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Chinese Journal of Cancer
窑Original Article窑 Establishment and biological characteristics of
oxaliplatin­resistant human colon cancer cell lines Zhen Liu 1 , Meng Qiu 1 , Qiu­Lin Tang 2 , Ming Liu 1 , Nan Lang 2 , Feng Bi 1 1 Department of Medical Oncology ,West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China; 2 The Laboratory of Signal Transduction & Molecular Targeting Therapy, State Key Laboratory of Biotherapy, West China Hospital,Sichuan University, Chengdu, Sichuan 610041, P. R. China
揖Abstract铱 Background and Objective:
Methods:
Results:
Conclusions:
Key words: Colon cancer is one of the common types of cancer in China. Chemotherapy is an important treatment for colon cancer [1] . Oxaliplatin (L­OHP), a third­generation platinum­ based drug, is one of the first­line drugs in colon cancer treatment. It has been reported in some clinical trials that as the standard first­line treatment of advanced colon cancer, the FOLFOX regimen [L­OHP, 5­fluorouracil (5­FU) plus Correspondence to: Feng Bi; Tel: +86­28­85423609; Fax: +86­28­85423609; Email: [email protected] This paper was translated from Chinese into English by Translation and edited by Wei Liu on 2010­04­30. Medical Received: 2009­11­16; Accepted: 2010­02­10 Grant: National Natural Science Foundation of China
30971519) www.cjcsysu.cn
(No. 30901719, No. leucovorin (LV)] can significantly increase the response rate up to 54% and the median survival to nearly two years. However, when switching to second­line treatment as the first­line chemotherapy fails, the response rate drops to only 4% [2] . Failure of chemotherapy greatly results from the acquired or congenital multidrug resistance (MDR) against various chemotherapeutic agents in a few cancer cells [3] . Certain cancer cells survive the chemotherapy and proliferate infinitely, which finally causes the death of the patient. Some researches showed that cancer stem cells may play an important role in this drug­resistant course [4] . Hence, it is necessary to investigate the mechanism of L­OHP­resistance in colon cancer cells and provide new methods of overcoming this resistance in clinical practice. In this study, we used L­OHP as an in vitro inducer to establish two L­OHP­resistant human colon cancer cell lines and explore the mechanism of this resistance. 661
Chinese Journal of Cancer RPMI­1640 culture medium and fetal bovine serum (FBS) were purchased from Gibco BRL company; CCK8 from Dojindo company; L­OHP from Sanofi­Aventis company; 5­FU from Jinyao Amino Acid company (Tianjin); cisplatin (DDP) from Gejiu Bio­Pharmaceutical (Yunnan); paclitaxel (PTX) from Taiji company; epirubicin (EPI) from Pfizer company; etoposide
(VP­16) from Sunnyhope Pharmaceutical company (Sichuan); and vincristine (VCR) from Minsheng Pharmaceutical company (Hangzhou). The human colon cancer cell lines SW620 and lovo were preserved by our own lab. Oxaliplatin­resistant cell lines SW620/L­OHP and LoVo/L­OHP were induced by continuous exposure to L­OHP of low and gradually increased concentrations. When the cells were in logarithmic growth phase, L­OHP was added to the medium to a final concentration of 0.01 mg/L. After a 24­hour incubation, the old medium was discarded and fresh medium was added. Cells were passed when they were 80% confluent and L­OHP of 0.01 mg/L was then added. The concentration of L­OHP was gradually increased after the cells had grown stably. Finally, a cell line resistant to L­OHP of 0.2 mg/L was derived from SW620 and kept in complete medium containing L­OHP of 0.2 mg/L; a cell line resistant to L­OHP of 2 mg/L was derived from lovo and kept in complete medium containing L­OHP of 2 mg/L. Single cell suspension (8 伊 10 3 /mL, 100 滋L) was dispensed in a 96­well plate. After a 24­hour pre­incubation, the old medium was replaced by medium containing L­OHP of 10 different concentrations, which was replaced in turn with fresh medium after another 24­hour incubation. After 72 h, CCK8 was added for another 4­hour incubation. Then the absorbance ( value) at 490/630 nm was measured using a UV spectrophotometer. An effect­dose curve was drawn to calculate 50% inhibition concentration (IC50) and resistance index (RI). RI = IC 50 of drug­resistant cell line / IC 50 of parent cell line. 662
Drug­resistant cells were dispended in a 96­well plate (800 cells/well). Three wells were taken each day and CCK8 was added in for a 4­hour incubation. A value at 490/630 nm was measured using a UV spectrophotometer. This procedure was repeated for 6 continuous days. Growth curves were drawn to calculate population doubling time according to the Patterson formula. Single cell suspension was collected and washed with cold PBS for 3 times, then fixed with 70% ethanol at 4益 overnight. After centrifugation, the supernatant was discarded and the cells were washed with cold PBS twice. Then the cells were stained with propidium iodide (PI). Flow cytometry (FCM) was performed to analyze cell cycle. After 8% sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS­PAGE), proteins were transferred onto a PVDF membrane. The membrane was blocked at room temperature for 2 h and then washed. Then the membrane was incubated with 1 颐 500
diluted P glycoprotein ( P­gp ) antibody, 1 颐 500
diluted multidrug­resistant protein 1 (MRP1) antibody and 1 颐 500
diluted MRP2 antibody at 4益 overnight, washed with TBST and then incubated with secondary antibody at room temperature for 2 h. After washed with TBST, the membrane was analyzed using an Odyssey Infrared Fluorescence Detector. After dissociation and centrifugation, the cells were washed with PBS once and incubated with FITC­labeled anti­CD133 and PE­labeled anti­CD44 in an ice bath in dark for 45 min. Then the cells were washed with FACS buffer for 3 times. FCM was performed, with anti­mouse IgG being the isotype control. The significance of difference between two paired groups was determined by the Student 爷 s ­test using the 2010; Vol. 29 Issue 7 Chinese Journal of Cancer
SPSS17.0 software. A value of significant. < 0.05 was considered Two L­OHP­resistant cell lines SW620/L­OHP (resistant to L­OHP of 0.2 mg/L) and LoVo/L­OHP (resistant to L­OHP of 2 mg/L) were successfully induced by continuous exposure to L­OHP of low and gradually increased concentrations after 10 months for more than 100 cell passages. The cell lines were used for experiments after another 2 months of culture in drug­free medium. Figure 1 shows the morphologic differences between the resistant cells and their parent cells under light microscope. According to the results of CCK8 assay, the IC 50 of Figure 1
A
B
L­OHP for SW620/L­OHP cells was 0.337 mg/L, and RI was 21.06. After 2 months of drug­free culture, the IC 50 was 0.316 mg/L and RI was 19.750. Thus 93.78% drug­resistance was preserved. The IC 50 for lovo/L­OHP was 3.513 mg/L, and RI was 13.780. After 2 months of drug­free culture, the IC 50 was 3.162 mg/L and RI was 12.4. Thus 90.00% resistance was preserved. In addition, the SW620/L­OHP and LoVo/L­OHP cells grew stably after they had been frozen for 3 months and then thawed, and the IC 50 then was 0.337 mg/L and 3.513 mg/L, respectively. Thus freezing and thawing did not influence the drug­resistance of these cell lines. The SW620/L­OHP and LoVo/L­OHP cells also showed cross­resistance to 5­FU, VP­16, DDP, VCR and EPI to various degrees, but were still sensitive to PTX (Table 1). The growth curves of the parent cells and the drug­resistant cells (Figure 2) show that the drug­resistant Morphologic appearance of parental cells and L鄄OHP鄄 resistant cells
C
D
SW620 cells (A) and lovo cells (C) have homogeneous size and are polygonal; SW620/L鄄OHP cells (B) and lovo/L鄄 OHP cells (D) are irregular, with large nuclei, and
some of them are enlarged ( 伊40).
cells grew more slowly. The population doubling time of SW620/L­OHP cells was significantly longer than that of SW620 cells by 5.41 h ( = 0.006), and the population doubling time of LoVo/L­OHP cells was significantly longer than that of lovo cells by 3.34 h ( = 0.005). After gaining drug­resistance by L­OHP induction, the cells proliferated more slowly in the logarithmic phase. The results of FCM showed an increase in the proportion of cells in G0 /G1 phase (38.9% vs. 29.0% , = 0.003) and a decrease in the proportion in G 2/M
phase
(15.6% vs. 31.5% , < 0.001) in SW620/L­OHP cells compared with SW620 cells, and an increase in the proportion of cells in G0 /G1 phase (61.2% vs. 50.1% , = 0.001) and a decrease in the proportion in G 2/M
phase (10.6% vs. 18.1% , = www.cjcsysu.cn
0.001) in LoVo/L­OHP cells compared with LoVo cells. All these changes were of statistical significance (Figure 3). Western blot analysis showed that comparing with their parental cells, the expression of MRP2 protein in the resistant cells was up­regulated, while those of P­gp and MRP1 had no significant change (Figure 4). The results of FCM showed that comparing with their parental cells, CD133 was overexpressed (9.6% vs. 4.6%)
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Chinese Journal of Cancer
Drug
L鄄 OHP
5鄄 FU
VP鄄 16
PTX
VCR
DDP
EPI
IC50 (mg/L)
SW620
0.016 依 0.004
0.018 依 0.005
0.175 依 0.084
0.015 依 0.001
0.014 依 0.002
0.122 依 0.065
0.004 依 0.001
RI
SW620/L鄄 OHP
0.337 依 0.084
0.105 依 0.017
0.400 依 0.027
0.015 依 0.002
0.027 依 0.007
0.634 依 0.168
0.034 依 0.003
IC50 (mg/L)
P
21.06
5.83
2.29
1.00
1.93
5.20
8.50
LoVo
0.255 依 0.025
0.085 依 0.007
1.241 依 0.251
0.738 依 0.142
0.289 依 0.027
0.525 依 0.113
0.252 依 0.020
0.022
0.001
0.011
0.815
0.038
0.008
< 0.001
LoVo/L鄄 OHP
3.513 依 0.329
0.249 依 0.018
2.354 依 0.484
0.847 依 0.114
0.478 依 0.058
3.188 依 0.725
1.271 依 0.041
RI
P
13.78
2.93
1.90
1.15
1.65
6.07
5.04
< 0.001
< 0.001
0.024
0.359
0.007
0.003
< 0.001
L鄄OHP, oxaliplatin; 5鄄FU, 5鄄fluorouracil; VP鄄16, etoposide; PTX, paclitaxel; VCR, vincristine; DDP, cisplatin; EPI, epirubicin. The sensitivity of colon cancer cell lines to seven
antitumor drugs was evaluated using the CCK8 assay as described in methods. The 50% inhibition concentration (IC50) of antitumor drugs was calculated. The IC50 values are
presented as mean 依 standard deviation. SW620/L鄄OHP and LoVo/L鄄OHP cells exhibit moderate cross鄄resistance to cisplatin and epirubicin, but are sensitive to paclitaxel.
while CD44 level did not significantly change (0.2% vs. 0.2% ) in SW620/L­OHP cells, and similarly CD133 was overexpressed (0.9% vs. 0.3% ) while CD44 level did not significantly change (0.2% vs. 0.2% ) in loVo/L­OHP cells (Figure 5). the only one p latinum­based drug that has anti­tumor activity in colon cancer. Its cytotoxicity is resulted from inhibition of DNA synthesis by cross­linking adjacent guanine bases or adjacent guanine and adenine [5] . Resistance occurs when this DNA damage is repaired by nucleotide excision repair (NER) mechanism [6] . However, recent
studies
on
the
mechanism
of
platinum drug­resistance mainly focus on cisplatin, but seldom on L­OHP. Studies on the establishment of in vitro L­OHP­resistant model are also rarely seen. Therefore, in
Oxaliplatin, a third­generation platinum­based drug, is 2.5
LoVo cells
LoVo/L鄄 OHP cells
2.0
SW620 cells
Figure 2 Growth curves of SW620,
SW620/L鄄OHP, LoVo and LoVo/L鄄OHP
cells
SW620/L鄄 OHP cells
1.5
1.0
The curves of L鄄OHP鄄resistant cells rise up slowly. The
doubling time of SW620 cells (银) is 29.53 h and that
of SW620/L鄄OHP cells (伊) is 34.94 h, on the other hand,
the doubling time of loVo cells (音) is 24.77 h and that
of loVo/L鄄OHP cells (姻) is 28.11 h.
0.5
0
A
100
1
CV G21 S0
80
2
CELL CYCLE
DATA
Mean G1=84.9
3
Time (/d)
B
100
4
CV G21 S0
5
CELL CYCLE
DATA
Mean G1=137.7
CV G1 = 8.4
80
60
Mean G2=148.6
60
Mean G2=241.0
40
% G2 = 18.1
40
% G2 = 10.6
%S
= 31.8
20
Chi Sq. = 1.8
0
% G1 = 50.1
CV G2 = 8.3
20
0
G2/G1 =1.750
0 64 128 192 256 320 384 448 512
DNA Content
Figure 3
CV G1 = 5.5
% G1 = 61.2
CV G2 = 5.5
%S
= 28.2
G2/G1 =1.750
Chi Sq. = 1.5
0 64 128 192 256 320 384 448 512
DNA Content
6
C
35
CV G21 S0
30
CELL CYCLE
DATA
Mean G1=122.2
CV G1 = 8.6
25
% G1 = 29.0
20
Mean G2=213.9
CV G2 = 8.6
15
% G2 = 31.5
10
%S
5
0
= 39.5
G2/G1 =1.750
Chi Sq. = 1.0
0 64 128 192 256 320 384 448 512
DNA Content
D
120
100
80
60
CV G21 S0
CELL CYCLE
DATA
Mean G1 =118.2
CV G1 = 7 .9
% G1 = 38 .9
Mean G2 =286.9
CV G2 = 7 .9
% G2 = 15 .6
40
20
0
% S
G2/G1
= 45. 5
=1 .750
Chi Sq. = 0 .9
0 64 128 192 256 320 384 448 512
DNA Content
Cell cycle distribution of parental cells and L鄄 OHP鄄resistant cells
Cell cycle determined by FCM shows a decrease of the proportion of cells in G2/M phase and an increase of the proportion in G0 /G1 phase in SW620/L鄄 OHP cells (D)
and LoVo/L鄄 OHP cells (B) compared with SW620 cells (C) and LoVo cells (A).
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2010; Vol. 29 Issue 7 Chinese Journal of Cancer Cancer
MRP2
Figure 4 Expressions of P鄄glycoprotein (P鄄gp),
multidrug鄄resistant protein 1 (MRP1) and MRP2
in SW620, SW620/L鄄OHP, LoVo and LoVo/L鄄
OHP cells.
MRP1
Up regulation of MRP2 is observed in SW620/L鄄 OHP and LoVo/L鄄
OHP cells compared with SW620 and LoVo cells, while the
expressions of P鄄 gp and MRP1 had no differences between
parental cells and oxaliplatin鄄 resistant cells.
P鄄 gp
茁鄄 actin
A
G: SSvsFS
1
A
3
E
2
PMT3 LOG
G: SSvsFS
1
.1
Figure 5
G: SSvsFS
1
4
.1
3
B
A
PMT3 LOG
1000
2
3
F
B
.1
PMT3 LOG
G: SSvsFS
1
4
1000
2
3
.1
B
PMT3 LOG
C
G: SSvsFS
4
1000
2
3
G
2
C
.1
PMT3 LOG
G: SSvsFS
1
4
1000
1
3
.1
A
PMT3 LOG
C G: SSvsFS
1
3
4
1000
2
D
.1
PMT3 LOG
H G: SSvsFS
1
3
4
1000
2
.1
B
PMT3 LOG
4
1000
2
4
1000
Expression of CD133 and CD44 in SW620 and SW620/L鄄OHP cells
The positive rates of CD133 were 4.6% in SW620 cells (A), 9.6% in SW620/L鄄 OHP cells (B), 0.9% in LoVo/L鄄 OHP cells (E), and 0.3% in LoVo cells (F); the
positive rates of CD44 were 0.2% in SW620 cells (C), SW620/L鄄 OHP cells (D), LoVo/L鄄 OHP cells (G), and LoVo cells (H). The expression of CD133 is increased
in oxaliplatin鄄 resistant cell lines compared with parental cells while CD44 expression shows no difference between parental cells and oxaliplatin鄄 resistant cells.
the current study, we established two in vitro L­OHP­resistant cell models to provide the basis for further studies on the mechanism of L­OHP­resistance in colon cancer. One of the major approaches of studying the mechanism of MDR in tumor cells is to establish drug­resistant cell lines in vitro. In this study, it took 10 months for us to successfully establish two L­OHP­resistant human colon cancer cell lines SW620/L­OHP and LoVo/L­OHP through continuous exposure to L­OHP of low www.cjcsysu.cn
and gradually increased concentrations. These two cell lines have shown stable drug­resistance and consistent biological characteristics. Cell cycle is arrested in G2 /M phase by platinum­based drugs through their inhibitory effect on cyclin­dependent . reported that kinase (CDK) activity. Carole Voland cells were arrested in G 2 phase by L­OHP platinum as a result of inhibition of G2 /M phase transition and thus inhibition of cell division [7] , an increase in cells in G 2/M phase finally led to apoptosis, which was showed as
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Chinese Journal of Cancer cytotoxicity of the drug [8] . In the current study, an increase in the proportion of cells in G 0/G 1 phase and S phase as well as a decrease in the proportion in G2 /M phase were observed in the two L­OHP­resistant cell lines. Activation of DNA repair mechanisms in the resistant cells might be the possible reason of these changes, which helped the cells pass through G 2 phase to M phase and subsequently divide and proliferate, which in turn resulted in a shift of cell cycle distribution. These findings may provide the basis for the selection of cell cycle­specific chemotherapeutic drugs for L­OHP­resistant patients in clinical treatment. The L­OHP­resistant cell lines also showed cross­resistance to platinum­based drugs such as DDP, as well as to some other anti­cancer drugs that act via different mechanisms. Some researchers have suggested a grading system depending on RI: low (< 5), medium (5­15) and high (> 15) [9] . According to this system, SW620/L­OHP and lovo/L­OHP cells both showed medium resistance to DDP and EPI and low resistance to VP­16 and VCR, but no resistance to PTX. In addition, SW620/L­OHP and LoVo/L­OHP cells showed medium and low resistance, respectively, to 5­FU. One common mechanism of drug­resistance is a decrease in the accumulation of platinum compounds in the cells, which is thought to be closely related to the exporter ATP7B but not associated with P­gp and MRP1 [10] . Liedert . [7] demonstrated an increase in both MRP2 mRNA and protein levels in cells resistant to platinum­based drugs, but no significant change in mRNA and protein levels of other ATP­binding cassette transporters, which is consistent with the L­OHP­resistant cell lines in our present study. It has also been reported that it is the ATP­dependent GS­X pump instead of P­gp that accounts for the enhanced exportation of platinum drugs in the platinum drug­resistant cells. MRP (ABCC) proteins are membrane transporters that can use the energy of ATP hydrolysis to carry out their
野drug pump 冶 function [11] . These proteins are 15% homologous to P­gp in amino sequence but do not act synergistically with P­gp in term of their 野 drug pump冶
function. P­gp acts on a variety of drugs, including doxorubicin (ADM), VCR, VP­16, PTX, and anthracene derivatives [12] , whereas MRP transfers cytotoxic drugs that are able to bind reduced glutathione (GSH), such as ADM, VP­16, and VCR [13] , and alters their intracellular distribution [14] . Resistance to EPI, VP­16, and VCR [15­17] is P­gp/MRP­ mediated; however, neither P­gp nor MRP1 was up­regulated in LoVo/L­OHP and SW620/L­OHP cells, which suggests that the cross­resistance to EPI, VP­16, and VCR in these two cell lines was possibly MRP2­mediated. Some researches showed that overexpression of MRP2 resulted in cross­resistance to VP­16, DDP, ADM, and EPI [18] . MRP2­mediated resistance to certain drugs is associated with GSH level, and the MRP2­mediated resistance to 666
VP­16, ADM, and DDP can be reversed by the GSH inhibitor BSA. Moreover, in our present study, the L­OHP­resistant cells also demonstrated low to medium resistance to 5­FU. 5­FU­resistance is believed to result from several mechanisms [19] , including changes in the activities of thymidylate synthetase (TS), dihydropyrimidine dehydrogenase (DPD) and folic acid metabolism­related enzymes, DNA mismatch repair (MMR) [20] , and so on, but not associated with the expressions of P­gp and MRP. These imply that aberration of DNA repair mechanisms may also play an important role in 5­FU­resistance. In addition, the two L­OHP­resistant cell lines in our study showed no cross­resistance to PTX, which may be because PTX­ resistance is mediated by P­gp rather than by MRP2 [21,22] . This provides an important basis for selecting sensitive second­line drugs when patients fail to respond to L­OHP as the first­line drug in clinical treatment. Cancer stem cells are stem cell­like cells that exist in the hematopoietic system or some solid tumors. The cancer stem cell hypothesis suggests that during chemotherapy tumor size reduces but a small number of stem cells survive, which cause relapse and metastasis [23] . The self­protective mechanism of stem cells against cytotoxic drugs remains to be elucidated. But it is currently believed that the major mechanisms involve the drug pump activity of ABC transporters [24] , overexpression of anti­apoptotic genes, DNA repair, increase in cells in G 0/G 1 phase, and slowdown of the proliferation of stem cells to evade the action of chemotherapeutic drugs [25] . Stem cells enriched from U87MG cells overexpress CD133 and are cross­resistant against ADM, VP­16, carboplatin and BCNU [26] . In our present study, MRP2 up­regulation, slowdown of cell proliferation, and an increase in G0/G1 phase cells were observed in the L­OHP­resistant cell lines, which implied a stem cell phenotype. Furthermore, the proportion of CD133 + cells also increased as the cells became resistant to L­OHP. These all may suggest an important role of cancer stem cells in the development of MDR in the L­OHP­resistant cell lines. Thus it is possible to sensitize the tumor tissue to L­OHP and reverse its resistance by using stem cell­targeting therapy. In the current study, we successfully established two L­OHP­resistant
human
colon
cancer
cell
lines SW620/L­OHP and lovo/L­OHP. They are stable in cell passage and drug­resistance, and their biological characteristics are highly consistent. These cell lines may serve as ideal models for the study of the mechanism and the way of reversal of L­OHP­resistance in colon cancer.
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