Cytotoxic effect of Rotheca serrata on cancer cell lines MCF-7 and neuroblastoma SH-SY5Y.

INTRODUCTION: Rotheca serrata (Lamiaceae), a highly medicinal plant is used as an antidote for snakebite and the plant possesses medicinal properties like hepatoprotective, antitussive, antioxidant, anticancer, neuro-protective, used in rheumatoid arthritis and is also a α-glucoside inhibitor. AIM OF THE STUDY: This work aimed to study the anticancerous effect of Rotheca serrata (root and leaf) on cancer cell lines MCF-7 (breast cancer cell line) and Neuroblastoma SH-SY5Y. MATERIALS AND METHODS: This investigation was a preliminary one which supported the retrospective and safe use of plants as described in Ayurveda. Dulbecco's Modified Eagle Medium with High Glucose (DMEM-HG) for culturing MCF-7- Human Breast cancer cell line and Minimum essential Medium (MEM)+F12 medium for culturing SH-SY5Y- Homo sapiens bone marrow neuroblast were used. MTT assay measured the cell proliferation rate and conversely, when metabolic events lead to apoptosis or necrosis, the reduction in cell viability. RESULTS: The results indicated that the Methanolic extract of Rotheca serrata (root and leaf) showed high anticancer activity. Different concentrations of plant extracts (25, 50, 100, 200, 400 µg/ml) were used to study the anticancerous activity, amongst which the significant results were obtained for 400 µg/ml concentration (both root & leaf). Effective anticancer activity against MCF - 7 breast cancer cells was shown in methanoilc extracts and were expressed as IC 50 values; in root (IC 50 value = 61.8259 ± 7.428 µg/ml) and in leaf (IC 50 value = 78.1497 ± 6.316 µg/ml). The MTT assay in case of neuroblastoma (SH-SY5Y) cell lines revealed that 400 µg/ml concentration of leaf methanolic extract showed effective inhibition of cancer cells with IC 50 value 37.8462 ± 2.957 µg/ml as compared to IC 50 value of root methanolic extract which was 57.0895 ± 2.351 µg/ml. CONCLUSION: R. serrata possess anticancer activity against breast cancer cell line (MCF-7) and neuroblastoma (SH-SY 5Y) cell lines. This study may to design plant-based drugs without side effects. Dosage compensation for specific type of cancer needs to be monitored in patients with 1st stage.

Network pharmacology analysis and clinical efficacy of the traditional Chinese medicine Bu-Shen-Jian-Pi. Part 2: Modulation of hypoxia, redox status, and mitochondrial protection in a neuroblastoma cell line, SH-SY5Y.

OBJECTIVE: To examine the mitochondrial protective effects of icariin, naringenin, kaempferol, and formononetin, potentially active agents in Bu-Shen-Jian-Pi formula (BSJP) identified using network pharmacology analysis. MATERIALS AND METHODS: Mitochondrial protection activity was determined using a hypoxia-reoxygenation in vitro model based on the neuroblastoma cell line SH-SY5Y and measurements of anti-ferroptotic activity. RESULTS: Icariin, naringenin, kaempferol, and formononetin showed mitochondrial protective activity involving diverse signaling pathways. The cytoprotective effects of formononetin depended on the inhibition of ferroptosis. Hypoxia-reoxygenation stimulation induced ferroptosis in SH-SY5Y cells. DISCUSSION: Ferroptosis is a key mechanism in nervous system diseases and is associated with hypoxia-reoxygenation injury. Naringenin and kaempferol were devoid of anti-ferroptotic activity. CONCLUSION: Evidence has been obtained showing that the core components: icariin, naringenin, kaempferol, and formononetin in BSJP formula have anti-hypoxic and mitochondrial protective activity of potential clinical importance in the treatment of amyotrophic lateral sclerosis and patients with symptoms of hypoxia.

Exploring the in vitro potential of royal jelly against glioblastoma and neuroblastoma: impact on cell proliferation, apoptosis, cell cycle, and the biomolecular content.

Neuroblastoma and glioblastoma are the most commonly seen nervous system tumors, and their treatment is challenging. Relatively safe and easy acquisition of nutraceutical natural products make them suitable candidates for anticancer research. Royal jelly (RJ), a superfood, has many biological and pharmacological activities. This study was conducted to, for the first time, elucidate its anticancer efficiency, even in high doses, on neuroblastoma and glioblastoma cell lines through cell viability, apoptosis, cell cycle and biomolecular content evaluation. We performed experiments with RJ concentrations in the range of 1.25-10 mg mL-1 for 48 h. Cell viability assays revealed a notable cytotoxic effect of RJ in a concentration-dependent manner. Treatment with a high dose of RJ significantly increased the apoptotic cell population of both cell lines. Furthermore, we observed G0-G1 phase arrest in neuroblastoma cells but G2-M arrest in glioblastoma cells. All these cellular changes are closely associated with the alterations of the macromolecular makeup of the cells, such as decreased saturated lipid, protein, DNA and RNA amounts, protein conformational changes, decreased protein phosphorylation and increased protein carbonylation. These cellular changes are associated with RJ triggered-ROS formation. The clear segregation between the control and the RJ-treated groups proved these changes, obtained from the unsupervised and supervised chemometric analysis. RJ has good anticancer activity against nervous system cancers and could be safely used with current treatment strategies.

Yoga as a non-pharmacologic therapy to reduce dinutuximab-induced pain in patients with neuroblastoma.

BACKGROUND: Anti-GD2 antibodies are key components of treatment for high-risk neuroblastoma; however, they cause neuropathic pain. Yoga therapy may help reduce pain and distress associated with anti-GD2 therapy. PROCEDURE: Children 3 years of age or older with neuroblastoma participated in individualized yoga therapy while receiving the anti-GD2 antibody dinutuximab (DIN). Yoga therapy was deemed feasible if patients participated during 60% or more of DIN admissions. Patients and caregivers assessed pain/distress before and after yoga therapy with a distress thermometer (DT) and Wong-Baker FACES pain rating scale and completed questionnaires regarding satisfaction with yoga therapy. Therapy was deemed efficacious if there was a ≥1 point pain score change and reduction in distress after yoga. RESULTS: Eighteen patients were enrolled; 52 encounters (admissions for DIN) were evaluable. Ten of 18 were female, three of 18 were Hispanic, and 10/18 were White. Median age at enrollment was 5.5 years (range: 3-11). Yoga therapy was feasible in 39/52 (75%) encounters. Significant reductions in caregiver-reported pain and distress and reductions in patient-reported pain and distress after yoga therapy were reported. Twelve of 18 caregivers completed questionnaires: seven agreed/strongly agreed that yoga was valuable, and nine agreed/strongly agreed to continued participation in yoga. Thirty-four of 36 clinicians reported that they would recommend yoga therapy for other patients receiving DIN. CONCLUSIONS: Yoga therapy was feasible during DIN therapy and may be effective in reducing DIN-associated pain and distress. Future studies are needed to evaluate changes in opioid usage with the addition of yoga therapy during anti-GD2 antibody therapy.

Dauricine attenuates Oct4/sonic hedgehog co-activated stemness and induces reactive oxygen species-mediated mitochondrial apoptosis via AKT/ß-catenin signaling in human neuroblastoma and glioblastoma stem-like cells.

Neuroblastoma and glioblastoma are primary malignant tumors of the nervous system, with frequent relapse and limited clinical therapeutic drugs. The failure of their treatment is due to the tumor cells exhibiting cancer stem-like cells (CSLCs) properties. Octamer binding transcription factor 4 (Oct4) is involved in mediating CSLCs, our previous work found that Oct4-driven reprogramming of astrocytes into induced neural stem cells was potentiated with continuous sonic hedgehog (Shh) stimulation. In this study, we aimed to study the importance of Oct4 and Shh combination in the stemness properties induction of neuroblastoma and glioblastoma cells, and evaluate the anti-stemness effect of dauricine (DAU), a natural product of bis-benzylisoquinoline alkaloid. The effect of Oct4 and Shh co-activation on cancer stemness was evaluated by tumor spheres formation model and flow cytometry analysis. Then the effects of DAU on SH-SY5Y and T98-G cells were assessed by the MTT, colony formation, and tumor spheres formation model. DAU acts on Oct4 were verified using the Western blotting, MTT, and so on. Mechanistic studies were explored by siRNA transfection assay, Western blotting, and flow cytometry analysis. We identified that Shh effectively improved Oct4-mediated generation of stemness in SH-SY5Y and T98-G cells, and Oct4 and Shh co-activation promoted cell growth, the resistance of apoptosis. In addition, DAU, a natural product, was found to be able to attenuate Oct4/Shh co-activated stemness and induce cell cycle arrest and apoptosis via blocking AKT/ß-catenin signaling in neuroblastoma and glioblastoma, which contributed to the neuroblastoma and glioblastoma cells growth inhibition by DAU. In summary, our results indicated that the treatment of DAU may be served as a potential therapeutic method in neuroblastoma and glioblastoma.

New metal-organic framework coated sodium alginate for the delivery of curcumin as a sustainable drug delivery and cancer therapy system.

The utilization of biocompatible drug delivery systems with extended drug release capabilities is highly advantageous in cancer therapy, as they can mitigate adverse effects. To establish such a biocompatible system with prolonged drug release behavior, researchers developed an innovative drug carrier. In this study, a sustainable approach was employed to synthesize a new zinc-based metal-organic framework (Zn-MOF) through the reaction between synthesized Schiff base ligands and zinc ions. Comprehensive analyses, including FT-IR, XRD, SEM, BET surface area, and TGA techniques, were employed to thoroughly characterize the frameworks. Following comprehensive characterization, curcumin (CUR) was loaded onto the Zn-MOF, resulting in CUR entrapment efficiency and loading capacity of 79.23 % and 26.11 %, respectively. In vitro evaluations of CUR release from CUR@MOF exhibited controlled release patterns, releasing 78.9 % and 50.0 % of CUR at pH 5.0 and pH 7.4, respectively. To mitigate initial burst release, a coating of the biopolymer sodium alginate (SA) was applied to CUR@Zn-MOF. In vitro CUR release tests indicated that SA/CUR@Zn-MOF outperformed pristine CUR@Zn-MOF. The release of CUR conformed to the Korsmeyer-Peppas model, displaying non-Fickian diffusion. Furthermore, an in vitro cytotoxicity study clearly demonstrated the potent anti-tumor activity of the synthesized CUR@Zn-MOF attributed to its controlled release of CUR. This led to the induction of apoptotic effects and cell death across HeLa, HEK293, and SH-SY5Y cell lines. These findings strongly suggest that the developed pH-sensitive carriers hold remarkable potential as targeted vehicles for drug delivery in cancer therapy.

Steroidal Alkaloids from Food Waste of Tomato Processing Inhibit Neuroblastoma Cell Viability.

Nowadays, there is considerable attention toward the use of food waste from food processing as possible sources of compounds with health properties, such as anticancer activity. An example is tomato processing, which is responsible for generating a remarkable amount of waste (leaves, peel, seeds). Therefore, our goal was to evaluate the potential anticancer property of tomato extracts, in particular "Datterino" tomato (DT) and "Piccadilly" tomato (PT), and to study their phytochemical composition. Liquid chromatography with tandem mass spectrometry (LC/MS-MS) results showed that these extracts are rich in alkaloids, flavonoids, fatty acids, lipids, and terpenes. Furthermore, their potential anticancer activity was evaluated in vitro by MTT assay. In particular, the percentage of cell viability was assessed in olfactory ensheathing cells (OECs), a particular glial cell type of the olfactory system, and in SH-SY5Y, a neuroblastoma cell line. All extracts (aqueous and ethanolic) did not lead to any significant change in the percentage of cell viability on OECs when compared with the control. Instead, in SH-SY5Y we observed a significant decrease in the percentage of cell viability, confirming their potential anticancer activity; this was more evident for the ethanolic extracts. In conclusion, tomato leaves extracts could be regarded as a valuable source of bioactive compounds, suitable for various applications in the food, nutraceutical, and pharmaceutical fields.

Finding Integrative Medication for Neuroblastoma and Glioblastoma through Zebrafish as A Model of Organism.

As far as malignant tumors of the central nervous system are concerned, glioblastoma (GB) and neuroblastoma (NB) are the most prevalent, aggressive, and fatal forms in adult and pediatric populations, respectively. NB is the most prominent childhood extracranial compact neoplasm in pediatrics when the embryo develops from undifferentiated neural crest cells. Regarding malignant primary brain tumors, GB is the most lethal and difficult to treat. Currently, there are few effective treatments available for either condition. Research using zebrafish is relatively new in the field of animal cancer studies, and the first results show promise. In particular, integrated genomic investigations of NB and GB have revealed the potential of the zebrafish model in elucidating the roles of specific genetic changes in the development of this fatal childhood malignancy. Hence, this study examines the possibility of zebrafish as a model organism for discovering integrative medicines for these types of cancer. This model is an excellent animal model for study due to its transparency, ease of genetic modification, ethics and financial benefits, and preservation of the primary brain areas andbloodbrain barrier (BBB). This review provides recent developments in the zebrafish model of NB and GB to illustrate the benefits of using them in cancer studies as a model of the organism. This approach provides novel insights into delivering individualized treatment and enhancing outcomes for people coping with central nervous system malignancies.

Ethanolic Extract of Polygonum minus Protects Differentiated Human Neuroblastoma Cells (SH-SY5Y) against H2O2-Induced Oxidative Stress.

Neuronal models are an important tool in neuroscientific research. Hydrogen peroxide (H2O2), a major risk factor of neuronal oxidative stress, initiates a cascade of neuronal cell death. Polygonum minus Huds, known as 'kesum', is widely used in traditional medicine. P. minus has been reported to exhibit a few medicinal and pharmacological properties. The current study aimed to investigate the neuroprotective effects of P. minus ethanolic extract (PMEE) on H2O2-induced neurotoxicity in SH-SY5Y cells. LC-MS/MS revealed the presence of 28 metabolites in PMEE. Our study showed that the PMEE provided neuroprotection against H2O2-induced oxidative stress by activating the Nrf2/ARE, NF-κB/IκB and MAPK signaling pathways in PMEE pre-treated differentiated SH-SY5Y cells. Meanwhile, the acetylcholine (ACH) level was increased in the oxidative stress-induced treatment group after 4 h of exposure with H2O2. Molecular docking results with acetylcholinesterase (AChE) depicted that quercitrin showed the highest docking score at -9.5 kcal/mol followed by aloe-emodin, afzelin, and citreorosein at -9.4, -9.3 and -9.0 kcal/mol, respectively, compared to the other PMEE's identified compounds, which show lower docking scores. The results indicate that PMEE has neuroprotective effects on SH-SY5Y neuroblastoma cells in vitro. In conclusion, PMEE may aid in reducing oxidative stress as a preventative therapy for neurodegenerative diseases.

Neuroprotective Effects of Zerumbone on H2O2-Induced Oxidative Injury in Human Neuroblastoma SH-SY5Y Cells.

Oxidative stress is recognized as one of the main reasons for cellular damage and neurodegenerative diseases. Zerumbone is one of the sesquiterpenoid compounds in the essential oil of Zingiber zerumbet Smith. Zerumbone exhibits various physiological activities, such as anticancer, antioxidant, and antibacterial effects. However, studies on the neuroprotective efficacy of zerumbone and the mechanism behind it are lacking. In this study, we explored the neuroprotective efficacy of zerumbone and its mechanism in hydrogen peroxide-treated human neuroblastoma SH-SY5Y cells. H2O2 treatment (400 µM) for 24 h enhanced the generation of intracellular reactive oxygen species (ROS) compared to untreated cells. By contrast, zerumbone treatment significantly suppressed the production of intracellular ROS. Zerumbone significantly inhibited H2O2-induced nitric oxide production and expression of inflammation-related genes. Moreover, zerumbone decreased H2O2-induced mitogen-activated protein kinase (MAPK) protein expression. Various hallmarks of apoptosis in H2O2-treated cells were suppressed in a dose-dependent manner through downregulation of the Bax/Bcl-2 expression ratio by zerumbone. Since activation of AMP-activated kinase (AMPK) is a promising therapeutic target for neurodegenerative diseases, we also investigated the mammalian target of rapamycin (mTOR) as part of the autophagy mechanism in H2O2-treated SH-SY5Y cells. In this study, zerumbone upregulated the expression of Sirtuin 1 (SIRT1) and p-AMPK (which were downregulated by the H2O2 treatment) and downregulated p-mTOR. Altogether, our results propose that inhibition of apoptosis and inflammation by autophagy activation plays an important neuroprotective role in H2O2-treated SH-SY5Y cells. Zerumbone may thus be a potent dietary agent that reduces the onset and progression, as well as prevents neurodegenerative diseases.

Glycosides of Buyang Huanwu decoction inhibits pyroptosis associated with cerebral ischemia-reperfusion through Nrf2-mediated antioxidant signaling pathway both in vivo and in vitro.

BACKGROUND: Glycosides are the pharmacodynamic substances of Buyang Huanwu Decoction (BYHWD) and they exert a protective effect in the brain by inhibiting neuronal pyroptosis of cerebral ischemia-reperfusion (CIR). However, the mechanism by which glycosides regulate neuronal pyroptosis of CIR is still unclear. PURPOSE: A significant part of this study aimed to demonstrate whether glycosides have an anti-pyroptotic effect on CIR by nuclear factor erythroid 2-related factor (Nrf2)-mediated antioxidative mechanism. METHODS: Rats were used in vivo models of middle cerebral artery occlusion and reperfusion (MCAO/R). Neuroprotective effect of glycosides after Nrf2 inhibiting was observed by nerve function score, Nissl staining, Nrf2 fluorescence staining and pyroptotic proteins detection. SH-SY5Y cells were used in vitro models of oxygen-glucose deprivation/reperfusion (OGD/R). Glycosides was evaluated for their effects by measuring cell morphology, survival rate, lactate dehydrogenase (LDH) rate and expression of pyroptotic proteins. Nrf2 si-RNA 54-1 interference with lentivirus was used to create silenced Nrf2 SH-SY5Y cells (si-Nrf2-SH-SY5Y). Glycosides were evaluated on si-Con-SH-SY5Y and si-Nrf2-SH-SY5Y cells based on the expression of Nrf2 signaling pathway, pyroptotic proteins and cell damage manifestation. RESULTS: In vivo, glycosides significantly promoted the fluorescence level of nuclear Nrf2, added more Nissl bodies, reduced neurological function scores and inhibited the pyroptotic proteins level. In vitro, glycosides significantly repaired the morphological damage of cells, promoted the survival rate, reduced the LDH rate, inhibited the pyroptosis. Moreover, antioxidant activity of glycosides was enhanced via Nrf2 activation. Both Nrf2 blocking in vivo and Nrf2 silencing in vitro significantly weakened the pyroptosis inhibitory and neuroprotective effects of glycosides. CONCLUSION: These results suggested for the first time that glycosides inhibited neuronal pyroptosis by regulating the Nrf2-mediated antioxidant stress pathway, thereby exerting brain protection of CIR. As a result of this study, This study improved understanding of the pharmacodynamics and mechanism of BYHWD, as well as providing a Traditional Chinese Medicine (TCM) treatment strategy for CIR .

Repurposing Two Old Friends to Fight Cancer: Caffeine and Statins.

Statins are a class of cholesterol-lowering drugs that inhibit 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme of the mevalonate pathway. Evidence suggests that certain cancers depend on the mevalonate pathway for growth and survival, and thus blocking the mevalonate pathway with statins may offer a viable therapeutic approach for treating cancer, or at least enhance the efficacy of existing cancer drugs. In this issue of Cancer Research, Tran and colleagues showed that caffeine works jointly with FOXM1 inhibition to enhance the antitumor activity of statins in neuroblastoma cells. They found that caffeine synergizes with statins by suppressing statin-induced feedback activation of the mevalonate pathway. Here, we reflect on the potential of combining caffeine and statin drugs as a strategy for potentiating anticancer activity. See related article by Tran et al., p. 2248.

Scrophularia buergeriana Extract (Brainon) Attenuates Neuroinflammation in BV-2 Microglia Cells and Promotes Neuroprotection in SH-SY5Y Neuroblastoma Cells.

Microglia-induced neuroinflammation is one of the causative factors in cognitive dysfunction and neurodegenerative disorders. Our previous studies have revealed several benefits of Scrophularia buergeriana extract (Brainon®) in the central nervous system, but the underlying mechanism of action has not been elucidated. This study is purposed to investigate the anti-inflammatory and neuroprotective mechanisms of Brainon in the BV-2 condition SH-SY5Y model. Lipopolysaccharide (LPS)-induced BV-2 conditioned media (CM) were used to treat SH-SY5Y cells to investigate neuroprotective effects of the extract against microglial cytotoxicity. Results demonstrated that pretreated Brainon decreased nitric oxide release, the inducible nitric oxide synthase expression level, and expression of cytokines like interleukin-6, interleukin-1ß, and tumor necrosis factor-α by blocking expression of TLR4/MyD88 and NLRP3 and suppressing nuclear factor κB/AP-1 and p38/JNK signaling pathways in LPS-induced BV-2 cells. In addition, when SH-SY5Y cells were treated with CM, pretreatment with Brainon increased neuronal viability by upregulating expression of antioxidant proteins like as SODs and Gpx-1. Increased autophagy and mitophagy-associated proteins also provide important clues for SH-SY5Y to prevent apoptosis by Brainon. Brainon also modulated mTOR/AMPK signaling to clear misfolded proteins or damaged mitochondria via auto/mitophagy to protect SH-SY5Y cells from CM. Taken together, these results indicate that Brainon could reduce inflammatory mediators secreted from BV-2 cells and prevent apoptosis by increasing antioxidant and auto/mitophagy mechanisms by regulating mTOR/AMPK signaling in SH-SY5Y cells. Therefore, Brainon has the potential to be developed as a natural product in a brain health functional food to inhibit cognitive decline and neuronal death.

SNS alleviates depression-like behaviors in CUMS mice by regluating dendritic spines via NCOA4-mediated ferritinophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Depression is one of the most common mood disturbances worldwide. The Si-ni-san formula (SNS) is a famous classic Traditional Chinese Medicine (TCM) widely used to treat depression for thousands of years in clinics. However, the mechanism underlying the therapeutic effect of SNS in improving depression-like behaviors following chronic unpredictable mild stress (CUMS) remains unknown. AIM OF THE STUDY: This study aimed to investigate whether SNS alleviates depression-like behaviors in CUMS mice by regulating dendritic spines via NCOA4-mediated ferritinophagy in vitro and in vivo. STUDY DESIGN AND METHODS: In vivo, mice were exposed to CUMS for 42 days, and SNS (4.9, 9.8, 19.6 g/kg/d), fluoxetine (10 mg/kg/d), 3-methyladenine (3-MA) (30 mg/kg/d), rapamycin(1 mg/kg/d), and deferoxamine (DFO) (200 mg/kg/d) were conducted once daily during the last 3 weeks of the CUMS procedure. In vitro, a depressive model was established by culture of SH-SY5Y cells with corticosterone, followed by treatment with different concentrations of freeze-dried SNS (0.001, 0.01, 0.1 mg/mL) and rapamycin (10 nM), NCOA4-overexpression, Si-NCOA4. After the behavioral test (open-field test (OFT), sucrose preference test (SPT), forced swimming test (FST) and tail suspension test (TST), dendritic spines, GluR2 protein expression, iron concentration, and ferritinophagy-related protein levels (P62, FTH, NCOA4, LC3-II/LC3-I) were tested in vitro and in vivo using immunohistochemistry, golgi staining, immunofluorescence, and Western blot assays. Finally, HEK-293T cells were transfected by si-NCOA4 or GluR2-and NCOA4-overexpression plasmid and treated with corticosterone(100 µM), freeze-dried SNS(0.01 mg/mL), rapamycin(25 nM), and 3-MA(5 mM). The binding amount of GluR2, NCOA4, and LC3 was assessed by the co-immunoprecipitation (CO-IP) assay. RESULTS: 3-MA, SNS, and DFO promoted depressive-like behaviors in CUMS mice during OFT, SPT, FST and TST, improved the amount of the total, thin, mushroom spine density and enhanced GluR2 protein expression in the hippocampus. Meanwhile, treatment with SNS decreased iron concentrations and inhibited NCOA4-mediated ferritinophagy activation in vitro and in vivo. Importantly, 3-MA and SNS could prevent the binding of GluR2, NCOA4 and LC3 in corticosterone-treated HEK-293T, and rapamycin reversed this phenomenon after treatment with SNS. CONCLUSION: SNS alleviates depression-like behaviors in CUMS mice by regulating dendritic spines via NCOA4-mediated ferritinophagy.

Caffeine Supplementation and FOXM1 Inhibition Enhance the Antitumor Effect of Statins in Neuroblastoma.

High-risk neuroblastoma exhibits transcriptional activation of the mevalonate pathway that produces cholesterol and nonsterol isoprenoids. A better understanding of how this metabolic reprogramming contributes to neuroblastoma development could help identify potential prevention and treatment strategies. Here, we report that both the cholesterol and nonsterol geranylgeranyl-pyrophosphate branches of the mevalonate pathway are critical to sustain neuroblastoma cell growth. Blocking the mevalonate pathway by simvastatin, a cholesterol-lowering drug, impeded neuroblastoma growth in neuroblastoma cell line xenograft, patient-derived xenograft (PDX), and TH-MYCN transgenic mouse models. Transcriptional profiling revealed that the mevalonate pathway was required to maintain the FOXM1-mediated transcriptional program that drives mitosis. High FOXM1 expression contributed to statin resistance and led to a therapeutic vulnerability to the combination of simvastatin and FOXM1 inhibition. Furthermore, caffeine synergized with simvastatin to inhibit the growth of neuroblastoma cells and PDX tumors by blocking statin-induced feedback activation of the mevalonate pathway. This function of caffeine depended on its activity as an adenosine receptor antagonist, and the A2A adenosine receptor antagonist istradefylline, an add-on drug for Parkinson's disease, could recapitulate the synergistic effect of caffeine with simvastatin. This study reveals that the FOXM1-mediated mitotic program is a molecular statin target in cancer and identifies classes of agents for maximizing the therapeutic efficacy of statins, with implications for treatment of high-risk neuroblastoma. SIGNIFICANCE: Caffeine treatment and FOXM1 inhibition can both enhance the antitumor effect of statins by blocking the molecular and metabolic processes that confer statin resistance, indicating potential combination therapeutic strategies for neuroblastoma. See related commentary by Stouth et al., p. 2091.

Essential oil of lemon myrtle (Backhousia citriodora) induces S-phase cell cycle arrest and apoptosis in HepG2 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Lemon myrtle (Backhousia citriodora F.Muell.) leaves, whether fresh or dried, are used traditionally in folk medicine to treat wounds, cancers, skin infections, and other infectious conditions. However, the targets and mechanisms related to anti-cancer effect of lemon myrtle are unavailable. In our study, we found that the essential oil of lemon myrtle (LMEO) showed anti-cancer activity in vitro, and we initially explored its mechanism of action. MATERIALS AND METHODS: We analyzed the chemical compositions of LMEO by GC-MS. We tested the cytotoxicity of LMEO on various cancer cell lines using the MTT assay. Network pharmacology was used also to analyze the targets of LMEO. Moreover, the mechanisms of LMEO were investigated through scratch assay, flow cytometry analysis, and western blot in the HepG2 liver cancer cell line. RESULTS: LMEO showed cytotoxicity on various cancer cell lines with values of IC50 40.90 ± 2.23 (liver cancer HepG2 cell line), 58.60 ± 6.76 (human neuroblastoma SH-SY5Y cell line), 68.91 ± 4.62 (human colon cancer HT-29 cell line) and 57.57 ± 7.61 µg/mL (human non-small cell lung cancer A549 cell line), respectively. The major cytotoxic chemical constituent in LMEO was identified as citrals, which accounted for 74.9% of the content. Network pharmacological analysis suggested that apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1), androgen receptor (AR), cyclin-dependent kinases 1 (CDK1), nuclear factor erythroid 2-related factor 2 (Nrf-2), fatty acid synthase (FASN), epithelial growth factor receptor (EGFR), estrogen receptor 1 (ERα) and cyclin-dependent kinases 4 (CDK4) are potential cytotoxic targets of LMEO. These targets are closely related to cell migration, cycle and apoptosis. Notley, the p53 protein had the highest confidence to co-associate with the eight common targets, which was further confirmed by scratch assay, flow cytometry analysis, and western blot in the HepG2 liver cancer cell line. LMEO significantly inhibited the migration of HepG2 cells in time-dependent and dose-dependent manner. Moreover, LMEO caused a S-phase blocking on HepG2 cells and promoted apoptosis in the meanwhile. Western blot results indicated that p53 protein, Cyclin A2 and Bax proteins were up-regulated, while Cyclin E1 and Bcl-2 proteins were down-regulated. CONCLUSION: LMEO showed cytotoxicity in various cancer cell lines in vitro. Pharmacological networks showed LMEO to have multi-component and multi-targeting effects that are related to inhibit migration of HepG2 cells, and affect cell cycle S-phase arrest and apoptosis through modulation of p53 protein.

LCMS Determination and Cytotoxicity of Abrus precatorius on L6 and SK-N-MC Cell Lines.

OBJECTIVE: The present study aimed to investigate the cytotoxic effect of various extracts derived from Abrus precatorius Linn. leaves on rat L6 and human SK-N-MC neuroblastoma cell lines and determine the secondary metabolites responsible for the cytotoxicity of Abrus precatorius. METHODS: Successive solvent extraction of A. precatorius leaves was carried out using the Soxhlet apparatus with solvents such as petroleum ether, chloroform, ethyl acetate, and ethanol. HPTLC fingerprinting and LC-MS studies were performed to assess the presence of secondary metabolites, such as flavonoids and phenols, in the ethyl acetate extract. Furthermore, the cytotoxic effect of extracts was tested on rat skeletal muscle cell line L6 and human neuroblastoma cell line SK-N-MC using MTT assay. RESULTS: The total phenolic content of ethyl acetate and ethanol extracts of A. precatorius were 72.67 and 60.73 mg, respectively, of GAE/g dry weight of the extract. The total flavonoid content of ethyl acetate and ethanol extract of A. precatorius were 107.33 and 40.66 mg of Quercetin equivalents/g dry weight of the extract. LCMS analysis demonstrated that the flavonoids in specific Naringenin, Diosmetin, Glycitin, and Genistein might play a prominent role in the cytotoxicity of A. precatorius. The cytotoxicity study revealed that the extracts of A. precatorius were non-toxic to rat L6 myotubes, and the IC50 values of the various extracts, such as APPE, APCH, APEA, and APET, were >100 µg/ml. The extracts exhibited cytotoxic activity against human neuroblastoma SK-N-MC cells, and the IC50 values of APPE, APCH, APEA, APET, and the standard drug "Cisplatin" were >100, >100, 64.88, >100, and 3.72 µg/ml, respectively. CONCLUSION: It was concluded from the study that the extracts of Abrus precatorius were cytotoxic to neuroblastoma cell lines but non-toxic to normal cell lines. HPTLC and LC-MS studies confirmed that flavonoids in the ethyl acetate extract could be responsible for the biological activity.

Loureirin C inhibits ferroptosis after cerebral ischemia reperfusion through regulation of the Nrf2 pathway in mice.

BACKGROUND: Ischemic stroke (IS) is considered as a serious cerebral vascular disease. Ferroptosis is a novel type of regulated cell death (RCD), that closely related to the occurrence and progress of IS. Loureirin C, a type of dihydrochalcone compound derived from the Chinese Dragon's blood (CDB). The effective components extracted from CDB have shown neuroprotective effects in ischemia reperfusion models. However, the role of Loureirin C in mice after IS is not well understood. Thus, it is worth to identify the effect and mechanism of Loureirin C on IS. PURPOSE: The present research aims to prove the existence of ferroptosis in IS and explore whether Loureirin C can inhibit ferroptosis by regulating nuclear factor E2 related factor 2 (Nrf2) pathway in mice and exert neuroprotective effects on IS models. METHODS: Middle cerebral artery occlusion and reperfusion (MCAO/R) model was established to evaluate the occurrence of ferroptosis and the potential Loureirin C brain-protective effect in vivo. The analysis of free iron, glutamate content, reactive oxygen species (ROS) and lipid peroxidation levels, along with transmission electron microscope (TEM) was applied to prove the existence of ferroptosis. The function of Loureirin C on Nrf2 nuclear translocation was verified by immunofluorescence staining. In vitro, primary neurons and SH-SY5Y cells were processed with Loureirin C after oxygen and glucose deprivation-reperfusion (OGD/R). ELISA kits, western blotting, co-immunoprecipitation (Co-IP) analysis, immunofluorescence, and quantitative real-time PCR were devoted to proving the neuroprotective effects of Loureirin C on IS via regulating ferroptosis and Nrf2 pathways. RESULTS: The results showed that Loureirin C not only dramatically alleviated brain injury and inhibited neurons ferroptosis in mice after MCAO/R, but also dose-dependently reduce ROS accumulation in ferroptosis after OGD/R. Further, Loureirin C inhibits ferroptosis by activating Nrf2 pathway, and promoting nuclear translocation of Nrf2. Besides, Loureirin C increases heme oxygenase 1 (HO-1), quinone oxidoreductase 1 (NQO1) and glutathione peroxidase 4 (GPX4) content after IS. Intriguingly, the anti-ferroptosis effect of Loureirin C is weakened by Nrf2 knockdown. CONCLUSION: Our discoveries first revealed that the inhibitory action of Loureirin C on ferroptosis may greatly depend on its adjusting effect on the Nrf2 pathway, suggesting that Loureirin C could act as a novel anti-ferroptosis candidate and play a therapeutic role in IS. These novel discoveries on the role of Loureirin C on IS models reveal an innovative method that may contribute to neuroprotection for the prevention of IS.

Characterization of the Antitumor Potential of Extracts of Cannabis sativa Strains with High CBD Content in Human Neuroblastoma.

Cannabis has been used for decades as a palliative therapy in the treatment of cancer. This is because of its beneficial effects on the pain and nausea that patients can experience as a result of chemo/radiotherapy. Tetrahydrocannabinol and cannabidiol are the main compounds present in Cannabis sativa, and both exert their actions through a receptor-mediated mechanism and through a non-receptor-mediated mechanism, which modulates the formation of reactive oxygen species. These oxidative stress conditions might trigger lipidic changes, which would compromise cell membrane stability and viability. In this sense, numerous pieces of evidence describe a potential antitumor effect of cannabinoid compounds in different types of cancer, although controversial results limit their implementation. In order to further investigate the possible mechanism involved in the antitumoral effects of cannabinoids, three extracts isolated from Cannabis sativa strains with high cannabidiol content were analyzed. Cell mortality, cytochrome c oxidase activity and the lipid composition of SH-SY5Y cells were determined in the absence and presence of specific cannabinoid ligands, with and without antioxidant pre-treatment. The cell mortality induced by the extracts in this study appeared to be related to the inhibition of the cytochrome c oxidase activity and to the THC concentration. This effect on cell viability was similar to that observed with the cannabinoid agonist WIN55,212-2. The effect was partially blocked by the selective CB1 antagonist AM281, and the antioxidant α-tocopherol. Moreover, certain membrane lipids were affected by the extracts, which demonstrated the importance of oxidative stress in the potential antitumoral effects of cannabinoids.

Protective effect of Xingnaojing injection on ferroptosis after cerebral ischemia injury in MCAO rats and SH-SY5Y cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Xingnaojing（XNJ）injection is a traditional Chinese medicine injection with neuroprotective effect, which has been widely used in the treatment of stroke for many years. AIM OF THE STUDY: This study aimed to explore the potential mechanism of XNJ in cerebral ischemia mediated by ferroptosis using proteomics and in vivo and in vitro experiments. MATERIALS AND METHODS: After the rat model of middle cerebral artery occlusion (MCAO) was successfully established, they were randomly divided into model, XNJ, and deferoxamine (DFO) group. Triphenyl tetrazolium chloride (TTC) staining, Hematoxylin and eosin (H&E), and Nissl staining were used to observe the infarct area, pathological changes and the degree of neuronal apoptosis of rat brain. Proteins extracted from rat brain tissues were analyzed by quantitative proteomics using tandem mass tags (TMT). Western blotting and immunohistochemical assessment were used to measure the expression of ferroptosis-related proteins. In vitro, the SH-SY5Y cells were subjected to hypoxia (37°C/5% CO2/1% O2) for 24 h to observe the survival rate, and detect the reactive oxygen species (ROS) content and ferroptosis-related proteins. RESULTS: In TTC and H&E experiments, we found that XNJ drug treatment reduced the infarct volume and brain tissue damage in MCAO rats. Nissl staining also showed that compared with MCAO group rats, the Nissl bodies of brain tissue after XNJ drug intervention were clear with a 3.54-fold increased times, suggesting that XNJ improved cerebral infraction, and neurological deficits in MCAO rats. Proteomics identified 101 intersected differentially expressed proteins (DEPs). According to the bioinformatics analysis, these DEPs were closely related to ferroptosis. Further research indicated that MCAO-induced cerebral ischemia was alleviated by upregulating recombinant glutathione peroxidase 4 (GPX4), ferroportin (FPN) expression, Heme oxygenase-1 (HO-1) expression, and downregulating cyclooxygenase-2 (COX-2), transferring receptor (TFR) and divalent metal transporter-1 (DMT1) expression after XNJ treatment. In addition, in vitro experiment indicated that XNJ improved the survival rate of hypoxia-damaged SH-SY5Y cells. XNJ increased the level of GPX4 and inhibited the protein expression of COX-2 and TFR after cell hypoxia. Moreover, different concentrations of XNJ (0.25%, 0.5%, 1%) reduced the ROS content of hypoxic cells, suggesting that XNJ could inhibit hypoxia-induced cell damage by regulating the expression of ferroptosis-related proteins and decreasing the production of ROS. CONCLUSIONS: XNJ could promote the recovery of neurological function in MCAO rats and hypoxia SH-SY5Y cells by regulating ferroptosis.