人類醛固酮還原酶家族1成員B10於口腔鱗狀細胞癌之表現

Abstract

中文摘要 背景： 人類醛固酮還原酶家族1成員B10 (Aldo-keto reductase family 1, member B10, AKR1B10)的量增加。醛糖還原酶(AKR)家族以NADPH為輔酶將來自食物和細胞內的脂肪類醛、酮和芳香類物質，還原成相對應的醇。AKR1B10蛋白分子量為36 kDa，最早在人類肝癌的研究中發現。AKR1B10 是一種分泌型蛋白，可經由分泌型lysosome 分泌到細胞外。AKR1B10在人的大多數組織並不表現或低度表現，它主要表現於小腸和結腸。最近研究指出，AKR1B10的表現與肺癌、乳癌、肝癌、胰臟癌的發生及發展有關。但並無文獻提到有關OSCC患者，其癌組織中AKR1B10蛋白值濃度及唾液中AKR1B10蛋白分泌量，與OSCC患者預後之相關性。因此，本研究進一步檢測OSCC患者癌組織中AKR1B10蛋白值濃度及唾液中AKR1B10蛋白分泌量，是否和OSCC患者之腫瘤發展、復發及存活有關。

材料及方法： 本研究第一部分利用口腔癌病患之組織病理切片進行免疫組織化學染色法(immunohistochemistry(IHC))，測量77例OSCC癌組織及32例正常口腔黏膜 (normal mucosa) 中AKR1B10蛋白表現量。AKR1B10蛋白表現量以染色記分(LSs，定義為染色強度與染色指標的乘積)定義，染色記分越高表示OSCC組織中AKR1B10蛋白表現量愈多。ANOVA、卡方檢定(Chi-square test)、Kaplan-Meier 存活率方法及Cox proportional hazard regression model，來分析癌組織中AKR1B10蛋白表現量與OSCC患者臨床病理參數及存活率之相關性。本研究第二部分利用酵素連結免疫吸附法(enzyme-linked immunosorbent assay, ELISA)，探討86位OSCC患者及30位具正常口腔黏膜者，其唾液中 AKR1B10 蛋白分泌量。同時利用 ANOVA、卡方檢定、Kaplan-Meier 存活率方法及Cox proportional hazard regression model，來分析唾液中 AKR1B10 蛋白分泌量與OSCC患者臨床病理參數及存活率之相關性，並試圖尋找預測存活時間的獨立預後因子。

結果： 本研究第一部分發現，AKR1B10蛋白的染色記分(labeling score, LS)較高和患者有較大腫瘤 (P = 0.041)、有較高臨床分期 (P = 0.037) 及有嚼食檳榔之口腔習慣 (P = 0.025) ，有統計學上有意義之相關。 以Cox regression model 進行多變數分析發現，腫瘤大小 (P = 0.013)、AKR1B10 labeling score> 1.16 (P = 0.001)為影響OSCC患者存活時間的獨立預測因子。Kaplan-Meier存活分析發現， AKR1B10 labeling score> 1.16比AKR1B10 labeling score ≤ 1.16之OSCC患者，有較差的無復發存活率 (log-rank test, P = 0.036)。 本研究第二部分發現，OSCC患者唾液中 AKR1B10 蛋白分泌量的平均值，比具正常口腔黏膜者的平均值高，且具統計學上有意義之差別 (25.01 ± 32.96 pg/mL, p < 0.001)。OSCC患者唾液中 AKR1B10 蛋白分泌量的平均值較高和較大腫瘤 (P = 0.033)、有較高臨床分期 (P = 0.030) 及有嚼食檳榔之口腔習慣 (P = 0.001)有統計學上有意義之相關。Cox regression model 進行多變數分析發現，唾液中 AKR1B10 蛋白分泌量為影響存活時間的獨立預測因子(P = 0.002)。Kaplan-Meier存活分析發現，唾液中AKR1B10蛋白 > 646 pg/mL比AKR1B10蛋白≦646 pg/mL者，會有較差的無復發存活率 (log-rank test，P = 0.026)。

結論： 本研究發現，OSCC患者組織中AKR1B10蛋白質表現量及唾液中 AKR1B10蛋白量，可以預測OSCC之腫瘤大小、臨床分期及其無復發存活率；同時，組織中AKR1B10蛋白質表現量及唾液中 AKR1B10蛋白量與OSCC患者嚼食檳榔之口腔習慣呈現正相關。因此OSCC患者組織中AKR1B10蛋白質表現量及唾液中 AKR1B10蛋白量，可以當作OSCC患者預後的指標。

Abstract Background/Purpose: Aldo-keto reductase family 1 member B10 (AKR1B10) is a member of the aldo-keto reductase superfamily. This member can efficiently reduce aliphatic and aromatic aldehydes, and it is less active on hexoses. AKR1B10 was originally identified as an up-regulated protein in hepatocellular carcinomas in 1998. AKR1B10 is a 36-kDa cytosolic reductase and secreted through a lysosome-mediated nonclassical pathway. In normal human tissues, AKR1B10 is primarily expressed in epithelial tissues of digestive tract such as colon, small intestine and stomach and barely detected in other tissues. Elevated expression of AKR1B10 is found in a wide variety of human tumors, such as hepatocellular, non-small cell lung, breast, cervical, and pancreatic carcinomas. However, there have been no previous reports on AKR1B10 protein expression in oral SCCs. In this study, we investigated whether the levels of AKR1B10 in the saliva and cancer tissue of OSCC patients can be used as a prognostic marker.

Materials and methods: In the first part of this study, we used immunohistochemical staining (IHC) to detect the AKR1B10 protein levels in 77 OSCC and 32 specimens of normal oral mucosa (NOM). The grade of AKR1B10 protein expression was evaluated semiquantitatively according to the proportion of positive-staining cells to the total number of counted cancer or epithelial cells (labeling index) and the staining intensity. In the second part of this study, serum samples were obtained from 72 OSCC patients and from 30 normal control subjects. Salivary AKR1B10 protein levels were determined by enzyme-linked immunosorbent assay (ELISA). The correlation between AKR1B10 LSs and salivary AKR1B10 level in OSCCs and clinicopathological parameters of OSCC patients was analyzed by Student’s t-test, ANOVA, Kaplan-Meier analyses and Multivariate Cox regression analyses. A p-value of less than 0.05 was considered statistically significant. Results: In the first part of this study, we found that the AKR1B10 labeling score (LS) for OSCCs (1.16±1.14) was significantly higher than that for normal oral mucosa (0.10±0.23, p < 0.0001). High expression of AKR1B10 significantly correlated with large tumor size (p = 0.041), advanced TNM stage (p = 0.037) and patient’s areca quid chewing habit (p = 0.025). Multivariate analysis revealed that high AKR1B10 LS>1.16 (Hazard ratio 3.647, P = 0.001) significantly correlated with mortality. In the second part of this study, we found that the mean salivary AKR1B10 levels were significantly higher in the OSCC patients than in the normal controls (p < 0.001). Higher salivary AKR1B10 levels were significantly associated with larger tumor size, more advanced clinical stage and areca quid chewing habit. Result also showed that OSCC patients with a higher salivary AKR1B10 level (> 646 pg/mL) had a significantly poorer survival than those with a lower (≤ 646 pg/mL) salivary AKR1B10 level (p = 0.026). Conclusion This study found that AKR1B10 protein level in OSCC tissues and salivary AKR1B20 protein level in OSCC patients could be used to predict the tumor size, clinical stage and the prognosis of OSCC patients. Therefore, we conclude that AKR1B20 protein level in OSCC tissues and salivary AKR1B10 protein level in OSCC patients may be valuable biomarkers for prediction of progression and prognosis of OSCC in Taiwan.