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Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Sakyo-ku, Kyoto, Japan
(Received June 28, 2008; Accepted August 14, 2008)
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Abstract |
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; Fuertes et al., 2003; Wang and Lippard, 2005). Oxaliplatin is used against colorectal cancers as a key drug in FOLFOX regimens, and its objective response rate for colorectal cancer is superior to that of cisplatin (Loehrer et al., 1988; Grem et al., 1993; de Gramont et al., 2000; Andre et al., 2004). However, the molecular mechanism(s) underlying the clinical results and that reason that oxaliplatin has such a potent anticolorectal cancer effect compared with cisplatin are unclear.
The organic cation transporters (OCT/SLC22A) and multidrug and toxin extrusion (MATE/SLC47A) family transport cationic drugs, toxins, and endogenous metabolites (Inui et al., 2000; Terada and Inui, 2008). Previously, we determined that the severity of the nephrotoxicity of platinum agents depends on the amount of platinum accumulated in the kidney, and OCT and MATE could play predominant roles in the renal handling of these agents (Yonezawa et al., 2005, 2006; Yokoo et al., 2007). We also reported that oxaliplatin, but not cisplatin, was selectively transported by rat (r) or human (h) organic cation transporter 3 (OCT3/SLC22A3) (Yonezawa et al., 2006; Yokoo et al., 2007). OCT3 mRNA was found in placenta, intestine, heart, brain, and kidney, but the distribution in the membrane and physiological role of OCT3 are not yet clearly understood (Kekuda et al., 1998).
We hypothesized that the substrate specificity and expression level of OCT3 affect the difference in the anticancer effect of platinum agents against colorectal cancer. In the present study, we examined and compared the levels of OCT3 mRNA in colorectal cancer and normal colorectum and among colorectal cancer-derived cell lines. In addition, the cytotoxicity and platinum accumulation in cultured cells caused by the treatment with oxaliplatin or cisplatin were examined.
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Materials and Methods |
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). Cell lines were cultured in an atmosphere of 5% CO2-95% air at 37°C. Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich, St. Louis, MO) with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA) was used for SW480, HT29, HCT116, Lovo, and HEK293. DMEM containing 10% FBS and 1% nonessential amino acid (Invitrogen) was used for SW837, and a 1:1 mixture of DMEM and nutrient mixture Ham's F12 medium (Sigma-Aldrich) with 10% FBS was used for T84. T84, HT29, and Lovo cells were seeded onto 24-well plates or 96-well plates at a density of 4.0 x 105 cells/ml. SW480 and HCT116 cells were seeded at 2.0 x 105 cells/ml, and SW837 cells were seeded at 5.0 x 105 cells/ml. Seventy-two hours after the seeding, the cells were used for the experiments.
Transfection. For a transient expression system, pCMV6-XL4 plasmid vector DNA (OriGene Technologies, Rockville, MD) containing hOCT3 cDNA was purified using an EndoFree Plasmid Mega Kit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer's instructions (Yonezawa et al., 2006). The day before transfection, HEK293 or SW480 cells were seeded onto poly-D-lysine-coated and noncoated 24-well plates, respectively. The cells were transfected with 800 ng of plasmid DNA per well in a combination of empty vector and hOCT3 cDNA using 2 µl of Lipofectamine 2000 (Invitrogen) per well according to the manufacturer's instructions. The amount of hOCT3 cDNA was 800 ng except in the experiment examining the transporter cDNA dependence. Forty-eight hours after the transfection, the cells were used for the experiments.
Uptake Experiment. Cellular uptake of [3H]1-methyl-4-phenylpyridium acetate (MPP) (2.7 TBq/mmol; PerkinElmer Life and Analytical Sciences, Waltham, MA) was measured with monolayer cultures grown on 24-well plates. The composition of the incubation buffer was as follows: 145 mM NaCl, 3 mM KCl, 1 mM CaCl2, 0.5 mM MgCl2, 5 mM D-glucose, and 5 mM HEPES (pH 7.4 adjusted with NaOH). As previously reported, experiments on the uptake were performed (Urakami et al., 2004).
For measurement of the cellular accumulation of oxaliplatin or cisplatin, cells seeded on 24-well plates were incubated with DMEM containing 10% FBS and oxaliplatin (Wako Pure Chemical Industries, Osaka, Japan) or cisplatin (Sigma-Aldrich) for 2 min or 1 h. After this incubation, the monolayers were rapidly washed twice with ice-cold incubation buffer containing 3% bovine serum albumin (Nacalai Tesque, Kyoto, Japan) and then washed three times with ice-cold incubation buffer. The cells were solubilized in 0.5 N NaOH, and the amount of platinum was determined using inductively coupled plasma-mass spectrometry (ICP-MS) by the Pharmacokinetics and Bioanalysis Center, Shin Nippon Biomedical Laboratories, Ltd. (Wakayama, Japan). The protein content of the cell monolayers solubilized in 0.5 N NaOH was determined with a Bio-Rad Protein Assay Kit (Bio-Rad, Richmond, CA).
Cytotoxicity Assay. The cytotoxicity of oxaliplatin was measured with cells seeded on 24-well plates for the lactate dehydrogenase (LDH) assay and on 96-well plates for the caspase 3/7 assay. Cells were incubated with medium containing oxaliplatin for 6 h for the LDH assay. After removal of the medium, a drug-free medium was added to the wells. After incubation for 24 h, the medium was collected, and the LDH activity in it was measured using a LDH Cytotoxicity Detection Kit (Takara Bio Inc., Shiga, Japan), according to the manufacturer's instructions. LDH release (percent) was calculated as described previously (Yonezawa et al., 2006). For the caspase assay, cells were incubated with medium containing oxaliplatin for 8 h. After the incubation, caspase 3/7 activity was determined by using a Caspase-Glo 3/7 Assay (Promega, Madison, WI), according to the manufacturer's instructions. Caspase activity (fold increase) represents (caspase 3/7 activity in oxaliplatin-treated cells)/(caspase 3/7 activity in cells without oxaliplatin).
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) was subjected to real-time PCR. Detailed information about the patients was given in the report of Terada et al. (2005). The conditions and primer-probe sets for real-time PCR were described previously (Motohashi et al., 2002; Masuda et al., 2006). The glyceraldehyde-3-phosphate dehydrogenase mRNA level was used as an internal control. This study was conducted in accordance with the Declaration of Helsinki and its amendments and was approved by the Kyoto University Graduate School and Faculty of Medicine Ethics Committee. Cancer Profiling Array. The cDNA cancer profiling array, Cancer Profiling Array I (Clontech, Mountain View, CA) was used. It includes normalized cDNAs from cancer and corresponding normal tissues from individual patients, amplified using SMART technology. Preparation of the cDNA probe for hOCT3, hybridization to the array, and signal detection on X-ray film were performed using the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche Ltd., Basel, Switzerland) according to the manufacturer's instructions. The relative intensity of each dot was determined densitometrically using ImageJ 1.38x (National Institutes of Health, Bethesda, MD).
Statistical Analysis. Data are expressed as means ± S.E. Data were analyzed statistically using the paired Student's t test. Probability values of less than 0.05 were considered statistically significant.
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Results |
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mRNA Expression of Organic Cation Transporters in Colorectal Cancer-Derived Cell Lines. We examined the expression of hOCT1, hOCT2, hOCT3, hMATE1, and hMATE2-K mRNA in colorectal cancer-derived cell lines, T84, SW480, HCT116, HT29, SW837, and Lovo, by real-time PCR. The hOCT3 transcript was found in all of these cells except HCT116 and was strongly detected in T84 and SW837 (Fig. 2). hMATE1 mRNA was only expressed in SW480. However, the mRNA expression of hOCT1, hOCT2, hMATE1, and hMATE2-K in these cells was almost negligible.
[3H]MPP Uptake by Colorectal Cancer-Derived Cell Lines. To check the functional activity of hOCT3 in cultured cells, we measured the cellular uptake of its typical substrate, [3H]MPP. The accumulation of [3H]MPP was greater in T84 cells, SW480 cells expressing hOCT3, and HEK293 cells expressing hOCT3 than in SW480 cells, SW480 cells transfected with vector cDNA, and HEK293 cells transfected with vector cDNA (Fig. 3A). In addition, we examined [3H]MPP uptake in other colorectal cancer-derived cell lines, HCT116, HT29, SW837, and Lovo. SW837 showed the highest level of activity to transport [3H]MPP among these six cell lines (Fig. 3B). The transport activity of the cells was confirmed, and then these cells and expression systems were used in subsequent experiments on the cytotoxicity and the cellular transport of platinum agents.
hOCT3 Expression and Oxaliplatin-Induced Cytotoxicity. We examined the effect of hOCT3 expression in a colon cancer-derived cell line, SW480. When SW480 cells transfected with 800 ng of empty vector or hOCT3 were treated with 500 µM oxaliplatin for 6 h and subsequently cultured in normal medium for 24 h, the release of LDH into the culture medium was increased by the expression of hOCT3 (Fig. 4A). In addition, we measured the amount of LDH released by treatment with 500 µM oxaliplatin in other colorectal cancer-derived cell lines. The amount of LDH released was greatest in SW837 and was also large in T84 and Lovo cells (Fig. 4B). SW480, HCT116, and HT29 cells showed little release of LDH with oxaliplatin treatment.
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In addition, caspase 3/7 activity induced by treatment with 50 µM oxaliplatin was examined in these cell lines. The most potent activation of caspase 3/7 was in SW837 but T84 and Lovo cells also showed strong caspase 3/7 activity (Fig. 4C). The results of caspase activity were consistent with those of LDH release.
Transport of Oxaliplatin. We examined the accumulation of oxaliplatin with the increase of hOCT3 cDNA on transfection of SW480 cells (Fig. 5A), because almost no hOCT3 mRNA was found in SW480 cells (Fig. 2). When SW480 cells transfected with 50 to 800 ng of hOCT3 cDNA per well were treated with 1000 µM oxaliplatin for 1 h, the level of platinum accumulated in the cells was increased, depending on the amount of hOCT3 cDNA transfected (Fig. 5A). Based on these results, we determined the platinum accumulation in T84 cells, SW480 cells, and SW480 cells transfected with 800 ng of hOCT3 cDNA. When treated with 100, 500, or 1000 µM oxaliplatin for 1 h, T84 cells and SW480 cells expressing hOCT3 transported oxaliplatin extensively in a concentration-dependent manner compared with SW480 cells or SW480 cells transfected with empty vector (Fig. 5B). Moreover, we examined the amount of platinum accumulated after the treatment with oxaliplatin in other colorectal cancer-derived cell lines, HCT116, HT29, SW837, and Lovo. Platinum was most abundant in SW837 cells at all three concentrations when the cells were incubated with the culture medium containing oxaliplatin for 2 min (Table 1). The same tendency was observed when they were treated for 1 h (Table 2). In HT29 and Lovo cells, the amount of platinum accumulated was approximately half of that in SW837 cells, and the levels in SW480 and HCT116 cells were low compared with those in other cultured cells.
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Relation among hOCT3 mRNA Expression, LDH Release, and Platinum Accumulation. When cultured cells were treated with 500 µM oxaliplatin, the release of LDH was increased by the hOCT3 mRNA expression (Fig. 6A). The accumulation of platinum in the cells after the incubation with 500 µM oxaliplatin was also dependent on hOCT3 mRNA expression (Fig. 6B). By combining the data from Fig. 6, A and B, the release of LDH was also comparable with the accumulation of platinum (Fig. 6C). On the other hand, when cells were treated with 500 µM cisplatin, the accumulation of platinum was independent of hOCT3 mRNA expression (Fig. 6D).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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; de Gramont et al., 2000). However, the molecular mechanism(s) that cause the difference in the effect has not been made clear. We previously determined that oxaliplatin but not cisplatin was transported by OCT3 (Yonezawa et al., 2006; Yokoo et al., 2007). Therefore, we hypothesized that the level of hOCT3 in cancerous tissues contributed to the superior anticancer effect of oxaliplatin. In the present study, the level of hOCT3 mRNA was significantly higher in cancerous colon than in normal colon tissues derived from Japanese patients (Fig. 1A). In addition, this tendency was reproduced in Caucasians using a cancer profiling array (Fig. 7, A and B). These findings indicated that the level of hOCT3 mRNA was heightened by colorectal cancerous transformation independent of ethnicity.
In human colorectal cancer-derived cell lines, hOCT3 mRNA expression was correlated with the release of LDH and accumulation of platinum induced by the treatment with oxaliplatin (Fig. 6, A and B). These results suggested that hOCT3 expression is a candidate marker for the efficacy of oxaliplatin treatment. Moreover, the release of LDH and accumulation of platinum caused by the incubation with cisplatin was independent of the hOCT3 mRNA level (Fig. 6D). This result was consistent with the report that cisplatin was not transported by hOCT3 (Yonezawa et al., 2006). Therefore, hOCT3 expression is suggested to be closely associated with the anticancer activity of oxaliplatin but not that of cisplatin.
Cisplatin plays an essential role in chemotherapy against solid tumors of the prostate, bladder, lung, testis, liver, and brain (Ho et al., 2003). However, the effect of cisplatin on colorectal cancer is weak. Loehrer et al. (1988) and Grem et al. (1993) reported rates of response of colorectal cancer to cisplatin-based chemotherapy of 22 and 19%, respectively. On the other hand, for oxaliplatin-based chemotherapy, de Gramont et al. (2000) reported that the response rate was 50%. The differences in molecular mechanisms whereby cisplatin has a weak effect but oxaliplatin has a strong effect on colorectal cancer have been unclear. The anticancer activity and resistance to platinum agents have been considered to be related to the DNA repair pathway, nucleotide excision repair, base excision repair, mismatch repair, and double-strand break repair, or the substrate specificity of copper transporters, CTR1, ATP7A, and ATP7B (Kelland, 2007). However, recently, we and others reported the contribution of organic cation transporters in the cellular transport of platinum agents (Ciarimboli et al., 2005; Yonezawa et al., 2005, 2006; Zhang et al., 2006; Yokoo et al., 2007; Kitada et al., 2008). Zhang et al. (2006) reported that the effect of oxaliplatin against colon cancer was related to the expression of hOCT1 and hOCT2. Kitada et al. (2008) reported that the levels of ATP7A and hOCT1 mRNA affect the sensitivity to oxaliplatin. However, we reported that oxaliplatin was transported by both human and rat OCT2 and OCT3, but not by OCT1 (Yonezawa et al., 2006; Yokoo et al., 2007). In the present study, only hOCT3 mRNA was found in the six cell lines derived from colorectal cancers, and the cytotoxicity of oxaliplatin was associated with the expression level. Based on these findings and the present results, at least in colorectal cancer, OCT3 is thought to be important for sensitivity to oxaliplatin.
In the six colorectal cancer-derived cell lines, hOCT3 mRNA levels were markedly higher than hOCT1, hOCT2, hMATE1, or hMATE2-K mRNA levels (Fig. 2). Previously, we reported that oxaliplatin was also transported by hOCT2 (Yonezawa et al., 2006). However, in these cell lines, the expression of hOCT2 mRNA was little detected by real-time PCR (Fig. 2). Therefore, the contribution of hOCT2 to the anticancer effect of oxaliplatin was suggested to be small. The transport activity of hOCT3 in these cells was confirmed by using [3H]MPP, a typical substrate of OCT3 (Fig. 3). Okuda et al. (2000) reported that the cytotoxicity of cisplatin differed at low and high doses, that is, 30 and 1000 µM cisplatin induced apoptosis and necrosis, respectively. In the present study, we used two indexes of cytotoxicity, LDH release and caspase 3/7 activity, as indicators of necrosis and apoptosis, respectively. Both LDH release and caspase activity showed a similar tendency; that is, values were high in cell lines expressing high levels of hOCT3 mRNA (Figs. 2 and 4, B and C). From these results, the hOCT3-mediated cellular accumulation of oxaliplatin might be a trigger for the subsequent cytotoxic effects.
Although the cytotoxicity of oxaliplatin in T84 cells was lower than expected, given the expression level of hOCT3 (Fig. 6A), the LDH release in these cells correlated quite well with the platinum accumulation (Fig. 6C). These results suggest some mechanisms including an unknown oxaliplatin efflux transporter to reduce the intracellular platinum concentration in T84 cells compared with that in SW837 cells.
We had reported that the nephrotoxicity caused by treatment with platinum agents was closely associated with their renal accumulation, which is determined by the substrate specificity of the OCT and MATE families (Yonezawa et al., 2005, 2006; Yokoo et al., 2007). There had also been a report that the uptake of imatinib, a tyrosine kinase inhibitor effective in the treatment of chronic myeloid leukemia, was mediated by hOCT1 (Thomas et al., 2004). Recently, two groups showed that hOCT1 was a determinant of outcome in imatinib-treated chronic myeloid leukemia (White et al., 2007; Wang et al., 2008). Patients with a high level of hOCT1 had a greater probability of achieving a cytogenetic response and superior progression-free and overall survival (Wang et al., 2008). These reports showed the participation of hOCT1 in the clinical effects of imatinib. Therefore, the results of this study, that the cytotoxicity of oxaliplatin depended on hOCT3 expression, may be expanded to include effectiveness in clinical cases.
OCT3 is widely distributed in many tissues (Kekuda et al., 1998), but its function has been examined mainly in the brain (Wu et al., 1998; Gasser et al., 2006). The results of this study suggested a new role for OCT3, as a determinant of the sensitivity of treatment with oxaliplatin against colorectal cancer. At present, oxaliplatin is used for colorectal cancer as a key drug of FOLFOX regimens (de Gramont et al., 2000). Other combinations including oxaliplatin for colorectal cancer or other cancers have been used in clinical trials (Goldberg et al., 2004; Zhu et al., 2006). The level of hOCT3 in cancerous tissue was significantly higher in colon, rectum, and stomach (Fig. 7, A-C). Conversely, the level was significantly lower in uterus, breast, ovary, and lung (Fig. 7, D-G). These changes in hOCT3 expression might contribute to the sensitivity and selectivity of oxaliplatin-based chemotherapy. Recently, there were several reports that oxaliplatin was effective against gastric cancer in phase II trials (Lordick et al., 2005; Park et al., 2006; Kim et al., 2008). Considering the present results, there is a possibility that the increase of hOCT3 expression in cancerous tissue affects the results of clinical trials. Therefore, taking a positive attitude to use of oxaliplatin-based chemotherapy for other cancers that express high levels of hOCT3 compared with normal tissue may lead to good clinical results.
In the present study, we clearly found selective induction of hOCT3 mRNA expression in colon cancer and colorectal cancer-derived cell lines. The cytotoxicity and accumulation of platinum caused by the treatment with oxaliplatin but not cisplatin depended on the expression of hOCT3 mRNA. In conclusion, the uptake of oxaliplatin into the cancer cells via hOCT3 was suggested to be an important mechanism for its cytotoxicity, and the expression of hOCT3 in cancers may became a marker for including oxaliplatin in cancer chemotherapy.
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Footnotes |
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ABBREVIATIONS: cisplatin, cis-diamminedichloroplatinum II; oxaliplatin, trans-L-1,2-diaminocyclohexaneoxalatoplatinum II; FOLFOX, folinic acid (leucovorin), 5-fluorouracil, and oxaliplatin; OCT, organic cation transporter; MATE, multidrug and toxin extrusion; r, rat; h, human; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; MPP, 1-methyl-4-phenylpyridium acetate; ICP-MS, inductively coupled plasma-mass spectrometry; LDH, lactate dehydrogenase; PCR, polymerase chain reaction; HEK, human embryonic kidney.
The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Ken-ichi Inui, Department of Pharmacy, Kyoto University Hospital, Sakyo-ku, Kyoto 606-8507, Japan. E-mail: inui{at}kuhp.kyoto-u.ac.jp
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