Li-xin Fan et al.: Significance of ABC Transporter in HCC Journal of Hard Tissue Biology 25[1] (2016) 81- 88 © 2016 The Hard Tissue Biology Network Association Printed in Japan, All rights reserved. Original CODEN-JHTBFF, ISSN 1341-7649 Clinical Significance of ABC Transporter Expression in Patients with Hepatocellular Carcinoma

Li-xin Fan1,3), Yong Zhang2), Yi Zhou3), Zhi-dong Wang2), Yong-mei Zhang3) and Hai-long Chen1)

1) Combine Traditional Chinese and Western Medicine Postdoctoral Station, Dalian Medical University, Dalian, China 2) Department of Hepatobiliary Surgery, Department of Pathology, The Sixth Hospital of Dalian, Dalian, China 3) Department of Medical Oncology, The Third Hospital of Dalian, Dalian, China (Accepted for publication, November 9, 2015)

Abstract: To gain insight into the clinical significance of expression of ABC transporter antigen in patients with hepatocellular carcinoma (HCC), we used immunohistochemistry to determine the patterns of ABCB1, ABCC1 and ABCG2 expression in 30 resected HCC tissues, 26 paired adjacent nonneoplastic tissues and 9 cirrhosis tissues accompanied with chronic hepatitis B virus infection in this study. It was found that the expression rates of ABCB1, ABCC1 and ABCG2 were 56.7 %, 93.3 % and 83.3 % in patients with HCC, respectively. ABCB1 and ABCC1 were expressed significantly differently among HCC, adjacent non-neoplastic and cirrhosis tissues. There were no significant differences in the clinical factors associated with the expression of these three ABC transporter antigens. Furthermore, preoperative TACE treatment didn’t affect the expression of these three ABC transporters. Moreover, patients with higher expression of both ABCC1 and ABCB1 showed significantly shorter disease-free survival (DFS) than those with lower expression of both antigens (P<0.01).Therefore, higher expression of ABCC1 and ABCB1 might be an unfavorable prognostic factor in patients of HCC, and represent an important hurdle to chemotherapy.

Key words: ABCB1, ABCC1, ABCG2, Hepatocellular carcinoma

Introduction critical role in drug efflux and chemoresistance in many Hepatocellular carcinoma (HCC) is the fifth most common malignancies, such as HCC7,8). It transports chemotherapy drugs, type of cancer worldwide. More than 50% of new cases diagnosed such as doxorubicin that is the routine medication of HCC9). The per year globally are in China1). Hepatitis B infection and followed overexpression of the ABCC1 transporter confers resistance to a cirrhosis contribute to higher incidence rates of HCC in China2). wide range of anticancer drugs, such as anthracyclines10) that is Poor disease process of HCC is caused by tumor metastasis and used to treat HCC. Decreasing ABCB1 expression and increasing recurrence. ABCC1 expression were associated with increasing Resistance to chemotherapy is the primary cause for HCC dedifferentiation and microvascular invasion11,12). Mitoxantrone, treatment failure3). Higher expression of ATP-binding cassette boron-dipyrromethene (BODIPY)-prazosin and almost all (ABC) transporters usually induces the multidrug resistance nucleoside inhibitors are substrates of ABCG213,14). As a progenitor (MDR) phenotype. As optimal candidates of biomarkers, ABC marker, ABCG2 was also used to identify the cancer stem cells transporter are used to predict chemotherapy resistance (CSCs) of HCC15).Although several ABC transporters have been and prognosis after chemotherapy4,5). Recently, 49 members of confirmed abnormally expression in HCC tissue16), there is the ABC transporter family have been identified and divided into discrepancy between studies aimed at the modulation of ABC seven subfamilies (ABCA through ABCG)6). Among them, transporters in HCC. ABCB1(MDR1/P-gp), ABCC1(MRP1), and ABCG2(BCRP/ A greater understanding of the role of ABC transporters in MXR) are major mediators of the efflux of anticancer drugs from HCC chemo-resistance will be important for the development of cancer cells6). more effective targeted therapies. In this study, we aimed to ABCB1 is the first identified ABC transporter. It plays a investigate the expression of ABCB1, ABCC1 and ABCG2 in patients with HCC. We determined the patterns of ABCB1, ABCC1 Correspondence to: Dr. Hai-long Chen, Combine Traditional Chinese and and ABCG2 expression in 30 resected HCC tissues, 26 paired Western Medicine Postdoctoral Station, Dalian Medical University, 9 West Section, Lvshun South Road, Dalian, 116044, China; Tel: +86-411-39052205; adjacent nonneoplastic tissues and 9 cirrhosis tissues accompanied Fax: +86-411-39052233; E-mail: [email protected] with chronic hepatitis B virus infection by immunohistochemistry. 81 J.Hard Tissue Biology Vol. 25(1):81 -88, 2016 The results achieved in this study will deepen our understanding Olympus, Tokyo, Japan). Two pathologists who were blind to of the expression and prognostic value of ABC transporters in diagnosis independently inspected the slices. The rate of agreement HCC. between these two pathologists was 95 %. The scores from both pathologists were averaged to provide the final score for each Materials and Methods case. Sections were scored combination of the percentage of Patients and samples positive cells and staining intensity. Positive cell percentage was Archival HCC tissues and cirrhosis samples from surgically scored based on the average percentage of positive cells per 100 resected specimens were obtained randomly from the patients at cells in 10 high-power fields, as follows: 0–10%: score 0; 11-25 the Sixth Hospital of Dalian (China) from 2010 to 2015 after %: score 1; 26-50 %: score 2; 51-75 %: score 3; and >75 %: score obtaining patients’ informed consent and approval by the Research 4. Staining intensity was scored as follows: negative, score 0; faint Ethics Board at the Sixth Hospital of Dalian. All of the samples yellow, score 1; yellow or deep yellow, score 2; brown or dark used in this study were cirrhosis accompanied with chronic brown, score 3. The final score was obtained by multiplying the hepatitis B virus infection. The tumor samples were taken from above two grades. For ABCB1, ABCC1 and ABCG2, final scoree 30 HCC patients and included 26 paired adjacent nonneoplastic ≧ 3 was defined as positive expression group and < 3 was defined tissues. As control, 9 cirrhosis tissues were obtained from the as negative expression. patients suffered decompensated liver cirrhosis. Among the 30 HCC patients, the 23 men and 7 women with a median age of 43 Statistical analysis years (range: 24-72 years). All HCC patients underwent curative SPSS software 18.0 (SPSS, Inc, Chicago, USA) was used to resection, and 11 patients among them received preoperative conduct the analysis. Significant differences between variables transcatheter arterial chemoembolization (TACE) therapy by were assessed by Chi-square test or Fisher’s exact probability test injecting a mixture of 10 ml iodipin, 1.0-1.5g 5-fluorouracil (5- when appropriate. The Kaplan-Meier method was used to FU) and 40-60mg Epirubicin. TACE was repeated two times every determine correlations between ABCB1, ABCC1 and ABCG2 four weeks. Tumor histological grade was divided into 3 groups, expression with Disease-free survival (DFS). Differences in including grade I (n=1), grade II (n=28) and grade III (n=1), Disease-free survival were analyzed using the log-rank test. In all according to Edmaondson Grading System. In addition, the clinical of the tests, p< 0.05 was considered statistically significant. and pathological data were also collected, such as serum alphafetoprotein (AFP) level, tumor size and Child-Pugh class. Results Expression of ABCB1, ABCC1 and ABCG2 in HCC, adjacent Immunohistochemistry nonneoplastic and cirrhosis tissues Immunohistochemical studies were performed using the Expression of ABCB1, ABCC1 and ABCG2 was identified by following antibodies: Mouse anti-ABCB1 (MDR1/P-gp) immunohistochemistry staining with different colors varying from polyclonal antibody (1:200 dilution, Bioworld technology, co, Ltd., faint yellow to dark-brown precipitate (Fig. 1). Significantly higher Nanjing, China), mouse anti-ABCC1 (MRP1) polyclonal antibody expression of ABCB1 and ABCC1 were detected among HCC, (1:100 dilution, Bioworld technology, co, Ltd.) and rabbit anti- adjacent non-neoplastic and cirrhosis tissues (P=0.006 and 0.001, ABCG2 (BCRP/MXR) polyclonal antibody (1:100 dilution, Table 1) while no difference in ABCG2 expression was detected Proteintech Technology Company, Chicago, USA). Paraffin- (P=0.816, Table 1). The final score of ABCB1 expression was embedded-4 m-thick serial sections were deparaffined with xylene significantly higher in tumor cells and adjacent live tissues than and rehydrated through graded alcohols and distilled water. The in cirrhosis tissues (HCC Vs CT: P=0.002; ANT Vs CT: P=0.030). endogenous peroxidase activity was blocked by immersing the No ABCB1 positive cells were detected in cirrhosis tissues (Table sections in 3 % hydrogen peroxide in methanol for 30min. After 1). Significantly more frequent expression of ABCC1 was detected treatment with block serum at room temperature, the in HCC than in adjacent non-neoplastic and cirrhosis tissues (HCC sections were incubated with primary antibodies at 4 °C overnight. Vs ANT: P=0.001; HCC Vs CT: P=0.070). For almost all of the The primary antibodies were omitted for negative controls. specimens, the staining of each ABC transporters was confined to Identification of immunoreactive sites was achieved by subsequent either membranes or cytoplasm of cells in HCC (Fig. 1) and incubation with biotinylated secondary antibodies followed by adjacent non-neoplastic tissues (data not shown). For cirrhosis Peroxidase/DAB Envision TM Detection Kit (GeneTech, tissues, ABCC1 and ABCG2 antigen presented a staining pattern Shanghai, China). After rinsed with distilled water, the sections similar to that observed in HCC tissues (data not shown). were then counterstained with Mayer’s hematoxylin (Sinopharm Interestingly, there was a case that the expression of ABCB1 in Group Co. Ltd., Shanghai, China) and mounted. The sections were HCC was not detected in tumor cells but in non-parenchymal cells observed using an inverted fluorescence microscope (DP73, (Fig. 2A), which was different from other 29 specimens. In this

82 Li-xin Fan et al.: Significance of ABC Transporter in HCC

Figure 1. Pattern and intensity of immunohistochemical staining for ABCB1, ABCC1 and ABCG2 in HCC specimens. (A) ABCB1, staining intensity score 0; (B) ABCB1, score 1; (C) ABCB1, score 2; (D) ABCC1, score 0; (E) ABCC1, score 1; (F) ABCC1, score 2; (G) ABCG2, score 0; (H) ABCG2, score 1; (I) ABCG2, score 2. Bar: 100µm.

Table 1. Expression of ABCB1, ABCC1 and ABCG2 in HCC, adjacent nonneoplastic and cirrhosis tissues.

Feature n ABCB1 P ABCC1 P ABCG2 P Pos Neg Pos Neg Pos Neg HCC 30 17 13 28 2 25 5 ANT 26 10 16 0.006 13 13 0.001 22 4 0.816 CT 9 0 9 6 3 7 2 HCC: hepatocellular carcinomas; ANT: adjacent nonneoplastic tissues; CT: cirrhosis tissues case, no ABCB1 positive cells were detected in adjacent live tissues in ABC transporter expression with tumor size (Table 2), high (Fig. 2B). Furthermore, the most strong staining (final score≧ 6) expression of ABCB1 tended to be more frequent in patients with of ABCC1 and ABCG2 was detected in HCC tissue of this case tumor size more than 2 cm (70 % vs 30 %, P = 0.056). (Fig. 2C and E). Relationships between the expression of ABCB1, ABCC1 and Association of ABCB1, ABCC1 and ABCG2 expression and ABCG2 and preoperative TACE clinicopathologic characteristics TACE has become one of the most popular and effective Since ABCB1, ABCC1 and ABCG2 were positive in more palliative methods for patients with HCC. Our results showed that than half of the HCC tissue samples, the association between these preoperative TACE treatment didn’t affect the expression of three ABC transporter expression and the clinicopathologic ABCB1, ABCC1 and ABCG2 in HCC, adjacent nonneoplastic characteristics of the patients were investigated. There was no and cirrhosis tissues (P>0.05, Table 3). This finding is different significant correlation with gender, age, AFP level, tumor from the recent observation that TACE treatment improved the differentiation, Child-Pugh class, tumor capsule and vascular expression of ABCB1 and ABCG2 in HCC tissues while didn’t invasion (P>0.05). Although no significant difference was detected have effect on ABCC1 expression17,18). In this study, most of the 83 J.Hard Tissue Biology Vol. 25(1):81 -88, 2016

Figure 2. Expression of ABCB1 in the special case with different expression pattern from other cases investigated in this study. (A) expression of ABCB1 in HCC; (B) expression of ABCB1 in ANT; (C) expression of ABCC1 in HCC; (D) expression of ABCC1 in ANT; (E) expression of ABCG2 in HCC; (H) expression of ABCG2 in ANT. Bar: 100µm. patients treated with TACE were in poor clinical status (including Overexpression of ABC transporters was considered to be 7 of the 8 [87.5 %] T3, 5 of the10 [50 %] poor differentiated related with MDR of HCC19). As major mediators of efflux of HCC). The variability of patients might result in the differences anticancer drugs from cancer cells, the expression of ABCB1, between our study and previous study17). ABCC1 and ABCG2 was investigated in primary liver tumors16). The expression of these three ABC transporters displays a high Impact of the expression of ABCB1, ABCC1 and ABCG2 on variability in tumors from different patients16), which might tumor recurrence contribute to variable response rates in chemotherapy of primary We next examined whether ABCB1, ABCC1 and ABCG2 liver tumors and their recurrence after initial therapy. expression correlated with tumor recurrence. All patients included Decreased ABCB1 expression was observed in the HCC with in this study were followed up until September 2015. The DFS poor dedifferentiation11,20), which was different from our results. was significantly shorter in the ABCB1-positive HCC (17 patients) The discrepancies in the differentiation of HCC on ABCB1 than in the ABCB1-negative HCC (13 patients) with reaching expression between this study and previous two studies11,20) might statistical significance (log-rank test P=0.004) (Fig. 3). Patients be due to the different differentiation status of samples whose tumors had high expression (final score ≧ 6) of ABCC1 investigated. In present study, most of the tumors were moderated (10 patients) had significantly shorter DFS compared with those differentiated (28 of the 30 cases). The expression pattern of whose tumors had low expression (final score <6) of ABCC1 (20 ABCB1 in HCC compared with adjacent nonneoplastic tissues is patients) (log-rank test P=0.001) (Fig. 3). There was no correlation heterogeneous. Increased21) and decreased22)expression of ABCB1 between ABCG2 expression and DFS (P=0.693, Fig. 3). in HCC compared with adjacent nonneoplastic tissues have been reported. In this study, no significant differences in ABCB1 Discussion expression were detected between HCC and adjacent

84 Li-xin Fan et al.: Significance of ABC Transporter in HCC Table 2. Relationships between the expression of ABCB1, ABCC1 and ABCG2 and clinicopathologic features.

ABCB1 ABCC1 ABCG2 Feature n P P P Pos Neg Pos Neg Pos Neg N(%) 30 17 (56.7) 13 (43.3) 28(93.3) 2(0.07) 25(83.3) 5(16.7) Gender Male 23 12 11 22 1 20 3 0.427 0.418 0.329 Female 7 5 2 6 1 5 2 Age ≦ 50 9 7 2 9 0 7 2 0.229 1.000 0.622 > 50 21 10 11 19 2 18 3 AFP(IU/ML) ≦ 400 23 13 10 21 2 19 4 1.000 1.000 1.000 >400 7 4 3 7 0 6 1 Tumor size ≦ 2cm 10 3 7 9 1 9 1 0.056 1.000 0.640 > 2cm 20 14 6 19 1 16 4 Differentiation grade I 1 1 0 1 0 1 0 grade II 28 15 13 1 . 0 0 0 26 2 1 . 0 0 0 24 4 0.310 grade III 1 1 0 1 0 0 1 Child-Pugh A 24 16 8 22 2 19 5 0.061 1.000 0.553 B 6 1 5 6 0 6 0 Tumor capsule complete 8 5 3 7 1 6 2 1.000 0.469 0.589 incomplete 22 12 10 21 1 19 3 Vascular invasion yes 3 2 1 3 0 3 0 1.000 1.000 1.000 no 27 15 12 25 2 22 5

Table 3 Relationships between the Expression of ABCB1, ABCC1 and ABCG2 and Preoperative TACE

ABCB1 ABCC1 ABCG2 Feature n P P P Pos Neg Pos Neg Pos Neg Preoperative TACE 11 6 5 11 0 9 2 1.000 0.52 1.000 NO TACE 19 11 8 17 2 16 3

Figure 3. Recurrence rate of HCC with ABCC1(A), ABCB1(B) and ABCG2 (C) expression.

85 J.Hard Tissue Biology Vol. 25(1):81 -88, 2016 nonneoplastic tissues (P=0.193). In 29 HCC cases investigated in 4. Fukuda Y and Schuetz J. ABC transporters and their role in this study, the localization of ABCB1 protein was in hepatic nucleoside and nucleotide drug resistance. Biochem parenchymal cells, while only one case was in nonparenchymal Pharmacol 83: 1073-1083, 2012 cells (Fig 2). In this special case, the patient showed higher AFP 5. Kim Y, Ishii G, Goto K, Ota S, Kubota K and Murata Y. level even after tumor resection and shorter DFS (44 days). In Expression of breast cancer resistance protein is associated normal human liver tissue, ABCB1 protein expression were with a poor clinical outcome in patients with small-cell lung observed in hepatocytes and cholangiocytes23), and responsible cancer. Lung Cancer 65: 105-111, 2009 for the elimination of drugs into bile24). So, next we plan to 6. Kathawala R, Gupta P, Ashby CJr and Chen Z. The modulation investigate the ABCB1 positive nonparenchymal cells. of ABC transporter-mediated multidrug resistance in cancer: In this study, higher ratio of the investigated HCC samples a review of the past decade. Drug Resist Update 18: 1-17, showed positive for ABCC1 antigen (28 of the 30 cases) compared 2015 with other studies (nearly 50 %)11,25). In this study, we found 7. Fojo A, Ueda K, Slamon D, Poplack D, Gottesman M and significantly higher expression of ABCC1 in HCC tissues Pastan I. Expression of a multidrug-resistance in human compared with adjacent nonneoplastic tissues and cirrhosis tissues. tumors and tissues. Proc Natl Acad Sci USA 84: 265-269, Different from our aforementioned findings, Sun et al.17) found 1987 that the expression of ABCC1 in HCC was similar to para- 8. Yakirevich E, Sabo E, Naroditsky I, Sova Y, Lavie O and carcimoma tissues. Resnick M. Multidrug resistance-related phenotype and In addition, an inverse correlation was observed between apoptosis-related protein expression in ovarian serous ABCB1 expression and DFS (Fig. 3) in this study, which was carcinomas. Gynecol Oncol 100: 152-159, 2006 similar to the previous study26). Moreover, the patients with positive 9. Veneroni S, Zaffaroni N, Daidone M, Benini E, Villa R and ABCC1 protein immunohistochemical expression in their tumors Silvestrini R. Expression of P-glycoprotein and in vitro or suffer shorter DFS, which was different with previous reports11). in vivo resistance to doxorubicin and cisplatin in breast and An inverse correlation between the expression of ABCB1, ABCC1 ovarian cancers. Eur J Cancer 30A: 1002-1007, 1994 and DFS suggested that these two ABC transporters might be 10. Anreddy N, Gupta P, Kathawala R, Patel A, Wurpel J and predictive markers for HCC prognosis. However, further studies Chen Z. Tyrosine kinase inhibitors as reversal agents for need to be done to assess the role of these two transporters in ABC transporter mediated drug resistance. Molecules 19: anticancer disposition of HCC. 13848-13877, 2014 11. Vander B, Komuta M, Libbrecht L, Katoonizadeh A, Aerts Acknowledgements R, Dymarkowski S, Verslype C, Nevens F and Roskams T. This work was supported by a grant from Dalian Science and Expression of multidrug resistance-associated protein 1 in Technology Foundation (2014E14SF156) and Dalian medical hepatocellular carcinoma is associated with a more university post-doctoral research subject to Li-xin Fan (123838). aggressive tumor phenotype and may reflect a progenitor cell origin. Liver Int 28: 1370-1380, 2008 Conflict of Interest 12. Borel F, Han R, Visser A, Petry H, van D, Jansen P, The authors have declared that no COI exists. Konstantinova P, Reseau C and Foie F. Adenosine triphosphate-binding cassette transporter up- Reference regulation in untreated hepatocellular carcinoma is mediated 1. McGlynn K and London W. Epidemiology and natural history by cellular microRNAs. Hepatology 55: 821-832, 2012 of hepatocellular carcinoma. Best Pract Res Clin 13. Sun Y, Patel A, Kumar P and Chen Z. Role of ABC transporters Gastroenterol 19: 3-23, 2005 in cancer chemotherapy. Chin J Cancer 31: 51-57, 2012 2. Li G, Harrison T, Yang J, Chen Q, Wang X and Fang ZL. 14. Nagai S, Takenaka K, Nachagari D, Rose C, Domoney K, Combined core promoter mutations and pre-S deletion of Sun D, Sparreboom A and Schuetz J. Deoxycytidine kinase HBV may not increase the risk of HCC: A geographical modulates the impact of the ABC transporter ABCG2 on epidemiological study in Guangxi, China. Liver Int 33: 936- clofarabine cytotoxicity. Cancer Res 71: 1781-1791, 2011 943, 2013 15. Hu C, Li H, Li J, Zhu Z, Yin S, Hao X, Yao M, Zheng S and 3. Zhang X, Jia Q, Lv L, Deng T and Gao J. Tumorspheres Gu J. Analysis of ABCG2 expression and side population derived from HCC Cells are enriched with cancer stem cell- identifies intrinsic drug efflux in the HCC cell line MHCC- like cells and present high chemoresistance dependent on 97L and its modulation by Akt signaling. Carcinogenesis the Akt pathway. Anticancer Agents Med Chem 15: 755- 29: 2289-2297, 2008 763, 2015 16. Wlcek K and Stieger B. ATP- binding cassette transporters

86 Li-xin Fan et al.: Significance of ABC Transporter in HCC in liver. Biofactors 40: 188-198, 2014 carcinoma and the peritumoral liver of patients with and 17. Sun Z, Zhao Z, Li G, Dong S, Huang Z, Ye L, Liang H, Qu without cirrhosis. Cancer Lett 186: 107-113, 2002 J, Ai X and Zhang W. Relevance of two genes in the 22. Richart J, Brunt E and Di B. Expression of P-glycoprotein multidrug resistance of hepatocellular carcinoma: in vivo and C-MOAT in human hepatocellular carcinoma: detection and clinical studies. Tumori 96: 90-96, 2010 by immunostaining. Dig Dis Sci 47: 2454-2458, 2002 18. Vander B, Van P, Malenstein, H, Cassiman, D and Renard, 23. Thiebaut F, Tsuruo T, Hamada H, Gottesman M, Pastan I M. Up-regulation of breast cancer resistance protein and Willingham M. cellular-localization of the multidrug- expression in hepatoblastoma following chemotherapy: a resistance gene-product P-glycoprotein in normal human- study in patients and in vitro. Hepatol Res 38: 1112-1121, tissues. Proc Natl Acad Sci USA 84: 7735-7738, 1987 2008 24. Zhou S. Structure, function and regulation of P-glycoprotein 19. Bonin S, Pascolo L, Croce L, Stanta G and Tiribelli C. Gene and its clinical relevance in drug disposition. Xenobiotica expression of ABC proteins in hepatocellular carcinoma, 38: 802-832, 2008 perineoplastic tissue, and liver diseases. Mol Med 8: 318- 25. Nies A, Konig J, Pfannschmidt M, Klar E, Hofmann W and 325, 2002 Keppler D. Expression of the multidrug resistance proteins 20. Effendi K, Mori T, Komuta M, Masugi Y, Du W and Sakamoto MRP2 and MRP3 in human hepatocellular carcinoma. Int J M. Bmi-1 gene is upregulated in early-stage hepatocellular Cancer 94: 492-499, 2001 carcinoma and correlates with ATP-binding cassette 26. Soini Y, Virkajarvi N, Raunio H and Paakko P. Expression transporter B1 expression. Cancer Sci 101: 666-672, 2010 of P-glycoprotein in hepatocellular carcinoma: a potential 21. Grude P, Conti F, Mennecier D, Louvel A, Houssin D, Weill marker of prognosis. J Clin Pathol 49: 470-473, 1996 B and Calmus. MDR1 in hepatocellular

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