Mol Cell Biochem DOI 10.1007/s11010-014-2117-0

Endocan, a potential prognostic and diagnostic biomarker of acute leukemia Zhe Xu • Sumei Zhang • Qing Zhou Yuan Wang • Ruixiang Xia



Received: 25 January 2014 / Accepted: 2 June 2014 Ó Springer Science+Business Media New York 2014

Abstract Recent evidence indicated that endocan may be a potential cell marker and a new target for cancers including acute leukemia since the serum endocan level in patients with acute leukemia was associated with the status of the disease, i.e., endocan was higly expressed in untreated acute leukemia, but decreased after chemotherapy and increased again during bone marrow regeneration. The present study showed that there was high level expression of endocan in cytoplasm of bone marrow blasts of patients with acute myeloid leukemia or acute lymphoblastic leukemia. The expression level of endocan was significantly decreased when the patients underwent remission after chemotherapy and re-bounces back when the acute leukemia relapsed. No obvious change in expression of endocan was observed before and after chemotherapy if the patients showed no remission after chemotherapy. (N-(4-Hydroxyphenyl) retinamide), a potent anti-angiogenic agent, could not only down-regulate the expression of vascular epithelial growth factor, but also decrease endocan transcription and expression in NB4

Zhe Xu and Sumei Zhang have contributed equally to this work. Z. Xu Department of Hematology, Anhui Provincial Children’s Hospital, Hefei 230051, Anhui, People’s Republic of China e-mail: [email protected] S. Zhang  Q. Zhou  Y. Wang Laboratory of Molecular Biology and Department of Biochemistry, Anhui Medical University, Hefei 230032, Anhui, People’s Republic of China R. Xia (&) Department of Hematology, Anhui Medical University, Hefei 230032, Anhui, People’s Republic of China e-mail: [email protected]

cells, a human acute promyelocytic leukemia cell line. These observations suggest that endocan could act as a predictor for the severity and the prognosis of acute leukemia. The findings could be used as the basis for future targeted therapy directed against bone marrow angiogenesis in acute leukemia treatment. Keywords 4-HPR

Endocan  Biomarker  Acute leukemia 

Introduction Acute leukemia is a common bone marrow malignancy with poor prognosis. Even with induction chemotherapy followed by consolidation therapy, long-time survival is still low. Anti-angiogenic therapy, in combination with chemotherapy and consolidation therapy, is now considered as a potential promising treatment strategy of acute leukemia [1]. Identification of biomarkers for leukemiaassociated angiogenesis, therefore, would be important for diagnosis and prognosis prediction of the disease. Endocan is an endothelial cell-specific molecule (ESM1) that was first characterized by Lassalle et al. [2] and was initially named as ESM-1. Endocan is regarded as a potential endothelial cell marker and a new target for malignancies [3]. It was reported that endocan level was increased in serum of untreated acute myeloid leukemia (AML), while the serum level of endocan decreased after AML was under control with chemotherapy. Increased endocan level was also observed during bone marrow regeneration [4]. Endocan level was associated with increased vascular epithelial growth factor (VEGF) level [5] and was regulated by VEGF [6].

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The current study examined the changes of endocan expression level in patients with AML or acute lymphoblastic leukemia (ALL) and determined the relationship between the expression level of endocan and the degree of malignancy of the acute leukemias. The results showed that endocan expression level was significantly decreased in the patients with acute leukemias who were under remission after chemotherapy but was increased again when the disease relapsed. No obvious change in the expression level of endocan was observed in the patients who showed no remission after chemotherapy. The findings are consistent with those from Hatfield KJ’s group and suggest that, in patients with acute leukemia, expression level of endocan could be used as a useful biomarker to monitor the degree of malignancy, disease progression, prediction of prognosis, and as a biomarker for diagnosis. Cell experiments were also performed in the present study to explore the effect of 4-HPR (N-(4-hydroxyphenyl) retinamide), a derivative of all-trans-retinoic acid (ATRA) and a potent anti-angiogenesis agent, on the expression of endocan and VEGF, in NB4 cells, a human promyelocytic leukemia cell line. Down-regulated endocan and VEGF levels were detected after the NB4 cells were treated with 4-HPR as compared to the untreated cells. These findings suggest that the anti-angiogenic property of 4-HPR may function as anti-leukemic roles by down-regulating expression of VEGF and endocan in acute leukemia cells.

Materials and methods Antibodies and reagents The sheep anti-endocan polyclonal antibody was from Santa Cruz. Donkey anti-sheep IgG–HRP and SuperSignalÒ West Pico Trial Kit were purchased from Beyotime Institute of Biotechnology. Immunohistochemistry SP kit and the DAB development reagents were from Zymed Company. RPMI-1640 medium was obtained from Gibco. 4-HPR was kindly provided by Institute of Pharmacology, Anhui Medical University, and was dissolved in DMSO.

Patients and clinical samples All studies were approved by the regional Ethics Committee, and the samples included in this study were collected after informed consent. The bone marrow samples were collected from 33 acute leukemia patients admitted to the first affiliated hospital of Anhui Medical University. The 33 patients consisted of 13 females and 20 males with a mean age of 39.3 years (range 14–71 years), 21 patients with acute myeloid leukemia (AML), and 12 patients with

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acute lymphoblastic leukemia (ALL). All patients were diagnosed according to clinic symptoms and laboratory detection. Immunocytochemistry staining for endocan in the bone marrow The bone marrow slides were fixed with 95 % ethanol and then treated with Triton X-100 to improve the membrane permeability. The slides were then blocked with normal sheep serum for 10 min at room temperature. A 12-h incubation at 4 °C with primary antibody against endocan was used to detect the endocan expression in the cells on bone marrow slides. After incubated in the biotin-labeled sheep anti-mouse IgG followed by a incubation in HRPlabeled streptavidin at 37 °C, the slides were dyed with DAB and redyed with brazilein. Control staining of endocan was performed with the same protocol but with omitting of the incubation of primary antibody. Western blot assay for endocan detection in the blasts of bone marrow The blasts in bone marrow were separated with Separation Medium according to the protocol. The collected cells were lysed and homogenized in RIPA lysis buffer supplemented with protease inhibitors on ice to get total protein. After centrifugation, the protein concentration in the supernatant was determined by BCA assay. Then, the protein was electrophoresed on a 12.5 % SDS-polyacrylamide gel and was transferred to PVDF membrane. After incubated with antiendocan multiclonal antibody and then with peroxidaseconjugated secondary antibodies in the second reaction, detection was performed with enhanced chemiluminescence sequentially reagent. Cell culture Human promyelocytic leukemia cell line NB4 was purchased from ATCC. Cells were maintained in RPMI-1640 medium supplemented with 10 % heat inactivated fetal calf serum, 1 mmol/l glutamine, 100 U/ml of penicillin, and 100 lg/ml of streptomycin at 37 °C in a humidified atmosphere of 5 % CO2 and 95 % air. RT-PCR assay to test the effect of 4-HPR on endocan transcription in NB4 Total RNA was prepared from the NB4 cells treated with 4-HPR at 0.1 lmol/l for 48 h, randomly primed, and reverse transcribed with Superscript (Gibco). The untreated cells and DMSO-treated cells were set as cell control and vehicle control. The sequences of specific primers used for

Mol Cell Biochem

Fig. 1 Endocan expression in bone marrow of the acute leukemia patients who got complete remission after chemotherapy. It was a positive expression of endocan protein in the bone marrow of the patients with acute leukemia. And endocan expression was obviously decreased when the patients got complete remission. But when the patients relapsed, endocan expression increased again. a Immunocytochemistry staining of endocan in bone marrow of the acute leukemia patients who got complete remission after chemotherapy. (A) Before chemotherapy, (B) complete remission, and (C) relapsed after remission. b Western blot detection of endocan level in the

blasts of bone marrow in acute leukemia patients who got complete remission after chemotherapy. Lane 1 before chemotherapy, Lane 2 complete remission, and Lane 3 relapsed after remission. c Column chart of the results showed in b. The gray value of each band was read using Quantity One software. And the relative ratio between endocan and b-actin was used to construct the column chart. *p \ 0.05 compared to endocan level in the patients before chemotherapy, # p \ 0.05 compared to endocan level when the patients got completed remission

endocan are as follows: sense 50 -GGTGGACTGCCCTC AACACT-30 , antisense 50 -AAGGTGCCGTAGGGACAG TCT-30 , the length of the PCR product is 246 bps. The sequences of the specific primers for G3PDH are sense 50 -TGAAGGTCGGAGTCAACGGATTTGGT-30 , antisense 50 -CATGTGGGCCATGAGGTCCACCAC-30 , the length of the PCR product is 1,000 bps. The PCR cycles consisted of denaturation at 94 °C for 1 min, annealing at 57 °C for 45 s, and extension at 72 °C for 45 s.

Data analysis Western blotting and RT-PCR datas were quantified using Quantity One software. Statistical analyses were carried out using SPSS16.0. One-way ANOVA was used to analyze the difference among the groups. A p \ 0.05 was considered statistically significant.

Results Western blot assay to test the effect of 4-HPR on endocan expression in NB4 The NB4 cells treated with 4-HPR at 0.1 lmol/l for 48 h were washed with PBS and lysed in RIPA lysis buffer. The cell control and vehicle control were set also. Western blot assay was performed as previously mentioned.

Expression of endocan in patients with acute leukemia In the bone marrow slides, the specific endocan protein was identified as scattered in the cytoplasm of the blasts by immunohistochemical staining. There is strong expression of endocan in the bone marrow of the patients with acute leukemia before chemotherapy. When the patients

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Mol Cell Biochem

Fig. 2 Endocan expression in the bone marrow of the acute leukemia patients who got non-remission after chemotherapy. Endocan was positively expressed in the bone marrow of the patients with acute leukemia. But there was no distinct change in the bone marrow of patients who got non-remission, almost the same level before and after chemotherapy. a Immunocytochemistry staining of endocan in the bone marrow of patients who got non-remission. (A) Before

chemotherapy and (B) after chemotherapy. b Western blot detection of endocan level in the karyocytes of bone marrow in acute leukemia patients who got non-remission. Lane 1 before chemotherapy, and Lane 2 after chemotherapy. c Column chart of the results showed in b. The gray value of each band was read using Quantity One software. And the relative ratio between endocan and b-actin was used to construct the column chart

underwent complete remission after received chemotherapy, there is significant decrease in expression of endocan protein the cytoplasm of the bone marrow blasts. Interestingly, when the patients were experiencing disease relapsed, the level of endocan expression increased again. The results got from Western blot assay were identical with the data from immunohistochemical staining (Fig. 1a–c). On the other hand, in the bone marrow of patients with acute leukemia who did not show remission after chemotherapy, there is no difference in endocan expression before and after the chemotherapy. The consistent results were obtained by immunocytochemistry staining and Western blot assay (Fig. 2a–c).

assay, respectively. As compared to untreated cells, the NB4 cells treated with 4-HPR showed significantly decreased levels of endocan transcription and expression (Fig. 3a–d). 4-HPR may play its anti-leukemia roles by down-regulating endocan expression. Effects of 4-HPR on expression of VEGF in NB4 cells The effects of 4-HPR on the expression of VEGF in NB4 cells were evaluated by Western blot analysis. After the NB4 cells were treated with 4-HPR for 48 h, the level of VEGF expression was significantly decreased as compared with the untreated cells (Fig. 4a, b).

Effects of 4-HPR on endocan expression in NB4 cells Discussion NB4 cells treated with 4-HPR were harvested for determination of endocan gene transcription with use of technique of RT-PCR and protein expression with Western blot

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Increase in bone marrow microvascularity is important for leukemogenesis and chemosensitivity in human acute

Mol Cell Biochem

Fig. 3 Endocan expression in NB4 cells was downregulated by 4-HPR treatment. Endocan protein and mRNA levels were reduced in the NB4 cells treated with 4-HPR for 48 h compared to cell control and vehicle control. Lane 1 cell control, Lane 2 vehicle control, and Lane 3 4-HPR-treated cells. a Western blot assay results for endocan protein detection in the cell lysates. b RT-PCR results for endocan mRNA detection in the cell lysates. c Column chart of the results

Fig. 4 VEGF expression was downregulated after the NB4 cells were treated with 4-HPR for 48. a Western blot assay results for VEGF protein detection in the cell lysates. Lane 1 cell control, Lane 2 vehicle control, and Lane 3 4-HPR-treated cells. b Column chart of the results showed in a. The gray value of each band was read using Quantity One software. And the relative ratio between VEGF and b-actin was used to construct the column chart. *p \ 0.05

leukemia. Identification of biomarkers for leukemia-associated angiogenesis is important for prediction of prognosis and early detection of post-treatment relapse [1]. The work of Ayala et al. [7] also indicated that leukemia-associated bone microenvironment markers could be used as

showed in a. The gray value of each band was read using Quantity One software. And the relative ratio between endocan and b-actin was used to construct the column chart. *p \ 0.05. d Column chart of the results showed in b. The gray value of each band was read using Quantity One software. And the relative ratio between endocan and G3PDH was used to construct the column chart. *p \ 0.05

prognostic or predictive indicators of disease progression and/or treatment outcome. Acute leukemia cells secrete mediators to induce angiogenesis [7–9]. Growth factors like vascular endothelial growth factor (VEGF), basic fibroblast growth factor, and angiopoietins are the main proangiogenic mediators in acute leukemia. Among them, vascular endothelial growth factor (VEGF) is one of the most potent angiogenic factors [10] and is considered to be one of the most important molecule in regulation of tumor angiogenesis [11–13]. A strong positive correlation between VEGF expression and MVD (microvessel density) was found in acute leukemia. Stronger expression of VEGF on blast cells indicates shorter overall survival in acute leukemia, and initial values of MVD had positive correlation with overall survival and leukemia-free survival [14]. VEGF plays a crucial role in promoting angiogenesis in acute leukemia [15]. VEGF as a therapeutic target in acute leukemia is suggested both by experimental and clinical observations. In acute leukemia, the serum levels of VEGF decreased after intensive chemotherapy and subsequent severe chemotherapyinduced cytopenia, and increased levels of VEGF were thereafter observed during bone marrow regeneration [4]. Endocan has shown to play a role in inducing tumor formation in vivo [5]. Increased levels of endocan mRNA have been reported in human renal tumors, which was associated with simultaneously increased VEGF mRNA levels [6]. Endocan gene expression is regulated by VEGFA since there is increased endocan protein expression in

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VEGF stimulated endothelial cells. Rennel et al. [16] indicate that endocan may itself represent a therapeutic target of the chemotherapy to control tumor angiogenesis and tumor growth. Endocan expression and its clinical implication in the blasts of acute leukemia patients were tested for the first time in the current study. Endocan expression in the bone marrow of acute leukemia patients, as determined by immunocytochemistry staining and Western blot, was significantly decreased after remission but no obvious change in the patient who got no remission; the endocan level was increased again when the patients relapsed. The findings suggest that endocan may be used as a prognostic biomarker as well as a biomarker for diagnosis of acute leukemia. N-(4-Hydroxyphenyl)-retinamide (4-HPR), a synthetic retinoid, is a derivative of ATRA with cytotoxic activity in a wide variety of cancer cells both in vitro and in vivo [17], including acute leukemia cell lines [18]. 4-HPR has antiangiogenic properties and exerts its cytotoxic effects through the induction of apoptosis and the inhibition of cell growth in the malignant cell line [19–21]. In the current study, it is hypothesized that 4-HPR may play anti-leukemia role by acting as anti-angiogenesis reagent. To test the hypothesis, the effects of 4-HPR on expression of the angiogenesis-related factors, namely, VEGF and endocan in cultured human promyelocytic leukemia cell line NB4, were determined. RT-PCR and Western blot assay were performed to reveal the effect of 4-HPR on the expression of endocan in NB4 cells. The levels of endocan mRNA and protein were both downregulated after the cells were treated with 4-HPR for 48 h. Simultaneously, VEGF level was decreased in the 4-HPRtreated NB4 cells as compared to untreated cells. The results demonstrated that 4-HPR indeed could inhibit the angiogenesis in acute leukemia, since treatment of the cultured NB4 cells with 4-HPR changed the expression of endocan and VEGF. The findings suggest that 4-HPR may play its anti-leukemia roles by down-regulating expression of endocan and VEGF. However, further in-depth studies are needed to explore the specific mechanisms. Acknowledgments We thank Professor Chen Feihu for kindly providing 4-HPR used in this study. This study was supported by the Fund for Young Talents in College of Anhui Province (No. 2012SQRL067), National Natural Science Foundation of China (No. 81201907), National Natural Science Foundation of China (No. 81272399), Research Fund for Doctor in Anhui Medical University (XJ201229).

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Endocan, a potential prognostic and diagnostic biomarker of acute leukemia.

Recent evidence indicated that endocan may be a potential cell marker and a new target for cancers including acute leukemia since the serum endocan le...
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