Dig Dis Sci DOI 10.1007/s10620-014-3111-9

ORIGINAL ARTICLE

Downregulating sCLU Enhances the Sensitivity of Hepatocellular Carcinoma Cells to Gemcitabine by Activating the Intrinsic Apoptosis Pathway Peng Xiu • Zongzhen Xu • Feng Liu • Ziqiang Li • Tao Li • Fang Zou • Xueying Sun Jie Li

•

Received: 31 July 2013 / Accepted: 10 March 2014 Ó Springer Science+Business Media New York 2014

Abstract Purpose The purpose of this study was to investigate whether the therapeutic activity of gemcitabine (GCB) in hepatocellular carcinoma (HCC) could be increased by the down-regulation of secretory clusterin (sCLU), a glycoprotein that is considered to play a cytoprotective role in the resistance to chemotherapy. Methods The expression of sCLU was detected in HCC tumor tissues and cell lines. A cell viability and apoptosis

Peng Xiu and Zongzhen Xu have contributed equally to this article. P. Xiu
Z. Xu
F. Liu
Z. Li
T. Li
J. Li (&) Department of General Surgery, Qianfoshan Hospital, Shandong University, Jinan 250014, China e-mail: [email protected] P. Xiu e-mail: [email protected] Z. Xu e-mail: [email protected] F. Liu e-mail: [email protected] Z. Li e-mail: [email protected] T. Li e-mail: [email protected] F. Zou Department of Emergency Surgery, The People’s Hospital of Linyi City, Linyi 276000, China e-mail: [email protected]

assay were performed in parental HCC cells or the same cells transfected with sCLU shRNA and treated with or without GCB. The potential downstream pathways were investigated using the Human Apoptosis RT2 ProfilerTM PCR Array. Results The expression levels of sCLU in HCC tissues were significantly higher than in adjacent non-tumor liver tissues and were associated with the histological grade and transarterial chemoembolization. sCLU overexpression was also found in three HCC cell lines and hepatocytes. The depletion of sCLU synergistically increased GCB sensitivity in Bel7402 and SMMC7721 cells and induced cell apoptosis. Based on the PCR array analysis, sCLU depletion also resulted in the up-regulation of BNIP1, GADD45A, TNFRSF10A, and TRADD and down-regulation of AKT1 in Bel7402 and SMMC7721 cells compared with the parental controls. These results were further supported by a Western blot analysis, which showed increased GADD45a protein expression and the decreased expression of phosphorylated AKT. GADD45a overexpression also increased the sensitivity to GCB in the Bel7402 and SMMC7721 cells. Conclusion Targeting sCLU may be a useful method to enhance the cytotoxic effect of GCB in hepatocellular carcinoma. Keywords Hepatocellular carcinoma
Clusterin
Apoptotic pathway
Gemcitabine

Introduction X. Sun The Hepatosplenic Surgery Center, Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin 150001, China e-mail: [email protected]

Hepatocellular carcinoma is the sixth most prevalent neoplasm and the third most frequent cause of cancer-related death after lung and colon cancer [1, 2]. As a characteristic

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of this particular cancer and a restriction of the diagnostic level, most patients are already in the mid–late stage of the disease at the time of diagnosis, with a consequent loss of the chance of surgery. Furthermore, HCC is extremely resistant to chemotherapy. Thus, an understanding of the mechanisms of drug resistance may improve the therapeutic efficacy of chemotherapy in combating HCC. Clusterin (CLU) is a glycoprotein ubiquitously expressed from a broad spectrum of tissues, including prostate [3], renal [4], bladder [5], breast [6], ovary [7], lung [8], and cervix uteri [9], which is playing roles in several physiological functions [10], such as lipid transport and maturation of sperm, and pathological functions, including Alzheimer’s disease, myocarditis, and cancer [11, 12]. And this can be attributed to the existence of the different spliced forms of the CLU gene. One protein form (sCLU) is an endoplasmic reticulum (ER)-targeted 449-amino acid polypeptide that represents the predominant translation product of the CLU gene and another protein form (nCLU) is translated from an alternatively spliced CLU transcript that bypasses the ER signal peptide [13, 14]. In many tumors, sCLU, which is a stress-activated, cytoprotective, small heat shock chaperone, has been defined as an antiapoptotic protein whose expression is increased after therapeutic stress and playing the role of sensitization, whereas the nuclear protein form nCLU was declared to have pro-apoptotic properties [3, 6, 7, 14]. A new recent research demonstrated that the role of sensitization to antitumor stress may be induced by the decrease of the sCLU/nCLU ratio by regulating the expression of sCLU or nCLU [15]. At present, an increasing number of reports have provided evidence that sCLU is promising as a useful biomarker for the diagnosis of HCC [16–18]. Indeed, we recently reported that sCLU contributes to oxaliplatin resistance by activating the Akt pathway in HCC [19]. We designed the present study to extend our understanding of sCLU in HCC by detecting the expression of sCLU in normal liver tissues, HCC tissues, and different cell lines and further analyzed its associations with the clinicopathologic parameters of HCC patients. In addition, we investigated whether sCLU suppression could enhance the sensitivity of HCC cells to gemcitabine (GCB) and the potential downstream pathways.

serum. All cultures were grown at 37 °C in a humidified atmosphere containing 5 % carbon dioxide. HepG2, Bel7402, Bel7404, SMMC7721, HuH-7, and SNU739 cells are the human hepatocellular carcinoma cell lines, and QSG7701 is a human hepatic cell line. The hepatocellular carcinoma cell lines were derived from the human hepatocellular carcinoma tissues, whereas QSG7701 from the tissues adjacent to cancer. All the cells were obtained from the type Culture Collection Cell Bank, Chinese Academy of Sciences Committee, Shanghai. The DMEM and RPMI1640 media were purchased from Thermo Scientific (Thermo Fisher Scientific, Shanghai, China). Antibodies Antibodies against clusterin (sc-5289), GADD45a (sc6850), Akt, phosphorylated Akt (p-Akt), BNIP1 (sc-1713), and goat anti-mouse IgG-HRP (sc-2005) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA. USA) and the GCB hydrochloride from Sigma-Aldrich Co. LLC (Sigma-Aldrich, Shanghai, China) (G6423). Human HCC Samples Fifty HCC specimens were obtained from Qianfoshan Hospital of Shandong Province between October 2007 to December 2010 with patients’ consent and approval from the Ethics Committee of Qianfoshan Hospital of Shandong Province. This study was performed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. Immunohistochemistry Immunohistochemistry was performed using Polink-2 plus R Polymer HRP Detection System (Zhongshan Golden Bridge Biotech, Beijing, China) according to the manufacturer’s instructions. The primary antibody against clusterin was diluted at 1:300. sCLU expression was scored as follows: negative (-) if weak or no staining was observed; positive (1?) if immunoreactivity was observed in \10 % of the tumor cells and moderate (2?) to strong (3?) in [10 % of the tumor cells. Plasmid Construction and Transfection

Methods Cell Culture HepG2 cells were cultured in DMEM culture medium and HuH-7 SNU739, Bel7402, Bel7404, SMMC7721, and QSG7701 in RPMI-1640 supplemented with 100 U/ml penicillin, 100 mg/ml streptomycin, and 10 % fetal bovine

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Four potentially effective targets of sCLU were designed, synthesized, and inserted into the pMAGic 7.1 vector to produce four shRNA vectors: CLU-1, CLU-2, CLU-3, and CLU-4 [19]. The GADD45a expression vector was constructed and termed pcDNA3.1-GADD45a-His. The plasmids were transfected into Bel7402 and SMMC7721 cells using the GenJet DNA in vitro transfection reagent

Dig Dis Sci

(SignaGen, MingRui. Biotech. Co. Ltd) according to the manufacturer’s instructions, and the protein expression level was detected using western blotting. Apoptosis, Western Blotting, and Cell Proliferation Assays These methods have been described previously [20, 21]. Total protein extracts were loaded on a 10 % SDS-PAGE and transferred to a PVDF membrane. Membranes were probed with the following primary antibodies specific for sCLU, GADD45a, AKT, pAKT, BNIP1. Human Apoptosis RT2 ProfilerTM PCR Array To profile the gene expression associated with cell apoptosis, we employed the Human Apoptosis RT2 ProfilerTM PCR Array (PAHS-012A) (QIAGEN, Shanghai, China, Co., Ltd.). RNA isolation, DNase treatment, RNA clean up, and RNA yield and quality assessments were performed according to the manufacturer’s protocol (QIAGEN, Shanghai, China). The isolated RNA was reverse transcribed into cDNA using High Capacity cDNA Reverse Transcription Kit (Invitrogen), and the PCR amplification was performed using SYBR Green Real-Time PCR Master Mixes (Invitrogen). The data analysis was performed based on the DDCt method to calculate the DCt for each pathway and for each gene across two PCR arrays. The fold-change for each gene was calculated as 2-DDCt. The data analysis was performed by Shanghai KangCheng Bio-tech Company, Shanghai, China.

with weak or no expression in normal hepatic cells (Fig. 1). Furthermore, the expression of sCLU was significantly higher in the tumor tissues (40/55, 72.7 %) than that in the surrounding non-tumorous tissues (12/55, 21.8 %) (Fig. 1; Table 1) and the expression of sCLU appeared to be related to the grade of HCC cell differentiation from well differentiated to poorly differentiated. We next investigated the relationship between sCLU expression and clinicopathologic parameters of HCC patients. The results shown in Table 1 demonstrate that there was a significant difference between sCLU expression and preoperative transarterial chemoembolization (TAE) (P = 0.019) and the progression of the histological grade (P = 0.034). Nevertheless, no relationship was found between sCLU expression and the serum AFP and HBsAg levels. Expression Level of sCLU in Hepatic Cells and HCC Cells The level of sCLU protein expression in a human hepatic cell line (QSG7701) and HCC cell lines (HepG2, Bel7402, Bel7404, SMMC7721, Huh-7, and SNU739) was detected using a western blot analysis. The highest expression was present in the Bel7402 cells, followed by the SMMC7721, HepG2, and HuH-7 cells. In contrast, there was no obvious band identified in the QSG7701, Bel7404, and SNU739 cells lysates (Fig. 2). Thus, the Bel7402 and SMMC7721 cells were chosen as the research model for the ensuing assays. Suppression of sCLU in HCC Cell Lines

Statistical Analysis The data are presented as the mean ± SD. The difference of the means of multiple groups was analyzed by the Wilcoxon rank sum test and Kruskal–Wallis H test. All of the statistical calculations were performed using SPSS 17.0 software. A difference was defined as significant at P \ 0.05 using Student’s t test and a one-way analysis of variance.

Figure 3a, b show that three of the four shRNA vectors were able to effectively suppress the expression of sCLU in SMMC7721 cells, with CLU-4 being the most notable. The appropriate time point for the effective suppression of sCLU expression was further investigated. CLU-4 could suppress the expression of sCLU in a time-dependent manner in the Bel7402 and SMMC7721 cells, with an obvious suppression effect at the 48 h (Fig. 3c, d). Figure 3e, f indicated the transfection efficiency of shRNA vector in Bel7402 and SMMC7721 cells.

Results Relationships Between sCLU Expression and Clinicopathologic Parameters of HCC Patients Immunohistochemistry using an anti-sCLU antibody was performed on a series of 55 pairs of human HCC specimens and adjacent non-tumorous liver tissues. In the nontumorous liver tissues, most of the cytoplasmic expression of sCLU was found localized to bile duct epithelial cells,

Effects of sCLU Suppression with GCB Treatment in HCC Cells To explore whether sCLU suppression is cytotoxic to HCC cells, we determined the cell viability of parental HCC cells or parental cells transfected with Sc or CLU-4. Figure 4a, b show that there was no significant change in Bel7402 and SMMC7721 cell viability by sCLU suppression. The dose at which GCB cytotoxicity develops in a

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Fig. 1 Immunohistochemical analysis of sCLU expression in HCC tissues and adjacent non-tumor liver tissues (9 200). HE staining in non-tumor liver tissues (a), and in well differentiated (c), poorly

differentiated (e) HCC. Representative images are from anti-sCLU Ab-stained sections from non-tumors liver tissues (b), well differentiated (d), or poorly differentiated (f) HCC

period of 48 h in the Bel7402 and SMMC7721 cells was then determined, and the cell density was significantly decreased by GCB in a dose-dependent manner, with the highest reduction at 170 nM (Fig. 4c, d). Based on this result, a dosage of 90 nM GCB was chosen for the subsequent experiments. GCB decreased the SMMC7721 and Bel7402 cell viability by 29.5 and 18.9 %, respectively; with the suppression of sCLU in the same cells, GCB decreased viability by 75.2 and 65.8 %, respectively (Fig. 4e, f). We then analyzed the effect of sCLU on cell apoptosis induced by GCB. Bel7402 and SMMC7721 cells were transfected with CLU4 and then cultured in the presence of

GCB (90 nM) for 24 h. Figure 5 shows that GCB significantly increased the apoptosis rates of the Bel7402 and SMMC7721 cells by 7.66 and 12.62 %, compared with the respective control cells. The combination of sCLU depletion plus GCB, however, dramatically increased cell apoptosis, resulting in the increase of apoptosis rate by 18.21 and 23.37 %, respectively.

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Modulation of the Expression of Apoptosis-Related Genes by sCLU in Bel7402 and SMMC7721 Cells To gain further insight into the functions of sCLU in HCC chemotherapy resistance, the expression profiles of

Dig Dis Sci Table 1 Relationship between sCLU overexpression and clinicopathologic characteristics Variables

N

CLU immunoreactivityu Negative

P value

Low

High

Sex Male

42

13

5

24

Female Age

13

2

2

9

\Median

25

7

2

16

[Median

30

8

5

17

0.352

0.727

Serum AFP level(lg/l) \20

26

6

4

16

[20

29

9

3

17

Negative

21

5

3

13

Positive

34

10

4

20

Done

45

11

3

31

Not done

10

4

4

2

0.699

Serum HBsAg 0.751

TAE 0.019*

Tumor size \2 cm

13

3

3

7

[2 cm 42 Child–Pugh stage

12

4

26

A

36

12

2

22

B

17

2

5

10

C

2

1

0

1

I

8

5

1

2

II

29

7

4

18

III

13

3

0

10

IV

5

0

2

3

0.820

0.806

Histological grades 0.034*

Liver cirrhosis Absent

14

3

2

9

Present

41

12

5

24

0.461

Intrahepatic metastasis Not observed

20

7

4

9

Observed

35

8

3

24

Fig. 2 Expression of sCLU in hepatic cells and HCC cells. Cell lysates a from SNU739, QSG7701, HepG2, SMMC7721, HuH-7, Bel7402, Bel7404 cells were subjected to Western blot analysis. b The density of each band in a was measured and normalized to that of GAPDH, respectively

TNFRSF25, TRADD, HRK, and LTA) of the 14 up-regulated genes are positive regulators of apoptosis. Four genes (Bag1 [22], NAIP [23], AKT1, and FASLG: the first three are negative regulators of apoptosis) were down-regulated after the suppression of sCLU expression. A western blot assay showed that sCLU depletion resulted in a significant increase in the GADD45a protein level and decrease in the level of phosphorylated AKT in the two HCC cell models (Fig. 6).

0.123

* Statistically significant or P \ 0.05

Overexpression of GADD45a Increases the Sensitivity to GCB

Bel7402-CLU (SMMC7721-CLU) were separately compared to Bel7402-Vec (SMMC7721-Vec) using the apoptosis pathway-specific Human Apoptosis RT2 ProfilerTM PCR Array (SABiosciences), which contains 84 genes. Using filtering criteria of a 1.5 or greater fold-change in expression, we analyzed the differentially expressed genes in the two types of cells. Of the 84 genes examined, 18 showed a [1.5-fold change in expression: 14 up-regulated and 4 down-regulated genes (Table 2). Interestingly, with the exception of the NOL3 gene, 13 (BAK1, Bcl2L1, BIK, BNIP1, Casp1, GADD45A, LTBR, TNF, TNFRSF10A,

Both the Bel7402 and SMMC7721 cells were transfected with the pcDNA3.1-GADD45a-His vector for GADD45a overexpression. The western blot results demonstrated a high level expression of GADD45a after pcDNA3.1GADD45a-His transfection than in the parental cells. The Bel7402 and SMMC7721 cells were treated with GCB (90 nM) after transfection with pcDNA3.1-GADD45a-His or CLU4, and a CCK-8 assay demonstrated that both the down-regulation of sCLU and up-regulation of GADD45a could increase the sensibility to GCB of cells compared with the parental cells. The up-regulation of GADD45a makes a more obvious effect (Fig. 7).

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Dig Dis Sci Fig. 3 The suppression of sCLU expression by shRNA. a Parental SMMC7721 cells (Control), or SMMC7721 cells transfected with Sc or CLU-1, CLU-2, CLU-3, and CLU-4 shRNA vectors, were subjected to Western blot analysis. c Parental Bel7402 or SMMC7721 cells (Control), and parental Bel7402 or SMMC7721 cells transfected with Sc or CLU-4, were incubated in the culture medium for 24 and 48 h, respectively, and then subjected to Western blot analysis. b, d The density of each band in a, c was measured and normalized to that of GAPDH, respectively. Parental Bel7402 or SMMC7721 cells were transfected with CLU-4, respectively, with a subsequently incubation in medium for 48 h and then observed under the fluorescent microscopy (e, f). A significant (P \ 0.05) difference from the respective control is denoted by ‘‘*’’ and a highly significant (P \ 0.001) difference by ‘‘**’’

Gemcitabine and depletion of sCLU induced apoptosis of Bel7402 cells at rates of 7.6 and 3.8 %, the combination of sCLU depletion plus GCB induced an even higher apoptosis rate (11.7 %). The Bel7402 cells overexpressed with GADD45a and treated with GCB had the highest apoptosis rate at 17.3 %. Similar results were obtained from SMMC7721 cells (Fig. 8).

Discussion Recent studies in numerous laboratories have demonstrated that sCLU is overexpressed in a broad range of human cancers, including breast [6], lung [8], ovarian [7], and cervical cancer [9]. However, due to the limited

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studies regarding the expression of sCLU in hepatocellular carcinoma, this role remains controversial. In Kang’s study, 89 of 100 surgically resected HCC samples exhibited clusterin overexpression, which was associated with a poor Edmondson’s histological grade and a high TNM stage [24]. CLU overexpression was considered to be an important factor for the metastasis and may be related to YKL-40 expression with regard to its metastatic role in hepatocellular carcinoma [25]. Our results confirmed that sCLU expression is significantly increased in HCC tissues compared with the adjacent non-tumorous liver tissues. Moreover, there was an increasing tendency for sCLU expression with the progression of the histological grade and TAE treatment (Fig. 1; Table 1).

Dig Dis Sci Fig. 4 Depletion of sCLU increased chemosensitivity to GCB. a, b Parental Bel7402 or SMMC7721 cells (control), or the cells transfected with Sc or CLU-4 shRNA vector, were cultured for 48 h, and the viability was measured using CCK-8 assay. c d Parental Bel7402 or SMMC7721 cells were seeded in a 96-well plate and allowed to overnight. Then, the next day, the media were replaced with the same media containing various concentrations of GCB. After 48 h incubation, the cell viability was measured using CCK-8 assay. e, f Parental Bel7402 or SMMC7721 cells, or the cells transfected with CLU-4 shRNA vector, were cultured in medium with 90nM GCB for 48 h and then the cell viability was measured. A significant (P \ 0.05) reduction from GCB-treated parental cells is denoted by ‘‘*’’ and a significant (P \ 0.001) increase by ‘‘**’’

sCLU, a 75–80 kDa glycosylated a–b heterodimer, consisting of two chains of about 40 kDa, linked by five disulfide bonds, is a multifunctional, stress-induced molecular chaperone that is expressed in human fluids and most tissues [26]. sCLU is also a cytoprotective protein that protect cells from numerous therapeutic factors, inducing androgen withdrawal [27], radiation therapy [28], oxidative

stress [12], and chemotherapy [29]. As an important anticancer drug, GCB has been used for the treatment of pancreatic, non-small cell lung, and breast cancers [30]. Although increasing recent clinical trials have shown that GCB could be used in advanced HCC patients [31, 32], it is unknown whether sCLU could render HCC resistant to GCB. In this study, two sCLU-overexpressing cell lines

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Dig Dis Sci Fig. 5 Cell apoptosis. Parental Bel7402 or SMMC7721 cells (control), and the cells transfected with CLU-4, were incubated in absence or presence of GCB (90 nM) for 24 h, and then cell apoptosis rates(%) were measured. a Representative dot plots from cytometrically analyzed cells. b Cell apoptosis rates were measured. A significant (P \ 0.05) difference from respective control is denoted by ‘‘*’’ and a highly significant (P \ 0.001) difference by ‘‘**’’. A significant (P \ 0.05) increase from respective GCBtreated parental cells is denoted by ‘‘ ’’

were chosen as the research model. The CLU-4 vector down-regulated sCLU expression in a time-dependent manner, and sCLU depletion increased GCB-induced cell growth inhibition and the cell apoptosis. Taking all the results together, targeting sCLU in the two HCC cell lines studied herein distinctly increased the sensitivity to GCB. Several molecular mechanisms mediating enhanced sensitivity to a range of therapeutic agents by regulating the expression of sCLU have been reported thus far [33–37]. For instance, increased sCLU levels promoted prostate tumor cell survival by increasing NF-kB nuclear translocation and the degradation of COMMD1 and I-kB [36]. Increased sCLU levels were also considered to enhance tumorigenesis by interfering with the Ku70-Bax complex [14] and Bax pro-apoptotic activities [37], thereby contributing to one of the major characteristics of cancer cells: the resistance to apoptosis. However, it is still of interest to explore the potential downstream apoptotic pathway of

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sCLU in HCC cells. Our study identified a series of genes downstream of sCLU in the apoptosis signaling pathway. BNIP1 [38], AKT1 [39], and GADD45a [40] are considered to be important for intracellular signaling in response to cell-death-inducing stimuli. The GADD45 gene family [40] encodes three related GADD proteins, GADD45a, GADD45b, and GADD 45c, which play important roles in cell survival and apoptosis. The up-regulation of GADD45a is considered to be an essential step following stressful growth arrest conditions and treatment with proapoptotic agents. Some groups have found that GADD45a up-regulation could increase the sensitivity of cells to paclitaxel, doxorubicin, and docetaxel [41, 42]. Furthermore, our results showed that the GADD45a protein was up-regulated and that the level of phosphorylated AKT was down-regulated after the depletion of sCLU in two HCC cell lines, in agreement with the results in our previous report [19]. GADD45a overexpression alone increased the

Dig Dis Sci Table 2 Gene expression differences in SMMC77721 (Bel7402) cells after down-regulation of sCLU Gene title

Gene bank ID

Fold expression SMMC7721

Bel7402

BAK1

NM_001188

1.69

1.72 ± 0.12

Bcl2L1

NM_138578

1.54 ± 0.15

1.69 ± 0.21

BIK BNIP1

NM_001197 NM_013978

1.58 ± 0.09 2.16 ± 0.19

1.96 ± 0.03 2.1 ± 0.03

Caspl

NM_033292

1.71 ± 0.1

2.38 ± 0.12

GADD45A

NM_001924

4.24 ± 1.07

7.09 ± 1.27

LTBR

NM_002342

2.69 ± 0.13

1.68 ± 0.01

NOL3

NM_003946

1.49 ± 0.08

1.63 ± 0.11

TNF

NM_000594

3.34 ± 0.84

1.59 ± 0.08

TNFRSF10A

NM_003844

2.23 ± 0.09

2.46 ± 0.27

TNFRSF25

NM_148970

1.49 ± 0.01

1.5 ± 0.008

TRADD

NM_003789

2.07 ± 0.06

2.04 ± 0.06

HRK

NM_003806

1.79 ± 0.01

5.93 ± 1.41

LTA

NM_000595

3.88 ± 0.43

1.57 ± 0.13

Bag1

NM_004323

-1.96 ± 0.06

-1.82 ± 0.04

NAIP

NM_004536

-1.69 ± 0.24

-1.78 ± 0.28

FASLG

NM_000639

-1.93 ± 0.02

-2.13 ± 0.18

AKT1

NM_005163

-2.65 ± 0.45

-2.85 ± 0.18

sensitivity of Bel7402 and SMMC7721 cells to GCB. Nevertheless, although some research groups [38] reported that BNIP overexpression increased the sensitivity to apoptotic stimuli, there was no significant relationship found between BNIP1 expression and sCLU protein depletion. These results are in accord with our finding that GADD45a and AKT1 may be downstream targets of the sCLU protein. However, it remains unclear whether there is any relationship between the two targets and the issue that which is the most important potential target of sCLU deserves us for further research. Recent research has demonstrated that clusterin might be deemed as a useful marker for the early detection and prediction of HCC progression [16, 18]. Hence, upcoming experiments, we will further explore the relationship between the serum clusterin level and its expression in HCC tissues. At the same time, we will explore whether custirsen (OGX-011, an inhibitor of sCLU) [4] can increase the sensitivity to GCB through the same signaling pathway to provide pre-clinical data for the use of OGX-011 in hepatocellular carcinoma. Indeed, these pre-clinical data could provide evidence to support the design of clinical studies of OGX-011 in combination with GCB or other therapeutic agents for patients with advanced HCC.

Fig. 6 Downstream protein expression after the depletion of the sCLU in Bel7402 and SMMC7721 cells. a, b Parental Bel7402 or SMMC7721 cells, or the cells transfected with Sc or CLU-4 shRNA vectors, were incubated in the medium for 48 h after the transfection and then subjected to Western blot analysis. c, d The density of each band in a and b was measured and normalized to that of GAPDH. A significant (P \ 0.05) difference from respective control is denoted by ‘‘*’’ and a highly significant (P \ 0.001) difference by ‘‘**’’

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Dig Dis Sci Fig. 7 Western blot results demonstrated that GADD45a protein was over expressed in Bel7404 and SMMC7721 cells after transfection with pcDNA3.1-GADD45a-His (a, b). Bel7402-CLU4 (SMMC7721-CLU4) cells and the parental Bel7402 (SMMC7721) cells, or the cells transfected with pcDNA3.1GADD45a-His, were incubated with or without GCB (90 uM) and the cell viability was measured (c, d). A significant (P \ 0.05) difference from respective control is denoted by ‘‘*’’ and a highly significant (P \ 0.001) difference by ‘‘**’’. A significant (P \ 0.05) decrease from respective GCBtreated parental cells is denoted by ‘‘ ’’

Fig. 8 Cell apoptosis. Representative dot plots were from cytometrically analyzed parental Bel7402 or SMMC7721 cells (control), and their stable transfectants depleted of sCLU protein, or cells

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transfected with pcDNA3.1-GADD45a-His, which were incubated in the absence or the presence of GCB (90 nM) for 24 h, and cell apoptosis rates (%) were measured

Dig Dis Sci Acknowledgments This research was supported by the National Natural Scientific Foundation of China (81172331, 30972890) and Shandong Provincial Science & Technology Development Planning, China (2010GSF10230). Conflict of interest

17.

None. 18.

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