Aspirin inhibits proliferation and promotes differentiation of neuroblastoma cells via p21Waf1 protein up‐regulation and Rb1 pathway modulation

Abstract Several clinical and experimental studies have demonstrated that regular use of aspirin (acetylsalicylic acid, ASA) correlates with a reduced risk of cancer and that the drug exerts direct anti‐tumour effects. We have previously reported that ASA inhibits proliferation of human glioblastoma multiforme‐derived cancer stem cells. In the present study, we analysed the effects of ASA on nervous system‐derived cancer cells, using the SK‐N‐SH (N) human neuroblastoma cell line as an experimental model. ASA treatment of SK‐N‐SH (N) dramatically reduced cell proliferation and motility, and induced neuronal‐like differentiation, indicated by the appearance of the neuronal differentiation marker tyrosine hydroxylase (TH) after 5 days. ASA did not affect cell viability, but caused a time‐dependent accumulation of cells in the G0/G1 phase of the cell cycle, with a concomitant decrease in the percentage of cells in the G2 phase. These effects appear to be mediated by a COX‐independent mechanism involving an increase in p21Waf1 and underphosphorylated retinoblastoma (hypo‐pRb1) protein levels. These findings may support a potential role of ASA as adjunctive therapeutic agent in the clinical management of neuroblastoma.

that this drug can prevent cell growth and induce apoptosis [6][7][8] in different tumour models, including nervous system-derived cancers. [9][10][11] Regarding NB, some studies showed that diclofenac, a non-steroidal anti-inflammatory drug, is able to induce apoptosis and cell growth inhibition in vitro and in xenografts in vivo. 12 In addition, ciclooxygenase-2 (COX-2) participates in the development and progression of different tumours, including neuroblastoma 13,14 and COX-2 overexpression is associated with resistance to apoptosis, induction of metastases and neo-angiogenesis in NB cell lines. 15 However, the effects of NSAIDs, and specifically of ASA, on these tumours are still poorly characterized. In the present study, we analysed the effects of ASA on NB cells growth and its putative underlying molecular mechanism(s), by using as a model the SK-N-SH (N) cells, a subpopulation of human neuroblastoma SK-N-SH cell line.
Since neuroblastoma cells retain the ability to undergo neuronal differentiation in the presence of appropriate signals 16,17 and the induction of the latter is a promising NB treatment approach, 18 we also used SK-N-SH (N) cells to study the effects of ASA on neuronal differentiation.

| Separation, purification and culture conditions of SK-N-SH (N) cells
Human neuroblastoma SK-N-SH (ATCC LCG Promochem, Milan, Italy, ATCCR No HTB-11) cells were cultured in 75 cm 2 tissue culture flasks in MEM with Earle's salts (Biochrom KG), supplemented with 10% foetal bovine serum (Biochrom KG), 2 mmol/L l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, non-essential amino acids and sodium pyruvate. The cells were incubated at 37°C in a humidified atmosphere consisting of 5% CO 2 and 95% O 2 . The culture medium was changed every 2 days, and cells were allowed to grow until they reached confluence. At this time, the culture medium was replaced with PBS without Ca 2+ and Mg 2+ plus antibiotics, and the cells were incubated for 15 minutes at 37°C. The flasks were then gently shaken to separate the weakly adherent neuroblast-like subpopulation, SK-N-SH (N), from the epithelial-like SK-N-SH (E) cells monolayer. The detached SK-N-SH (N) cells were collected and re-cultured in MEM with Earle's salts containing 10% foetal bovine serum, 2 mmol/L l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, non-essential amino acids and sodium pyruvate. At confluence, cells were again purified as described earlier. This separation procedure was repeated at least 15 times, until the appearance of the cellular neuroblast-like phenotype became uniform. 19

| Drug treatment and analysis of cell growth
ASA was purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions (100 mmol/L) were prepared by dissolving the substance in ethanol (vehicle) and diluted to the desired concentrations in incubation medium. Prostaglandin E 2 (PGE 2 ) was purchased from Sigma-Aldrich. Stock solutions (5 mmol/L) were prepared by dissolving the substance in ethanol (vehicle) and diluted to the desired concentrations in incubation medium. Cells were cultured in 60 mm Petri dishes (50 000 cells/dish) and treated with 2 mmol/L ASA or vehicle (control). The drug was added to the cultures starting from the second day after plating and thereafter every 48 hours, and growth was evaluated, every 24 hours, by counting the cells in quadruplicate. In experiments with PGE 2 , the latter was added to cell cultures together with ASA. Cell viability was determined by trypan blue dye exclusion.

| LDH release assay
Lactate dehydrogenase released in the incubation media was quantified using the CytoTox-96 cytotoxicity assay kit from Promega, according to the manufacturer's instructions. Plates were incubated at room temperature for 30 min in the dark, and adsorbance was then measured at 490 nm with a microimmunoanalyzer (Labsystems Oy).

| Wound healing assay
Cells were cultured in 35 mm Petri dishes to confluence, in order to form a monolayer covering the surface of the entire plate. Wounds were created with a pipette tip as previously described 19 and washed extensively with MEM to remove cell debris. Cells were then treated with 2 mmol/L ASA or with vehicle. The open gaps were microscopically assessed over time as the cells moved in and filled the damaged area. Images were taken at 6 and 24 hours during cell migration, and the latter was quantified by measuring the distance between two defined points on either side of the gap with the program Image J.
For statistical evaluation, at least three measurements at different points were performed at each image.

| Microscopic analysis of cell death
The morphological features of apoptotic degeneration were analysed through the use of fluorescence microscopy with the nuclear dye Hoechst 33 258. 19 Cells were plated at a density of Normal and apoptotic cells were counted from three fields per dish in a fixed pattern.  Research Laboratories). The proteins were then visualized using a chemiluminescence system (ECL Millipore). The densitometric analyses of bands normalized to β-actin protein levels were performed using the ImageMasterR VDS and the Imagesystem software package (Amersham-Pharmacia Biotech).

| Statistical analysis
Data were analysed by one-way ANOVA, followed by post hoc Newman-Keul test for multiple comparisons among group means, using a PrismTM software (GraphPad), and differences were considered statistically significant if P < .05. All results are presented as the mean ± SEM of at least three different experiments performed in duplicate, unless otherwise specified.  (Table 1). These results suggest that ASA can induce a G 0 /G 1 arrest and hence delays cell cycle progression in neuroblastoma cells, exerting a cytostatic, rather than a cytotoxic effect.

| ASA inhibits the proliferation of SK-N-SH (N) cells in a COX-independent manner
Acetylsalicylic acid is a non-selective irreversible inhibitor of both COX-1 and COX-2. 21 COX-2 and PGE 2 receptors are usually expressed in neuroblastoma cells, and inhibition of COX-2-derived PGE 2 signal results in a modulation of cell growth. 22 In addition, COX-1 is constitutively expressed in SK-N-SH (N) cells. 23

| ASA reduces SK-N-SH (N) cells motility
An increase in cell motility is crucial for the metastatic potential of tumour cells; therefore, we performed a wound healing assay to investigate the effect of ASA on SK-N-SH (N) cells motility. To this purpose, a line was formed by scratching the cell monolayer with a tip ( Figure 4A). In this model at the early time-points, the gap is mainly filled by cells that move into it rather than cells that prolifer-

| ASA increases p21 Waf1 and underphosphorylated retinoblastoma (Rb1) protein levels, and decreases the expression survivin in SK-N-SH (N) cells
Since aspirin inhibits growth and causes G 0 /G 1 cell cycle arrest in SK-N-SH (N) cells, we studied its effects on the expression of the cell cycle-related proteins p21 Waf1 and p27 Kip1 that are highly expressed in these cells. 17 ASA rapidly and selectively increased the endogenous protein levels of p21 Waf1 in a time-dependent manner. p21 Waf1 accumulation might impair CDK2 and CDK4 functions, which promote G 1 -to-S transition by phosphorylating the retinoblastoma (Rb1) protein 25 ; therefore, we exposed the SK-N-SH (N) cells to ASA and analysed its effects on Rb1, by using a specific antibody, which recognizes the underphosphorylated form of this protein (hypo-pRb1). As shown in Figure 5, ASA produced a significant up-regulation of hypo-pRb1 levels after 1 day. These results suggest that Rb1 pathway may be directly involved in the G 0 /G 1 cell cycle arrest in neuroblastoma cells induced by aspirin ( Figure 6).
Interestingly, we found that ASA significantly decreased the amounts of the anti-apoptotic protein survivin in a time-dependent manner ( Figure 5). This protein, highly expressed in tumour cells, is

| D ISCUSS I ON
There is considerable evidence that ASA exhibits powerful anticancer properties. In fact, an increasing number of observational, epidemiological and clinical studies have demonstrated that prolonged, daily use of ASA correlates with a reduced risk for different types of cancer. In addition, ASA can prevent cell growth, via COXdependent and COX-independent mechanisms, in several tumour models in vitro and in vivo. [28][29][30][31] Neuroblastoma (NB), a paediatric cancer derived from primordial neural crest precursors, is the most common extracranial solid tumour of childhood. Because of its proliferative potential, resistance to apoptosis and high biological heterogeneity, which accounts for variable clinical behaviour, NB standard treatment requires a combined multimodal approach, including chemotherapy, surgery, radioand immunotherapy, as well as bone marrow transplant. 1 In some cases, the tumour may regress completely, or spontaneously differentiate, but generally, patients affected by NB have a poor prognosis and may develop resistance to conventional therapy. 1  COX-independent mechanism. This result is consistent with the effects of ASA observed in glioblastoma cancer stem cells, 11 as well as in many other tumour models. 28 We also found that ASA reduced SK-N-SH (N) cells motility, which is a key feature of a tumour propagation potential. It should be noted that the scratch test usually measures both cell replication and growth, but these phenomena may be relevant only in the latephase of experiments, whereas within the first 6 hours the observed effects are generally related to motility alone. 37 Acetylsalicylic acid did not cause cell death, but produced an accumulation of cells in the G 0 /G 1 phase of the cell cycle, with a parallel decrease of cells in G 2 phase. Time course of the effects of aspirin on SK-N-SH (N) cell cycle. Cells were incubated with vehicle (control) or with 2 mmol/L aspirin for the indicated times. Results are from two independent experiments performed in duplicate. *P < .05; **P < .01 (vs control).
TA B L E 1 Aspirin causes a G 0 /G 1 cell cycle arrest in SK-N-SH (N) cells  CKIs are grouped into two distinct families: the INK proteins,   including p15 Ink4b , p16 Ink4a , p18 Ink4c and p19 Ink4d , and the CIP/KIP proteins, which include p21 Waf1 , p27 Kip1 and p57 Kip2 . 38 Interestingly, CKIs are also potent tumour suppressor agents 38 ; p18 Ink4c , p21 Waf1 and p27 Kip1 , for instance, can induce cell cycle arrest and differentiation of many neoplastic cell lines. [39][40][41] Since p21 Waf1 and p27 Kip1 are highly expressed in SK-NSH (N) cells which, conversely, do not express p15 Ink4b , p16 Ink4a , p18 Ink4c and p19 Ink4d mRNAs, 17 we investigated the effects of ASA on the expression patterns of these proteins. We found that ASA greatly and selectively increased the p21 Waf1 levels. Downstream, we also observed an accumulation of the Rb1 tumour suppressor protein in its hypo-phosphorylated form (hypo-pRb1). Phosphorylated Rb1 predominates in rapidly proliferating cells, while the hypo-phosphorylated form blocks the progression from G 1 to S phase and directs the cell towards differentiation ( Figure 6). [42][43][44][45] Interestingly, the retinoblastoma family of proteins plays a central role not only on cell cycle regulation but has other relevant actions, that is regulation of apoptosis, cellular differentiation, preservation of genome stability and ultimately the cell fate. 46 Apart from p21 waf1 , other CKIs are generally involved in cell cycle control through the modulation of the Rb pathway. In particular, p16 Ink4a is a negative regulator of mammalian cell cycle in late G 1 phase and can induce repression of pRb1 synthesis. 47 However, in human cells, p16 Ink4a seems to be mainly associated with the progression towards the senescent phenotype, [48][49][50]   The inhibition of survivin may represent an additional advantage in neoplastic patients, since through this action aspirin might enhance the effect of cytotoxic drugs to cancer cells.
In conclusion, here we show that ASA greatly inhibits neuroblastoma cells proliferation and motility, and promotes their differentiation in vitro. These effects are mediated, at least in part, by the increase in p21 Waf1 levels, associated with an accumulation of hypo-pRb1, suggesting that ASA might be potentially useful as an adjuvant therapy in the treatment of NB patients. However, this potential does not take in account the side effects of ASA; therefore, the use of this compound as an adjuvant therapy, especially for paediatric patients, would require caution. 58 Moreover, a very recent study, while confirming that the chronic use of ASA correlates with a reduced risk for different cancers, has shown that, unexpectedly, it could also have opposite effects, in particular by increasing the risk of breast cancer. 59 In this light, further in vivo preclinical investigation, possibly involving human tumour xenograft of NB in nude mice, will be required, in order to confirm the efficacy of ASA and to consider a careful risk-benefit assessment.

CON S ENT FOR PUB LI C ATI ON
Approved by all authors.

ACK N OWLED G EM ENTS
We thank the Catholic University del Sacro Cuore of Rome and CNR staff for technical assistance.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.

AUTH O R CO NTR I B UTI O N S
GP and CC conceived the study, supervised the experiments, collected the data, conceived the paper and wrote the primary draft.  Carlo Cenciarelli https://orcid.org/0000-0001-7480-4608