Background
Neuroblastoma, a pediatric cancer of the developing sympathetic nervous system, is one of the most common solid tumors in infancy and early childhood [
1‐
3]. The tumor emerges in tissues of the sympathetic nervous system, typically in the paraspinal ganglia or adrenal medulla, and thus can present as mass lesions in the chest, neck, pelvis, and abdomen [
4]. The prognosis is highly variable and is associated with a number of parameters including tumor stage, age at diagnosis, and grade of differentiation of the tumor [
5,
6]. Although the survival of children with neuroblastoma has significantly improved during recent years, patients with advanced-stage disease still show a poor prognosis, despite intensive and advanced treatments, with overall survival probabilities of less than 40% [
7‐
9]. Therefore, it is urgent to clarify the molecular and genetic properties of neuroblastoma that will greatly improve the therapeutic effect of this complex heterogeneous disease [
5,
10].
The PEST motif is a peptide sequence which is rich in proline (P), glutamic acid (E), serine (S), and threonine (T) [
11‐
13]. It is well known that the PEST sequence functions as a proteolytic signal to target proteins for degradation via the proteasome pathway or calpain proteolysis [
11,
14,
15]. The PEST sequence is considered the unstructured region in a number of protein sequences, possibly serving as a phosphodegron for the recruitment of F-box-containing ubiquitin E3 ligases that result in ubiquitination and degradation [
16,
17]. A novel PEST-containing nuclear protein (PCNP) has been identified in the nucleus through database mining. Np95/ICBP90-like RING finger protein (NIRF) is a nuclear protein with a ubiquitin-like domain, a YDG/SRA domain, a PHD finger, and a RING finger. PCNP could interact with NIRF and modulate the transcriptional activity of NIRF [
18]. PCNP and NIRF may be involved in the signaling pathway concerned with cell cycle regulation and/or genome stability [
19]. In addition, it has been shown that PCNP mRNA can be detected in many types of cancer cells, such as HT-1080 fibrosarcoma cells, HepG2 hepatoma cells, and U-937 myeloid leukemia cells, indicating that PCNP might play an important role in cell proliferation and tumorigenesis [
18,
19]. However, the expression level of PCNP in neuroblastoma cells is unknown, and the effect of PCNP on the growth of neuroblastoma cells has not yet been elucidated.
In the present study, we investigated the effects and mechanisms of PCNP on the proliferation, migration, and invasion of human neuroblastoma cells. We further examined the effects of PCNP on tumor growth and angiogenesis in nude mice xenografted with human neuroblastoma.
Methods
Cell culture
Human neuroblastoma cell lines SH-SY5Y and SK-N-SH were purchased from CoBioer Biosciences Co., Ltd. (Nanjing, Jiangsu, China) and cultured in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were grown in an incubator with a humidified atmosphere of 95% air and 5% CO2 at 37 °C.
Over-expression and knockdown of PCNP
Human PCNP complementary deoxyribonucleic acid (cDNA) (NM_020357) was sub-cloned into the Xho I and Kpn I restrictive sites of GV230 (Genechem, Shanghai, China), validated by sequencing and transfected into tumor cells with Lipofectamine 3000 Transfection Reagent (Life Technologies, Carlsbad, CA, USA). The empty vector (Mock group) or GV230-PCNP construct (PCNP group) was transfected into tumor cells, and stable cell lines were screened by administration of G418 (Solarbio, Shanghai, China). The oligonucleotides encoding short hairpin ribonucleic acid (shRNA) specific for PCNP and their scramble sequences were sub-cloned into the Age I and EcoR 1 restrictive sites of GV248 (Genechem, Shanghai, China). The PCNP shRNA (sh-PCNP group) and scramble shRNA (sh-Scb group) were verified by DNA sequencing and transfected into tumor cells with Lipofectamine 3000 Transfection Reagent. Stable tumor cell lines transfected with shRNAs were screened by administration of puromycin (Solarbio, Shanghai, China). The untransfected tumor cells were used as controls. Seventy-two hours post-transfection, the localization of PCNP within tumor cells was detected under a fluorescent microscope (Eclipse Ti, Nikon, Melville, NY, USA).
Reverse transcription-polymerase chain reaction (RT-PCR)
Seventy-two hours post-transfection, total RNA was isolated from the cells using TRIzol reagent, treated with DNase I, and purified using an RNA clean-up kit (Cwbiotech, Beijing, China). Total RNA (1 μg) was applied for cDNA synthesis using a cDNA reverse transcription kit (Cwbiotech, Beijing, China). Primers were designed according to the primer design principles with Primer Premier 5.0 (Premier Biosoft, Palo Alto, CA, USA): PCNP, forward 5’-ATAGGATCCAAAATGGCGGACGGGAAGGCG-3′ and reverse 5′- CCGAAGCTTTTAATTGTCTTGGTCATGGAC-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward 5’-TATGACAACGAATTTGGCTACAG-3′ and reverse 5’-GATGGTACATGACAAGGTGC-3′. The reactions were performed in a total volume of 20 μl using the following thermal cycling parameters: 95 °C for 10 min, 40 cycles of 95 °C for 15 s, 60 °C for 60 s, and 72 °C for 1 min. The results were normalized to the level of GAPDH.
Cell proliferation and viability assays
The 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay was performed using the Cell-Light EdU Apollo 567 In Vitro Imaging Kit (RiboBio, Guangzhou, Guangdong, China). After incubation with 10 mM EdU for 2 h, SH-SY5Y and SK-N-SH cells were fixed with 4% paraformaldehyde, permeabilized with 0.3% Triton X-100, and stained with fluorescent dyes. 4′, 6-diamidino-2-phenylindole (DAPI) was used to stain the cell nuclei (blue) at a concentration of 5 mg/ml at room temperature for 10 min. Cells were observed under a fluorescent microscope (Eclipse Ti, Nikon, Melville, NY, USA). Cell proliferation rate (%) = (EdU-positive cells)/(total number of cells) × 100 [
20]. The cell viability was detected using the CellTiter 96 AQ
ueous One Solution Cell Proliferation Assay kit (MTS; Promega, Madison, WI, USA) according to the manufacturer’s protocols.
Cells (4 × 102 per well) were seeded in 6-well plates and cultivated in culture medium at 37 °C for a week. At that point, colonies were washed with phosphate-buffered saline (PBS) buffer for three times before subjected to cell fixation using 1 ml of methanol at room temperature for 15 min. Then, 1 ml of crystal violet was added into each well and incubated for 30 min at room temperature. Plates were gently washed with water and air-dried at room temperature. Finally, the 6-well plate was scanned for colony counting and analysis.
Wound healing assay
Confluent cells were scratched with a sterile micropipette tip and subsequently washed twice with PBS. The migration distance was photographed under an Olympus CKX41 microscope and then measured using Image J software (National Institute for Health, Bethesda, MD, USA). The migration rate (MR) was calculated as MR (%) = [(A - B)/A] × 100, where A is the width at 0 h, and B is the width at 24 h.
Soft agar assay
Cells were suspended in 0.6% agarose and medium supplemented with 10% FBS, and the mixture was seeded in 6-well plates containing a basal layer of 1.2% agarose at 1 × 104 cells/well. The medium was replaced twice per week. After 2 weeks of routine culture, colonies were photographed under an Olympus CKX41 microscope. Viable colonies larger than 0.1 mm in diameter were counted.
Migration and invasion assays
For migration and invasion assays, 1 × 105 cells in serum-free medium were seeded into the upper chamber uncoated or coated with Matrigel (BD Biosciences, San Jose, CA, USA). 500 μl corresponding medium containing 10% FBS was added to the lower chamber. After incubation for 24 h, remaining cells were scrubbed off with cotton swabs, while cells on the bottom surface of the membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The cell number was counted using a Zeiss Axioskop 2 plus microscope (Carl Zeiss, Thornwood, NY, USA).
TUNEL assay was conducted using an In Situ Cell Death Detection Kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s protocols. Cells were observed under a fluorescent microscope (Eclipse Ti, Nikon, Melville, NY, USA). The percentage of TUNEL-positive cells was calculated using Image J software.
Western blotting
Seventy-two hours post-transfection, total protein was extracted from SH-SY5Y and SK-N-SH cells. Western blotting was performed to detect the expression of target proteins. The primary antibodies, including anti-extracellular signal-regulated protein kinase 1/2 (ERK1/2), anti-phospho (p)-ERK1/2 (Thr202/Tyr204), anti-c-Jun N-terminal kinase (JNK), anti-p-JNK (Thr183/Tyr185), anti-p38, anti-p-p38 (Thr180/Tyr182), anti-phosphatidylinositol 3-kinase (PI3K), anti-p-PI3K (Tyr458/Tyr199), anti-Akt, anti-p-Akt (Ser473), anti-mammalian target of rapamycin (mTOR), and anti-p-mTOR (Ser2448) antibodies were purchased from Cell Signaling Technology (CST, Danvers, MA, USA). Anti-PCNP, Anti-B-cell lymphoma-2 (Bcl-2), anti-Bcl-2-associated X protein (Bax), anti-B-cell lymphoma-extra large (Bcl-xl), anti-Bcl-xl/Bcl-2-associated death promoter (Bad), anti-cleaved caspase-3, anti-cleaved caspase-8, anti-cleaved caspase-9, anti-Cleaved poly adenosine diphosphate-ribose polymerase (PARP), and anti-GAPDH antibodies were purchased from ProteinTech (Chicago, IL, USA). The horseradish peroxidase-conjugated secondary antibody was purchased from Cell Signaling Technology. The results were normalized to the level of GAPDH. The reaction was visualized using an enhanced chemiluminescence system (Thermo Fisher Scientific, Rockford, IL, USA). The bands were semi-quantified with Image J software.
Animal study
Animal experiments were approved by the Committee of Medical Ethics and Welfare for Experimental Animals of Henan University School of Medicine (HUSOM-2017-196) in compliance with the Experimental Animal Regulations formulated by the National Science and Technology Commission, China. Animal studies were conducted as previously described with slight modifications [
21]. Thirty 4-week-old male BALB/C nude mice (
n = 6 per group) were obtained from Beijing HFK Bioscience Co., Ltd. (Certificate No. SCXK (Jing) 2014–0004, Beijing, China). SH-SY5Y and SK-N-SH cells (1 × 10
7 cells in 200 μl PBS) with over-expression and knockdown of PCNP were implanted by subcutaneous injection into the right flanks of mice. The mice were weighed and the tumor volumes were measured daily during the experiment. The tumor volumes were calculated as volume = L × W
2/2, where L is the longest dimension parallel to the skin surface and W is the dimension perpendicular to L and parallel to the surface [
22]. Then the tumor volume doubling time (TVDT) was calculated. TVDT = (T – T
0) × log2/log(V2/V1), where (T – T
0) represents the time interval and V2 and V1 indicate the volumes of tumor at the two measurement times [
23]. At the end of the experiment, mice were sacrificed and tumors were excised and weighted to evaluate the inhibition rate (IR). The IR of tumor growth was calculated as IR (%) = [(A - B)/A] × 100, where A is the average tumor weight of the control group, and B is that of the treatment group [
21].
Hematoxylin and eosin (HE) staining
After sacrifice, a necropsy examination was immediately performed. Tumor samples were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 5 μm thickness, and processed according to the HE staining protocols. Tumor tissues were observed using a Zeiss Axioskop 2 plus microscope.
Immunohistochemistry (IHC) and evaluation
Tumor tissues were stained with anti-Ki67 antibody (CST, Danvers, MA, USA), followed by incubation with secondary antibody. Ki67-positive tumor cells were photographed using a Zeiss Axioskop 2 plus microscope and the proliferation index (PI) was determined by the number of Ki67 positive cells among the total number of counted tumor cells [
24]. Cluster of differentiation 31 (CD31) has been considered an ideal biomarker for vascular endothelial cells, and its immunostaining density is represented by the tumor microvessel density (MVD) [
25]. To determine the tumor MVD, tumor tissues were stained by IHC using CD31 antibody (CST, Danvers, MA, USA). Stained vessels with a clearly defined lumen or well-defined linear vessel shape were observed using a Zeiss Axioskop 2 plus microscope and counted from the representative tumor zone, and the mean value was regarded as MVD.
Statistical analysis
Data are presented as means ± standard error of the mean. The differences between multiple groups were analyzed by one-way analysis of variance using SPSS 17.0 software, followed by Tukey’s test. A P value of less than 0.05 was considered to be statistically significant.
Discussion
PCNP is a novel nuclear protein that could interact with NIRF and modulate the transcriptional activity of NIRF [
18]. Recent studies suggest that PCNP may play an important role in cell proliferation and tumorigenesis [
18,
19]. However, the precise mechanism of action of PCNP in the proliferation, migration, and invasion of cancer cells has not yet been fully elucidated. The human neuroblastoma cell lines SH-SY5Y and SK-N-SH possess several properties of neuronal cells and have been widely used as cellular models to investigate the intracellular mechanisms of action of therapeutic agents [
36]. In the present study, SH-SY5Y and SK-N-SH cells were used to evaluate the effects of PCNP in vitro and in vivo. The results demonstrated that the expression of PCNP can be detected in human neuroblastoma cells, in addition to fibrosarcoma cells, hepatoma cells, and myeloid leukemia cells [
18]. PCNP over-expression attenuated the proliferation and viability, as well as decreased the migration and invasion capabilities of SH-SY5Y and SK-N-SH cells, whereas PCNP knockdown exhibited completely opposite effects, suggesting that PCNP could play important roles in the growth, migration, and invasion of human neuroblastoma cells.
Apoptosis, also known as programmed cell death, is a critical process for the normal development and maintenance of tissue homeostasis in multicellular organisms [
37]. There are two apoptotic signaling pathways: an intrinsic pathway that occurs through the mitochondria and an extrinsic pathway initiated by death receptors [
38]. The Bcl-2 family of proteins, such as Bax, Bcl-2, Bad, and Bcl-xl, could function as central regulators of apoptosis in mammals [
39]. Caspases could be activated in response to apoptotic stimuli and cleaved caspase-3 could inactivate PARP, thus eventually resulting in the occurrence of apoptotic cascade [
40]. Our results showed that PCNP over-expression remarkably increased the apoptotic index, protein expressions of cleaved caspase-3, 8, 9, as well as Bax/Bcl-2 and Bad/Bcl-xl ratios, suggesting the activation of mitochondria-mediated pathway. However, PCNP knockdown dramatically decreased the level of apoptosis, indicating that PCNP has pro-apoptotic function in neuroblastoma.
MAPKs regulate a variety of cellular events, including proliferation, differentiation, and apoptosis, and three major MAPK subfamilies have been identified, ERK 1/2, p38, and JNK [
28‐
30]. Increased expression of p-ERK has been found in many cancers, which can induce cancer cell proliferation and cancer progression [
41]. However, many studies have shown that increased p-ERK could promote the apoptosis process in SH-SY5Y cells [
42‐
44]. These controversial results may be attributed to the differences in cell lines [
45]. Furthermore, p38 and JNK can be phosphorylated in rotenone-induced apoptosis in SH-SY5Y cells [
46]. Our results indicated that PCNP over-expression could induce apoptosis by triggering phosphorylations of p38 (Thr180/Tyr182), JNK (Thr183/Tyr185), and ERK1/2 (Thr202/Tyr204) in both SH-SY5Y and SK-N-SH cells. However, PCNP knockdown could promote the growth, migration, and invasion of neuroblastoma cells by decreasing phosphorylations of p38 (Thr180/Tyr182), JNK (Thr183/Tyr185), and ERK1/2 (Thr202/Tyr204). These results together suggest that PCNP can regulate the growth process of human neuroblastoma cells via the MAPK signaling pathway.
The PI3K/Akt/mTOR signaling pathway plays important roles in promoting cell survival, growth, motility, and protein synthesis [
32,
47,
48]. PI3K activates the serine/threonine kinase Akt, which in turn phosphorylates and activates mTOR through a cascade of regulators [
47]. Activation of the PI3K/AKT/mTOR pathway is involved in tumor progression and reduced patient survival [
49]. It has been widely accepted that PI3K/AKT/mTOR pathway is a promising therapeutic target for the treatment of cancer [
32,
47,
50]. A recent study indicates that alectinib could suppress cell proliferation and induce apoptosis through the inhibition of PI3K/Akt/mTOR signaling in neuroblastoma cells [
51]. Moreover, afatinib exhibits anti-tumor efficacy by inducing apoptosis and blocking the PI3K/AKT/mTOR signaling in a neuroblastoma xenograft mouse model [
52]. Similarly, our results showed that PCNP over-expression significantly induced apoptosis by inhibiting phosphorylations of PI3K (Tyr458/Tyr199), AKT (Ser473), and mTOR (Ser2448), suggesting that PCNP-associated agents can be developed as anti-cancer drugs. Nevertheless, PCNP knockdown promoted the growth, migration, and invasion of neuroblastoma cells via increasing phosphorylations of PI3K (Tyr458/Tyr199), AKT (Ser473), and mTOR (Ser2448). These data reveal that PCNP can regulate the growth, migration, and invasion of human neuroblastoma cells through the PI3K/Akt/mTOR signaling pathway.
A number of studies indicate that SH-SY5Y and SK-N-SH cells have been widely adopted to establish subcutaneous xenograft models [
33‐
35]. We therefore examined the effect of PCNP on the growth of neuroblastoma xenograft tumors in BALB/c nude mice. PCNP over-expression significantly decreased the growth of neuroblastoma xenograft tumors, whereas PCNP knockdown notably promoted tumor growth. However, the tumor inhibitory rate of PCNP in SH-SY5Y cells was higher than that in SK-N-SH cells, which can be attributed to the difference of the level of GD2 ganglioside expression between SH-SY5Y and SK-N-SH cells [
53,
54]. Ki67, a nuclear non-histone protein, can be detected in proliferating cells in all stages of the cell cycle except G0 [
55]. The expression of Ki67 closely associates with the proliferation, invasiveness, and clinical outcome of a variety of malignant tumors [
56]. Ki67 is considered an important marker and has been widely used in detecting the proliferation of malignant cells [
23,
55,
56]. The results showed that the expression of Ki67 was decreased in the PCNP group and increased in the sh-PCNP group, which was in good agreement with the above findings. CD31 is an ideal biomarker for vascular endothelial cells and its density is represented by the tumor MVD [
24,
25]. The results indicated that PCNP over-expression reduced the expression of CD31, while PCNP knockdown promoted the expression of CD31 in neuroblastoma xenograft tumors, suggesting that PCNP could modulate the growth of human neuroblastoma xenograft tumors by regulating angiogenesis.