Biomedical Research (Tokyo) 36 (4) 253-261, 2015
Protein expression profile related to cisplatin resistance in bladder cancer cell lines detected by two-dimensional gel electrophoresis Yoshinori TAOKA1, Kazumasa MATSUMOTO1, Kazuya OHASHI2, Satoru MINAMIDA1, Masahiro HAGIWARA1, Shoji NAGI1, Tatsuya SAITO2, Yoshio KODERA2, and Masatsugu IWAMURA1 1
Department of Urology, Kitasato University School of Medicine, Sagamihara, Kanagawa 252-0374, Japan and 2 Department of Physics, Kitasato University School of Science, Sagamihara, Kanagawa 252-0373, Japan (Received 9 June 2015; and accepted 17 June 2015)
ABSTRACT We used a proteomic approach to compare the differentially regulated protein expression profiles of cisplatin-naïve and cisplatin-resistant bladder cancer cell lines to screen candidate molecules related to cisplatin resistance. The cisplatin-resistant cell line T24 was established by the stepwise exposure of T24 cells to up to 40 μM of cisplatin. We performed a comprehensive study of protein expression in bladder cancer cell lines that included cisplatin-naïve (T24) and cisplatin-resistant cells (T24CDDPR) by means of agarose two-dimensional gel electrophoresis followed by analysis of liquid chromatography tandem mass spectroscopy. We identified 25 obviously different spots for T24 and T24 CDDPR. Seven spots had increased expression and 18 spots had decreased expression in T24CDDPR compared to those in T24. Cytoskeletal proteins and enzyme modulators were prominent among differential proteins. Of the 25 proteins, we selected HNRNPA3, PCK2, PPL, PGK1, TKT, SERPINB2, GOT2, and EIF3A for further validation by Western blot. HNRNPA3, PGK1, TKT, and SERPINB2 had more than 1.5-times incremental expression in T24CDDPR compared to that in T24. PCK2 and PPL expressions were decreased less than 20% in T24CDDPR compared to that in T24. The results of 25 new proteins in this study could be valuable and could lead to the development of a new molecular marker.
Bladder cancer is the second most common malignant tumor in the urologic field. Patients with locally advanced bladder cancer at diagnosis will subsequently have development of local progression or distant metastases. In patients with advanced cancer, chemotherapy is the treatment of choice. Since cisdiamminedichloroplatinum, known as cisplatin, for the treatment of testicular and bladder cancers was approved by the Food and Drug Administration in 1978, platinum-based drugs have been the main treatment administered against a variety of cancers (3). Address correspondence to: Kazumasa Matsumoto, M.D. Department of Urology, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan Tel: +81-42-778-9091, Fax: +81-42-778-9374 E-mail:
[email protected]
Cisplatin-based chemotherapy, including methotrexate, vinblastine, doxorubicin, and cisplatin (MVAC) or gemcitabine and cisplatin (GC), is an effective and frequently used modality for advanced and metastatic bladder cancer (21). However, resistance to chemotherapeutic agents is a major clinical problem in the effective management of cancer. Long-term follow-up has been reported in patients with advanced bladder cancer, but progression-free survival and overall survival have not been satisfactory (16). To overcome drug resistance, alteration of chemotherapy has been performed, including increased dose, shortened intervals, or a variety of combination strategies. Unfortunately, in terms of bladder cancer, there are no established therapeutic modalities for patients with progression or recurrence after cisplatin-based treatment (5, 8). Proteomics using cancer specimens is promising
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for detecting clinically available biomarkers, which eventually provide the information for an appropriate clinical choice and development of innovative therapy. Such a proteomic approach has been used not only for a host of tumor tissue biopsy specimens but also for cultured tumor cells. To identify appropriate proteins for biomarkers, we utilized a unique two-dimensional gel electrophoresis (2-DE) method that uses an agarose gel for isoelectric focusing on the first dimension. Agarose 2-DE can analyze much larger quantities of proteins (up to 1.5 mg) and a wider dynamic range than conventional 2-DE (14). In our previous studies, proteomic analyses were continuously investigated in urothelial carcinoma to identify the candidates for biomarkers, including proteins, sera, and autoantibodies (11, 14, 19, 20). In this present study, we used a proteomic approach to compare the differentially regulated protein expression profiles of cisplatin-naïve and cisplatinresistant bladder cancer cell lines to screen candidate molecules related to cisplatin resistance. MATERIALS AND METHODS Cell line and cell culture. Human bladder cancer cell lines T24, RT4, 5637, and TCCSUP were purchased from American Type Culture Collection. EJ was kindly gifted from the Kitasato Institution Hospital (Dr. Akira Irie). The cisplatin-resistant cell line T24 (T24CDDPR) was established by the stepwise exposure of T24 cells to 13.3 μM, 26.6 μM, and 40 μM of cisplatin (12). RT4ρ0, which was deleted in mitochondrial DNA, was established from RT4 using continuous exposure of ethidium bromide in conditional medium additionally supplemented with 50 μg/mL uridine and 100 μg/mL sodium pyruvate (1). These cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (GIBCO BRL), penicillin (100 U/mL), and streptomycin (100 μg/mL) at 37°C in a 5% CO2 humidified incubator. Sample preparation. Bladder cancer cell lines approximately 20 mg in weight were homogenized using a Teflon glass homogenizer in an extracting solution with 30-fold volume. The extraction solution contained 7 M urea, 2 M thiourea, 0.1 M DTT, 2.5% w/v Pharmalyte (pH 3–10), 2% w/v CHAPS, and Complete Mini EDTA-free protease inhibitor (Roche Diagnostics, Mannheim, Germany), one tablet of which was dissolved in 10 mL of the solution. After homogenization, each homogenate was subjected to rapid agitation in the presence of 0.35-mm
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to 0.50-mm glass beads (As One Corporation, Osaka, Japan) and centrifuged at 112,000×g for 20 min. The supernatants were subjected to agarose 2-DE. Agarose 2-DE and MS/MS analysis. Protein extracts were separated by agarose 2-DE. The isoelectric focusing of the first dimension used an agarose gel, and that of the second dimension used an SDS-PAGE gel. We used 12% SDS-PAGE gel for the proteins weighing between 30 kDa and 100 kDa, and we used a linear gradient of 6% to 10% SDS-PAGE gel for the proteins weighing more than 50 kDa. Agarose 2-DE was performed on three sets of T24 and T24CDDPR bladder cancer cell lines. The slab gel was stained with CBB R-350 (PhastGel Blue R; GE Healthcare, Little Chalfont, UK). We calculated the molecular weight of the protein spot using CS analyzer 2.0 (Atto, Tokyo, Japan) computer software. We subjected each spot to protein digestion and tandem mass spectroscopy (MS/MS) analysis using a liquid chromatography tandem mass spectroscopy (LC-MS/MS) system, which consisted of a Nanospace SI-2 HPLC system (Shiseido Fine Chemicals, Tokyo, Japan) and an ion-trap mass spectrometer (LCQ Deca; Thermo Fisher Scientific, San Jose, CA, USA). We used the SEQUEST search program to identify each protein. SEQUEST referenced the nr.Z and Swiss-Prot.Z protein sequence databases downloaded from ftp://ftp.ncbi.nih.gov/blast/db/. The two criteria used to judge whether our identification was successful were as follows: 1) the SEQUEST score was more than 50; and 2) at least two peptide fragments were reliably detected. If the SEQUEST score was less than 50, then we confirmed that there were more than two high-quality and reliable MS/MS spectra available. If there were several names for a single protein, then the most popular one was applied for analysis. Theoretical molecular weight was calculated from the amino acid sequence. Accession numbers were obtained from the protein database on the UniProt Web page (http://www.uniprot.org/). Sequence coverage shows the accordance rate of peptides confirmed by MS/MS analyses in amino-acid sequences of candidate proteins. Peptide-spectrum matches were obtained from the total number of identified peptide sequences for the protein, including those redundantly identified. Select proteins. To select proteins, we first excluded myosin or cytokeratin, which were broadly investigated in the field of protein analysis. Then, proteins with expression that obviously increased or de-
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Fig. 1 Two-dimensional electrophoresis gels (2-DE) shows 30 proteins with differential expression of cisplatin-naïve T24 (a, c) and cisplatin-resistant T24 (b, d). The 2-DE gels show 20 spots in 12% gels (a, b) and 10 spots in a linear gradient of 6% to 10% gels (c, d). Red arrow shows increased cisplatin-resistant T24 and blue arrow shows decreased cisplatin-resistant T24.
creased in 2-DE were selected. Finally, we searched PubMed for relevant articles published up until February 2015. The following search terms and their combinations were used: each protein name and “bladder cancer”; “urothelial carcinoma”; or “cisplatin.” Inclusion criteria for further immunoblotting were as follow: less than 10 articles matched protein names with “bladder cancer” or “urothelial carcinoma” and less than five articles matched protein names with “cisplatin.” From these searches, we selected eight proteins for immunoblotting. Immunoblotting. Cell line samples were homogenized with a Teflon glass homogenizer in sample buffer (50 mM Tris-HCl, pH 6.8, 0.1 M DTT, 10% glycerol, 2% SDS, and 0.1% bromophenol blue). Protein extracts were separated under denaturing conditions in 10% to 20% polyacrylamide slab gel (DRC, Tokyo, Japan). The SDS-PAGE gel was run with a constant voltage of 300 V. After completion of electrophoresis, proteins were transferred onto HybondP PVDF membranes (GE Healthcare) and detected with mouse monoclonal antibodies against phosphoenolpyruvate carboxykinase (PCK2) (1 : 100; Abgent, San Diego, CA, USA), heterogeneous nuclear
ribonucleoprotein A3 (HNRNPA3) (1 : 200; Abcam, Cambridge, UK), glutamate oxaloacetate transaminase 2 (GOT2) (1 : 1000; Abnova, Taipei City, Taiwan), phospoglycerate kinase 1 (PGK1) (1 : 200; Abgent, San Diego, CA, USA), transketolase (TKT) (1 : 100; Abcam), plasminogen activator inhibitor 2 (SERPINB2) (1 : 1000; Novusn Biologicals, Littleton, CO, USA), eukaryotic translation initiation factor 3 subunit A (EIF3A) (1 : 5000; Santa Cruz Biotech, Dallas, TX, USA), or periplakin (PPL) (1 : 1000; Novusn Biologicals). Horseradish peroxidase–conjugated secondary antibodies (Dako, Glostrup, Denmark) were diluted 1 : 10,000 (2% normal swine serum/TBS). For the calibration of each lane, we obtained rabbit anti-β actin monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA). Antigens on the membrane were detected with Immobilon Western detection reagents (Millipore, Billerica, MA, USA). RESULTS Identification of cisplatin-related protein in cisplatinnaïve and cisplatin-resistant bladder cancer cell lines Protein expressions were quite different for T24 and
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T24CDDPR on 2-DE gels (Fig. 1). We compared 2-DE gels of both cell lines and selected 20 spots in 12% gels and 10 spots in a linear gradient of 6% to 10% gels. There were 30 obviously different spots for T24 and T24CDDPR. Eight spots had increased expression and 22 spots had decreased expression in T24CDDPR compared to that in T24. Characteristics of these spots are shown in Table 1. Because five of these spots were duplicated, 25 spots were finally selected. The 25 proteins, which were differentiated protein expressions of T24 and T24CDDPR, were categorized as 10 functions. The categories were as follows: seven cytoskeletal proteins (Spot No. 8(24), 9(25), 10(28), 12, 13, 26, 27); five enzyme modulators (Spot No. 16, 17, 18, 22, 23); four transferases (Spot No. 3, 4, 11(29), 15); two nucleic acid–binding proteins (Spot No. 2, 14); two transporter proteins (Spot No. 7, 20); one oxidoreductase (Spot No. 1); one protein synthesis (Spot No. 5); one lyase (Spot No. 6); one coagulation factor (Spot No. 19(30)); and one translation factor (Spot No. 21). Of the 25 proteins, we selected HNRNPA3, PCK2, PPL, PGK1, TKT, SERPINB2, GOT2, and EIF3A for further validation by Western blot. Validation of differentiated protein expression To further verify the identified proteins, Western blotting analysis was performed to determine the existence of these eight proteins in T24 or T24CDDPR. Representative results are shown in Fig. 2. The results showed that HNRNPA3, PGK1, TK, and SERPINB2 had more than 1.5-times the incremental expression in T24CDDPR compared to that in T24. PCK2 and PPL had less than 20% decreased expressions in T24CDDPR compared to that in T24. GOT2 and EIF3A had comparable expressions in the two cell lines. Western blot analysis was also performed to determine six protein expression patterns in various bladder cancer cell lines (Fig. 3). HNRNPA3, TKT, SERPINB2, and PPL were expressed in all bladder cancer cell lines. PCK2 was expressed in two other cell lines. In terms of PGK1, there was slight expression or loss of expression in bladder cancer cell lines (except for T24). Expression levels of four proteins in T24CDDPR in three different concentrations of cisplatin were investigated (Fig. 4). HNRNPA3 and PGK1 showed increased expression irrespective of the exposure of cisplatin concentration. PCK2 and PPL demonstrated gradually decreased expression, which correlated with the exposure of cisplatin concentration.
DISCUSSION Proteomics research is a new biological modality that contributes greatly to our understanding of gene functions in the post-genomic era and that focuses on protein expression and post-translational modification. There is an increasing interest in proteomic techniques because the cells could express the function of proteins in various ways, but DNA sequence information provides a static frame. The detection of proteins was based on the consideration that cells could produce specific proteins to exert specific functions. When cells encounter unusual situations, they try to adjust by expressing proteins that may help to deal with the new situation. Such proteins, specifically synthesized on demand, may indicate characteristic disease states and thus may serve as diagnostic markers. Identifying anti-cancer drug resistance proteins using proteomic techniques in bladder cancer has been performed in recent reports. Miura et al. (13) identified 36 differentially expressed proteins; 21 proteins were upregulated and 15 were downregulated in HT1376 cisplatin-resistant bladder cancer cell lines exposed to 5 μM cisplatin. They reported that adseverin (SCIN), which is a calcium-dependent actin-binding protein, was upregulated four-fold in cisplatin-resistant cells compared to cisplatin-naïve cells. The mitochondrial fraction was more increased than the cytoskeletal fraction among the differentiated proteins. Meng et al. (10) identified 30 proteins (19 upregulated and 11 downregulated proteins) in pumc91 bladder cancer cell lines exposed to 1.0 mg/L adriamycin. They demonstrated that annexin A2 (ANXA2), a calcium-dependent phospholipid-binding protein, and nucleophosmin (NPM1), a nonribosomal nucleolar phosphoprotein, were upregulated in adriamycin-resistant cells compared to adriamycin-naïve cells. Transferase and oxidoreductase were prominent fractions among differential proteins. These proteins, described in previous reports (10, 13), play important roles in apoptosis and cell proliferation. Once cancer cells acquire a resistance to anti-cancer drugs because of a changing protein profile, they show unregulated and aggressive behavior, resulting in patients suffering from bulky tumors after local progression and distant metastasis. In this study, we performed a proteomics analysis to identify differential protein expressions of cisplatin-naïve and cisplatin-resistant bladder cancer cell lines. We successfully identified 25 proteins with significantly altered expression levels. Based on their functions, these proteins are mainly involved
80,000
42,000
30
46,567
77,209
48,029
204,526
226,392
51,304
30,760
47,140
57,901
166,469
71,413
46,567
37,074
67,835
44,587
47,446
39,595
32,658
44,066
51,304
30,760
44,840
70,592
95,278
40,397
55,955
44,939
Plasminogen activator inhibitor 2
Protein-glutamine gamma-glutamyltransferase 2
Keratin, type I cytoskeletal 18
Periplakin
Myosin-9
Keratin, type II cytoskeletal 7
Keratin, type II cytoskeletal 8
Alpha-enolase
Pyruvate kinase PKM
Eukaryotic translation initiation factor 3 subunit A
Cytoplasmic dynein 1 intermediate chain 2
Plasminogen activator inhibitor 2
Aldo-keto reductase family 1 member C4
Transketolase
Phosphoglycerate kinase 1
Aspartate aminotransferase, mitochondrial
Heterogeneous nuclear ribonucleoprotein A3
Tropomyosin alpha-1 chain
Keratin, type I cytoskeletal 19
Protein-glutamine gamma-glutamyltransferase 2
Keratin, type I cytoskeletal 18
Keratin, type II cytoskeletal 7
Keratin, type II cytoskeletal 8
DnaJ homolog subfamily A member 1
Phosphoenolpyruvate carboxykinase
Elongation factor 2
Phosphoserine aminotransferase
Serine hydroxymethyltransferase
Plasminogen activator inhibitor 1 RNA-binding protein
Sulfide:quinone oxidoreductase
Protein Name
SERPINB2
TGM2
KRT18
PPL
MYH9
KRT7
KRT8
ENO1
PKM
EIF3A
DYNC1I2
SERPINB2
AKR1C4
TKT
PGK1
GOT2
HNRNPA3
TPM1
KRT19
TGM2
KRT18
KRT7
KRT8
DNAJA1
PCK2
EEF2
PSAT1
SHMT2
SERBP1
SQRDL
G.N.
108.2
208.26
266.27
72.20
268.26
222.22
272.28
178.23
202.22
220.27
110.2
108.2
60.2
186.21
190.25
110.23
142.3
58.3
204.3
168.3
160.2
154.2
206.2
168.3
104.2
268.28
190.2
200.3
182.24
146.2
Score
P05120
P21980
P05783
O60437
P35579
P08729
P05787
P06733
P14618
Q14152
Q13409
P05120
P17516
P29401
P00558
P00505
P51991
Q9Y427
P08727
P21980
P05783
P08729
P05787
P31689
Q16822
P13639
Q9Y617
P34897
Q8NC51
Q9Y6N5
Accession No.
29.74
25.32
58.57
5.81
5.81
37.87
52.39
16.3
44.03
16.3
23.23
29.74
9.49
25.68
38.25
26.82
31.78
12.45
53.65
25.25
42.57
30.65
52.83
41.15
16.93
27.42
37.38
32.09
35.71
27.58
Seq. Cov. (%)
11
21
27
8
27
23
28
18
21
24
11
11
6
19
19
11
15
6
21
18
16
16
21
17
11
27
20
21
20
16
PSMs
Increase
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Increase
Increase
Increase
Increase
Increase
Increase
Increase
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Decrease
Status
Spot No., spot number; M.W. (Obs.), observed molecular weight; M.W. (Theo.), theoretical molecular weight; G.N., gene name; Score, SEQUEST score; Accession No., accession number; Seq. Cov. (%), sequence coverage; PSMs, peptide-spectrum matches; Status, change in the expression levels of the protein in cisplatin-resistant T24 compared to that in T24.
45,000
29
42,000
19
28
40,000
18
210,000
70,000
17
220,000
45,000
16
27
45,000
15
26
43,000
14
49,000
41,000
13
40,000
42,000
25
80,000
11
12
24
45,000
10
45,000
49,000
09
55,000
40,000
08
23
45,000
07
22
70,000
06
70,000
95,000
05
150,000
41,000
04
21
77,209
47,000
03
20
48,029
45,000
02
49,929
43,000
01
M.W. (Theo.)
M.W. (Obs.)
Spot No.
Table 1 Protein expression profile in cisplatin-resistant T24 compared to that in T24
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258
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Fig. 2 Differentially expressed representative proteins, PCK2 (A) and HNRNPA3 (B). (a) Two-dimensional electrophoresis gel. (b) Gel images, enlarged in triplicate, of the area containing protein spots. Red arrow shows increased expression and blue arrow shows decreased expression. (c) Western blot. (d) Protein expression levels calculated from Western blot.
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Fig. 3 (a) Protein expression in various bladder cancer cell lines using Western blot. Protein was applied to each column (20 μg). Lane 1: cisplatin-naïve T24, Lane 2: cisplatin-resistant T24, Lane 3: RT4, Lane 4: RT4ρ0, Lane 5: 5637, Lane 6: EJ, Lane 7: TCCSUP. (b) Table shows protein expression levels. Each protein level is calculated from Western blot and compared to the level of T24.
in cytoskeletal proteins and enzyme modulators. Some of these proteins have been reported to be associated with chemotherapy resistance in previous proteomic studies (10, 13). Our study also found several novel candidate resistance-associated proteins. Using PubMed, HNRNPA3 and PCK2 showed no match with “bladder cancer” or “urothelial carcinoma” and only showed one match with “cisplatin”; there was one study of HNRNPA3 with cisplatin, reporting that the expression of HNRNPA3 was upregurated in cisplatin-resistant breast cancer cells (18). These proteins need to validate whether proteins would contribute to responding to anti-cancer therapy. The analysis of protein changes after treatment may aid in clinical decisions and may possibly facilitate optimized personalized treatments. More than 20 distinct HNRNPs have been identified in human cells, designated HNRNPA1 to HNRNPU in increasing molecular size from 32 to 110 kDa. HNRNPs are RNA-binding proteins (RBP) that are essential players in mRNA metabolism, acting as coordinators of post-transcriptional events by participating in an extensive network of RNA–RBP inter-
action. Individual HNRNPs also function in several other cellular processes, like transcription, DNA repair, telomere biogenesis, and cell signaling (2, 4, 6). As a consequence of their multiple roles in the regulation of gene expression, expression levels of HNRNP are tightly linked to cell proliferation (4). Our findings showed HNRNPA3 overexpression, which may acquire biological aggressiveness after exposure to cisplatin in bladder cancer cells. PCK activity, which catalyzes the conversion of oxaloacetate to phosphoenolpyruvate, is distributed in cytosol and mitochondria as a result of two enzymatically indistinct isozymes, PCK1 and PCK2 (9). PCK2 is important for maintaining cell progression and survival, especially under stress conditions. Our results showed downregulation of PCK2 using cisplatin-resistant bladder cancer cell lines. Park et al. (15) investigated the association of PCK2 expression with chemoradiation response using 5-fluouracyl (5-FU). They reported that downregulation of PCK2 may be linked to induced 5-FU resistance, but not to intrinsic 5-FU susceptibility, in colon cancer cells. Because low energy metabolism and slow
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Fig. 4 (a) Protein expression in various resistance levels of bladder cancer cell lines using Western blot. Protein was applied (20 μg) in each column. Lane 1: cisplatin-naïve T24, Lane 2: cisplatin-resistant T24 using 13.3 μM of cisplatin, Lane 3: cisplatin-resistant T24 using 26.6 μM of cisplatin, Lane 4: cisplatin-resistant T24 using 40.0 μM of cisplatin. (b) Table shows protein expression levels. Each protein level is calculated from Western blot and compared to the level of T24.
proliferation can decrease the susceptibility to chemotherapy (17, 22), PCK2 downregulation could lead to slow energy metabolism and may subsequently reduce the susceptibility to anti-cancer drugs. Cisplatin resistance is most often multifactor, multistep, and multipathway (3). In our study, we found proteins possibly correlated with cisplatin resistance and showed that each protein demonstrated each behavior in various concentrations of cisplatin. For example, periplakin, which is involved in cellular movement and attachment (7), showed gradually decreased expression correlated with the concentration of cisplatin and, on the contrary, HNRNPA3 showed incremental expressions. This finding suggests that each protein would be preferentially expressed at the time of exposure level of cisplatin, and that it might be difficult to completely inhibit a single mechanism regulating cisplatin resistance to restore the susceptibility. The results could lead to the development of a new molecular marker. This would be an important step in establishing a treatment strategy for patients with advanced cancer. However, it is unlikely that
all the detected proteins would be related to cisplatin resistance. We observed that cancer cells chemically changed the protein profile, and we think of this as “cisplatin resistance.” In addition, our results demonstrated that a variety of protein characteristics contributed to resistance, including high molecular weight and complex protein profile (10, 13). Finally, the observed proteins were not noted to be fully shared by other cancer models because of the heterogeneity of cancerous cells. Further studies are warranted to determine the potential application of altered proteins that are possibly connected to cisplatin resistance. Acknowledgment This study was supported in part by a Grant-in-Aid for Scientific Research C (15K10607) from The Japan Society for the Promotion of Science (to K. Matsumoto).
Cisplatin-related proteins
REFERENCES 1. Cook CC, Kim A, Terao S, Gotoh A and Higuchi M (2012) Consumption of oxygen: a mitochondrial-generated progression signal of advanced cancer. Cell Death Dis 3, e258. 2. Dreyfuss G, Kim VN and Kataoka N (2002) MessengerRNA-binding proteins and the messages they carry. Nat Rev Mol Cell Biol 3, 195–205. 3. Galluzzi L, Senovilla L, Vitale I, Michels J, Martins I, Kepp O, Castedo M and Kroemer G (2012) Molecular mechanisms of cisplatin resistance. Oncogene 31, 1869–1883. 4. He Y, Brown MA, Rothnagel JA, Saunders NA and Smith R (2005) Roles of heterogeneous nuclear ribonucleoproteins A and B in cell proliferation. J Cell Sci 118, 3173–3183. 5. Ikeda M, Matsumoto K, Tabata K, Minamida S, Fujita T, Satoh T, Iwamura M and Baba S (2011) Combination of gemcitabine and paclitaxel is a favorable option for patients with advanced or metastatic urothelial carcinoma previously treated with cisplatin-based chemotherapy. Jpn J Clin Oncol 41, 1214–1220. 6. Krecic AM and Swanson MS (1999) hnRNP complexes: composition, structure, and function. Curr Opin Cell Biol 11, 363–371. 7. Matsumoto K, Ikeda M, Sato Y, Kuruma H, Kamata Y, Nishimori T, Tomonaga T, Nomura F, Egawa S and Iwamura M (2014) Loss of periplakin expression is associated with pathological stage and cancer-specific survival in patients with urothelial carcinoma of the urinary bladder. Biomed Res (Tokyo) 35, 201–206. 8. Matsumoto K, Mochizuki K, Hirayama T, Ikeda M, Nishi M, Tabata K, Okazaki M, Fujita T, Taoka Y and Iwamura M (2015) Gemcitabine plus nedaplatin as salvage therapy is a favorable option for patients with progressive metastatic urothelial carcinoma after two lines of chemotherapy. Asian Pac J Cancer Prev 16, 2483–2487. 9. Mendez-Lucas A, Hyrossova P, Novellasdemunt L, Vinals F and Perales JC (2014) Mitochondrial phosphoenolpyruvate carboxykinase (PEPCK-M) is a pro-survival, endoplasmic reticulum (ER) stress response gene involved in tumor cell adaptation to nutrient availability. J Biol Chem 289, 22090– 22102. 10. Meng Q, Lei T, Zhang M, Zhao J, Zhao XH and Zhang M (2013) Identification of proteins differentially expressed in adriamycin-resistant (pumc-91/ADM) and parental (pumc-91) human bladder cancer cell lines by proteome analysis. J Cancer Res Clin Oncol 139, 509–519. 11. Minami S, Nagashio R, Ueda J, Matsumoto K, Goshima N, Hattori M, Hachimura K, Iwamura M and Sato Y (2014) Detection of tumor-associated antigens in culture supernatants using autoantibodies in sera from patients with bladder cancer. Biomed Res (Tokyo) 35, 25–35. 12. Misawa T, Kikkawa F, Maeda O, Obata NH, Higashide K, Suganuma N and Tomoda Y (1995) Establishment and char-
261 acterization of acquired resistance to platinum anticancer drugs in human ovarian carcinoma cells. Jpn J Cancer Res 86, 88–94. 13. Miura N, Takemori N, Kikugawa T, Tanji N, Higashiyama S and Yokoyama M (2012) Adseverin: a novel cisplatin-resistant marker in the human bladder cancer cell line HT1376 identified by quantitative proteomic analysis. Mol Oncol 6, 311–322. 14. Okusa H, Kodera Y, Oh-ishi M, Minamida S, Tsuchida M, Kavvoussi PK, Matsumoto K, Satoh T, Iwamura M, Maeda T and Baba S (2008) Searching for new biomarkers of bladder cancer based on proteomics analysis. J Electrophoresis 52, 19–24. 15. Park JW, Kim SC, Kim WK, Hong JP, Kim KH, Yeo HY, Lee JY, Kim MS, Kim JH, Yang SY, Kim DY, Oh JH, Cho JY and Yoo BC (2014) Expression of phosphoenolpyruvate carboxykinase linked to chemoradiation susceptibility of human colon cancer cells. BMC Cancer 14, 160. 16. Saxman SB, Propert KJ, Einhorn LH, Crawford ED, Tannock I, Raghavan D, Loehrer PJ, Sr. and Trump D (1997) Longterm follow-up of a phase III intergroup study of cisplatin alone or in combination with methotrexate, vinblastine, and doxorubicin in patients with metastatic urothelial carcinoma: a cooperative group study. J Clin Oncol 15, 2564–2569. 17. Shin YK, Yoo BC, Chang HJ, Jeon E, Hong SH, Jung MS, Lim SJ and Park JG (2005) Down-regulation of mitochondrial F1F0-ATP synthase in human colon cancer cells with induced 5-fluorouracil resistance. Cancer Res 65, 3162–3170. 18. Smith L, Welham KJ, Watson MB, Drew PJ, Lind MJ and Cawkwell L (2007) The proteomic analysis of cisplatin resistance in breast cancer cells. Oncol Res 16, 497–506. 19. Tabata K, Matsumoto K, Minami S, Ishii D, Nishi M, Fujita T, Saegusa M, Sato Y and Iwamura M (2014) Nestin is an independent predictor of cancer-specific survival after radical cystectomy in patients with urothelial carcinoma of the bladder. PLoS One 9, e91548. 20. Tsumura H, Matsumoto K, Ikeda M, Yanagita K, Hirano S, Hagiwara M, Nagashio R, Fujita T, Sato Y and Iwamura M (2015) High expression level of preoperative serum uroplakin III is associated with biologically aggressive bladder cancer. Asian Pac J Cancer Prev 16, 1539–1543. 21. von der Maase H, Hansen SW, Roberts JT, Dogliotti L, Oliver T, Moore MJ, Bodrogi I, Albers P, Knuth A, Lippert CM, Kerbrat P, Sanchez Rovira P, Wersall P, Cleall SP, Roychowdhury DF, Tomlin I, Visseren-Grul CM and Conte PF (2000) Gemcitabine and cisplatin versus methotrexate, vinblastine, doxorubicin, and cisplatin in advanced or metastatic bladder cancer: results of a large, randomized, multinational, multicenter, phase III study. J Clin Oncol 18, 3068–3077. 22. Yoo BC, Ku JL, Hong SH, Shin YK, Park SY, Kim HK and Park JG (2004) Decreased pyruvate kinase M2 activity linked to cisplatin resistance in human gastric carcinoma cell lines. Int J Cancer 108, 532–539.
