Capsaicin and dihydrocapsaicin extracted from Capsicum chinenses de-crease cell viability of neuroblastoma SH-SY5Y cells in vitro

: Neuroblastoma is an extra-cranial solid cancer that primarily affects children. Aggressive neuroblastoma tumors typically demonstrate resistance to conventional chemotherapeutic and radiotherapeutic regimens. Interestingly, the use of dietary supplements in the control of cancers has gained ascendance in recent scientific investigations. Capsaicin and dihydrocapsaicin are bioactive components of Capsicum chinenses fruit. Qutenza (a high-dose capsaicin patch) is used in the management of neuropathic pain from postherpetic neuralgia and HIV-associated neuropathy. Research on the potency of capsaicin as an anticancer agent has been demonstrated on several cancer cell lines and in in vivo models. The possibility of conventional cancer therapies having long-term developmental and other side effects on pediatric patients invokes the need to search for other less toxic agents against neuroblastoma. In this study, we tested if Capsicum chinenses fruit extract has therapeutic potential against neuroblastoma. To carry out this study, capsaicin and dihydrocapsaicin extract were made from Capsicum chinenses red fruits via hexane extraction method. Then, a range of concentrations (1pg/mL – 100 mg/mL) of the extract was administered to cultured SH-SY5Y neuroblastoma cells and their viabilities assessed. The potency of capsaicin in destroying neuroblastoma cells indicated that it might act via multiple routes, hence we screen for possible receptors in and on neuroblastoma cells that might interact with capsaicin using molecular docking techniques. Our findings showed that capsaicin and dihydrocapsaicin extracted from Capsicum chinenses reduced neuroblastoma cell viability in a concentration-dependent man-ner with an IC 50 of 69.75 µg/mL. Our in-silico analysis determined that capsaicin might potentially bind to other receptors on the surface of neuroblastoma cells. We demonstrated a stronger binding affinity of capsaicin to human D4 Dopamine receptor (DRD4) than to the known vanilloid receptor TRPV1 using molecular docking. In conclusion, these results illustrated that Capsicum chinenses extract containing capsaicin and dihydrocapsaicin is effective in reducing viability of neuroblastoma cells in vitro and may serve as a naturally derived treatment source for this pediatric cancer, secondly, capsaicin may have multiple targets, and its strong binding to human D4 Dopamine receptors may point to different pathways by which capsaicin exerts its cancer killing effects. study the interaction between capsaicin and all receptors at the and starting point for in vitro binding kinetic studies, activity assays be between capsaicin and these receptors.


Reagents
All chemicals (Hexane, methanol -LC-MS (≥ 99.9%), water, and ethanol absolute proof (≥ 99.5%) were all high-performance liquid chromatography (HPLC) grade from Sigma Aldrich, MO, USA. Capsaicin and dihydrocapsaicin standards were obtained from Santa Cruz Biotechnology Inc., CA, USA. The concentrations of the capsaicin and dihydrocapsaicin standards were evaluated using a stock solution of 6 mg/mL capsaicin and a stock solution of 5 mg/mL dihydrocapsaicin. The standards were dissolved completely 10:1 hexane-ethanol solution.

Extraction and Isolation
Red fruits of C. chinenses were removed from -20 º C and defrosted at room temperature. The procedure used by Abugri et al. was adopted with little modifications (2012). The Capsicum chinenses fruits of mass (10 g) were weighed into a mortal and then 10 mL hexane was added. The content was ground using a pestle to release the capsaicin and dihydrocapsaicin into the solvent. Samples were collected into a large pyrex culture test tube and vortexed for three min and incubated at 30 °C for 20 min with automated shaking at 100 rpm using a clinical bench top centrifuge (IEC Centra, CL2, International Equipment Company, Needham Heights, MA, USA). Extracts were cooled at room temperature for five min, filtered into a pre-weighed amber bottle, and then dried in an incubator at 37 º C (Fisher Scientific, Model 146E, USA). Finally, the dry weight was measured.

Validation and quantification of capsaicin in extract using HPLC/HPTLC
To identify the presence of capsaicin and dihydrocapsaicin in the extract, samples were resuspended into solution using 1mL of Hexane-ethanol and tested using high performance thin layer chromatography (HPTLC) as a qualitative validation (Figures 1-4). High performance thin layer chromatography (HPTLC) analysis of the extract was performed using a developing chamber (29 cm x 24.5 cm). The mobile phase constituted a 1:1 ratio of ethyl acetate-hexane (total volume was 20 mL). The mobile solvent was poured into the chamber and allowed for 20-30 min total time to saturate under a closed taped lid in a fume hood. A silica gel TLC DC-glasplatten-Kieselgel with fluorescence indicator at 254 nm (size = 2.25 μm, layer thickness = 0.25 nm medium pore diameter = 60 A, 10 x 20 cm glass plate) was used to run the sample. A 1 cm height was chosen as the reference point for spotting of compounds, and a straight line was marked across the plate. Samples were spotted in 1 cm interval gaps with that of capsaicin and dihydrocapsaicin standards on the same fluorescent TLC plate. Standards consisted of capsaicin and dihydrocapsaicin were obtained from Santa Cruz biotechnology Inc. CA, USA. The plates were then placed into the chamber and allowed to sit for approximately 16 min for total run time for complete separation of compounds. After development, retention factors were calculated for the pure capsaicin and dihydrocapsaicin in the C. chinenses fruits. Bands that were similar in the plate were further verified using high performance liquid chromatography (HPLC) to confirm the presence of capsaicin and dihydrocapsaicin. Extracts were analyzed concurrently with external standards in the same sequential run. The samples were analyzed using Agilent 1100 series high performance liquid chromatography (HPLC) equipped with a diode array detector (DAD) coupled with UV -Visible spectrometer. The mobile phases used were 95% methanol and 5% HPLC water at a pH of 3.0, all HPLC grade. The injection volume and pressure was set up to 20 µL/mL and maximum pressure of 400bar. Compounds were detected at UV wavelengths of 254 and 280 nm with reference wavelength of 530 nm. Identification and quantification were done using external standards by comparison of the retention time of samples to standards using Agilent ChemStation software version B.03.01.
2.5. Cell culture The neuroblastoma SH-SY5Y cells were purchased from Sigma, with a passage number of 24. Neuroblastoma cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) with 15% fetal bovine serum (FBS). SH-SY5Y neuroblastoma cells were cultured in T-25 flasks and maintained at 37 ºC with 5% CO2. Cells were allowed to grow to 100% confluency. Next, cells were trypsinized and transferred onto six-well plates for microscopy analysis. Another subset of neuroblastoma cells were utilized for the trypan blue cell viability assay and were cultured in 60 mm petri dishes. Once wells or petri dishes were 70% confluent with SH-SY5Y cells, they were utilized for testing with different concentrations of Capsicum chinenses extract. The cell viability test was conducted in triplicate.
2.6. Extract administration A 10 mg/mL concentration of the extract was initially prepared in 0.1% dimethyl sulfoxide (DMSO) as a stock solution. From this stock solution, 1 pg/mL, 1 ng/mL, 10 ng/mL, 1 μg/mL, 10 μg/mL, 100 μg/mL, 1 mg/mL, 10 mg/mL, and 100 mg/mL concentrations were prepared by adding the proper volume of 0.1% DMEM. Control preparations were prepared by using equivalent volumes of the DMSO mixed with DMEM. Twenty microliters of each extract concentration or control were administered to each well or petri dish. Photomicrographs and cell viability measures were obtained three hours after treatment administration.
2.7. Microscopy Differential interference contrast (DIC) microscopy was performed, and images of neuroblastoma cells were captured. The photomicrographs were taken and digitized using an Olympus 1X2-UCB microscope and the 7.5 version of Metamorph software. Each plate of cells was viewed under the microscope at 100X magnification using the DIC setting. Next, three separate photomicrographs were taken from every well in different fields and stored as a TIFF file. All cell treatments and image capturing were performed in parallel.

Trypan blue assay
To measure cell viability, a trypan blue assay was performed. Briefly, wells were trypsinized to create a cell suspension. Next, 0.1 mL of 0.4% trypan blue stain was added and mixed thoroughly with 0.5 mL of SH-SY5Y cells and then allowed to stand for 5 min at 25°C. Next, ten microliters of the resulting mixture were used to fill a hemocytometer (LW Scientific Hemacytometer Neubauer Bright Line, Double-Counting Chamber) for cell counting. All viability evaluations were conducted under the microscope at magnification of 100X.

Molecular Docking of capsaicin to receptors
The crystal structure of squirrel TRPV1 (PDB ID: 7lqz), human TRPV2 (PDB ID: 2f37), human prohibitin 2 (PDB ID: 6iqe), human dopamine receptor DRD1-Gs signaling complex (DRD1) (PDB ID: 7ckz), human dopamine D3 receptor (DRD3) (PDB ID: 3pbl), and human D4 Dopamine receptor (DRD4) (PDB ID: 5wiv) were extracted from the RCSB website (https://www.rcsb.org/) for the docking analysis. These receptors were used in the virtual screening process against capsaicin molecule (Pubchem CID: 1548943) using PyRx (an open-source software for performing virtual screening that combines AutoDock Vina, AutoDock 4.2, Mayavi, Open Babel etc.) ( 41 , 42 , 43 ). The receptor and the capsaicin molecule were prepared using AutoDock Vina wizard, and Biovia Discovery Studio software (version; 21.1.0.278). Both the receptors and the capsaicin structures were minimized and converted to a pdbqt format. In the case of the receptors, their bond orders were assigned, and charged hydrogen atoms added to the proteins. The structures minimizations were carried out using the AutoDock Vina wizard. The receptor grid box was generated in PyRx using the build-in Vina Wizard module, the dimensions of the grid boxes were maximized to covered entire molecule in each case. The docking process were carried out using the Auto-Dock wizard in-built in PyRx program with an exhaustiveness of 8. The best receptor-capsaicin complex that showed the highest relative free binding energy released was saved as a pdb file and exported into Biovia Discovery Studio software for specific atomic interaction analysis between the capsaicin and the receptors.

Data Analysis
Data obtained from trypan blue assays are displayed as mean ± S.E.M. An analysis of variance (ANOVA) was utilized for assessing statistical significance of the different treatment group. A Tukey's multiple comparison posthoc test was performed if warranted. The alpha value was set at < 0.05. Statistical comparisons and graphs of viability assays were prepared using GraphPad Prism version 6.0.

Phytochemical analysis of C. chinenses fruits by HPTLC/ HPLC
Comparison of band heights for identification of capsaicin and dihydrocapsaicin from the extract were done based on matching extracts' retention values with external standards retention values using HPTLC. The retention factor (RF) values for the pure capsaicin, pure dihydrocapsaicin, and the C. chinenses extract were calculated to be 0.59, 0.61 and 0.57, respectively. To further confirm for the presence of capsaicin and dihydrocapsaicin we ran a portion of the air-dried extract on a high-performance liquid chromatography apparatus equipped with a diode array detector. The verification of dihydrocapsaicin in C. chinenses extract was done by overlaying the chromatograms of both standard compounds with that of the extract. These chromatograms were obtained by running the HPLC at 280 nm and 254 nm. At 280 nm, the HPLC detected pure dihydrocapsaicin (blue line for dihydrocapsaicin standard in Figure 4) at a retention time of 4.45 min. Running the extract samples at 280 nm revealed a retention time of 4.40 min (green line representing sample from extract in Figure 4), indicating the presence of dihydrocapsaicin. The extract sample (green line) also contained other peaks at retention times 5.75 min and 6.25 min.

Effects of C. chinenses extract containing capsaicin and DHC on neuroblastoma cell morphology
We tested whether capsaicin and dihydrocapsaicin extracted from the C. chinenses would affect the structure of neuroblastoma cells. Immediately after administration, the cells treated with the extract were not visually distinct from cells treated with vehicle control. However, within 24 hours, cells administered the capsaicin and dihydrocapsaicin extract had differential changes in morphology ( Figure 5). Cells treated with vehicle control exhibited a cell confluency of approximately 95% with no clear disruption of cellular morphology. All these cells were well adhered to the culture plate. Cells treated with either pure capsaicin (1mg/mL) or the extract (1 mg/mL) had 0% confluency measurements within 24 hours. Moreover, these cells were rounded and had no extensions (compared to vehicle controls; see Figure 5). After washing with media to remove floating dead cells, we confirmed that the extract and standard capsaicin-or dihydrocapsaicin-treated samples contained no living cells.

Response of increasing concentrations of standard capsaicin, standard dihydrocapsaicin and C. capsicum extract on neuroblastoma cell viability
After detecting that the C. chinenses extract killed all cells within 24 hours, we focused on determining the effects of the extract, standard capsaicin and standard dihydrocapsaicin on a shorter time scale (3 hours) and at various concentrations. The SH-SY5Y cells treated with vehicle control were confluent, spread, fibroblastic in appearance and contained neurite extensions associated with healthy neuroblastoma cells. Cells treated with 1 ng/mL and 1 µg/mL of the extract had similar cellular morphologies to those given vehicle. Cytopathology was evident however in cells that were administered with greater than 100 µg/mL of the C. chinenses extract (Figure 6).  As shown in Figure 7, the mean viability of cells post extract administration at different concentrations was calculated using the trypan blue assay. The percentages of viable cells were the following: 93.37% (vehicle control), 94.03% (1 pg/mL), 93.82% (1 ng/mL), 92.12% (10 ng/mL), 89.06% (100 ng/mL), 93.91% (1 µg/mL), 90.81% (10 µg/mL), 60.42% (100 µg/mL), 32.32% (1 mg/mL), 1.23% (10 mg/mL), and 0% (100 mg/mL). The results demonstrated significant decreases in cell viability among the control treatment and the 100 µg/mL, 1 mg/mL, 10 mg/mL, and 100 mg/mL concentrations of the C. chinenses extract (Figure 7). Next, as shown in Figure 8, we compared cell viabilities between pure capsaicin and dihydrocapsaicin treatments with that of the C. chinenses extract. After three hours of the various treatments, cell viability was quantified via the trypan blue assay. Pure capsaicin produced the following cell viabilities: 75.37% (10 µg/mL), 64.70% (50 µg/mL), 12.43% (100 µg/mL), and 1.25% (1 mg/mL). Standard dihydrocapsaicin resulted in 73.95% (10 µg/mL), 62.45% (50 µg/mL), 15.35% (100 µg/mL) and 0.5% (1 mg/mL) cell viabilities. The extract treatment yielded the following mean viabilities: 77.92% (10 µg/mL), 67.22% (50 µg/mL), 50.31% (100 µg/mL), and 5.21% (1 mg/mL). SH-SY5Y neuroblastoma cells treated with vehicle control (DMEM) had mean viabilities all greater than 95% in all treatments (Figure 8). While there were no statistically significant differences between the standard dihydrocapsaicin and capsaicin treatments at any of the concentrations, cell viabilities for the two pure treatments and the C. chinenses extract were statistically significant from the vehicle control group at all concentrations (p<0.05). At 100 µg/mL, both pure standards reduced neuroblastoma cell viability at a greater degree than the extract (p<0.05). There were no significant differences among cell viabilities of cells treated with the pure standards or the extract at 1 mg/mL. As shown in

Molecular docking of capsaicin to receptors
The chemical structure of capsaicin is shown in Figure 9. Capsaicin has a molecular weight of 305.4 Da, an XLogP3_AA of 3.6, a rotatable bond count of 9, hydrogen bond acceptor count of 3, hydrogen bond donor count of 2, heavy atom count of 22, and a topological surface of 58.6 Å².
Capsaicin-receptor interactions were studied in this work using docking simulations similar to works carried out by Wu et al (Wu et al., 2021 44 ). The binding sites of capsaicin in these receptors were identified after blind docking process. The conformation that showed the strongest binding energy in each receptor was chosen for further ligand-receptor interaction analysis. As shown in Figures 10, 11, 12, 13, 14 and 15 as well as in Table 2, the 3D and 2D structural depiction of the strongest binding conformation of capsaicin to the respective receptors studied are shown. Whereas Figure 10 shows the interaction between capsaicin and the TRPV1 receptor which produced a net negative enthalpy of -6.3 kcal/mol, the TRPV2 receptor capsaicin interaction produced a comparatively less strong affinity to capsaicin (-5.9 kcal/mol; Figure 11). Prohibitin 2 and capsaicin interaction resulted in the release of -4.8 kcal/mol of free binding energy (Figure 12). DRD1 and DRD3 interaction with capsaicin recorded -6.6 and -6.0 kcal/mol respectively (Figure 13 and 14 respectively). The interaction between capsaicin and DRD4 was the strongest (-8.3 kcal/mol). Capsaicin formed three strong hydrogen bonds with DRD4 by binding to SER196, ASP115 and VAL193 of DRD4. Hydrophobic Pi-sigma bonding was registered between VAL116 and capsaicin, whereas pi-pi T-shaped hydrophobic interaction was recorded between amino acid PH410 and capsaicin. Other hydrophobic interactions depicted occurred between capsaicin and the amino acids VAL87, LEU111, VAL193, PHE91, PHE411 and CYS119 ( Table 2) Rohm et al., 2013 demonstrated that nonivamide (an analog of capsaicin) at sub micro molar concentrations stimulated the Ca 2+ -dependent release of serotonin and dopamine in SH-SY5Y cells. They showed that nonivamide-treated SH-SY5Y cells upregulated the expression of dopamine D1 and D2 receptors. As shown in Figure 13, 14 and 15, we demonstrate by molecular docking analysis the binding of capsaicin to dopamine D1, D3, and D4 receptors (labeled as (DRD1, DRD3 and DRD4 respectively). Also in Table 2, we showed the specific residues involved in the binding of capsaicin to these receptors. They revealed that that capsaicin induced serotonin and dopamine release (Rohm et al., 2013 47 ).

Capsaicin interactions with its receptors
Capsaicin inhibits the degeneration of dopamine neurons by blocking glial activation and oxidative stress (Chung et al., 2017 48 ). The transient receptor potential receptor vanilloid type 1 (TRPV1), is a cation channel that is highly expressed in sensory neurons (Gunthorpe et al., 2018 49 ) and in the brain and plays a crucial role in the central nervous system (Kauer et al., 2009 50 ; Starowicz et al., 2008 51 ). TRPV1 activation is showed to regulates neuroinflammation and contributes to mesencephalic dopaminergic neuronal survival by blocking oxidative stress (Park et al., 2012 52 ) and modulates neuronal function (Kauer et al., 2009 53 ). In a rat model of Parkinson's disease, nandamide was demonstrated to activate TRPV1 and modulates dopamine transmission in basal ganglia (Morgese et al., 2007 54 ). In this study, we demonstrated that capsaicin strongly binds to TRPV1 (Table 2 and Figure 10).
TRPV2 is a capsaicin receptor homologue and is highly expressed in primary sensory neurons (Tamura et  al., 2005 55 ). TRPV2 has been demonstrated experimentally to respond to high-threshold noxious heat (Julius and Basbaum, 2001 56 ). In this work via molecular docking we showed capsaicin can bind to TRPV2 (Figure 11) and hence could be a molecular target of capsaicin.
Another molecular target of capsaicin is prohibitin 2 which is localized to the inner mitochondrial membrane (Wei et al., 2017 57 ). Capsaicin binds to prohibitin 2 and translocate it from the mitochondria to the nucleus (Kuramori et al., 2009 58 ). Prohibitin 2 has been revealed to play a crucial role in the maintenance of mitochondrial morphology and in governing apoptotic processes of the cell (Zhang et al., 2020 59 ). Downregulation of prohibitin 2 shows a reduced parkin-mediated mitophagy and suppressed proliferation and migration of lung cancer cells (Zhang et al., 2020 60 ). In Figure 12, we showed prohibitin 2 bound to capsaicin at the active binding site with residues ALA196, VAL197, ALA199, ALA203, PHE189, TYR193, LYS200 and ALA203.     Table 2.

Virtual analysis of thermodynamic parameters and type of binding forces
Capsaicin interactions with the cell surface receptors such as TRPV1, TRPV2, Prohibitin 2, DRD1, DRD3 and DRD4 were analyzed via in silico method. These interactions are characterized by forces such as hydrogen bonding, hydrophobic interactions, van de Waals forces and other electrostatic interactions which ultimately determines the thermodynamics of the interaction events (Ross and Subramanian , 1981 61 ). The capsaicin solvent penetration of hydration layers possibly caused slight disordering of the solvent inaccessible regions of the proteins, subsequent short-range interactions of capsaicin with the receptors further exacerbates this process. In the final event, the net ΔG for the short-range interactions between capsaicin and these receptors produced negative enthalpy change ( Table 2), hinting at thermostable complexes between the receptors and capsaicin. As indicated in Table 2, the strengthening of hydrogen bonds in the binding pocket and hydrophobic interactions between capsaicin and these receptors led to the observed negative enthalpies. The highest net negative enthalpy was observed between capsaicin binding to DRD4 receptor (-8.3 kcal/mol). The interaction between capsaicin and its receptor (the vanilloid receptor TRPV1) produced (-6.3 kcal/mol). The 2.0 kcal/mol energy differences between these two interactions possibly indicates that capsaicin might interact with higher affinity to DRD4 compared to TRPV1 than previously understood. This new finding highlights a novel approach that could be explored as capsaicin target in drug formulation process. This study however is not substantive enough to completely inform of the interaction between capsaicin and all receptors studied in this work, hence this results only provide a hint at the direction and starting point for in vitro binding kinetic studies, activity assays etc. that can be conducted between capsaicin and these receptors.   Table 2.

Discussion
Capsaicin inhibits various oncogenic signaling pathways and have been shown to be a potential anti-cancer agent in both in vitro and in vivo models (Min et  To decipher the mechanism by which capsaicin inhibited neuroblastoma cell growth, we hypothesized that capsaicin reduces neuroblastoma viability by the induction of DNA damage, activation of oxidative stress mechanism, or by strongly binding to the TRPV1 and DRD4 receptors leading to MAP kinase activation and subsequently apoptosis. Capsaicin has been associated with DNA damage (Nagabhushan and Blide, 1985 77 ; Singh et al., 2001 78 ). In the presence of Cu (II) and molecular oxygen, capsaicin was reported to cause strand excision in DNA through an oxidative stress mechanism (Oikawa et al., 2006 79 ). For example, capsaicin has been revealed to induce apoptosis by generating reactive oxygen species and disrupting mitochondrial transmembrane potential in human colon cancer cell lines. (Yang et al., 2009 80 ). This could imply that capsaicin may also be able to induce neuroblastoma cell death either through oxidative stress or blocking transcription and translation by DNA excision. The interaction between capsaicin and its receptor (the vanilloid receptor TRPV1) as well as the interaction between capsaicin and the dopamine receptors showed comparable enthalpies, indicating the possibility that capsaicin might interact potently with other receptors of neuroblastoma cells that could potentiate the apoptotic cascades of these cells than previously understood. These new findings highlight novel approaches that could be explored as capsaicin targets in drug formulation processes. This study however is preliminary and not substantive enough to completely inform of the in-depth actual interaction between capsaicin and all receptors studied in this work, hence this work can only provide the starting point for in vitro binding kinetic studies, activity assays etc. that can be conducted between capsaicin and these receptors. While future studies are required to determine the mechanism by which the C. chinenses extract kills neuroblastoma cells, the present work indicates that the natural extract is effective in reducing neuroblastoma cell viability. Neuroblastoma's affinity to pediatric patients highlights the need to develop treatment methods that have few side effects and more importantly fewer developmental consequences. Capsaicin in its natural state is commonly used as food additives and for spices in food preparation. Thus, it could serve as an ideal candidate for nutritional medicine to prevent neuroblastoma. Moreover, coupling capsaicin and dihydrocapsaicin-containing extracts of C. chinenses with innovative medicinal delivery systems may serve well to treat neuroblastoma, a cancer primarily of the peripheral nervous system. Overall, our results point to C. chinenses extract with capsaicin and dihydrocapsaicin as a potential therapy for neuroblastoma, a pediatric cancer.

Conclusion
In conclusion, our data revealed that C. chinenses red fruits extracts are effective in limiting the growth of pediatric cancer cells (neuroblastoma) in vitro. Furthermore, the study supports that capsaicin and dihydrocapsaicin obtained from C. chinenses fruits could be a leading source of natural therapies for preventing or treating of neuroblastoma, a pediatric cancer. Future study plans includes testing the mechanism(s) of C. chinenses extract-induced inhibition of neuroblastoma cells in order to understand the antitumor effects of capsaicin. Secondly, further studies to understand the signaling pathways implicated in capsaicin induction of cell death will be undertaken, by looking at the relationship between capsaicin administration and the induction of STAT3 phosphorylation. This will help decipher if capsaicin is implicated in the STAT3 activation pathway, which is known to have potential role in the prevention and treatment of multiple myeloma and other cancers. Hopefully, the relevant molecular mechanisms underlying capsaicin induction of cell death would be fully assessed to enhance our understanding of the effects and mechanisms of capsaicin on neuroblastoma. Finally, we also hope to explore specific cellular targets of capsaicin for therapeutic application.