Lung Resistance Protein and Multidrug Resistance Protein in Non-small Cell Lung Cancer and Their Clinical Significance

The Journal of International Medical Research 2011; 39: 1693 1700 Lung Resistance Protein and Multidrug Resistance Protein in Non-small Cell Lung Cancer and Their Clinical Significance ZJ CHEN 1, HB LE 1, YK ZHANG 1, LY QIAN 2, K RAJA SEKHAR 3 AND WD LI 4 1 Department of Thoracic Surgery, and 2 Department of Pathology, The First Hospital, Zhoushan City, China; 3 Department of General Surgery, and 4 Department of Cardiothoracic Surgery, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China This study examined lung resistance protein (LRP) and multidrug resistance protein (MRP) in lung tumour tissue from 92 patients with non-small cell lung cancer (CLC) and normal lung tissue from 20 patients with benign lung tumours. The rates for LRP- and MRPpositive tumours among the CLC cases were 54% and 59%, respectively, and their combined positive rate was 45%. These rates were significantly higher than in normal lung tissue. The rates of LRP- and MRP-positive tumours were significantly higher among cases of adenocarcinoma than in cases of squamous cell carcinoma, and in highly differentiated tumours compared with tumours of low or moderate differentiation. There was a significant association between LRP- and MRP-positive tumours and a decrease in overall survival. In conclusion, LRP and MRP play a role in multidrug resistance in CLC and are related to prognosis in patients with CLC. KEY WORDS: NON-SMALL CELL LUNG CANCER; LUNG RESISTANCE PROTEIN; MULTIDRUG-RESISTANCE PROTEIN Introduction Lung cancer is the leading cause of cancerrelated mortality worldwide, and chemotherapy is important in combination therapy for this disease. 1 Postoperative chemotherapy is an important adjuvant treatment in non-small cell lung cancer (CLC), used to prevent tumour recurrence and prolong survival. 2 In spite of treatment with adjuvant chemotherapy, tumour recurrence and metastasis can occur shortly after surgery. 2 The primary cause of tumour recurrence and metastasis is multidrug resistance (MDR). 3 Drug resistance is likely to be multifactorial 3 and molecular markers that predict treatment response would be extremely useful in selecting treatment protocols for patients. Multidrug resistance protein (MRP) and lung resistance protein (LRP) are involved in drug resistance in CLC cell lines. 4 7 MRP is a member of the ATP-binding cassette (ABC) transport protein superfamily. 4 Overexpression of MRP has been detected in 1693

a variety of tumour types 5 and is associated with drug resistance or poor patient outcome in lung cancer. 8 LRP is identical to human major vault protein, and is associated with in vitro resistance to anticancer drugs (including etoposide, doxorubicin and paclitaxel) and also to non-classical MDR to drugs such as cisplatin and carboplatin. 6,7 The present study examined the relationship between MRP and LRP in CLC tissue samples (assessed by immunohistochemistry), and adjuvant chemotherapy response and patient survival, in order to investigate the predictive value of these chemotherapy resistancerelated proteins. Patients and methods STUDY POPULATION AND SAMPLE COLLECTION Patients with CLC (squamous cell carcinoma or adenocarcinoma) who underwent surgical resection at the First Hospital, Zhoushan City, China, between January 2007 and June 2009 were enrolled sequentially in the study. Patients who had received radiotherapy or chemical therapy prior to surgery were excluded. Tumours were categorized according to the tumour, node, metastasis (TNM) staging system for lung cancer. Patients with stage I or II tumours were combined into the earlystage group and those with stage III and IV tumours were combined into the late-stage group. Patients with stage II or higher CLC received postsurgical chemotherapy every 21 days that consisted of 30 mg/m 2 vinorelbine on days 1 and 8 and 80 mg/m 2 cisplatin on day 1, both administered intravenously. Patients with benign lung tumours (hamartoma, inflammatory pseudotumour or sclerosing haemangioma) formed the control group. Tumour tissue from patients and normal lung tissue from controls (taken at a distance of 15 cm away from lesions) were excised for examination. The study was approved by the Ethics Committee of The First Hospital, Zhoushan City, and all patients provided written informed consent prior to their participation in the study. IMMUNOHISTOCHEMICAL ANALYSIS OF LRP AND MRP Formalin-fixed paraffin-embedded tissue sections (4 µm) were stained according to the Envision method 8 using Envision immunohistochemistry kits (Shanghai Bioleaf Biotech Co. Ltd, Shanghai, China). Briefly, the tissue sections were deparaffinized, rehydrated and then microwaved in 10 mm citrate buffer, ph 6.0, for 15 min. Endogenous peroxidase activity was blocked with hydrogen peroxide in methanol for 10 min at 24 C in order to avoid background staining. The sections were then incubated with a mouse monoclonal anti-mrp antibody or an anti- LRP antibody (1 : 100 dilution; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) for 1 h at 24 C. The slides were washed three times with 10 mm phosphate-buffered saline (PBS), ph 7.4, for 5 min, then incubated with a horseradish peroxidase-conjugated rabbit antimouse secondary antibody (1 : 500 dilution; Shanghai Bioleaf Biotech Co. Ltd) for 30 min at 24 C. The slides were washed again three times with 10 mm PBS for 5 min and incubated with 10 µg/ml streptavidin biotin for 30 min at 24 C. The colour reaction was developed in 3,3 - diaminobenzidine tetrahydrochloride (DAB; Sigma-Aldrich, St Louis, MO, USA), and slides were counterstained with haematoxylin and eosin. Stained tissue sections were examined at a magnification of 200 under a light microscope. LRP and MRP staining was seen 1694

as yellow or brown orange, with a granulated, pieced or zonal arrangement without any background staining. MRP was located in the cytoplasm and cell membrane, and LRP was located in the cell membrane. For each slide, 100 tumour cells in each of 10 fields were examined and the mean number of positive cells was recorded. LRP and MRP staining was evaluated according to a weighting method, using both the percentage and intensity of tumour cells within the tumour. 9 The percentage of tumour cells was recorded as follows: 0 points, 0 1%; 1 point, 2 25%; 2 points, 26 50%; 3 points, 51 75%; and 4 points, > 75%. Tumour intensity was recorded as follows: 1 point, faint yellow; 2 points, yellow; and 3 points, brown orange. The final weighting score was calculated by multiplying the percentage and intensity scores. A final score of 0 4 was considered to be negative and a score of > 4 as positive. Each section was evaluated by three independent observers who were blinded to the clinical status of the patients. STATISTICAL ANALYSES Statistical analyses were performed using the SPSS statistical software, version 16.0 (SPSS Inc., Chicago, IL, USA) for Windows. The Pearson s χ 2 -test was used for between-group comparisons of LRP and MRP. Cumulative survival rates were calculated by Kaplan Meier survival analysis and compared using the log-rank test. A value of P < 0.05 was considered to be statistically significant. Results The study included 92 patients with CLC; 51 with squamous cell carcinoma and 41 with adenocarcinoma. According to TNM staging criteria, 16 patients were stage I, 37 were stage II, 36 were stage III and three were stage IV. The control group included 20 patients with benign lung tumours, comprising five cases of hamartoma, seven of inflammatory pseudotumour, and eight of sclerosing haemangioma. Rates of positive staining for MRP and LRP in CLC and normal lung tissue are given in Table 1. CLC tissue had significantly higher levels of MRP-positive, LRP-positive, and MRP/LRP-positive cells than normal lung tissue (P < 0.001 for all). The total positive rate for either of the two proteins (i.e. the percentage of patients positive for MRP or LRP) was 68% (63/92 patients). The association between LRP and MRP and clinical features is shown in Table 2. Across subgroup comparison showed that the percentages of LRP and MRP immunopositive tumours were significantly higher for adenocarcinomas and bronchoalveolar carcinoma than for squamous cell carcinoma TABLE 1: Proportion of cases that were positive for multidrug resistance protein (MRP) and lung resistance protein (LRP) in tumour tissue from patients with non-small cell lung cancer (CLC) and normal lung tissue from patients with benign lung tumours MRP-positive and Tissue type n LRP-positive MRP-positive LRP-positive CLC 92 50 (54) 54 (59) 41 (45) Normal lung tissue 20 2 (10) 3 (15) 1 (5) Statistical significance a P < 0.001 P < 0.001 P < 0.001 Data presented as n (%) of patients. a Pearson s χ 2 -test. 1695

TABLE 2: The association between multidrug resistance protein (MRP) or lung resistance protein (LRP) and clinical features in the patients with non-small cell lung cancer (n = 92) LRP MRP (%) Positive Negative Statistical Positive Negative Statistical Variable n n (%) n (%) significance a n (%) n (%) significance a Age 35 55 years 53 27 (51) 26 (49) 27 (51) 26 (49) 56 76 years 39 22 (56) 17 (44) 21 (54) 18 (46) Gender Male 53 30 (57) 23 (43) 30 (57) 23 (43) Female 39 19 (49) 20 (51) 18 (46) 21 (54) Differentiation High 35 25 (71) 10 (29) 23 (66) 12 (34) Moderate 21 13 (62) 8 (38) P = 0.002 12 (57) 9 (43) P = 0.039 Low 36 11 (31) 25 (69) 13 (36) 23 (64) Pathology Squamous cell carcinoma 51 22 (43) 29 (57) 21 (41) 30 (59) Adenocarcinoma other than P = 0.038 bronchoalveolar carcinoma 20 11 (55) 9 (45) 11 (55) 9 (45) P = 0.025 Bronchoalveolar carcinoma 21 16 (76) 5 (24) 16 (76) 5 (24) Clinical stage I 16 7 (44) 9 (56) 7 (44) 9 (56) II 37 17 (46) 20 (54) 18 (49) 19 (51) III 36 23 (64) 13 (36) 20 (56) 16 (44) IV 3 2 (67) 1 (33) 3 (100) 0 (0) Lymph node metastases N0 37 18 (49) 19 (51) 18 (49) 19 (51) N1 29 15 (52) 14 (48) 16 (55) 13 (45) N2 26 16 (62) 10 (38) 14 (54) 12 (46) a Pearson s χ 2 -test; statistically analysed across subgroup comparisons for LRP- and MRP-positive tumours., not statistically significant (P > 0.05). 1696

or adenocarcinomas (P = 0.038 and P = 0.025 for LRP and MRP, respectively). Tumours that were highly differentiated had a significantly higher immunopositive rate for LRP and MRP compared with low or moderately differentiated tumours (P = 0.002 and P = 0.039 for LRP and MRP, respectively). There were no significant differences in the incidence of MRP and LRP positive tumours with respect to age, gender, TNM clinical stage, or lymph node metastasis (Table 2). The mean ± SD duration of follow-up was 23.8 ± 6.5 months (range 8 36 months), and 30 (33%) patients died during the follow-up period. Overall survival rates for LRP- and MRP-positive cases were significantly lower than for LRP- or MRPnegative cases (P = 0.038 [Fig. 1] and P = 0.004 [Fig. 2], respectively). Overall survival rates were significantly lower in patients with both LRP- and MRP-positive tumours compared with patients with either LRP- or MRP-positive tumours (but not both), and compared with patients with both LRP- and MRP-negative tumours (P = 0.023; Fig. 3). Discussion The mechanism of clinical drug resistance in CLC is likely to be multifactorial. 3 LRP and MRP are encoded by drug resistance-related genes 10,11 that have been shown to play an important role in drug resistance in CLC. 12,13 The present study found significantly higher rates of LRP- and MRP-positive cells in CLC than in normal tissue from patients with benign lung tumours. The total positive rate in CLC patients for the two proteins was 68%. A previous study found three types of drug resistance-related protein in CLC, with a total positive rate of 90%. 14 This previous report, together with the present study, show that drug resistance is common in CLC. 100 80 LRP-negative Survival (%) 60 40 LRP-positive P = 0.038 20 0 0 6 12 18 24 Survival after operation (months) 30 36 FIGURE 1: Cumulative survival rates in the 92 patients with non-small cell lung cancer according to whether their tumours were positive or negative for lung resistance protein (LRP) (Kaplan Meier survival analysis compared by the log-rank test) 1697

100 80 MRP-negative Survival (%) 60 40 MRP-positive P = 0.004 20 0 0 6 12 18 24 Survival after operation (months) 30 36 FIGURE 2: Cumulative survival rates in the 92 patients with non-small cell lung cancer according to whether their tumours were positive or negative for multidrug resistance protein (MRP) (Kaplan Meier survival analysis and compared by the log-rank test) 100 80 Both LRP-negative and MRP-negative Survival (%) 60 40 Both LRP-positive and MRP-positive Either LRP-positive or MRP-positive P = 0.023 20 0 0 6 12 18 24 Survival after operation (months) 30 36 FIGURE 3: Cumulative survival rates in the 92 patients with non-small cell lung cancer according to whether their tumours were positive or negative for both lung resistance protein (LRP) and multidrug resistance protein (MRP) (Kaplan Meier survival analysis compared by the log-rank test) The variation in LRP- and MRP-positive cases of CLC in the present study suggests that the mechanism of MDR differs between the various histological subtypes. The 1698

incidence of LRP- and MRP-positive tumours was significantly higher in adenocarcinoma compared with squamous cell carcinoma, suggesting that a higher MDR phenotype exists in adenocarcinoma. This is consistent with a previous report that chemotherapy is more effective in squamous cell carcinoma than adenocarcinomas. 15 The significantly higher rates of LRP- and MRP-positive bronchoalveolar carcinoma is consistent with reduced effectiveness of chemotherapy; this mechanism needs further study. The present finding that highly differentiated tumours were significantly more likely to be LRP- and MRP-positive compared with those with low or moderate differentiation is consistent with the low sensitivity to chemotherapy drugs found in highly differentiated forms of lung cancer. 16 Consistent with previous reports, 17 there was no significant difference in the rate of LRP- and MRP-positive tumours with respect to patient age, gender, TNM clinical stage or lymph node metastasis in the present study. This indicates that LRP and MRP cannot be used to estimate the progression of lung cancer. Increased LRP or MRP levels have been shown to be associated with decreased survival, indicating a role for MRP and LRP in the prognosis of lung cancer. 18 Similarly, survival rates among LRP- and MRP-positive cases in the present study were significantly lower than for LRP- or MRP-negative cases. Multidrug resistance in lung cancer is complex and is associated with multiple proteins. 19,20 Understanding the mechanism of tumour drug resistance and recurrence will help to evaluate the effectiveness of chemotherapy and patient prognosis, and assist in selecting individualized chemotherapy regimens according to drug resistance mechanisms. MDR modulators, such as dexniguldipine and cyclosporine A, may be used to alter the chemosensitivity of tumour cells, thereby improving the effect of chemotherapy and prolonging patient survival time. 21 In conclusion, LRP and MRP play a role in MDR in CLC and are related to prognosis in patients with CLC. Conflicts of interest The authors had no conflicts of interest to declare in relation to this article. Received for publication 7 March 2011 Accepted subject to revision 5 May 2011 Revised accepted 1 September 2011 Copyright 2011 Field House Publishing LLP References 1 Molina JR, Yang P, Cassivi SD, et al: Non-small cell lung cancer: epidemiology, risk factors, treatment, and survivorship. Mayo Clin Proc 2008; 83: 584 594. 2 Okamoto T, Ichinose Y: Adjuvant chemotherapy for non-small cell lung cancer. Gan To Kagaku Ryoho 2006; 33: 1985 1990 [in Japanese, English abstract]. 3 Monzo M, Rosell R, Taron M: Drug resistance in non-small cell lung cancer. Lung Cancer 2001; 34(suppl 2): S91 S94. 4 Young LC, Campling BG, Cole SP, et al: Multidrug resistance proteins MRP3, MRP1, and MRP2 in lung cancer: correlation of protein levels with drug response and messenger RNA levels. Clin Cancer Res 2001; 7: 1798 1804. 5 Young LC, Campling BG, Voskoglou-Nomikos T, et al: Expression of multidrug resistance protein-related genes in lung cancer: correlation with drug response. Clin Cancer Res 1999; 5: 673 680. 6 Laurençot CM, Scheffer GL, Scheper RJ, et al: Increased LRP mrna expression is associated with the MDR phenotype in intrinsically resistant human cancer cell lines. Int J Cancer 1997; 72: 1021 1026. 7 Berger W, Elbling L, Micksche M: Expression of the major vault protein LRP in human nonsmall-cell lung cancer cells: activation by shortterm exposure to antineoplastic drugs. Int J Cancer 2000; 88: 293 300. 1699

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