Download Reversal of multidrug resistance in renal cell carcinoma by short

Reversal of multidrug resistance in renal cell carcinoma by short hairpin RNA targeting MDR1 gene
HAO Yi-xin, HE Zheng-wen, ZHU Jian-hua, SHEN Qian, SUN Jun-zhong, DU Nan, XIAO Wen-hua
Department of Oncology, The First Affiliated Hospital of the PLA General Hospital, Beijing 100048,
China(Hao YX, Zhu JH, Sun JZ, Dun, Xi ao WH); Department of Laboratory Diagnosis, Changhai
Hospital, The Second Military Medical University, Shanghai 200433, China(He ZW, Shen Q)
Correspondence to: HAO Yi-xin, Department of Oncology, The First Affiliated Hospital of the PLA
General
Hospital,
No.51,
Fucheng
Road,
Haidian
District,
Beijing
100048,
China(Email:
[email protected])
Key words: multidrug resistance; P-glycoprotein; short hairpin RNA；renal cell carcinoma
Abstract
Background Over-expression of P-glycoprotein(P-gp), encoded by the MDR1 gene, confers
multidrug resistance (MDR) in renal cell carcinoma (RCC) and is a major reason for unsuccessful
chemotherapy. Recent developments on the inhibition usage of RNA interference (RNAi) to specific
protein expression have highlighted the potential usage as therapeutic agents.
Methods In this study, we have designed and selected one short hairpin RNA (shRNA) targeting
MDR1 gene, which is stably expressed from integrated plasmid and transfected by lentivirus fluid in
human RCC A498 cell.
Results The MDR1-targeted RNAi resulted in decreased MDR1 gene mRNA level, almost abolished
P-glycoprotein expression and reversed MDR to different chemotherapy drugs in the RCC A498 cell line.
Conclusions These results demonstrated that MDR may be reversed by RNAi in human RCC A498
cell line, which may be used for clinical application in future.
INTRODUCTION
Renal cell carcinoma (RCC) accounts for 2-3% malignant carcinoma, of which the conventional type
is clear cell type.1Surgical resection is the most effective treatment for organ-confined RCC, and
combined therapy is made for progressed RCC. But RCC is not sensitive and resistant to multiple
chemotherapeutic agents, which is known as multidrug resistance(MDR).2 In the treatment of malignant
carcinoma, MDR is a common clinical problem and may occur in primary therapy (intrinsic)or be
acquired during or after treatment.3 A number of mechanisms have been implicated in MDR generation,
which includes reduced drug accumulation due to the over-expression of transport proteins, increased
detoxification, altered targets such as reduced topoisomeraseI level and impaired apoptosis pathways. 4
One of the most widely studied aspects of MDR is the reduction of intracellular drug accumulation, in
which P-glycoprotein(P-gp, MDR1) is the first identified and most widely studied. Over-expression of
P-gp, the 170-kDa plasma membrane glycoprotein, has been implicated in both intrinsic and acquired
multidrug resistance. And as one of the adenosine triphosphate(ATP)-binding cassette(ABC) transporter
efflux pumps for cytotoxic drugs, P-gp presents in the proximal tubule cell of the normal kidney and also
is important for the RCC. Experimental data supported that high P-gp mRNA level was detected in the
RCC tissues and cell lines.5,6 And for a high P-gp expression rate of 30% to 89%, RCC usually display an
intrinsically high degree of chemo-resistance leading to the failure of chemotherapeutic treatment.7,8 How
can MDR be reversed? Drugs? Or others? Because of the severe side-effects, drugs for MDR reversal
have been abandoned in clinical usage. Thought of the gene silencing effect, RNA interference (RNAi)
for MDR reversal has been widely studied. So, the aim of this study was to determine the effect of RNAi
on the expression of MDR1 gene and the reversal of MDR in human RCC.
METHODS
Chemicals and reagents pSIH1-H1-copGFP shRNA Vector was purchased from System
Biosciences Inc. MiniBEST Plasmid Purification Kit Ver 2.0, Reverse Transcriptase M-MLV and
Ribonuclease Inhibitor were purchased from Takara Company. Plasmid mini kit was purchased from
Qiagen Inc and Lipofectamine TM 2000 was purchased from Invitrogen Corporation Company. UNIQ-10
column DNA gel extraction kit was purchased from Shanghai Sangon Biotech Inc.
Construction of recombinant plasmid Targeting the MDR1 gene sequence (NM 000927), 3 small
interfering RNA (siRNA) were designed using online software of the Invitrogen company:
a)siRNA-1:5'-GACATGACCAGGTATGCCT-3'
b)siRNA-2:5'-GGAGATAGGCTGGTTTGAT-3'
c)siRNA-3: 5'-GCTGGAGCAGTAGCTGAAG-3'. In addition, a random siRNA sequence (siRNA-neg)
was designed as negative control: 5'-CGTTTAACTCTCCCAACCA-3'. Then the corresponding
single-strand DNA templates of short hairpin RNA (shRNA) were designed based on each siRNA
sequence: the sense and anti-sense strands of siRNA were connected by a loop structure in the middle
(5'-CTTCCTGTCAGA-3') and followed by a termination signal (TTTTT), BamH I and EcoR I restriction
sites were respectively added at both 5' and 3' ends (synthesized by Shanghai Invitrogen Inc). The two
single DNA strands were annealed and linearized by restriction enzyme digestion, linked to linearized
vector and transformed into E. coli DH5α competent cells. Ampicillin-resistant single colon was picked
for DNA sequence amplification and detection, and colonies with the correct sequence were used for
large scale culture. Qiagen plasmid mini kit was used for endotoxin-free plasmid extraction, and agarose
gel electrophoresis was performed to check the integrity of the plasmid DNA. UV spectrophotometry was
applied to test the purity and concentration of the plasmid DNA.
Transfection and culturation of cell Human renal clear cell carcinoma A498 cell line, purchased
from the Cell Institute of Chinese Academy of Sciences, was seeded into 6-well plate. Cell concentration
was adjusted to 1.6 × 106 / well in DMEM+10% fetal bovine serum medium, cultured at 37℃ under 5%
CO2 for 24 hours. Upon 70% confluence of the attached cells, plasmid transfection was conducted
following instructions of the Lipofectmaine2000 kit. Empty plasmid was used as control.
MDR1 mRNA level detection 48h after transfection, MDR1 mRNA level of RCC A498 cell was
detected. Total RNA was extracted and mixed with Oligo(dT)18 primer and RNase-free dH2O to make
RNA/primer denaturation solution. The cDNA was synthesized through reverse transcription reaction,
which was performed with Reverse Transcriptase M-MLV at 42℃ for 1 h and 70℃ for 15 min. Primers
used for MDR1 cDNA amplification were fw: 5’-CCGTGGGGCAAGTCAGTTCA-3’and rev:
5’-CCGGTCGGGTGGGATAGTTG-3’. A house-keeping gene (β-actin) was amplified as internal
control(fw:
5’-CCTGTACGCCAACACAGTGC-3’ and rev: 5’-ATACTCCTGCTTGCTGATCC-3’).
Real-time PCR amplification cycling conditions for MDR1 and β-actin were as follows: initial enzyme
activation at 95℃ for 10 min, followed by 40 cycles at 95℃ for 10 sec, 57℃ for 20 sec and 72℃ for
20 sec. Gene-specific fluorescence was measured at 85℃ and confirmed by melting curve analysis. For
analysis, MDR1 mRNA level was evaluated by the fomula: MDR1/β-actin=2-[Ct(MDR1 )- Ct(β-actin)]. The
RNA interference effect was calculated to select the most effective RNAi sequence through the gene
inhibition rate.
shRNA packaged by lentivirus 293T cells were plated into culture dishes, with cell concentration
adjusted to 1.0 × 106/plate, and cultured in DMEM + 10% FBS medium for 24 h. 10μg lentiviral
packaging plasmid and 2 μg recombinant shRNA plasmid were co-transfected into 293T cells. 48 h after
co-transfection, culture supernatant was collected, centrifuged at 2,800 × g for 5 min, filtered through
0.45 μm PVDF membrane, then the virus particle concentration was adjusted to 1 × 105 pfu/μl.
Establishment of RCC A498-RNAi cell line RCC A498 cells were seeded into 96-well plates,
adjusted to 2 × 103 cells / well, and cultured in DMEM +10% FBS medium for 24 h before changed into
fresh medium. 96 h after added by lentiviral packaging shRNA solution , attached RCC A498 cells
reached confluence of above 90%. Then single cell suspension solution was prepared to be plated into
96-well plates. After culture for 48 h, 3 well cells with the strongest fluorescence expression were chosen
for clone amplification. RT-PCR method was applied for detection of the MDR1 mRNA level to screen
the cell clone with the most effective gene silencing rate, and to establish the stable shRNA expression
RCC A498 cell line (RCC A498-RNAi cell line). Cryopreservation and recovery were repeated for three
times and Western blot was performed to verify the P-gp expression level in this selected cell clone.
Cell growth detection The cell growth state was detected in RCC A498-RNAi cell line and
untransfected A498 cell line by the MTT assay method. Cells were plated into 96-well plates, adjusted to
4 × 103 cells / well, and cultured in DMEM + 10% FBS medium for 24 h before changing the culture
medium. 10 μl MTT and 0.1 ml PBS were added and co-cultured with cells for 6 h at 37℃. After 0.1 ml
acidified isopropanol were added into cells and fully dissolved, the optical density (OD) values were read
at 570 nm using MultiSkan FC ELISA device. Then the cell growth curves of RCC A498-RNAi cell line
and untransfected A498 cell line were constructed.
Cytotoxicity studies The anti-proliferative effects of different chemotherapeutic drugs were
assessed in RCC A498-RNAi cell line and untransfected A498 cell line by the MTT assay method. Cells
were plated into 96-well plates, adjusted to 4 × 103 cells / well, and cultured in DMEM +10% FBS
medium for 24 h before changing the culture medium. Gradient concentrations of different
chemotherapeutic drugs (Table 1) were added into cell culture plates. After 24 h, 10μl MTT were added
and co-cultured with cells for 6 h at 37℃. The optical density (OD) values, including the cells added with
and without chemotherapeutic drugs, were read at 570 nm using MultiSkan FC ELISA device. IC50
values were calculated by the median effect equation.
Data analysis Statistical comparisons between RCC A498 cell and RCC A498-RNAi cell line were
performed using the Student’s t-test using SPSS 10.0 software. P< 0.05 was considered statistically
significant.
RESULTS
MDR1 mRNA level in RCC A498-RNAi cell To confirm the most effective RNAi sequence, the
MDR1 mRNA level was detected by real-time PCR method and repeated for 3 times in untransfected
RCC A498 cell, RCC A498 cells transfected with empty vector and with different shRNA recombinant
plasmids. As shown in Figure 1, almost all RCC A498 cells transfected with different MDR1-targeted
RNAi recombinant plasmids expressed a lower level of MDR1 mRNA, compared to the RCC A498 cells
untransfected and transfected with empty vector and random RNAi recombinant plasmid. And the
shRNA-1 sequence had the highest interference efficiency (P = 0.001<0.01) in these three shRNA
sequences.
Establishment of RCC A498-RNAi cell line To produce the most effective RCC A498-RNAi cell
line, shRNA-1 recombinant plasmid was packaged into lentivirus and the RCC A498 cells were infected
by the lentiviral fluid. 96h after infection, RCC A498 cells were observed under microscope. The healthy
RCC A498-RNAi cells were spindle-shaped, cytoplasm-enriched, close to 100% of green fluorescent
protein expression and 90% of attached cells. Then single cell suspension solution was prepared and
cloned. Three cell clones were selected of the strongest green fluorescent protein expression. Through
detecting the MDR1 mRNA levels of these three cell clones, the cell clone-3 was selected with the
highest RNA interference efficiency to construct stable RCC A498-RNAi cell line for the following
experiments (Figure 2).
MDR1 protein expression in stable RCC A498-RNAi cell The selected stable RCC A498-RNAi
cell (clone-3) and control cell (RCC A498 cell transfected by random RNAi recombinant plasmid,
clone-n)were repeatedly cryopreservated and recovered. Three generations of clone-3 and clone-n
(P1,P2,P3) were randomly selected to be detected of the MDR1 protein expression level using grey
values analysis of Western-blot detection results. As shown in Figure 3 and Table 2, MDR1 protein
expression in stable RCC A498-RNAi cell line was significantly decreased (P<0.01). In addition, no
significant difference in MDR1 protein expression level was identified among different generations in the
same cell line (P = 0.959>0.01 for clone-n and P = 0.861>0.01 for clone-3).
Cell growth state of stable RCC A498-RNAi cell and untransfected RCC A498 cell To confirm
the growth state, the stable RCC A498-RNAi cell and untransfected RCC A498 cell were applied to
analyze the growth curve using MTT assay method. As shown in Figure 4, the growth curve of stable
RCC MDR1-RNAi A498 cell was almost consistent with that of the untransfected RCC A498 cell(P =
0.612> 0.05).
IC50 of different chemotherapeutic drugs To confirm the reversal of MDR by MDR1 gene
targeted RNAi, the stable RCC A498-RNAi cell and untransfected RCC A498 cell were applied to
analyze the sensitivity to different chemotherapeutic drugs using MTT assay method. As shown in Table
3, the IC50 values of a variety of chemotherapeutic drugs were significantly different between the stable
RCC A498-RNAi cell and untransfected RCC A498 cell. Compared to the untransfected RCC A498 cells,
IC50 of stable RCC A498-RNAi cell was decreased 83.25% of vincristine(VCR) (P<0.01), 81.79% of
paclitaxel(PTX) (P<0.01), 67.17% of gemcitabine(dFdC) (P<0.01), 71.73% of 5-fluororacil(5-FU)
(P<0.01), 83.37% of cisplatin(DDP) (P<0.01), 79.67% of etoposide(VP-16) (P<0.01), 86.47% of
cyclophosphamide(CTX) (P<0.01). But the IC50 for adriamycin (ADM) was not significantly different
between the two cell lines (P = 0.087>0.05).
DISCUSSION
Multidrug resistance (MDR) includes intrinsic and acquired drug resistance, intrinsic drug resistance
preexists in the naive tumor, acquired drug resistance is defined as the development of insensitivity to
chemotherapeutic drugs to which previous sensitivity existed. The mechanisms of MDR include
increased drug efflux or decreased influx by transporters, activation of detoxifying systems, activation of
DNA repair systems and evasion of apoptosis, which may operate singly or in concert. Although several
different mechanisms contribute to MDR in malignant carcinoma, the most widely studied mechanism is
P-glycoprotein(P-gp), which is a member of the ATP-binding cassette (ABC) superfamily of active
transporters and functions by effluxing drugs, reducing their intracellular concentrations and toxicity.9,10
RCC is widely known as one of the intrinsic and acquired MDR malignant carcinoma. Several
studies have suggested that P-gp is highly associated with the MDR of RCC, and MDR1 mRNA is
over-expressed and the degree of P-gp expression may correlate with tumor grade and resistance to
chemotherapeutic drugs in RCC.11,12Many compounds for reversing MDR have been developed to
modulate P-gp activity, such as dexverapamil, dexniguldipine or the cyclosporineD analogue PSC833,
and so on. But these drugs have been difficult to be used in clinical therapy, because of their serious side
effects.13 In recent years, many new methods of MDR reversal were developed, in which the RNAi is the
most important and deeply studied.
RNA interference (RNAi) is a conserved cellular mechanism where double-stranded RNA silences
the corresponding homologous cellular gene.14 The mechanism of RNAi-triggered mRNA destruction
represents a novel and powerful tool for the application in gene therapy of cancer. Two major discoveries
have highlighted its potential application in gene therapy: the synthetic small interfering RNA (siRNA)
resulted in efficient gene silencing, and short hairpin RNA (shRNA) expressed from RNA promoters
within expression vectors.15-17 In MDR reversal, the modulation by transient expression of
MDR1-targeted siRNA has been reported with the short duration and the partial reduction of P-gp.18,19
Stable expression of shRNA targeting MDR1 gene was extensively used for the more effective against
P-gp from vector that permit sustained expression. Effective shRNA gene silencing with lentiviral vector
has been demonstrated in cycling, primary immune, embryonic stem cells, and transgenic mice. 20,21 Then
the lentiviral vector probably offer the most promise in future preclinical studies for gene therapy.22
In this study, the RCC A498 cell line was selected for the greater P-gp level and three different
shRNAs targeting MDR1 gene were designed and synthesized, then the most effective shRNA of gene
silencing was selected and recombined into the expression vector to be packed by lentivirus. As a result,
the RCC A498-RNAi cell line was produced and the MDR1 mRNA
level, P-gp exprsssion and IC50 of
different drugs was examined. The effect of RNAi on MDR reversal was assessed by comparisons of
IC50 values between the primary RCC A498 cell line and the RCC A498-RNAi cell line. In the RCC
A498-RNAi cell, MDR1 mRNA level was highly reduced, P-gp expression was almost completely
abolished and the IC50 was highly decreased. Thus, pronounced MDR reversal effect of MDR1-targeted
RNAi could be expected by stable transfection of shRNA-containing expression vectors. This work
presented the evidence for silencing MDR associated gene by shRNA in MDR RCC cell, which
represents a novel approach for the treatment of RCC. The utilized MDR-targeted shRNA construct used
in this study not only appear to be effective laboratory tool, but also have implications for the prevention
and
reversal of MDR to prolong the survival time in certain cases by gene therapeutic approaches. The
only unsuccessful reversal of ADM resistance suggested that other mechanisms of MDR existed in RCC,
but the main MDR-associated gene is still the MDR1.
But it is important to note that the MDR cell line used in this study is highly resistant to multidrug
and exhibit an enormously enhanced MDR1 mRNA expression level. But in the clinical situation, a
two-fold or three-fold increased resistance level is already suffcient to inhibit a successful antineoplastic
drug-based cancer therapy. And it is not known whether the RNAi is same effective in clinical condition.
Additional problems may arise by the physiological expression of P-gp in several epithelial and
endothelial cells, suggesting that P-gp plays an important role in the blood barrier and is crucial for
limiting the potential toxicity of many drugs. So for clinical usage, many problems have to be solved, e.g.
delivery of shRNA, specific for cancer cells and effective sustained gene silencing.
Acknowledgements
We would like to thank Dr Zhang Jun and Dr Sun Jun-zhong and Dr Du Nan for their assistance in
this study.
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Table 1. The gradient concentrations of different chemotherapeutic drugs for IC50 detection
Drugs
Cells
VCR
RCC A498 cell
32
16
8
4
2
1
RCC A498-RNAi cell
3.2
1.6
0.8
0.4
0.2
0.1
RCC A498 cell
32
16
8
4
2
1
RCC A498-RNAi cell
3.2
1.6
0.8
0.4
0.2
0.1
RCC A498 cell
128
64
32
16
8
4
RCC A498-RNAi cell
64
32
16
8
4
2
RCC A498 cell
32
16
8
4
2
1
RCC A498-RNAi cell
3.2
1.6
0.8
0.4
0.2
0.1
RCC A498 cell
32
16
8
4
2
1
RCC A498-RNAi cell
3.2
1.6
0.8
0.4
0.2
0.1
RCC A498 cell
160
80
40
20
10
1
RCC A498-RNAi cell
32
16
8
4
2
1
RCC A498 cell
32
16
8
4
2
1
RCC A498-RNAi cell
3.2
1.6
0.8
0.4
0.2
0.1
RCC A498 cell
3.2
1.6
0.8
0.4
0.2
0.1
RCC A498-RNAi cell
3.2
1.6
0.8
0.4
0.2
0.1
PTX
dFdC
5-Fu
DDP
VP-16
CTX
ADM
Final drug concentration in the well (mg/l)
VCR: vincristine, PTX: paclitaxel, dFdC: gemcitabine, 5-FU: 5-fluororacil, DDP: cisplatin, VP-16: etoposideM, CTX:
cyclophosphamide, ADM: adriamycin
Table 2. Grey value of MDR1 protein expression in stable RCC A498- RNAi cell line of different generations
MDR-1
GAPDH
MDR-1/GAPDH
Clone-n-P1
269915.41
293110.37
0.92
Clone-n-P2
271650.75
305865.52
0.89
Clone-n-P3
244334.83
310140.43
0.79
X±S
0.87±0.07
P
0.959
Clone-3-P1
84454.97
313988.34
0.27
Clone-3-P2
82177.86
311669.38
0.26
Clone-3-P3
71886.01
319119.11
0.23
X±S
0.25±0.02
P
0.861
t
14.480
P
0.000
The clone-3 is the stable RCC A498-RNAi cell and clone-n is the RCC A498 cell trandfected by shRNA-neg. Both of
these cell lines were repeatedly cryopreservated and recovered. Three generations of clone-3 and clone-n (P1,P2,P3)
were randomly selected to be detected of the MDR1 protein expression level using grey values analysis of WB results.
Table 3. IC50 values of different chemotherapeutic drugs in different RNAi cells
Drugs
VCR
PTX
dFdC
5-Fu
DDP
VP-16
CTX
ADM
(mg/l)
(mg/l)
(mg/l)
(mg/l)
(mg/l)
(mg/l)
(mg/l)
(mg/l)
6.14±0.60
5.38±0.12
48.42±1.10
6.40±1.17
5.76±0.40
46.88±0.29
7.24±0.54
1.09±0.21
1.03±0.13
0.98±0.10
15.89±0.59
1.81±0.20
0.96±0.12
9.53±0.75
0.98±0.05
0.80±0.08
83.25
81.79
67.17
71.73
83.37
79.67
86.47
26.40
t
14.405
49.759
45.151
6.710
19.889
81.056
20.059
2.253
P
0.000
0.000
0.000
0.003
0.000
0.000
0.002
0.087
Cells
RCC A498 cell
RCC
A498-RNAi cell
decreased
rate %
IC50 values of different chemotherapeutic drugs in stable RCC A498- RNAi cell and untransfected RCC A498 cell
were detected by MTT method and repeated for 3 times. The average values were calculated and shown in this table.
Statistical comparisons were performed using the Student’s t-test. P< 0.05 was considered statistically significant.
MDR1/β-actin
0.6
0.5
0.4
0.3
0.2
0.1
0
untransfected
transfected by
vector
transfected by
shRNA-neg
transfected by
shRNA-1
transfected by
shRNA-2
transfected by
shRNA-3
Figure 1. MDR1 mRNA levels in different RCC A498 cells were detected by real-time PCR method and repeated for 3
times. The average values were calculated and shown in this plot.And the shRNA-1 sequence had the highest
interference efficiency in these three shRNA sequences.
MDR1/β-actin
0.6
0.5
0.4
0.3
0.2
0.1
0
untransfected
transfected by
shRNA-neg
transfected by
shRNA-1 clone-1
transfected by
shRNA-1 clone-2
transfected by
shRNA-1 clone-3
Figure 2. MDR1 mRNA levels in different RCC A498 cell clones were detected by real-time PCR method and repeated
for 3 times. The average values were calculated and shown in this plot. The cell clone-3 had the highest RNA
interference efficiency.
Clone-n-P1 Clone-n-P2 Clone-n-P3
Clone-3-P1 Clone-3-P2 Clone-3-P3
MDR1→
GAPDH→
Figure 3. WB of MDR1 protein in stable RCC A498-RNAi cell line of different generations. The clone-3 is the selected
stable RCC A498-RNAi cell and clone-n is the RCC A498 cell trandfected by shRNA-neg. Both of these cell lines were
repeatedly cryopreservated and recovered. Three generations of clone-3 and clone-n (P1,P2,P3) were randomly selected
to be detected of the MDR1 protein using WB. GAPDH is the internal control.
OD(570-630)
0.7
0.6
0.5
A498 cell
0.4
A498-RNAi cell
0.3
0.2
0.1
0
0h
24h
48h
72h
96h
120h
144h
Figure 4. Growth curves of the stable RCC A498-RNAi cell and untransfected RCC A498 cell were constructed
according to the OD values using MTT assay method.