Anticancer and apoptotic activity in neuroblastoma SK‐N‐SH using phospholipid extract from bone of Scomberomorus niphonius

Among various types children's health challenges, neuroblastoma is the most serious solid neoplasm forming outside the cranium. Scomberomorus niphonius is a valuable edible fish that has been widely used for a meal. In this study, we obtained phospholipid extract from the bone of S. niphonius with the supercritical CO2 extraction method and tested anticancer activity with a cell viability assay. The phospholipid showed anticancer activity on neuroblastoma SK‐N‐SH cells, and the anticancer activity was presented with an IC50 of 710.25 ± 28.31 μg/ml, but did not show a significant toxicity on HUVEC cell lines. Western blot was used to detect signaling proteins; Bak, caspase‐9, caspase‐8, caspase‐3, Bax, and IκBα were increased, whereas IKKβ and NFκB were downregulated in experimental groups compared to untreated groups. Gene expression was revealed by RT‐qPCR, and the fold ratio of Apaf‐1, cytochrome‐c, caspase‐9, caspase‐3, and Bax genes' expression was raised in treated groups, implying apoptosis. Gel electrophoresis revealed that the experimental groups had more fragmented DNA than the control group. The study shows that a phospholipid extract from S. niphonius' bone could be used as a biological origin of anticancer activity in neuroblastoma SK‐N‐SH cells.


| INTRODUCTION
Cancer is currently one of the world's most serious health problems and the top cause of mortality. The predominant etiological factors in the incidence of cancer include environmental/physical, some virus infections/biological, and diets/chemical carcinogens (Kiddane et al., 2022). Neuroblastoma is a solid extracranial tumor that has a wide range of clinical manifestations in children (Castel et al., 2007) and the third most common pediatric cancer (Swift et al., 2018). It is a neuroblastic tumor of the primordial neural crest (Riley et al., 2004). Neuroblastoma is a cancer that develops as a result of abnormal sympathetic nervous system maturation (Bowen & Chung, 2009). It is a postganglionic sympathetic nervous system embryonal cancer that mostly develops in the adrenal gland. In addition, it is the most prevalent cancer observed in children under the age of one (Stiller & Parkint, 1994). Tumors can regress on their own due to triggering of apoptosis or division, or they might be exceedingly malignant with limited cure chances (Riley et al., 2004). Neuroblastoma is known for its clinical variability, with the chance of tumor growth varied greatly depending on the patient's age and anatomic level at the time of diagnosis (Castel et al., 2007). Neuroblastoma presents an extensive range of clinical symptoms that are dependent on the location, size, and physiological characteristics of the main tumor, as well as the existence of distant metastatic illness. Abdominal distension, generalized skeletal pain or even arthritis type complaints, effects of hormone production and nonspecific findings from bone marrow involvement, such as weight loss, malaise, anemia, fever, and irritability, can be encountered (Papaioannou & McHugh, 2005). The cancers develop in sympathetic nervous system tissues, most commonly in the adrenal medulla or paraspinal ganglia, and can manifest as tumor nodules in the neck, chest, abdomen, and pelvis (Bowen & Chung, 2009). One of the most common symptoms associated with cervical neuroblastoma is a neck lump, stridor, or difficulty swallowing (Castellote et al., 1999). The diagnosis of neuroblastoma may come as early as the neonatal stage in certain cases (Castel et al., 2007). Prior to recent discoveries, there was a paucity of information on the genetic underpinnings of this illness (Bowen & Chung, 2009). This might be an underestimate if certain genetic variants are more likely to coincide with fatal birth abnormalities or if the undiscovered tumor is more likely to go into remission on its own without medical intervention (Heck et al., 2009). There is limited evidence to establish a causal relationship between maternal consumption of tobacco during pregnancy and the development of neuroblastoma (Müller-Schulte et al., 2018). It is widely recognized that drinking alcohol while pregnant causes disturbance in the normal development of the neural pathways in the brain. Early exposure to ethanol prevents neuronal cells from migrating and multiplying normally, which can lead to the death of neuronal cells (Guerri, 2002) , (Guerri, 1998). Case studies that identified the tumor as co-occurring with fetal alcohol syndrome prompted researchers to speculate about the potential of a connection between neuroblastoma and the consumption of alcohol. On the contrary, the illness has the greatest spontaneous regression rate of any malignancy (Heck et al., 2009). The condition is identified in 90% of children who have it within the first 5 years of their lives (Berthold & Hero, 2000). The majority of children who have neuroblastoma appear with a palpable abdominal mass between the ages of 1 and 5 years old, with the median age being 2 years (Park et al., 2008). The spectrum of endeavors may be useful to stratify the most appropriate prevention and management with this disease.
The discovery of novel medications to treat neuroblastoma with a high risk of recurrence has been the focus of a large body of research and the efforts of several groups (Bowen & Chung, 2009). Anti-tumor compounds work through a variety of mechanisms, including inducing apoptosis via DNA cleavage mediated by topoisomerase I or II inhibition, mitochondrial permeabilization, inhibition of key enzymes involved in signal transduction or cellular metabolism, and inhibition of tumor-induced angiogenesis (Demain & Vaishnav, 2011). The conventional treatments for cancer, such as surgery, radiation, and chemotherapy, all have undesirable side effects. As a consequence of this, there is a growing need for natural substances that have fewer adverse effects, are less toxic, and have a higher degree of effectiveness in the prevention and treatment of cancer (Suphachai, 2014). Almost 50% of the most popular medications are natural or derived from natural ingredients (Brown, 2008). Marine sources are used in functional foods and medicines, accomplish a wide range of biological functions, and provide a significant contribution to items that promote health. However, the ocean is home to a diverse array of living forms teeming with natural substances having potential therapeutic use. Because marine exploration is still a relatively recent phenomena, the marine life that exists is still a largely untapped area of possibility (Demain & Vaishnav, 2011). They have an enticing nutritional content and numerous classes of bioactive compounds, such as alkaloids, steroids, polyphenols, polysaccharides, fatty acids, micronutrients, minerals, vitamins, and a great deal of other types of bioactive compounds. Japanese Spanish mackerel, Scomberomorus niphonius, is one of the marine species that includes numerous compounds that are ideal for a healthy diet. Some examples of these compounds are high-quality protein and polyunsaturated fatty acids (docosahexaenoic, eicosapentaenoic, and linolenic acid), essential amino acids, vitamins, and minerals (Wu et al., 2021). Marine phospholipid carrying mainly omega-3 fatty acid. Phospholipids are amphiphilic lipids. Furthermore, phospholipids are essential component for the synthesis of unsaturated fatty acids, which are required as precursors for the synthesis of eicosanoids. In vitro, the expansion of neurites drives up the need for phospholipids, and the activity of nerve growth factor stimulates the manufacture of phosphatidylcholine (PC) in response to this demand (Gimenez et al., 2011). From the vast number of investigations, it has become obvious that dietary phospholipids have a good influence on a number of disorders, presumably without serious adverse effects. Moreover, they have been proven to lessen the adverse effects of certain medications. Due to their ability to transfer fatty acid residues to cells implicated in many illnesses, it is possible to explain both of these findings, for example, immune or cancer cells. Researchers have investigated the effectiveness of phospholipids in inhibiting the growth of cancer in several ways. Numerous studies have shown that phospholipids possess qualities that are both anti-cancer and anti-metastatic. By blocking the phosphorylation of IκBα and NFκB, omega-6 PL inhibit the activation of NFκB in SH-SY5Y neuroblastoma cells. Dilinoleoylphosphatidylcholine (DLPC) totally inhibits TNF-αinduced IκBα phosphorylation. It is anticipated that a decrease in IκBα phosphorylation will inhibit NFκB phosphorylation. Other research has shown that DLPC may prevent NFκB from being activated by LPS and TNF in HepG2 and Caco-2 cell lines. As a result, DLPC significantly inhibits NFκB in many tissues (Pandey et al., 2009). In addition, the treatment of marine PLs (PC derived from squid meal and starfish, both of which include n-3 FAs) inhibited the development of in vitro colon cancer that was chemically produced. Rats that were given PC derived from squid (which is rich in DHA) and PC derived from starfish (which is rich in EPA) were found to have a significantly increased apoptosis rate. This was explained by an increased lipid peroxidation rate, which was the result of structural and functional changes in the cellular membrane. This was likewise shown to be the case after mixing DHA or EPA with sodium butyrate (NaBt) in either free fatty acid form or bound to PLs. Furthermore, they examined the effect of additional marine PLs (PC along with PS derived from starfish) as a chemotherapeutic treatment on Caco-2 tumors in vivo, and their results showed a suppression of growth as well as a promotion of cell differentiation (Fukunaga et al., 2008).
Various marine species and their extracts, notably S. niphonius and phospholipid, have been the subject of countless scientific study thus far. This research aims to ascertain the neuroblastoma SK-N-SH cells' in vitro apoptotic pathway and anticancer activity in response to the phospholipid extract of S. niphonius.

| Removal of oil by SC-CO 2 extraction
For oil extraction, a supercritical fluid extraction method was applied. In particular, 100 g of freeze-dried, crushed fish bone was added to a 200 ml stainless steel extractor. Using SC-CO 2 extraction at 45°C and 25 MPa pressure for 3 h, the oil was extracted from the sample. Completion of oil extraction was ensured by repeated weighing of the extracted oil for 3 consecutive times at 5 min intervals which showed consistent weight. Around 35.15 ± 1.14% oil was recovered at this stage. Then, the residues were left in the extractor so that SNPL could be extracted using SC-CO 2 and ethanol as a co-solvent. Organic solvent was utilized to extract SNPL from the de-oiled residue.
2.1.2 | Extraction of phospholipid by SC-CO 2 with ethanol as co-solvent Phospholipid was extracted from the residue left behind after de-oiling fish bones using SC-CO 2 with ethanol as the co-solvent. This process was carried out at 45°C and between 22.5 MPa and 30 MPa, and varied CO 2 densities were maintained throughout. Several studies have proposed the temperature and pressure required for phospholipid extraction. The flow rate of carbon dioxide was maintained at 27 g per min during the whole 2.5 h that it took to complete the extraction process. With the help of a solvent delivery pump, the flow rate of the cosolvent ethanol was pre-optimized and kept at 3 ml/min throughout the process (Model: M930, Young Lin Co., Korea). After using a rotary evaporator heated to 45°C to remove the solvent from the ethanol-phospholipid combination, the SNPL that was extracted and kept at a temperature of −20°C.

| Solution preparation
After dissolving 30 mg of SNPL in 150 μl of DMSO, the final concentration of the SNPL stock was brought up to 200 mg/ml. To treat SK-N-SH and HUVEC cells, stock was diluted in the appropriate medium and then prepared at a range of concentrations (600, 700, 800, and 900 μg/ml).

| Cell viability assay
To assess the cytotoxicity of SNPL, control and experimental group cells were sown in a 96-well plate at a density of 1 × 10 4 cells/well, and blank wells containing 100 μl of medium with no cells was incubated for 24 h at 37°C with 5% CO 2 . SK-N-SH and HUVEC experimental group cells were loaded with varying doses (600, 700, 800, and 900 μg/ ml) of the SNPL and then incubated for another 24 h. Next, media was changed and replaced with fresh media containing 10 μl EZ-Cytox solution (DoGenBio, Seoul, Korea) in blank, control and experimental group wells, also protected from light and incubated for 2 h at 37°C 5% CO 2 . These wells were likewise shielded from light and incubated at 37°C with 5% CO 2 for 2 h. Using an ELISA plate reader (Varioskan Lux, Vantaa, Finland) at 460 nm, the cell viability was computed. Cell viability calculated as [(experimental -blank) ÷ (control -blank)] × 100. This experiment was conducted through repetition.

| Protein extraction and Western blot
A total of 20 ml of MEM constituted SK-N-SH cells (adjusted: 1 × 10 5 cells/ml) was prepared; then, 5 ml was put into the 100 mm cell culture plate and incubated at 37°C with 5% CO 2 for 24 h. The media were subsequently switched, and the control group received no treatment, while the experiment group received 600, 700, and 800 μg/ ml of SNPL and was incubated at 37°C with 5% CO 2 for 12 h. After 12 h, the cells were washed and scraped with cold PBS buffer and then transfer into 15 ml tubes, followed by 5 min of centrifugation at 390 g. By adding 35 μl lysis buffer (Cat. No. 17081;iNtRON BIOTECHNOLOGY, Korea) to the centrifuged cells, the cells are lysed. After 10 min of incubation on ice, lysates were obtained and cleared by centrifuging at 18700 g for 20 min at 4°C. Using a 2 mg bovine albumin standard (Thermo Scientific, Rockford, IL, USA) and Bradford reagent (Biosesang, Gyeonggi-do, Korea) for an ELISA microplate reader (Varioskan Lux, Vantaa, Finland) at 595 nm, protein concentration was determined. 12% SDS-PAGE was used to separate aliquots of whole cell lysates or cytosolic fractions, which were subsequently transferred to nitrocellulose membranes. PBST (PBS buffer with 0.5% Tween-20; Sigma-Aldrich, St. Louis, MO, USA) with 5% skim milk (BioShop, Burlington, Canada) was used to block the membranes. Following the blocking of any non-specific sites, the membranes were probed with primary antibodies (Cell Signaling Technology, Danvers, MA, USA), washed three times in PBST, and then subjected to an incubation period of 1 h with horseradish peroxidaseconjugated anti-rabbit IgG (Cell Signaling Technology, Danvers, MA, USA) and anti-mouse IgG as secondary antibodies (Cell Signaling Technology, Danvers, MA, USA). The membranes were then rinsed with PBST and observed using an improved chemiluminescent detection solution (Abfrontier, Lot.QJN28, Seoul, Korea) and apparatus (Thermo Fisher Scientific, iBrightCL1000, Waltham, MA, USA). Adobe Photoshop CS6 was used to conduct the measurement of the band density.

| RNA extraction
After preparing 15 ml of MEM containing constituted HeLa cells suspension (with an adjusted concentration of 1 × 10 5 cells/ml), a volume of 5 ml was plated onto each 100 mm cell culture dish, and the dishes were placed in an incubator at 37°C containing 5% CO 2 for 24 h. The medium was then switched out, the control group did not get any of the extract, and the experimental groups were given 700 and 800 μg/ml of SNPL before being incubated at 37°C with 5% CO 2 for 12 h. After a period of 12 h, the media was discarded. Both the control and experimental cells were rinsed with 1 ml of cold PBS, scraped, and collected into 1.5 ml Eppendorf tubes. 10 min were spent centrifuging at 8000 RPM while the temperature was maintained at 4°C. For extraction of RNA, the RNeasy® Mini Kit (50; Qiagen, Hilden, Germany) was used according to the manufacturer's protocol.

| Reverse transcriptase PCR
cDNA was synthesized using extracted RNA, a PCR machine (SampliAmp Thermal cycler, Singapore) and a SuPrimeScript RT Premix (2X with oligo dT; GeNet Bio, Global Gene Network, Daejeon, Korea) with the appropriate amount of RNA and RNase-free water. PCR condition: (50°C for 60 min, then 70°C for 10 min) 1-cycle. Eventually maintained at 4°C.

| DNA fragmentation
Fifteen milliliter of MEM containing a suspension of SK-N-SH cells (1 × 10 5 cells/ml) was made. Then, 5 ml of the suspension was put on each 100 mm culture dish and left to grow in the incubator for 24 h. The medium was then changed, and the control and experimental groups containing 700 and 800 μg/ml SNPL were incubated for 48 h. Kits (AccuPrep® Genomic DNA Extraction Kit; Bioneer, Korea) were used to extract genomic DNA from the control and experimental groups. An imaging device (Thermo Fisher Scientific, iBrightCL1000, USA) is used to assess for DNA fragmentation after gel electrophoresis.

| Statistical analysis
The presentation and display of all data is done as Mean ± SE. Microsoft Excel was used for the statistical analysis, which included a comparison of each treatment group's mean value to that of the control group using an ANOVA and a t-test. The p-value was utilized to determine statistical significance.

| RESULTS
Cell viability was done on SK-N-SH and HUVEC cell lines in vitro; results obtained and expressed as 50% inhibitory concentration (IC 50 ) of SNPL for SK-N-SH were 710.25 ± 28.31 μg/ml (Figure 1) when compared with control group and did not show a significant sign of toxicity on HUVEC cells (Figure 2). Additionally, DMSO did not show significant toxicity on SK-N-SH (Figure 3). Figure 4 depicts a Western blot analysis of the signaling protein expression variations responsible for the difference in proliferation between the treated and control groups, Bak, Bax, caspase-3, caspase-8, caspase-9, and IκBα were upregulated; IKKβ and NFκB were downregulated, related to dose-dependent manner. RT-qPCR ( Figure 5) was also used to look at the degree to which the genes that cause apoptosis, or cell death, are expressed, and the fold change ratio of genes expression for control, 700 and 800 μg/ml treated SK-N-SH cells was 1.01 ± 0.11, 4.82 ± 0.45, and 18.44 ± 6.41 for caspase-3; 1.03 ± 0.19, 5.38 ± 0.61, and 24.02 ± 6.84 for Apaf-1; 1.03 ± 0.16, 5.29 ± 0.91, 22.52 ± 10.6 for cytochrome-c; 1.03 ± 0.16, 6.43 ± 0.88, and 35.54 ± 7.14 for caspase-9; 1.30 ± 0.68, 6.13 ± 1.73, and 32.02 ± 12.88 for Bax, respectively. Apoptotic cell death may be identified by a number of telltale characteristics, one of which is the fragmentation of nuclear DNA into nucleosomal pieces. Gel electrophoresis revealed that the experimental groups treated with 700 and 800 μg/ml had more fragmented DNA than the control group ( Figure 6).

| DISCUSSION
This study has been conducted with the intention of assisting and serving mankind in the process of finding an acceptable and efficient therapeutic source for cancer. This research focuses on employing phospholipid extract from the bone of S. niphonius as a natural substance to treat and cure neuroblastoma/nerve cancer. Neuroblastoma is a kind of cancer that affects youngsters and is both invasive and life-threatening. It consists of neuroblasts, which are immature, undifferentiated, small, round-shaped sympathetic cells, with little cytoplasm, dark nuclei and small indistinct nucleoli (Papaioannou & McHugh, 2005). The presence of typical histopathologic features, in addition to elevated urine levels of one of the catecholamines, is required to make a diagnosis of neuroblastoma (Park et al., 2008). It has been shown that extracts from natural sources exhibit a wide variety of biological activities, one of which is the ability to fight cancer, and research reports on this topic are also growing. The phospholipid extract derived from the bone of S. niphonius own anticancer activity on SK-N-SH. Anticancer activity of the phospholipid extract on SK-N-SH cell was observed by causing death of 50% of the cells at 710.25 ± 28.31 μg/ml concentration amounts in vitro. The cytotoxic and antiproliferative effects of the phospholipid on SK-N-SH cells were shown by this finding. Apoptosis is a sort of planned cell death that may be produced by either activation of death receptors, which is referred to as the extrinsic apoptotic pathway, or by intrinsic apoptosis, which occurs naturally inside the cell (Shakeri et al., 2017). The molecular mechanisms that lead to programmed cell death in mammals include regulation of apoptotic signals by Bcl-2 family of proteins and initiator caspases, cytochrome-c release, downstream caspase activation, chromatin condensation, DNA fragmentation, and phagocytosis of dead cells by scavenger cells (Zhang & Xu, 2000). The Bcl-2 family proteins, which include both pro-and anti-apoptotic members, are important players in apoptosis; interactions between them may control mitochondrial integrity and play a crucial role in controlling the release of cytochrome-c from mitochondria . The intracellular Bcl-2 family of molecules is crucial for transmitting the apoptotic signal. Early in neural development, apoptosis-suppressing genes like Bcl-2 and Bclx are abundantly expressed. In the majority of neuroblastoma cell lines and actual tumors, the level of Bcl-2 expression is negatively correlated with the proportion of cells undergoing apoptosis (Li & Nakagawara, 2013). The F I G U R E 1 Cell viability (%) results of SK-N-SH cells; control group (C = 100% viability) was received no treatment, and various concentrations were administered to the experimental groups. (600, 700, 800, and 900 μg/ml) of SNPL. IC 50 of SNPL for SK-N-SH was 710.25 ± 28.31 μg/ml. The data are given as mean ± SE. Significant mean difference (*p < .05) from control. outer mitochondrial membrane's holes are considered to be created by the homo-oligomerization of Bax and Bak (Bratton & Salvesen, 2010). The release of cytochrome-c results from the oligomerization of Bax on mitochondria. Apoptosis is brought on by a variety of intracellular stresses that cause the release of cytochrome-c from the mitochondria into the cytoplasm. In the intrinsic route, cytochrome-c that has been liberated from the mitochondria interacts with the adaptor protein Apaf-1 to start the caspase-9 activation process that leads to the activation of caspase-3 via the apoptosome. Apaf-1 is a crucial protein in the intrinsic mechanism of apoptosis, also known as the mitochondrial pathway. It oligomerizes in reaction to the release of cytochrome-c and forms a huge complex that is known as the apoptosome. The apoptosome recruits and activates procaspase-9, a mitochondrial pathway initiator caspase, which results in the processing of caspase-3 downstream. Caspase-3 is consequently regarded to be an important marker of apoptotic cells. An adjustment to the Bcl-2 family, such as an uptick in pro-apoptotic protein Bax expression and a downtick in anti-apoptotic protein Bcl-2 expression, coincides with an increase in caspase-3 activation . The expression of apoptotic genes, Apaf-1, Bax, caspase-3, caspase-9, and cytochrome-c F I G U R E 4 Results of the Western blot analysis of the protein signals produced by SK-N-SH cells in the control (C) and test groups incubated with varying amounts (600, 700, and 800 μg/ml) of SNPL for 12 h.  F I G U R E 6 After being exposed to SNPL (700 and 800 g/ml) for 24 h, the genomic DNA from SK-N-SH cells from the control (C) and experimental groups was extracted, and the 1.5% agarose gel with 50 V electrophoresis was used to separate the DNA for 30 min.
up-regulation was observed after treating SK-N-SH cells with SNPL. The sensitivity of cells to apoptosis can be modulated by regulating the gene expression of caspases (Jin & El-Deiry, 2005). These data show that apoptosis occurred inside the cells in response to the therapy, because these genes that produce apoptosis have a unique character and capacity to generate apoptotic cell death signaling via intrinsic mitochondrial pathway.
The proteasome quickly phosphorylates, ubiquitinates, and degrades IκB, and the liberated NFκB dimer is then transported to the nucleus, where it may control particular genes (Karin & Ben-Neriah, 2000). As crucial procedures in the control of NFκB complexes, the phosphorylation and degradation of IκB have attracted a lot of interest (Kanarek & Ben-Neriah, 2012). In order to prevent NFκB from moving to the nucleus following treatment, a number of molecules maintain large levels of IκB protein in the cytoplasm (Wu et al., 2008). Some of these compounds stimulate IκB production, others inhibit IκB ubiquitination, and yet others prevent IκB breakdown (Gilmore & Herscovitch, 2006). As a result, inhibitors of any stage of the ubiquitin-proteasome decrease NFκB activation by stabilizing IκB (Yang et al., 2007). IKK protein induces the phosphorylation of IκBα at Ser 32/36 (Khaleel et al., 2020). Innate immune regulators like inflammatory cytokines and DNA transcription are both controlled by the heterodimer protein complex known as NFκB, which is essential for cell survival (Albensi, 2019). Because of its fundamental effect on cellular proliferation and differentiation in cancers, an increasing body of data suggests that activation of NFκB is related to resistance to apoptosis, production of angiogenic proteins, and carcinogenesis (Ito et al., 2015). A potential strategy for increasing tumor cells' susceptibility to TNF-induced apoptosis is inhibition of the NFκB pathway (Chakraborty et al., 2012). The downregulation of NFκB protein was seen in a dosedependent way in this investigation, and this finding revealed one of the apoptotic possibilities. The study's findings suggest that the overexpression of IκBα and the downregulation of IKKβ and NFκB were connected in a dose-dependent way.
Apoptotic features can be created in the cell in a number of ways, depending on the apoptosis inducing compounds used. One of the characteristics of apoptotic cell death and the most efficient method of cell death is the breakdown of nuclear DNA into nucleosomal fragments (Nagata, 2000). After treatment with SNPL, chromosomal DNA is cleaved into oligonucleosomal-sized fragments, indicating apoptosis in SK-N-SH. DNA fragmentation may hasten the process of cell death in response to apoptotic stimuli, which may also induce cell death without causing DNA degradation. DNA fragmentation occurred in the later stages of the apoptotic process in SK-N-SH. In addition, caspase family is primarily involved in apoptotic signaling, which leads to DNA fragmentation. Executioner caspases like caspase-3 need to be activated after caspase-8 or caspase-9 have been activated in order to carry out apoptosis (Gogada et al., 2011). The activation of certain proteases known as caspases seems to be linked to the beginning of all intracellular activities. Caspase-3 seems to be involved in how apoptosis is carried out in this process. If the membrane potential is lowered, it is anticipated that the expression of the caspase-3 protein would change in drug-treated cells. Caspase-3, which actively participates in the proteolytic cleavage of the PARP protein, is the most significant of the several caspases. PARP is involved in a number of cellular processes, including DNA repair (Jagtap & Szabó, 2005). Additionally, throughout the apoptosis process, caspases break more than 100 proteins. They are proteins for DNA replication, transcription, or translation, cytoskeletal proteins, and kinases and phosphatases (Nagata, 2000). Following the treatment, SK-N-SH may undergo apoptotic DNA fragmentation in a manner that is either caspase-dependent or caspase-independent. In this experiment, the DNA ladder gel electrophoresis technique was carried out, and the results showed that the SNPL treated groups had much more DNA fragmentation than the control group had.

| CONCLUSION
After treating SK-N-SH cells with the phospholipid extract from the bone of S. niphonius, this research found that there were discernible changes detected on cell survival, genes expression, proteins expression, and the outcomes of DNA fragmentation studies. The intrinsic apoptotic pathways, NFκB pathway and mitochondrial pathway, are both involved in the process of apoptosis, as stated in the results and discussion section. These significant results help to support the conclusion that the phospholipid extract possesses natural therapeutic potential in the treatment of neuroblastoma by inducing intrinsic suicidal apoptotic signals, inhibiting transcription factors that control DNA survival and proliferation, and fragmenting DNA, which stops the growth and metastatic potential of the cancerous cells.