Introduction
Pancreatic cancer has the worst prognosis of all major cancers, with an overall 5-year survival rate of around 5%[
1]. The current clinical standard of care for advanced pancreatic cancer is gemcitabine, a cytotoxic nucleoside analogue. Gemcitabine results in a tumor response rate of 12% and offers a median survival time of 5 months[
2]. Unfortunately, this means that the best current treatment offers very modest benefits. Recent studies have indicated that targeted therapies in combination with gemcitabine can have statistically significant benefits[
3]. However, the results to date remain meager, and new approaches to improving the effectiveness of gemcitabine are needed. One of the targets considered for combination therapy that has generated wide attention is clusterin[
4].
Clusterin, also known as testosterone-repressed prostate message-2 (TRPM-2), sulfated glycoprotein-2 (SGP-2), apolipoprotein J (Apo J) or SP40, is a ubiquitous heterodimeric-secreted glycoprotein of 75–80 kDa. A single-copy gene in humans of nine exons, spanning over 16 kb and located on chromosome 8p21-p12, encodes an mRNA of approximately 2 kb, which directs the synthesis of a 449-amino acid primary polypeptides chain[
5]. Recent focus has turned to clusterin as a key contributor to chemoresistance to anticancer agents. Its role has been documented in prostate cancer for paclitaxel/docetaxel resistance[
6] as well as in renal[
7], breast[
8], and lung tumor cells[
9]. Moreover, it is abnormally upregulated in numerous advanced stage and metastatic cancers spanning gastric cancer[
10], bladder[
11], cervical[
12], breast[
13],ovarian[
14], hepatocellular[
15], colorectal[
16], renal[
17], prostate[
18], head and neck[
19], lung carcinomas[
20], melanoma[
21]and lymphoma[
22].It is noteworthy that only the cytoplasmic/secretory clusterin form (sCLU), and not the nuclear form, is expressed in aggressive late stage tumors, which is in line with its antiapoptotic function[
23].
Many reports also document that sCLUc inhibits mitochondrial apoptosis. For example, sCLUc suppresses p53-activating stress signals and stabilizes cytosolic Ku70-Bax protein complex to inhibit Bax activation[
24]. sCLUc specifically interacts with conformationally altered Bax to inhibit apoptosis in response to chemotherapeutic drugs[
25]. sCLU sliencing alters the ratio of anti-apoptotic Bcl-2 family members, disrupting Ku70/Bax complexes and Bax activation[
24,
25]. In addition, sCLU increases Akt phosphorylation levels and cell survival rates[
26]. sCLU induces epithelial-mesenchymal transformation by increasing Smad2/3 stability and enhancing TGF-β-mediated Smad transcriptional activity[
27]. sCLU also promotes prostate cancer cell survival by increasing NF-κB nuclear transactivation, acting as a ubiquitin-binding protein that enhances COMMD1 and I-kB proteasomal degradation via interaction with E3 ligase family members[
28]. sCLU sliencing stabilized COMMD1 and I-κB, suppressing NF-κB translocation to the nucleus, and suppressing NF-κB-regulated gene signatures. Thus, sCLU has a key role in preventing apoptosis induced by cytotoxic agents and has the potential to be targeted for cancer therapy.
It has recently reported sCLU was overexpressed in pancreatic cancer tissues and sCLU overexpression confered gmcitabine resistance in pancreatic cancer cells,. Furthermore,sCLU silencing sensitized pancreatic cancer cells to gemcitabine chemotherapy, however the mechanism is still unclear[
29].
ERK1/2 is an important subfamily of mitogen-activated protein kinases that control a broad range of cellular activities and physiological processes. ERK1/2 can be activated transiently or persistently by MEK1/2 and upstream MAP3Ks in conjunction with regulation and involvement of scaffolding proteins and phosphatases[
30]. There is abundant evidence that survival factors can use the ERK1/2 pathway to increase the expression of several pro-survival BCL-2 proteins, notably BCL-2, BCL-xL and MCL-1, by promoting de novo gene expression in a variety of cell types[
31]. Clearly the ERK1/2 pathway can regulate several members of the BCL-2 protein family to achieve cell survival. ERK1/2 signalling can provide protection against chemotherapeutic cytotoxic drugs.
It has shown previously sCLU plays an important role in astrogliosis by stimulating the proliferation of astrocytes through activation of the extracellular signal-regulated kinase 1/2 signaling pathway[
32]. Shim and Chou et al. also found significant relation between sCLU and ERK1/2 expression[
33,
34]. We therefore suggested that sCLU silencing sensitized pancreatic cancer cells to gemcitabine chemotherapy may via ERK1/2 signaling pathway.
sCLU is not a traditional druggable target and can only be targeted at mRNA levels. An antisense inhibitor targeting the translation initiation site of human exon II CLU (OGX-011) was developed at the University of British Columbia and out-licensed to OncoGeneX Pharmaceuticals Inc. OGX-011, or custirsen, is a second-generation antisense oligonucleotide with a long tissue half-life of ~ 7 days, which potently suppresses sCLU levels in vitro and in vivo. OGX-011 improved the efficacy of chemotherapy, radiation, and hormone withdrawal by inhibiting expression of sCLU and enhancing apoptotic rates in preclinical xenograft models of prostate, lung, renal cell, breast, and other cancers[
35‐
39].
In this study, we study the effect of sCLU silencing by OGX-011 on sensitizion of pancreatic cancer cells to gemcitabine chemotherapy, and eluated the mechanisms.
Materials and methods
Cell culture
The human pancreatic cancer MIAPaCa-2 cells resistant to gemcitabine and BxPC-3 cells sensitive to gemcitabine[
38] were purchased from American Type Culture Collection. They were routinely cultured in DMEM supplemented with 10% fetal bovine serum in a 37°C incubator in a humidified atmosphere of 5% CO
2.
Reagents and antibodies
OGX-011 was purchased from OncoGenex Technologies. The antisense oligonucleotides were second-generation 21-mer antisense oligonucleotides with a 2′-O-(2-methoxy)ethyl modification. The antisense oligonucleotide clusterin sequence corresponding to the human clusterin initiation site was 5′-CAGCAGCAGAGTCTTCATCAT-3′ and designated OGX-011 (OncoGenex Technologies). The MEK inhibitor PD98059 was products of Calbiochem (San Diego, CA, USA), Antibodies for sCLU, and phospho-specific or the total form of antibodies against ERK1/2,GAPDH were purchased from Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Construction of transient transfection with a plasmid expressing human wt-pERK
Total RNA was extracted from PANC-1 cells using TRIzol reagent (Invitrogen, CA, United States), according to the manufacturer’s protocol. The cDNAs were synthesized using the TaKaRa RNA polymerase chain reaction (PCR) Kit (TaKaRa, Japan). A full-length cDNA encoding human wt-pERK was cloned by PCR using 500 ng cDNA as a template and primers containing HindIII and BamHI restriction enzyme sites. The PCR products were ligated into pcDNA3.1 (Invitrogen, CA, United States) to create the plasmid pcDNA3.1- wt-pERK. MIA PaCa-2 and BxPC-3 cells were transfected with the pcDNA3.1 vector or pcDNA3.1- wt-pERK using FuGENE (Roche Diagnostic GmbH, Mannheim, Germany), according to the manufacturer’s protocol.
Transient transfection
MIA PaCa-2 and BxPC-3 cells were treated with OGX-011(400,800,1000,1200 nM) for 24 h, then the cells were cultured overnight in 6-well plates and transfected with pcDNA3.1- wt-pERK using Lipofectamine Plus (Invitrogen) in 1 ml serum-free medium according to the manufacturer’s instructions. Four hours post-transfection, each well was supplemented with 1 ml of medium containing 20% FBS. Twenty-four hours post-transfection, media were removed and the cells were harvested or treated with gemcitabine for a further 24 hours.
Western blotting assay
About 25 μg protein was extracted, separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride membranes, and then reacted with primary rabbit antibodies against sCLU(1:100), pERK1/2(1:100) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)(1:200). After being extensively washed with PBS containing 0.1% Triton X-100, the membranes were incubated with alkaline phosphatase-conjugated goat anti-rabbit antibody for 30 minutes at room temperature. The bands were visualized using 1-step™ NBT/BCIP reagents (Thermo Fisher Scientific, Rockford, IL, USA) and detected by the Alpha Imager (Alpha Innotech, San Leandro, CA, USA).
RT-PCR assay
The mRNA extraction and RT reaction for synthesizing the first-strand cDNA was carried out according to the manufacturer’s instructions. Primer sequences were below: 5′-CCAACAGAATTCATACGAGAAGG-3′ and 5′-CGTTGTATTTCCTGGTCAACCTC-3′ for sCLU;5′-TGATGGGTGTGAACCACGAG-3′, 3′-TTGAAGTCGCAGGAGACAACC-5′for GAPDH. The PCR conditions consisted of an initial denaturation at 95°C for 3 min, followed by 28 cycles of amplification (95°C for 15 s, 58°C for 15 s, and 72°C for 20 s) and a final extension step of 5 min at 72°C. PCR products were analyzed on a 1.2% agarose gel. The significance of differences was evaluated with Student’s t-test. The mean ± SD are shown in the figures. P < 0.05 was considered to be statistically significant.
FACS analysis
To identify the induction of apoptosis, cells underwent propidium iodide (PI) staining and fluorescence-activated cell sorting (FACS) as to the manufacture’s instruction. In brief, cells were plated at a density of 1 × 105 cells/ml. After allowing 24 hours for cell adherence, cells were transfected and/or treated. Cells were collected by gentle trypsinization, washed in phosphate-buffered saline (PBS), pelleted by centrifugation and fixed in 70% ethanol. Immediately prior to staining, cells were washed twice in PBS and resuspended in PBS containing RNAse A (20 μg/ml). Cells were stained with propidium iodide (final concentration 10 μg/ml) for 10 min at room temperature. Samples were analyzed by FACS (FL-3 channel) using a Beckman Coulter Counter Epics XL flow cytometer (Beckman Coulter, Miami, FL, USA). For each sample, 50,000 events were collected and stored for subsequent analysis using EXPO software (version 2.0; Applied Cytometry Systems, Sheffield, UK). The percentage of cells in the sub-G0 phase was quantitated as an estimate of cells undergoing apoptosis.
MTT assay
Cells were plated at 2 × 103 cells per well in 96-well plates for six days. Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT, Trevigen, Inc., Gaithersburg, MD) in accordance with the manufacturer’s instructions. Plates were read using a Vmax microplate spectrophotometer (Molecular Devices, Sunnyvale, CA) at a wavelength of 570 nm corrected to 650 nm and normalized to controls. Each independent experiment was done thrice, with 10 determinations for each condition tested. At identical time points,cells were trypsinized to form a single cell suspension. Intact cells, determined by trypan blue exclusion, were counted using a Neubauer hemocytometer (Hausser Scientific, Horsham, PA). Cell counts were used to confirm MTT results.
Antitumor study
MIAPaCa-2 or BxPC-3 cells (107) were injected into the pancreas of SCID mice. Four weeks after tumor implantation, the mice were assigned to one of the following four treatment groups (n = 10 each): (a) vehicle control; (b) gemcitabine, biweekly treatment 80 mg/kg/injection; (c) OGX-011, biweekly treatment 0.35mg/kg/injection; (d) gemcitabine plus OGX-011, with gemcitabine on Monday and Thursday and OGX-011 on Wednesday and Saturday. All groups received treatment via i.p. injection. Mice in all groups were killed after 5 weeks of treatment. Orthotopic tumors were harvested and weighed.
In vivo apoptosis assay
Five serial sections (5 um thick) were obtained for each frozen tumor, mounted on glass slides, and then fixed in 4% paraformaldehyde. The first section was processed for H&E staining. Apoptosis was evaluated by terminal transferase dUTP nick end labeling [TUNEL] staining using the Apoptag Peroxidase In Situ Detection Kit S7100 [Chemicon] according to the manufacturer’s instructions.
Statistical analysis
All statistical analyses were performed using the SPSS13.0 software. The results were presented as means ± SD of two-three replicate assays. Differences between different groups were assessed using X2 or t-test. A P value of <0.05 was considered to indicate statistical significance.
Discussion
Pancreatic cancer is one of the most difficult human cancers to treat due to the inability to detect disease at an early stage and the lack of effective therapies. Although there has been some progress in the use of improved diagnostic methods and development of novel targeted therapies, the overall survival rate has not improved over the last decade[
39]. The most commonly used chemotherapy for pancreatic cancer, gemcitabine, has modest clinical benefit and may not improve overall survival to a clinically meaningful degree[
40,
41]. The lack of significant clinical response of pancreatic cancer patients to chemotherapy is likely due to the inherent chemoresistance of pancreatic cancer cells as well as impaired drug delivery pathways[
42]. Understanding the underlying mechanisms of drug resistance in pancreatic cancer is critical to develop new effective treatments for this deadly disease.
sCLU expression has been implicated in chemoresistance in several other cancer types[
43‐
45], including pancreatic cancer[
29]. Because the resistance of tumor cells to various available chemotherapeutic agents has been one of the major factors leading to poor survival in pancreatic cancer patients, we therefore hypothesized that sCLU confers chemoresistance to pancreatic cancer cells.
In this study, we demonstrated that sCLU was correlated with inherent resistance both in vitro and in vivo. We found that high levels of sCLU in pancreatic cancer MIAPaCa-2 cell line was correlated with gemcitabine resistance, low levels of sCLU in BxPC-3 cells was sensitive to gemcitabine .To demonstrate the role of sCLU in gemcitabine resistance, we manipulated the endogenous level of sCLU in a gemcitabine -sensitive BxPC-3 cell line and a gemcitabine -resistant MIAPaCa-2 cell line. We found that gemcitabine -sensitive BxPC-3 cells became more resistant to gemcitabine when endogenous sCLU expression was up-regulated. Conversely, gemcitabine -resistant MIAPaCa-2 cells became more sensitive to gemcitabine and more apoptotic in vitro and in vivo when endogenous sCLU expression was down-regulated by GOX-011 treatment. These results indicated that high levels of endogenous sCLU were involved in the gemcitabine resistance of ovarian cancer cells.
Acquired drug resistance is also thought to be a reason for the limited benefit of most pancreatic cancer therapies.In the present study, we found treatment by gemcitabine increased sCLU expression in BxPC-3 cells, suggesting that sCLU upregulation is likely to be an adaptative response that mediates chemoresistance.We also investigated whether anticlusterin treatment sensitized BxPC-3 cells to gemcitabine.
GOX-011 efficiently inhibited sCLU expression in BxPC-3 cell lines, and this activity was associated with a increase in cell apoptosis in gemcitabine-treated BxPC-3 cells in vivo and vitro. This was indicated that increased sCLU, expression was correlates with gemcitabine resistance in pancreatic adenocarcinoma cells. These results provide preclinical proof of principle for the use of OGX-011 as a novel therapeutic strategy for gemcitabine resistance in the treatment of pancreatic cancer.
Though sCLU confers gmcitabine resistance in pancreatic cancer cells, however, the signaling pathway was unclear. ERK activation has been identified as a potential survival pathway in several tumor types[
46], and recent studies show that ERKs may also be activated in response to chemotherapeutic drugs[
47‐
50], and pERK1/2 played critical roles in drug resistance[
51]. Our in vitro and in vivo studies here indicated that pERK1/2 play significant roles in gemcitabine resistance to pancreatic cancer cells. Most importantly, we demonstrated that blocking pERK1/2 enhanced the chemotherapeutic potential of gemcitabine in pancreatic cancer cells in vitro. ERK1/2 inhibitors in combination with chemotherapeutic drugs might be a better option to treat patients with pancreatic cancer than drugs alone.
It has shown previously sCLU plays an important role in regulating ERK1/2 signal[
32‐
34].We next study whether sCLU silencing sensitized pancreatic cancer cells to gemcitabine chemotherapy may via ERK1/2 signal. Our results shown sCLU sliencing by OGX-011 sensitizes pancreatic cancer cells to gemcitabine treatment,followed by inhibition of pERK1/2 activation. Conversely, transfection with a constitutively active wt-pERK1/2 construct promotes gemcitabine resistance. These data demonstrated sCLU sliencing sensitizes pancreatic cancer cells to gemcitabine via pERK1/2 dependent signaling pathway.
In conclusion, gemcitabine may influence pancreatic cancer behavior via the upregulation of sCLU, which might play a major role in the effects of gemcitabine, protecting pancreatic cancer cells from the effects of gemcitabine. Inherent chemoresistance of pancreatic cancer cells to gemcitabine may be correlated to sCLU. Blocking sCLU, on the other hand, reverses the drug’s unwanted effects on cancer cell apoptosis and survival. In addition, our studies have firmly established a role for sCLU as a cell survival gene that is increased after gemcitabine chemotherapy to inhibit tumor cell death. The inhibition of sCLU, using OGX-011, enhances the cytotoxic effects of chemotherapy agents via pERK1/2 dependent signaling pathway.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
TY, LFH, ZCN and JYS performed the majority of experiments; SSC and TY designed the study and wrote the manuscript; TY and JYS edited the manuscript. All authors read and approved the final manuscript.