Background
Gastric cancer is the one of the most common causes of cancer-related death all over the world. The survival of gastric cancer patients has improved owing to improvement in diagnostic and therapeutic tools [
1]. However, overall patient prognosis and long-term survival has not significantly improved due to diagnosis of advanced stages and the high rate of recurrence [
2]. In addition, the biologic heterogeneity of gastric cancer is one of the main causes of cancer therapy failure [
3]. Therefore, it is necessary to find a novel strategy to overcome gastric cancer.
Cyclin D1, a cell cycle regulator, is closely related to several human cancers. Dysregulation of cyclin D1 immediately contributes to tumor progression in some animal models, further demonstrating a connection between cyclin D1 and carcinogenesis. Accordingly, inhibition of cyclin D1 may be an innovative strategy for cancer treatment [
4,
5].
Targeted therapy is becoming an important cancer therapy. Although gene targeting has emerged as an important treatment strategy of various malignant tumors in the past decade, fewer targeted therapies for gastric cancer have been studied compared to other cancers [
6]. RNAi is one of the most powerful targeted therapies to inhibit specific gene expression at the post-transcriptional level. There are two types of artificial RNAi induction: siRNA and shRNA. Previous reports have demonstrated the following limitations of siRNA: insufficient cellular uptake, instability, rapid clearance
in vitro, and inefficient delivery
in vivo and in clinical settings [
7‐
10]. However, viral vector-mediated shRNA permit long-term stable and consistent gene silencing [
11]. Lentiviral vectors of shRNA have been derived from HIV; the vectors can express shRNA in mammalian cells [
12,
13]. These vectors have been validated in clinical databases in animal models of relevant cancers. Therefore, the lentivirus-mediated shRNA strategy is an appealing candidate therapy for cancer treatment [
14].
In this study, we hypothesized that inhibition of cyclin D1 in human gastric cancer could suppress cancer progression in vitro and in vivo. To test this hypothesis, a third-generation lentiviral vector was used to integrate the shRNA sequence targeting cyclin D1 (ShCCND1) in the human gastric cancer cell line NCI-N87. ShRNA was expressed stably and consistently inhibited cyclin D1 expression. In vitro, the effect of silencing cyclin D1 on gastric cancer cell function, including cell proliferation, cell cycle, apoptosis, and cell motility was examined. In vivo, stably ShCCND1-expressing NCI-N87 xenografts were used to compare the growth rate with ShSramble (shRNA-targeting scramble) cell xenografts. Next, the effect of intratumoral injection of these recombinant lentiviruses targeting cyclin D1 on attenuation of the growth of pre-existing cancer was assessed.
Methods
Cell lines and cell culture
NCI-N87 gastric carcinoma cells were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). Cells were maintained in RPMI 1640 with 10% FBS and 1% penicillin/streptomycin at 37°C, 5% CO2 within a humidified chamber. The 293TN Human Kidney producer cell line was purchased from System Biosciences (SBI, Mountain View, CA, USA). This cell line was maintained in DMEM high-glucose medium supplemented with 10% FBS, Glutamax, and 1% penicillin/streptomycin at 37°C, 5% CO2 within a humidified chamber.
ShRNA-expressing plasmid DNA
Three different targeted sequences designed to be homologous to cyclin D1 were designed using the lentiviral expression vector (pGreenPuro™ Vector, SBI) [
15]. Target sites in human genes encoding cyclin D1 were as follows: ShCCND1_1 sense strand, 5′-GCCCTCGGTGTCCTACTTCAAAT-3′, ShCCND1_1 antisense strand, 5′-ATTTGAAGTAGGACACCGAGGGC-3′; ShCCND1_2 sense strand, 5′-GCACGATT TCATTGAACACTTCC-3′, ShCCND1_2 antisense strand, 5′- GGAAGTGT TCAATGAAATCGTGC-3′; ShCCND1_3 sense strand, 5′-GAAGTTCATTTCCAATCCGCCCT-3, ShCCND1_3 antisense strand, 5′-AGGGCGGATTGGAAATGAACTTC-3′. These plasmid DNAs transcribed shRNA with loop sequences of 5′-CTTCCTGTCAGA-3′. A negative control sequence was also designed by the same process, which had no homology with human proteins (ShScramble sense strand, 5′-GACTTCATAAGGC GCATGC-3′, ShScramble antisense strand, 5′-GCATGCGCCT TATGAAGTC-3′) [
16]. Each DNA was used to transform the
E. coli strain DH5α and purified with a plasmid purification kit (Qiagen, Valencia, CA, USA). The ligation product was confirmed by PCR and sequencing.
Lentivirus generation and establishment of the NCI-N87 cell line stably expressing shRNA
293TN cells were seeded at a density of 3 × 106 cells on 10-cm culture plates. After 24 h, successful co-transfection of plasmid including the ShCCND1 or ShScramble with lentiviral vector expression construct using lipofectamine reagent was demonstrated. After 48 h, supernatant was collected and added to PEG-it™ virus precipitation solution. The supernatant/PEG-IT mixture was centrifuged 1,500 × g for 30 min at 4°C and then resuspended in RPMI medium at 1/100 of the original volume. One day prior to transduction, NCI-N87 cells were plated in 24-well plates at 5 × 104 cells. After 24 h, NCI-N87 cells were infected with lentiviral particles containing ShScramble or ShCCND1. The next day, NCI-N87 cells were cultured in RPMI 1640 medium including puromycin (1 μg/ml). The expanded cells were then used for further experiments. Transduction efficiency of NCI-N87 cells with GFP signals were determined by flow cytometry.
Western blotting
Stable cancer cells (NCI-N87, ShScramble, and ShCCND1) were lysed in RIPA buffer including protease inhibitor (Sigma-Aldrich, St. Louis, MO, USA). The protein concentration of cell lysate was determined using the BCA™ protein assay kit (Thermo Scientific, Rockford, IL, USA). Equal amounts of total protein were electrophoresed by 10% SDS-PAGE, transferred onto nitrocellulose membranes, and incubated with antibodies against cyclin D1 (1:500, Santa-Cruz Biotechnology, Santa Cruz, CA, USA), pRB (1:200, Cell Signaling Technology, Beverly, MA, USA), and β-actin (1:500, Santa-Cruz Biotechnology) at 4°C. Membranes were then incubated with HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (1:1000, Santa-Cruz Biotechnology). Signals were detected using an ECL Test Kit (KPL, Gaithersburg, MD, USA). β-actin served as the internal standard. Densitometric analysis was performed using ImageJ software.
Cell proliferation assay and colony formation assay
The in vitro cell viability was analyzed using the CCK-8 assay (Dojindo Laboratories, Kumamoto, Japan). NCI-N87, ShScramble, and ShCCND1 were seeded at a density of 104 cells/well in 96-well plates. After 24 h, 10 μl of CCK-8 was added to each well and incubated for 2 h. The absorbance value was observed at 1, 2, 3, and 4 days using an enzyme-linked immunosorbent assay (Tecan Sunrise, Sunnyville, CA, USA).
To assess colony formation ability, NCI-N87 cells containing shRNA were seeded at a density of 500 cells/well in 6-well plates. After 3 weeks, cells were stained with 1% crystal violet. The number of colonies (≥25 cells) was counted under a microscope. The relative number of colonies in ShCCND1 was adjusted to the number in ShScramble. Image analysis was conducted using Metamorph version 7.5.6.0 software (Molecular Devices, Sunnyvale, CA, USA).
Scratch-wound healing assay
The scratch-wound healing assay was performed to determine the role of cyclin D1 in cell migration [
17]. NCI-N87 cells were plate on 60-mm plates at 5 × 10
4 cells. A scratch was made with a pipette tip when the plate was almost filled with cells. After 48 h, the image of cells that had migrated into the wounded area was obtained by a Zeiss Axiovert 200 inverted microscope (Carl Zeiss MicroImaging, Thornwood, NY, USA) with a 10× objective lens. The wound area was analyzed by Metamorph version 7.5.6.0 software.
Cell cycle and apoptosis analysis
To determine the cell cycle distribution, 106 NCI-N87 cells were washed twice with ice-cold PBS and resuspended in PBS containing RNase A (Sigma-Aldrich) and PI (propidium iodide, Sigma-Aldrich). Cells were incubated at 37°C in the dark for 1 h. The percentage of the cell population in each phase of the cell cycle was measured using FACSCalibur (Becton-Dickinson, Rutherford, NJ, USA), and results were analyzed with the CELLQUEST software (Becton-Dickinson).
To assess the apoptosis rate, NCI-N87 cells were starved in FBS-free culture medium for 48 h. Then, aliquots (100 μl) of 106 cells/ml were incubated with Annexin-V-APC (allophycocyanin, BD Biosciencls Pharmingen, San Diego, CA, USA) and PI (BD Biosciences Pharmingen) for 15 min in the dark. After incubation, 400 μl of the binding buffer was added to the sample and then, samples were analyzed by flow cytometry. The flow cytometric data were analyzed with the CELLQUEST software.
Tumorigenecity in nude mouse
Male athymic BALB/c nude mice at 5 weeks of age were purchased from the Nara Bio animal center (NARA Biotech, Seoul, Korea). The mice were bred and maintained at the laboratory facility in Konkuk University (Seoul, Korea). Tumorigenicity in nude mice was determined. The NCI-N87 cell line including ShScramble or ShCCND1 was cultured with RPMI 1640 medium. Cells (8 × 106) in 100 μl saline were subcutaneously injected into the posterior right thigh. Tumor growth was measured by the tumor diameter with a digital vernier caliper three times a week from the 9th day until the 35th day after tumor injection. Tumor volume (mm3) was calculated according to the following equation: tumor volume = d2 × D/2, where d and D represent the shortest and the longest diameter, respectively. At the end of the experiment (the 35th day), mice were sacrificed. Tumorigenecity study was approved by the IACUC (Institutional Animal Care and Use Committee) of Konkuk University (KU12036).
Titration and intratumoral injection of lentivirus-mediated ShCCND1 into established tumors
NCI-N87 cells were seeded at a density of 4 × 105 cells per well in 6-well plates. After 24 h, cell culture medium was replaced by RPMI 1640 medium including hexadimethrine bromide. Then, lentiviral stock in serial dilution was added to pre-plated NCI-N87 cells. After 48 h incubation, analysis was performed by flow cytometer. GFP-positive NCI-N87 cells were detected by flow cytometry. Then, the virus titer was calculated by the following equation: lentivirus titer in IU (infectious unit)/ml = {(cell number at starting time) × (dilution factor) × (percent infection)}/(added virus volume expressed in ml).
To assess the inhibition effects of virus particle including ShCCND1 on established tumors, male athymic BALB/c nude mice (NARA Biotech, N = 20) at 5 weeks of age were injected subcutaneously into the posterior right thigh with 100 μl saline containing 4 × 106 NCI-N87 cells. The mice were divided randomly into three groups. Once tumors reached a volume of 80–100 mm3 (usually 6–8 days after injection of the cells), mice received an intratumoral injection of 50 μl (4 × 107 IU/ml) of saline (Saline, N = 6), virus containing ShScramble (Virus_Scramble, N = 7), or virus containing ShCCND1 (Virus_CCND1, N = 7). After 1 week, Saline, Virus_Scramble, or Virus_CCND1 was reinjected into the tumor mass. In vivo therapeutic study was approved by the IACUC of Konkuk University (KU13049).
H&E and TUNEL staining
At day 13 after the first intratumoral injection, the mice were sacrificed and subcutaneous tumors were fixed in 10% neutral-buffered formalin. Fixed tumors were routinely processed for paraffin sectioning and H&E staining for histopathological examination. For quantification of the mitotic count, mitotic figures were counted in five random high-fields (400×) per slide and expressed as the average number of mitotic figures per field. The detection of apoptosis was performed according to the instructions of the manufacturer (ApopTag, Chemicon International Inc., Temecula, CA, USA). Briefly, the sections were deparaffinized and hydrated, and then treated with proteinase K (20 μg/ml) in PBS (pH 7.5) at 37°C for 20 min followed by treatment with 3% hydroperoxidase in PBS to block endogenous peroxidases. After washing with PBS, the slides were incubated with terminal deoxynucleotidyl transferase and nucleotide mixture at 37°C for 1 h, and then anti-digoxigenin conjugate was added for 30 min. The slides were treated with DAB before counterstaining with hematoxylin. To measure the number of apoptotic cells, five fields at 400× magnification per slide were quantitatively analyzed with the image analyzer (Metamorph version 7.5.6.0 software). The data were expressed as the average number of cells per field.
Statistical analysis
For statistical analysis, all data obtained were analyzed using Prism 5 for Windows software (GraphPad Software, San Diego, USA). Statistically significant differences between studied groups were evaluated using the unpaired Student’s t-test and Fisher’s exact test. The results were determined to be significantly significant when P < 0.05 was obtained.
Discussion
Cyclin D1 is one of the three D-type cyclins and has been implicated in neoplastic function. The gene for cyclin D1 is located on chromosome 11q13 and is related to various cancers [
20,
21]. The increased expression of cyclin D1 has been associated with decreased G1 phase distribution and with decreased survival in many common cancers including gastric cancer [
22‐
25]. Inhibition of cyclin D1 expression markedly suppressed cell proliferation and the beginning of S phase in the cell cycle of many cancers [
25,
26].
RNAi has been used as a therapeutic tool [
27]. However, it is important to determine the most efficient and straightforward delivery strategy of the siRNA. The present study highlights the use of the shRNA-based technique, specifically the use of shRNA-based recombinant lentiviral vector to produce siRNA [
3,
28].
Lentiviruses are in the genus of the retroviridae family, which is an efficient vehicle for gene transduction and integration. In contrast to other viral vectors, lentiviral vectors have many advantageous. They have a lower immunogenicity, decreased likelihood of insertion mutagenesis, the ability to efficiently infect both primary and non-dividing cells, and a high capability of transgene insertion. In addition, they have been used as efficient preclinical models [
14,
29]. Therefore, they are ideally suited as a silencing technique in human cancer cells [
11].
In this study, ShCCND1 resulted in stable integration of virus into the cellular genome and marked inhibition of cyclin D1 protein levels. Our previous study also reported that ShCCND1 significantly inhibited the expression levels of cyclin D1 and pRB, the major downstream molecule of cyclin D1, in another gastric cancer cell, AGS [
30]. Downregulated of cyclin D1 was related to decreased cell proliferation, colony formation potential, and cell motility
in vitro. Downregulation of cyclin D1 was associated with both the cell cycle and apoptosis; therefore, ShCCND1 significantly induced apoptosis and G1 arrest. G1 phase arrest allows cells to either undergo repair or enter the apoptotic pathway to maintain tissue homeostasis and eliminate the mutated neoplastic and hyperproliferating neoplastic cells from the system. In recent years, the concept of cell cycle regulation-mediated apoptosis has gained increasing attention, and it has emerged as a potential way to inhibit cell growth [
3,
31]. As such, ShCCND1 may be a potent therapeutic agent by causing G1 phase arrest and inducing apoptosis [
32]. Collectively, these results clearly demonstrate that the anticancer effect of ShCCND1 NCI-N87 cells is mediated by induction of cell cycle arrest and apoptosis.
To analyze the therapeutic potential of the lentivirus expressing ShCCND1, in vivo experiments were performed. The recombinant lentivirus including ShCCND1 was intratumorally injected into established tumors. Intratumoral injection of the recombinant lentivirus suppressed the growth of established tumors arising from NCI-N87 cells. This result demonstrates that downregulation of cyclin D1 by the lentivirus-mediated shRNA strategy resulted in a prolonged biological effect in vivo. These results were also supported by the H&E and TUNEL staining data. Mitotic figure is a marker of proliferation and TUNEL-positive staining is a marker of apoptosis; results from these experiments suggested that silencing of cyclin D1 inhibited cancer cell growth in vivo. The decrease of mitotic figure count and increase in the number of TUNEL-positive cells are consistent with the known effects of silencing cyclin D1 and with the in vitro data presented in this study.
There are several reasons that intratumoral injection of lentivirus-mediated ShCCND1 may have diverse advantageous effects. Targeting cyclin D1 could hinder progress toward advanced cancer because cyclin D1 levels increase in the relatively early stages of tumorigenesis [
33]. Furthermore, cyclin D1 is an essential component for carcinogenesis in some cancers, which is supported by the fact that cyclin D1 knockout mice do not make mammary carcinomas in spite of the overexpression of c-
neu and v-Ha-
ras[
34]
. Gastric cancer is characterized by the interaction between cancer cells and stromal cells that contain proliferating cancer-associated activated fibroblasts, endothelial cells, and inflammatory cells. These stromal cells have been suggested to confer a growth advantage to gastric cancer cells within the tumor mass [
35,
36]. Direct injection of Virus_CCND1 into gastric cancer would result in a downregulation of cyclin D1 not only in cancer cells but also in non-cancerous stromal cells. Therefore, direct injection of Virus_CCND1 effectively inhibited gastric cancer growth due to growth inhibition by simultaneously blocking cancer cell and non-cancer cell growth. While intravenous injection of lentivirus showed histological damage to the liver cells, intratumoral injection predominantly infected the tumor instead of the liver and other organs. In view of this fact, intratumoral injection of the lentiviral vector is more advantageous than intravenous injection [
37]. Intratumoral injection of lentivirus should be generally applicable to human solid tumors [
38]. Therefore, direct injection of lentivirus-mediated ShCCND1 is an efficient tool to significantly suppress cyclin D1 in gastric cancer cells. But because this study has some limitations, such as off-target effect or side-effect of lentivirus-mediated ShCCND1
in vivo, further study will be necessary to elucidate the accurate balance between therapeutic efficacy and safety of lentivirus-mediated ShCCND1 [
39].
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JHS carried out in vivo and in vitro experiments, participated in the design of the study and drafted the manuscript. ESJ carried out in vivo experiments and critically revised the manuscripts. YKC participated in the study supervision and critically reviewed the manuscript. All authors read and approved the final manuscript.