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Concentration-dependent collateral sensitivity of cisplatin-resistant gastric cancer cell sublines
BBRC Biochemical and Biophysical Research Communications 328 (2005) 618–622 www.elsevier.com/locate/ybbrc
Concentration-dependent collateral sensitivity of cisplatin-resistant gastric cancer cell sublines Haidong Xua, Seok-Min Choib, Chun-San Anb, Young-Don Minc, Kweon-Cheon Kimc, Kyung-Jong Kimc, Cheol-Hee Choia,b,* b
a Research Center for Resistant Cells, Chosun University, Gwangju 501-759, Republic of Korea Department of Pharmacology, Chosun University Medical School, Gwangju 501-759, Republic of Korea c Department of Surgery, Chosun University Medical School, Gwangju 501-759, Republic of Korea
Received 26 December 2004 Available online 13 January 2005
Abstract The cisplatin-resistant gastric cancer cell sublines, SNU-601/Cis2 and /Cis10, were 49 and >530 times more resistant to cisplatin, respectively, compared with the drug-sensitive cells, SNU-601/WT. The SNU-601/Cis2 showed cross-resistance to carboplatin, heptaplatin, doxorubicin, mitomycin C, and 5-fluorouracil compared with the SNU-601/WT whereas the SNU601/Cis10 displayed collateral sensitivity to these drugs with the exception of cisplatin compared with the SNU-601/Cis2, suggesting that the cross-resistance and collateral sensitivity of cisplatin-resistant gastric cancer cells are dependent upon cisplatin concentrations. Altered expression of the antioxidant and transporter genes (metallothionein, catalase, superoxide dismutases, P-glycoprotein, and the breast cancer resistance protein) was involved in these phenotypes of the cisplatin-resistant gastric cancer cell lines. 2005 Elsevier Inc. All rights reserved. Keywords: Cisplatin; Gastric cancer; Cross-resistance; Collateral sensitivity; Antioxidant; Transporter
Gastric cancer is the most frequently diagnosed and the second leading cause of cancer-related death in Korea. However, the treatment response rate has been <30% and cases of complete remission are rare. Several combination chemotherapy regimens such as FAM (5-fluorouracil, adriamycin, and mitomycin C) have been used to improve the treatment outcomes [1]. In a nonrandomized Phase II study on advanced gastric cancer, the FAM regimen achieved an objective partial response rate of 42% [2]. Some cisplatin-based combination chemotherapy regimens have shown high response rates [3,4]. However, the intrinsic or acquired resistance to cisplatin reduces its efficacy [5]. The
*
Corresponding author. Fax: +82 62 232 4045. E-mail address: [email protected] (C.-H. Choi).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.01.015
mechanism of resistance to cisplatin includes reduced cisplatin accumulation, such as the canalicular multispecific anion transporter cMOAT (MRP2/ABCC2) and the copper-transporting P-type ATPase ATP7B [6,7], an increased intracellular thiol level, such as glutathione or metallothionein (MT) [8–10], and increased DNA repair [11]. The cisplatin-resistant gastric cancer cell lines, SNU601/Cis2 and /Cis10, which were selected by chronic exposure to 2 and 10 lg/ml cisplatin, respectively, were 49 times and >530 times more resistant to cisplatin than the parental SNU-601 cells. SNU-601/Cis2 showed cross-resistance to carboplatin, heptaplatin, doxorubicin, mitomycin C, and 5-fluorouracil compared with SNU-601/WT whereas SNU-601/Cis10 displayed collateral sensitivity to these drugs, except for cisplatin compared with SNU-601/Cis2.
H. Xu et al. / Biochemical and Biophysical Research Communications 328 (2005) 618–622
This paper reports the molecular mechanisms of the cross-resistance and collateral sensitivity of two cisplatin-resistant gastric cancer sublines to anticancer drugs, which are dependent upon cisplatin concentrations.
Materials and methods Cell culture and the selection of the gastric cancer cell sublines for cisplatin resistance. The human gastric cancer cell line, SNU-601, was obtained from the Cancer Research Center in Seoul National University (South Korea). The cells were cultured in RPMI 1640 (GibcoBRL, Grand Island, NY, USA) supplemented with 10% FBS (Sigma Chemical, St. Louis, MO, USA). The cells were maintained as a monolayer culture and were subcultured at confluence. The cisplatinresistant gastric cancer cell subline was selected by exposing them to gradually increasing cisplatin concentrations ranging from 200 ng/ml (approximate IC50 value) to 2 and 10 lg/ml on an intermittent dosage schedule. Cytotoxicity assay. The in vitro cytotoxicity of the drugs was determined using an MTT (Sigma Chemical, St. Louis, MO, USA). The 50% inhibitory concentration (IC50) for a particular agent was defined as the drug concentration that causes a 50% reduction in the number of cells compared with the untreated control. The IC50 values were determined directly from the semilogarithmic dose–response curves. All the cytotoxic drugs with the exception of heptaplatin (Sunpla, SK Pharmaceutical, Seoul, Korea) were obtained from the Sigma Chemical (St. Louis, MO, USA). RNA extraction and reverse transcription-polymerase chain reaction assay. The total RNA was prepared from the cells using a RNeasy mini kit (Quiagen, Hilden, Germany). The steady-state levels of each mRNA transcript were determined using a reverse transcriptionpolymerase chain reaction (RT-PCR) assay, where the expression levels were normalized to those of the b-actin expression level as a control. Information on the primer pairs and corresponding genes is summarized in Table 1. The RNAs from each sample were reverse transcribed using units of Moloney murine leukemia virus reverse transcriptase (GibcoBethesta Research Laboratory, Grand Island, NY, USA) and the oligo(dT) primer for 1 h at 37 C. The resulting cDNA was diluted 1:5 with water and was amplified with 2.5 U Taq polymerase (Promega, Madison, WI, USA) and 10 pmol of each primer in a GeneAmp PCR9600 (Perkin-Elmer, Boston, MA, USA) at each PCR condition shown in Table 1. After the final cycle, all the PCR products were subjected to a final extension for 5 min at 72 C. For quantitation,
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5 lCi of [a-32P]dCTP was added to each reaction mixture. The PCR products were electrophoresed on 7.5% nondenaturing polyacrylamide gels. The bands were scanned using a densitometer. Determination of the level of reactive oxygen species using a fluorometric probe. Dichlorofluorescein (DCFH) was used to measure the concentration of reactive oxygen species (ROS). After 2 0 ,7 0 -dichlorofluorescein diacetate (DCFH-DA) crosses the membrane, it is deesterified to DCFH, which is oxidized to fluorescent DCF by the ROS [12]. Phosphate-buffered saline containing 1 · 105/ml cells was incubated with 30 lg/ml paraquat, an intracellular superoxide generator, in the presence of 1 lM DCFH-DA at 37 C for 2 h. After incubation, the DCF fluorescence intensity was determined using a fluorometer at an excitation and emission wavelength of 485 and 530 nm, respectively. Statistical analysis. Statistical significance of the data was determined using StudentÕs t test. P values <0.05 were considered significant.
Results Sensitivity of cisplatin-resistant gastric cancer cell sublines to various anticancer drugs Of the eight human gastric cancer cell lines (SNU-1, SNU-5, SNU-16, SNU-484, SNU-601, SNU-620, SNU-638, and SNU-668), the SNU-601 cells were chosen owing to their having the lowest MT mRNA expression level. The cisplatin-resistant gastric cancer cell lines, SNU-601/Cis2 and /Cis10, which were selected by the chronic exposure to 2 and 10 lg/ml cisplatin, respectively, were 49 and >530 times more resistant to cisplatin than the parental SNU-601 cells (SNU-601/WT), respectively (Fig. 1). As shown in Table 2, the SNU601/Cis2 cells showed cross-resistance to carboplatin (10.68-fold), heptaplatin (18.49-fold), doxorubicin (44.95-fold), mitomycin C (56.77-fold), and 5-fluorouracil (64.20-fold) compared with SNU-601/WT whereas SNU-601/Cis10 displayed collateral sensitivity to these drugs with the exception of cisplatin compared with the SNU-601/Cis2 cells (Table 3).
Table 1 PCR primers used in this study Gene
Length of PCR products (bp)
No. of cycles
Hot start
Denaturation
Annealing
Extension
MDR 1 MRP LRP BCRP MT ATB7B sod-1 sod-2 cat b-Actin
295 355 390 475 220 833 462 260 440 501
30 22 23 22 21 22 20 22 23 17
94 C, 12 min
94 C, 30 s
65 C, 53 C, 53 C, 53 C, 57 C, 53 C, 53 C, 57 C, 53 C, 53 C,
72 C, 1 min
30 s 30 s 30 s 30 s 30 s 30 s 30 s 30 s 30 s 30 s
MDR1, multidrug resistance 1; MRP, multidrug resistance-associated protein; LRP, lung resistance-related protein; BCRP, breast cancer resistance protein; MT, metallothionein; ATB7B, copper-transporting ATPase; sod-1, Cu/Zn superoxide dismutase; sod-2, Mn superoxide dismutase; cat, catalase; b-actin (PCR control).
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H. Xu et al. / Biochemical and Biophysical Research Communications 328 (2005) 618–622
Fig. 1. Sensitivity of the SNU-601/WT cells and their cisplatinresistant cell sublines to cisplatin. Cytotoxicity was determined using an MTT assay.
Table 2 Comparison of the sensitivity of the SNU-601/WT and SNU-601/Cis2 cells to various anticancer drugs Drug
SNU-601/WT, IC50 (lg/ml)
SNU-601/Cis2, IC50 (lg/ml)
Relative resistance (fold)
Cisplatin Carboplatin Heptaplatin Mitomycin C Doxorubicin 5-Fluorouracil
0.19 7.5 0.76 0.046 0.015 0.5
9.2 80.1 14.0 2.7 0.67 32.1
48.75 10.68 18.49 56.77 45.02 64.20
Table 3 Comparison of the sensitivity of the SNU-601/Cis2 and /Cis10 cells to various anticancer drugs Drug
SNU-601/Cis2, IC50 (lg/ml)
SNU-601/Cis10, IC50 (lg/ml)
Relative resistance (fold)
Cisplatin Carboplatin Heptaplatin Mitomycin C Doxorubicin 5-Fluorouracil
9.2 80.1 14.0 2.7 0.67 32.1
>100 42.2 5.0 0.2 0.06 19.5
>10.87 0.53 0.36 0.07 0.09 0.61
Expression profiles of the resistance-related genes in the SNU-601/WT cells and their cisplatin-resistant cell sublines SNU-601/Cis2 and /Cis10 In this study, the expression profiles of the resistancerelated genes were examined in the SNU-601/WT cells and their cisplatin-resistant cell sublines, and compared. The resistance-related genes examined included metallothionein, catalase, copper/zinc and manganese superoxide dismutases, P-glycoprotein, the lung resistance protein, the multidrug resistance-associated protein, the breast cancer resistance protein, and ATP7B. With the exception of the lung resistance protein, the multidrug resistance-associated protein, and ATP7B, the steady-state mRNA levels of the other genes in the
Fig. 2. Expression profiles of the drug efflux pumps and antioxidant enzymes in the SNU-601/WT cells and their cisplatin-resistant cell sublines. Gene expression was determined using a RT-PCR assay. b-Actin was used as the control for RNA. Abbreviations for gene names are described in Table 1.
SNU-601/Cis2 cells were higher than in the SNU-601/ WT cells whereas the expression levels in the SNU601/Cis10 cells were lower than those of the SNU-601/ Cis2 cells (see Fig. 2). Comparison of scavenging activity against paraquatinduced ROS between SNU-601/WT and its cisplatinresistant cell sublines The scavenging activities against paraquat-induced ROS were determined using DCFH-DA. After the incubation of cells with 30 lg/ml paraquat in the presence of 1 lM DCFH-DA at 37 C for 2 h, the ROS-scavenging effect of the SNU-601/Cis2 cells were higher than those of either the SNU-601/WT or SNU-601/Cis10 cells (Fig. 3).
Discussion The cisplatin analogs are among the most active and widely used cytotoxic anticancer drugs. However, the acquisition or presence of resistance significantly undermines the curative potential of these drugs against many malignancies [5]. In this study, two cisplatin-resistant gastric cancer cell sublines, SNU-601/Cis2 and /Cis10, were found to be resistant to cisplatin and various anticancer drugs including carboplatin, heptaplatin, mitomycin C, doxorubicin, and 5-fluorouracil. Interestingly, although the SNU-601/Cis10 cells, which were more than 10 times more resistant to cisplatin than the SNU-601/Cis2 cells, they were more sensitive to other
H. Xu et al. / Biochemical and Biophysical Research Communications 328 (2005) 618–622
Fig. 3. Generation of ROS by paraquat in the SNU-601/WT cells and their cisplatin-resistant cell sublines. Phosphate-buffered saline containing 1 · 105/ml cells was incubated with 30 lg/ml paraquat, an intracellular superoxide generator, in the presence of 1 lM DCFH-DA at 37 C for 2 h. The fluorescence intensity was determined using a spectrofluorometer with an excitation wavelength at 485 nm and an emission wavelength at 530 nm. *P < 0.05.
anticancer drugs than the SNU-601/Cis2 cells. The collateral sensitivity indicates that different cellular alterations in the two cisplatin-resistant gastric cancer cell sublines would be induced during the process of developing cisplatin resistance. Extensive resistance, cross-resistance, and collateral-sensitivity patterns have been established with most sublines of the various drug-resistant cells [13–16]. This is the first report on the collateral sensitivity in cisplatin-resistant gastric cancer cells in a concentration-dependent manner. The mRNA expression profiles of the transporter genes as well as the ROS-scavenging genes were compared in the SNU-601/WT and its two cisplatin-resistant cell sublines. The RT-PCR assay showed that metallothionein, catalase, the copper/zinc or manganese containing superoxide dismutases, P-glycoprotein and the breast cancer resistance protein but not the lung resistance protein, the multidrug resistance-associated protein, and ATP7B might be involved in cisplatin resistance in the SNU-601/Cis2 cells. The administration of antineoplastic agents such as cisplatin and anthracyclines causes oxidative stress, i.e., a production of free radicals and other ROS [17]. The involvement of ROS in the cytotoxicity induced by cisplatin was confirmed not only by measuring the level of dichlorofluorescein (DCF) production but also by measuring the levels of the glutathione (GSH) depletors and N-acetylcysteine, a GSH precursor [18]. In this study, antioxidant mole-
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cules including metallothionein, catalase, and superoxide dismutases were found to play important roles in the cisplatin resistance of SNU-601/Cis2 cells. In addition, the scavenging activity against paraquat-induced ROS of the SNU-601/Cis2 cells was higher than that of the SNU-601/WT or SNU-601/Cis10 cells. This result can be interpreted by overexpression of antioxidant genes, especially superoxide dismutases, in SNU-601/ Cis2 cells as compared with SNU-601/WT or SNU601/Cis10 cells. The decreased expression of the antioxidant genes in the SNU-601/Cis10 cells compared with that of the SNU-601/Cis2 cells might account for the collateral sensitivity of SNU-601/Cis10 cells to the various cytotoxic drugs. Regarding the transporters, the P-glycoprotein and the breast cancer resistance protein but not the multidrug resistance-associated protein and ATP7B were implicated in the cisplatin resistance of SNU-601/Cis2 cells. The involvement of ATP7B in cisplatin resistance has been reported in various cancer cells including epidermoid cancer, prostate cancer [7], ovarian cancer [19], breast cancer [20], gastric cancer [21], oral squamous cell cancer [22], esophageal cancer [23], and hepatocellular cancer [24]. In this study, the lack of involvement of ATP7B in cisplatin resistance in the SNU-601/Cis2 and /Cis10 cells indicates that ATP7B is not always involved in cisplatin resistance. The increased mRNA levels of the resistance-related genes in the SNU-601/Cis2 cells were lower in the SNU-601/Cis10 cells, which might be responsible for the collateral sensitivity of the SNU601/Cis10 cells compared with SNU-601/Cis2 cells. These results suggest that both the cisplatin dose and cisplatin-based combination chemotherapy for gastric cancer patients need to consider the cross-resistance and collateral sensitivity.
Acknowledgments This work was supported, in part, by grants from the Ministry of Science and Technology, Korea, and the Korea Science and Engineering Foundation through the Research Center for Resistant Cells (R13-2003-009).
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Concentration-dependent collateral sensitivity of cisplatin-resistant gastric cancer cell sublines Haidong Xua, Seok-Min Choib, Chun-San Anb, Young-Don Minc, Kweon-Cheon Kimc, Kyung-Jong Kimc, Cheol-Hee Choia,b,* b
a Research Center for Resistant Cells, Chosun University, Gwangju 501-759, Republic of Korea Department of Pharmacology, Chosun University Medical School, Gwangju 501-759, Republic of Korea c Department of Surgery, Chosun University Medical School, Gwangju 501-759, Republic of Korea
Received 26 December 2004 Available online 13 January 2005
Abstract The cisplatin-resistant gastric cancer cell sublines, SNU-601/Cis2 and /Cis10, were 49 and >530 times more resistant to cisplatin, respectively, compared with the drug-sensitive cells, SNU-601/WT. The SNU-601/Cis2 showed cross-resistance to carboplatin, heptaplatin, doxorubicin, mitomycin C, and 5-fluorouracil compared with the SNU-601/WT whereas the SNU601/Cis10 displayed collateral sensitivity to these drugs with the exception of cisplatin compared with the SNU-601/Cis2, suggesting that the cross-resistance and collateral sensitivity of cisplatin-resistant gastric cancer cells are dependent upon cisplatin concentrations. Altered expression of the antioxidant and transporter genes (metallothionein, catalase, superoxide dismutases, P-glycoprotein, and the breast cancer resistance protein) was involved in these phenotypes of the cisplatin-resistant gastric cancer cell lines. 2005 Elsevier Inc. All rights reserved. Keywords: Cisplatin; Gastric cancer; Cross-resistance; Collateral sensitivity; Antioxidant; Transporter
Gastric cancer is the most frequently diagnosed and the second leading cause of cancer-related death in Korea. However, the treatment response rate has been <30% and cases of complete remission are rare. Several combination chemotherapy regimens such as FAM (5-fluorouracil, adriamycin, and mitomycin C) have been used to improve the treatment outcomes [1]. In a nonrandomized Phase II study on advanced gastric cancer, the FAM regimen achieved an objective partial response rate of 42% [2]. Some cisplatin-based combination chemotherapy regimens have shown high response rates [3,4]. However, the intrinsic or acquired resistance to cisplatin reduces its efficacy [5]. The
*
Corresponding author. Fax: +82 62 232 4045. E-mail address: [email protected] (C.-H. Choi).
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.01.015
mechanism of resistance to cisplatin includes reduced cisplatin accumulation, such as the canalicular multispecific anion transporter cMOAT (MRP2/ABCC2) and the copper-transporting P-type ATPase ATP7B [6,7], an increased intracellular thiol level, such as glutathione or metallothionein (MT) [8–10], and increased DNA repair [11]. The cisplatin-resistant gastric cancer cell lines, SNU601/Cis2 and /Cis10, which were selected by chronic exposure to 2 and 10 lg/ml cisplatin, respectively, were 49 times and >530 times more resistant to cisplatin than the parental SNU-601 cells. SNU-601/Cis2 showed cross-resistance to carboplatin, heptaplatin, doxorubicin, mitomycin C, and 5-fluorouracil compared with SNU-601/WT whereas SNU-601/Cis10 displayed collateral sensitivity to these drugs, except for cisplatin compared with SNU-601/Cis2.
H. Xu et al. / Biochemical and Biophysical Research Communications 328 (2005) 618–622
This paper reports the molecular mechanisms of the cross-resistance and collateral sensitivity of two cisplatin-resistant gastric cancer sublines to anticancer drugs, which are dependent upon cisplatin concentrations.
Materials and methods Cell culture and the selection of the gastric cancer cell sublines for cisplatin resistance. The human gastric cancer cell line, SNU-601, was obtained from the Cancer Research Center in Seoul National University (South Korea). The cells were cultured in RPMI 1640 (GibcoBRL, Grand Island, NY, USA) supplemented with 10% FBS (Sigma Chemical, St. Louis, MO, USA). The cells were maintained as a monolayer culture and were subcultured at confluence. The cisplatinresistant gastric cancer cell subline was selected by exposing them to gradually increasing cisplatin concentrations ranging from 200 ng/ml (approximate IC50 value) to 2 and 10 lg/ml on an intermittent dosage schedule. Cytotoxicity assay. The in vitro cytotoxicity of the drugs was determined using an MTT (Sigma Chemical, St. Louis, MO, USA). The 50% inhibitory concentration (IC50) for a particular agent was defined as the drug concentration that causes a 50% reduction in the number of cells compared with the untreated control. The IC50 values were determined directly from the semilogarithmic dose–response curves. All the cytotoxic drugs with the exception of heptaplatin (Sunpla, SK Pharmaceutical, Seoul, Korea) were obtained from the Sigma Chemical (St. Louis, MO, USA). RNA extraction and reverse transcription-polymerase chain reaction assay. The total RNA was prepared from the cells using a RNeasy mini kit (Quiagen, Hilden, Germany). The steady-state levels of each mRNA transcript were determined using a reverse transcriptionpolymerase chain reaction (RT-PCR) assay, where the expression levels were normalized to those of the b-actin expression level as a control. Information on the primer pairs and corresponding genes is summarized in Table 1. The RNAs from each sample were reverse transcribed using units of Moloney murine leukemia virus reverse transcriptase (GibcoBethesta Research Laboratory, Grand Island, NY, USA) and the oligo(dT) primer for 1 h at 37 C. The resulting cDNA was diluted 1:5 with water and was amplified with 2.5 U Taq polymerase (Promega, Madison, WI, USA) and 10 pmol of each primer in a GeneAmp PCR9600 (Perkin-Elmer, Boston, MA, USA) at each PCR condition shown in Table 1. After the final cycle, all the PCR products were subjected to a final extension for 5 min at 72 C. For quantitation,
619
5 lCi of [a-32P]dCTP was added to each reaction mixture. The PCR products were electrophoresed on 7.5% nondenaturing polyacrylamide gels. The bands were scanned using a densitometer. Determination of the level of reactive oxygen species using a fluorometric probe. Dichlorofluorescein (DCFH) was used to measure the concentration of reactive oxygen species (ROS). After 2 0 ,7 0 -dichlorofluorescein diacetate (DCFH-DA) crosses the membrane, it is deesterified to DCFH, which is oxidized to fluorescent DCF by the ROS [12]. Phosphate-buffered saline containing 1 · 105/ml cells was incubated with 30 lg/ml paraquat, an intracellular superoxide generator, in the presence of 1 lM DCFH-DA at 37 C for 2 h. After incubation, the DCF fluorescence intensity was determined using a fluorometer at an excitation and emission wavelength of 485 and 530 nm, respectively. Statistical analysis. Statistical significance of the data was determined using StudentÕs t test. P values <0.05 were considered significant.
Results Sensitivity of cisplatin-resistant gastric cancer cell sublines to various anticancer drugs Of the eight human gastric cancer cell lines (SNU-1, SNU-5, SNU-16, SNU-484, SNU-601, SNU-620, SNU-638, and SNU-668), the SNU-601 cells were chosen owing to their having the lowest MT mRNA expression level. The cisplatin-resistant gastric cancer cell lines, SNU-601/Cis2 and /Cis10, which were selected by the chronic exposure to 2 and 10 lg/ml cisplatin, respectively, were 49 and >530 times more resistant to cisplatin than the parental SNU-601 cells (SNU-601/WT), respectively (Fig. 1). As shown in Table 2, the SNU601/Cis2 cells showed cross-resistance to carboplatin (10.68-fold), heptaplatin (18.49-fold), doxorubicin (44.95-fold), mitomycin C (56.77-fold), and 5-fluorouracil (64.20-fold) compared with SNU-601/WT whereas SNU-601/Cis10 displayed collateral sensitivity to these drugs with the exception of cisplatin compared with the SNU-601/Cis2 cells (Table 3).
Table 1 PCR primers used in this study Gene
Length of PCR products (bp)
No. of cycles
Hot start
Denaturation
Annealing
Extension
MDR 1 MRP LRP BCRP MT ATB7B sod-1 sod-2 cat b-Actin
295 355 390 475 220 833 462 260 440 501
30 22 23 22 21 22 20 22 23 17
94 C, 12 min
94 C, 30 s
65 C, 53 C, 53 C, 53 C, 57 C, 53 C, 53 C, 57 C, 53 C, 53 C,
72 C, 1 min
30 s 30 s 30 s 30 s 30 s 30 s 30 s 30 s 30 s 30 s
MDR1, multidrug resistance 1; MRP, multidrug resistance-associated protein; LRP, lung resistance-related protein; BCRP, breast cancer resistance protein; MT, metallothionein; ATB7B, copper-transporting ATPase; sod-1, Cu/Zn superoxide dismutase; sod-2, Mn superoxide dismutase; cat, catalase; b-actin (PCR control).
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Fig. 1. Sensitivity of the SNU-601/WT cells and their cisplatinresistant cell sublines to cisplatin. Cytotoxicity was determined using an MTT assay.
Table 2 Comparison of the sensitivity of the SNU-601/WT and SNU-601/Cis2 cells to various anticancer drugs Drug
SNU-601/WT, IC50 (lg/ml)
SNU-601/Cis2, IC50 (lg/ml)
Relative resistance (fold)
Cisplatin Carboplatin Heptaplatin Mitomycin C Doxorubicin 5-Fluorouracil
0.19 7.5 0.76 0.046 0.015 0.5
9.2 80.1 14.0 2.7 0.67 32.1
48.75 10.68 18.49 56.77 45.02 64.20
Table 3 Comparison of the sensitivity of the SNU-601/Cis2 and /Cis10 cells to various anticancer drugs Drug
SNU-601/Cis2, IC50 (lg/ml)
SNU-601/Cis10, IC50 (lg/ml)
Relative resistance (fold)
Cisplatin Carboplatin Heptaplatin Mitomycin C Doxorubicin 5-Fluorouracil
9.2 80.1 14.0 2.7 0.67 32.1
>100 42.2 5.0 0.2 0.06 19.5
>10.87 0.53 0.36 0.07 0.09 0.61
Expression profiles of the resistance-related genes in the SNU-601/WT cells and their cisplatin-resistant cell sublines SNU-601/Cis2 and /Cis10 In this study, the expression profiles of the resistancerelated genes were examined in the SNU-601/WT cells and their cisplatin-resistant cell sublines, and compared. The resistance-related genes examined included metallothionein, catalase, copper/zinc and manganese superoxide dismutases, P-glycoprotein, the lung resistance protein, the multidrug resistance-associated protein, the breast cancer resistance protein, and ATP7B. With the exception of the lung resistance protein, the multidrug resistance-associated protein, and ATP7B, the steady-state mRNA levels of the other genes in the
Fig. 2. Expression profiles of the drug efflux pumps and antioxidant enzymes in the SNU-601/WT cells and their cisplatin-resistant cell sublines. Gene expression was determined using a RT-PCR assay. b-Actin was used as the control for RNA. Abbreviations for gene names are described in Table 1.
SNU-601/Cis2 cells were higher than in the SNU-601/ WT cells whereas the expression levels in the SNU601/Cis10 cells were lower than those of the SNU-601/ Cis2 cells (see Fig. 2). Comparison of scavenging activity against paraquatinduced ROS between SNU-601/WT and its cisplatinresistant cell sublines The scavenging activities against paraquat-induced ROS were determined using DCFH-DA. After the incubation of cells with 30 lg/ml paraquat in the presence of 1 lM DCFH-DA at 37 C for 2 h, the ROS-scavenging effect of the SNU-601/Cis2 cells were higher than those of either the SNU-601/WT or SNU-601/Cis10 cells (Fig. 3).
Discussion The cisplatin analogs are among the most active and widely used cytotoxic anticancer drugs. However, the acquisition or presence of resistance significantly undermines the curative potential of these drugs against many malignancies [5]. In this study, two cisplatin-resistant gastric cancer cell sublines, SNU-601/Cis2 and /Cis10, were found to be resistant to cisplatin and various anticancer drugs including carboplatin, heptaplatin, mitomycin C, doxorubicin, and 5-fluorouracil. Interestingly, although the SNU-601/Cis10 cells, which were more than 10 times more resistant to cisplatin than the SNU-601/Cis2 cells, they were more sensitive to other
H. Xu et al. / Biochemical and Biophysical Research Communications 328 (2005) 618–622
Fig. 3. Generation of ROS by paraquat in the SNU-601/WT cells and their cisplatin-resistant cell sublines. Phosphate-buffered saline containing 1 · 105/ml cells was incubated with 30 lg/ml paraquat, an intracellular superoxide generator, in the presence of 1 lM DCFH-DA at 37 C for 2 h. The fluorescence intensity was determined using a spectrofluorometer with an excitation wavelength at 485 nm and an emission wavelength at 530 nm. *P < 0.05.
anticancer drugs than the SNU-601/Cis2 cells. The collateral sensitivity indicates that different cellular alterations in the two cisplatin-resistant gastric cancer cell sublines would be induced during the process of developing cisplatin resistance. Extensive resistance, cross-resistance, and collateral-sensitivity patterns have been established with most sublines of the various drug-resistant cells [13–16]. This is the first report on the collateral sensitivity in cisplatin-resistant gastric cancer cells in a concentration-dependent manner. The mRNA expression profiles of the transporter genes as well as the ROS-scavenging genes were compared in the SNU-601/WT and its two cisplatin-resistant cell sublines. The RT-PCR assay showed that metallothionein, catalase, the copper/zinc or manganese containing superoxide dismutases, P-glycoprotein and the breast cancer resistance protein but not the lung resistance protein, the multidrug resistance-associated protein, and ATP7B might be involved in cisplatin resistance in the SNU-601/Cis2 cells. The administration of antineoplastic agents such as cisplatin and anthracyclines causes oxidative stress, i.e., a production of free radicals and other ROS [17]. The involvement of ROS in the cytotoxicity induced by cisplatin was confirmed not only by measuring the level of dichlorofluorescein (DCF) production but also by measuring the levels of the glutathione (GSH) depletors and N-acetylcysteine, a GSH precursor [18]. In this study, antioxidant mole-
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cules including metallothionein, catalase, and superoxide dismutases were found to play important roles in the cisplatin resistance of SNU-601/Cis2 cells. In addition, the scavenging activity against paraquat-induced ROS of the SNU-601/Cis2 cells was higher than that of the SNU-601/WT or SNU-601/Cis10 cells. This result can be interpreted by overexpression of antioxidant genes, especially superoxide dismutases, in SNU-601/ Cis2 cells as compared with SNU-601/WT or SNU601/Cis10 cells. The decreased expression of the antioxidant genes in the SNU-601/Cis10 cells compared with that of the SNU-601/Cis2 cells might account for the collateral sensitivity of SNU-601/Cis10 cells to the various cytotoxic drugs. Regarding the transporters, the P-glycoprotein and the breast cancer resistance protein but not the multidrug resistance-associated protein and ATP7B were implicated in the cisplatin resistance of SNU-601/Cis2 cells. The involvement of ATP7B in cisplatin resistance has been reported in various cancer cells including epidermoid cancer, prostate cancer [7], ovarian cancer [19], breast cancer [20], gastric cancer [21], oral squamous cell cancer [22], esophageal cancer [23], and hepatocellular cancer [24]. In this study, the lack of involvement of ATP7B in cisplatin resistance in the SNU-601/Cis2 and /Cis10 cells indicates that ATP7B is not always involved in cisplatin resistance. The increased mRNA levels of the resistance-related genes in the SNU-601/Cis2 cells were lower in the SNU-601/Cis10 cells, which might be responsible for the collateral sensitivity of the SNU601/Cis10 cells compared with SNU-601/Cis2 cells. These results suggest that both the cisplatin dose and cisplatin-based combination chemotherapy for gastric cancer patients need to consider the cross-resistance and collateral sensitivity.
Acknowledgments This work was supported, in part, by grants from the Ministry of Science and Technology, Korea, and the Korea Science and Engineering Foundation through the Research Center for Resistant Cells (R13-2003-009).
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