycin was added to cells in log phase to a final concentration of 5 lgÆmL)1 After 1 h, cells were harvested (for detection of adriamycin accumulation) or cultured in drug-free
RPMI-1640 for another 30 min, followed by harvesting (for detection of adriamycin retention) The fluorescence intensity of intracellular adriamycin was determined using flow cytometry with an excitation wavelength of 488 nm and an emission wavelength of 575 nm (A) Fluorescence intensity analysis of intracellular adriamycin in gastric cancer cells (B) Adriamycin releasing index of gastric cancer cells The releasing index = (accumulation value ) retention value)/accumulation value.
Fig 4 PI3K/Akt is involved in the activation of P-gp by PrPCin gastric cancer (A) Expression of P-gp at both the mRNA and protein levels was investigated in PrP C -transfected gastric cancer cells treated with LY294002 (10 lM) or Akt siRNA (0.2 lg) b-actin was used as a loading control Autoradiograms were quantified by densitometry, and gene or protein levels were calculated relative to the amount of b-actin gene
or protein (B) Luciferase reporter assay to determine the regulatory effect of PI3K/Akt on MDR-1 promoter activity in PrPC-transfected cells.
A dual luciferase system was used in this assay The promoter activity was measured in terms of relative luminescence units (RLU), calculated using the following formula: RLU = luminescence intensity of the Firefy luciferase/luminescence intensity of the Renilla luciferase Control, cells co-transfected with empty pcDNA3.1 vector or parental cells SGC7901, pGL-MDR vector and pRL-TK vector; PrP C , cells co-transfected with empty pcDNA3.1B/PrPCvector treated with or without LY294002 (10 lM) or Akt siRNA (0.2 lg), pGL-MDR vector and pRL-TK vector The luciferase activities of each reporter plasmid were measured in triplicate and are expressed as fold inductions after normalization.
Trang 6Multidrug resistance (MDR) is one of the major
causes of failure of chemotherapy of human
malignan-cies Studies have shown that cellular drug resistance is
mediated by various mechanisms operating at various
steps in the cytotoxic activity of the drug, from a
decrease of drug accumulation in the cell to abrogation
of apoptosis induced by the chemical substance Often
several different mechanisms were switched on in the
cells, but usually one major mechanism was operating
MDR-1 (ABCB1, P-glycoprotein) is known to
signifi-cantly alter the extent of drug absorption and
excre-tion, which play a key role in the MDR of cancers
[17] Previous studies identified P-gp as an important
molecule in gastric cancer [4–6,18–20] and leukemia
MDR [20]
PI3K/Akt signal transduction pathway has been
reported to be involved in the neuroprotective effect of
humanin on cerebral ischemia/reperfusion injury [21]
Activation of the PI3K/Akt pathway is one of the
critical steps in cell survival and MDR [22] The
present study focuses on whether the PI3K/Akt
path-way contributes to the PrPC-induced MDR phenotype
in gastric cancer cells We show that PrPC and Akt
are co-expressed in gastric carcinoma, and whose
expression was related to MDR in gastric cancer
PrPC-transfected cell lines showed increased expression
of phosphorylated Akt, indicating that PrP may serve
as a positive upstream regulator of PI3K/Akt in gastric
cancer cells By inhibiting the PI3K/Akt pathway using
LY294002 or co-transfection with Akt siRNA, the
drug sensitivity and accumulation in SGC7901/PrPC
cells were significantly increased The results indicate
that inhibition of the PI3K/Akt signaling pathway
may lead to inhibition of the MDR induced by PrPC
in gastric cancer cells
The mechanism underlying PI3K/Akt-mediated
PrPC-induced MDR in gastric cancer was further
inves-tigated PrPC-transfected gastric cancer cell lines and
corresponding controls were treated with LY294002 or
Akt siRNA as described previously [6] PrPC-transfected
cells treated with an inhibitor of Akt exhibited
downre-gulation of P-gp at both the mRNA and protein levels;
this was proven to be due to transcriptional inhibition
of MDR-1 using a luciferase reporter assay Thus the
PI3K/Akt pathway may be involved in transcriptional
activation of P-gp in PrPC-transfected gastric cancer
cells, inhibition of which would reverse gastric cancer
MDR, partly by decreasing the transcriptional
activa-tion of P-gp This model of a PrPC/PI3K/P-gp signal
agrees with other reported findings on prion protein
sig-naling It was found that the Fyn protein governed a
number of the PrPC-induced pathways that converged
to the PI3K module in neurons [23] PI3K is known to
physically associate with Fyn to transducing differen-tiation signals [24] It has also been demonstrated that constitutively activated PI3K enhances activation of the MDR-1 promoter by 2-acetylaminofluorene [25]
In the present study, we report for the first time that activation of PI3K/Akt signaling pathway plays an essential role in PrP-induced MDR in gastric cancer cells Inhibition of the PI3K/Akt signaling pathway by LY2940002 or Akt siRNA leads to inhibition of PrPC -induced cell drug resistance and P-gp upregulation in gastric cancer cells, which indicated a possible novel mechanism by which PrPC regulates gastric cancer cell survival
Experimental procedures
Antibodies and reagents SP-9000 Histostain-Plus kits were obtained from Zhong-shan Goldbridge Biotechnology (Beijing, China) The drugs vincristine (VCR) and adriamycin (ADR) were purchased from Farmitalia Carlo Erba (Milan, Italy) and Minsheng Pharmaceutical Company (Hangzhou, China), respectively The antibodies rabbit anti-human t-Akt, mouse anti-human p-Akt and mouse anti-human P-gp, and the PI3K/Akt-specific inhibitor LY294002 were obtained from Cell Signal-ing Technology (Beverly, MA, USA) Mouse anti-human monoclonal PrPC serum (3F4) was purchased from Sigma (St Louis, MO, USA) and rabbit anti-human polyclonal PrP was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) The Sp1 immunohistochemistry kit and fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit and goat anti-mouse IgG were purchased from Zhongshan Goldbridge Biotechnology (Beijing, China)
Clinical samples Eighty-five gastric carcinoma patients (34 females, 51 males; mean age 56.1 ± 11.5 years; range 29–78 years) were included from a population-based case-controlled study conducted in Xijing Hospital, Xi’an, China Patients whose surgical tissue was used for the study signed informed consent All cases of gastric cancer were clini-cally and pathologiclini-cally proved The protocols used in the study were approved by the hospital’s Protection of Human Subjects Committee Formalin-fixed paraffin-embedded tissue sections were retrieved from archives at the Department of Pathology Demographic and clinico-pathological information was obtained from our popula-tion-based database, and tumor staging was carried out for each case according to the classification at the time of surgery Thirty cases were classified as well-differentiated,
43 as moderately differentiated and 12 as poorly differen-tiated gastric cancers
Trang 7Immunohistochemistry analysis
Tissue sections from gastric carcinoma were immunostained
using SP-9000 Histostain-Plus kits The slides were
depa-raffinized, rehydrated, blocked and then primary antibody
was added as described previously [7] Biotinylated goat
anti-mouse IgG was applied to the cell sections, which were
then incubated at room temperature for 30 min After
washing with NaCl/Pifor 10min, the tissues were incubated
with avidin dehydrogenase/biotinylated peroxidase for
30 min Color was developed by immersion of the sections
in a peroxidase substrate diaminobezidin solution
Evaluation of immunostaining
All sections were examined independently by two observers
with respect to the various histopathological characteristics
and specific immunoreactivity (IR) The staining was
semi-quantitatively evaluated by assigning a score for the
inten-sity of the IR and for the proportion of cells positively
stained The intensity of IR (intensity score) was divided
into four categories: 0, no IR; 1, weak IR (+); 2, moderate
IR (++); 3, strong IR (+++) The proportion of
posi-tive cells was classified into five groups: 0, no tumor cells
exhibiting IR; 1, < 25% of the tumor cells exhibiting IR;
2, 25–50% of the tumor cells exhibiting IR; 3, 50–75% of
the tumor cells exhibiting IR; 4, > 75% of the tumor cells
exhibiting IR The overall score was the multiplication of
the two The scores were then classified as negative (score 0
or 1) or positive (score‡ 2)
Cell culture
The human gastric adenocarcinoma cell line SGC7901 was
obtained from Academy of Military Medical Science
(Beij-ing, China) SGC7901/ADR was selected from SGC7901 by
increasing ADR stepwise from 0.01 to 0.8 lgÆmL)1, and
has been characterized previously [26] The cells were
maintained in RPMI-1640 containing 10% heat-inactivated
newborn bovine serum (SiJiQing, Hangzhou China) at 37 C in a humidified atmosphere of 5% CO2and 95% air
Double immunofluorescence staining Double immunostaining for antigen co-localization was per-formed using FITC-conjugated goat anti-mouse IgG (Chem-icon, Billerica, MA, USA; 1 : 80) and tetraethylrhodamine isothiocyanate-labeled rabbit anti-goat IgG (Chemicon,
1 : 100) Cells were nuclear-stained using 4¢,6-diamidino-2-phenylindole (DAPI) The primary antibody combinations consisted of monoclonal antibody against p-Akt and mono-clonal antibody against PrP SGC7901/ADR cells were incu-bated on glass coverslips for 24 h and fixed with 4% paraformaldehyde in NaCl/Pi The fixed cells were stained and examined using a FLUOVIEW FV1000 laser scanning confocal microscope (Olympus, Tokyo, Japan)
Plasmid construction and transfection The target sequences were aligned to the human genome database by a blast search to ensure that the chosen sequences were not highly homologous to those of other genes The primers were designed using primer.5 software (Premier Co., Edmonton, Canada) or the siRNA target finder at https://www.genscript.com/ssl-bin/app/rnai, and are listed in Table 2 siRNAs for PrP, Akt and control were chemically synthesized (Invitrogen, Carlsbad, CA, USA) and the target sequences were as previously reported [5– 7,9,13,15] SGC7901 cells were transfected with the plasmids and maintained in medium supplied with 400 lgÆmL)1of the neomycin analogue G418 (Life Technologies Inc., Gaithers-burg, MD, USA) The expression levels of PrPC in G418-resistant clones were evaluated by Western blot analysis
RNA extraction and semi-quantitative RT-PCR Total RNA was extracted from SGC7901/PrPC and SGC7901/pcDNA, and DNase was used to decrease the
Table 2 Primers for plasmid construction.
Trang 8contamination of genomic DNA The PCR primers and
reaction parameters used for MDR-1 amplification are
listed in Table 1 The reaction conditions for PCR of
MDR-1 were as follows: initial denaturation at 94 C for
10 min, and 35 cycles of denaturation at 94 C for 30 s,
annealing at 55 C for 30 s and extension at 72 C for 30 s
on a Touchgene gradient thermal cycler (Techne,
Cam-bridge, UK) Appropriate numbers of cycles were chosen to
ensure completion of PCR amplification before reaching
the stable stage in each reaction Gene expression was
quantified as the relative yield of the PCR product from
target sequences compared to that from the b-actin gene
PCR products were loaded onto a 1.5% agarose gel and
separated electrophoretically The gel was then visualized
under ultraviolet light following ethidium bromide staining
Autoradiograms were quantified by densitometry Relative
RNA levels were calculated relative to the levels for the
b-actin gene
Western blot analysis
Cells in log phase were harvested and washed twice for
10 min with NaCl/Pi, then lyzed in lysis buffer (150 mmolÆ
L)1 Tris/HCl pH 8.0, 50 mmolÆL)1 NaCl, 0.2 mmolÆL)1
EDTA, 0.1 mmolÆL)1 phenylmethanesulfonyl fluoride and
10 gÆL)1 Nonidet P-40) Fifty micrograms of protein from
each cell lysate were separated by 12% SDS–PAGE under
denaturing conditions, and transferred to nitrocellulose
membrane (Amersham, Pittsburgh, PA, USA) The
mem-brane was blocked with 10% non-fat dry milk in NaCl/
Tris-T (20 mm Tris/HCl, 100 mm NaCl, 0.1% Tween-20)
for 2 h at room temperature, probed with the primary
anti-bodies against Akt (1 : 1000), p-Akt (Thr308; 1 : 1000),
P-gp (1 : 1000), and PrPC3F4 (1 : 1000) overnight at 4 C
and subsequently incubated with horseradish
peroxidase-linked secondary antibodies in NaCl/Tris-T (with 5%
non-fat dry milk) Bound antibodies were visualized by
chemiluminescent substrate as described by the
manufac-turer (Zhongshan Goldbridge Biotechnology)
Autoradio-grams were quantified by densitometry Protein levels were
calculated relative to the amount of b-actin protein
In vitro drug sensitivity assay
The P-gp-affecting drugs adriamycin (ADR) and vincristine
(VCR) were both freshly prepared before each experiment
Drug sensitivity was evaluated using the
3-(4,5-dim-ethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)
assay Cells in the log growth phase were diluted with
200 lL RPMI-1640 supplemented with 100 mLÆL)1
new-born bovine serum, and seeded into 96-well plates (Costar,
New York, NY, USA) at a density of 8· 103
cells/well
After 24 h, the medium was replaced with fresh growth
medium containing various concentrations of drugs and 10,
20 or 40 lm LY294002 or dimethylsulfoxide (solvent
control) After 48 h growth in the presence of drugs, 20 lL MTT reagent (final concentration 5 gÆL)1) was added to each well, the supernatant was discarded after 4 h, and
150 lL dimethylsulfoxide was added to melt the crystals The absorbance of the formazan product was measured using an ELISA reader (Bio-Rad, Hercules, CA, USA) at a wavelength of 492 nm The drug concentration that pro-duced 50% inhibition of growth (IC50) was estimated using relative survival curves The survival rate was calculated as mean A490 of treated wells/mean A490 of untreated wells· 100%, where A490 indicates the absorbance of the solution at 490 nm Finally, dose–effect curves for the anti-cancer drugs were plotted on semi-logarithmic coordinate paper and IC50 values were determined Each study was performed in triplicate and repeated three times
Flow cytometric analysis Cells were cultured in six-well culture plates at 37 C for
24 h, and ADR was added to a final concentration of
5 mgÆL)1 After further culture for 1 h, various concentra-tions of LY294002 (10, 20 or 40 lm) were added and the mixtures were incubated for 1 h Dimethylsulfoxide was used for the negative control Cells were harvested, or cul-tured in drug-free medium for another 30 min and then harvested The harvested cells were suspended in cold NaCl/Pi, and the intracellular adriamycin fluorescence intensity was determined by flow cytometric analysis with excitation and emission wavelengths of 488 and 575 nm, respectively
Luciferase reporter assay The promoter sequence of MDR-1 ()136 to 10) was ampli-fied from the genomic DNA of peripheral blood mono-nuclear cells by PCR [27] The primers used are shown in Table 1 The promoter sequences were then cloned into a pGL3 enhancer vector (Promega, Madison, WI, USA) to construct the reporter vector pGL-MDR SGC7901 cells were passaged into 24-well plates at a density of
5· 105cells/well, and incubated until they reached 90% confluence pcDNA3.1/PrPC or empty pcDNA3.1/V5-his B plasmids were transfected into SGC7901 cells with pGL-MDR using Lipofectamine 2000 reagent (Invitrogen) pRL-TK was used as a control for transfection efficiency Luciferase reporter assays were performed using the dual-luciferase reporter assay system (Promega) according to the manufacturer’s instructions Each experiment was per-formed in triplicate and repeated three times
Statistical analysis Each experiment was repeated at least three times Numerical data are presented as the means ± SD The significance of the difference between means was
Trang 9deter-mined using Student’s t test The Wilcoxon signed-rank
test was used to evaluate the intensity of IR and the
pro-portion of cells staining positively in the
immunohisto-chemical assay Correlation between two groups was
assessed by Spearman analysis All statistical analyses
were performed using spss11.0 software (SPSS, Chicago,
IL, USA) A P value < 0.05 was considered statistically
significant
Acknowledgements
This study was supported in part by grants from the
Chinese National Foundation of National Sciences
(30572134 and 30872965) and the National Basic
Rsearch Program of China (2009CB521703) We thank
Professor Jie Liu for the pSilencer vector plasmid used
for siRNA construction We are also grateful to Bo
Huang (Department of Biochemistry, Fourth Military
Medical University, Xi’an, Shaanxi, China) for help
with the luciferase activity analyses, Dan Chen
(Department of Microscope, Fourth Military Medical
University, Xi’an, Shaanxi, China) for help with
confo-cal microscope detection, and technicians Taidong
Qiao, Zhen Chen, Baojun Chen and Baohua Song for
their excellent technical assistance
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