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Original Clinical Significance of ABC Transporter Expression in Patients 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 proteins 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 protein 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
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