EGCG-induced selective death of cancer cells through autophagy-dependent regulation of the p62-mediated antioxidant survival pathway.

The effects of EGCG on the selective death of cancer cells by modulating antioxidant pathways through autophagy were explored in various normal and cancer cells. EGCG positively regulated the p62-KEAP1-NRF2-HO-1 pathway in normal cells, while negatively regulating it in cancer cells, leading to selective apoptotic death of cancer cells. In EGCG-treated MRC5 cells (EGCG-MRC5), autophagic flux was blocked, which was accompanied by the formation of p62-positive aggregates. However, EGCG-treated HeLa cells (EGCG-HeLa) showed incomplete autophagic flux and no aggregate formation. The levels of P-ULK1 S556 and S758 increased in EGCG-MRC5 through AMPK-mTOR cooperative interaction. In contrast, EGCG treatment in HeLa cells led to AMPK-induced mTOR inactivation, resulting in abrogation of P-ULK1 S556 and S758 levels. AMPK knockout in EGCG-HeLa restored positive regulation of the p62-mediated pathway, which was accompanied by increased P-mTOR S2448 and P-ULK1 S758 levels. Knockdown of 67LR in EGCG-HeLa abolished AMPK activity but did not restore the p62-mediated pathway. Surprisingly, both AMPK knockout and 67LR knockdown in EGCG-HeLa markedly increased cell viability, despite differential regulation of the antioxidant enzyme HO-1. In conclusion, EGCG induces the selective death of cancer cells through the modulation of at least two autophagy-dependent and independent regulatory pathways: negative regulation involves the mTOR-ULK1 (S556 and S758)-p62-KEAP1-NRF2-HO-1 axis via AMPK activation, whereas positive regulation occurs through the 67LR-AMPK axis.

KPNA3 promotes epithelial-mesenchymal transition by regulating TGF-ß and AKT signaling pathways in MDA-MB-231, a triple-negative breast cancer cell line.

Karyopherin-α3 (KPNA3), a karyopherin- α isoform, is intimately associated with metastatic progression via epithelial-mesenchymal transition (EMT). However, the molecular mechanism underlying how KPNA3 acts as an EMT inducer remains to be elucidated. In this report, we identified that KPNA3 was significantly upregulated in cancer cells, particularly in triple-negative breast cancer, and its knockdown resulted in the suppression of cell proliferation and metastasis. The comprehensive transcriptome analysis from KPNA3 knockdown cells indicated that KPNA3 is involved in the regulation of numerous EMTrelated genes, including the downregulation of GATA3 and E-cadherin and the up-regulation of HAS2. Moreover, it was found that KPNA3 EMT-mediated metastasis can be achieved by TGF-ß or AKT signaling pathways; this suggests that the novel independent signaling pathways KPNA3-TGF-ß-GATA3-HAS2/E-cadherin and KPNA3-AKT-HAS2/E-cadherin are involved in the EMT-mediated progress of TNBC MDA-MB-231 cells. These findings provide new insights into the divergent EMT inducibility of KPNA3 according to cell and cancer type. [BMB Reports 2023; 56(2): 120-125].

Gold nanostructure-integrated conductive microwell arrays for uniform cancer spheroid formation and electrochemical drug screening.

Cancer spheroids, which mimic distinct cell-to-cell and cell-extracellular matrix interactions of solid tumors in vitro, have emerged as a promising tumor model for drug screening. However, owing to the unique characteristics of spheroids composed of three-dimensionally densely-packed cells, the precise characterizations of cell viability and function with conventional colorimetric assays are challenging. Herein, we report gold nanostructure-integrated conductive microwell arrays (GONIMA) that enable both highly efficient uniform cancer spheroid formation and precise electrochemical detection of cell viability. A nanostructured gold on indium tin oxide (ITO) substrate facilitated the initial cell aggregation and further 3D cell growth, while the non-cytophilic polymer microwell arrays restricted the size and shape of the spheroids. As a result, approximately 150 human glioblastoma spheroids were formed on a chip area of 1.13 cm2 with an average diameter of 224 µm and a size variation of only 5% (±11.36 µm). The high uniformity of cancer spheroids contributed to the stability of electrical signals measuring cell viability. Using the fabricated GONIMA, the effects of a representative chemotherapeutic agent, hydroxyurea, on the glioblastoma spheroids were precisely monitored under conditions of varying drug concentrations (0-0.3 mg/mL) and incubation times (24-48 h). Therefore, we conclude that the newly developed platform is highly useful for rapid and precise in vitro drug screening, as well as for the pharmacokinetic analyses of specific drugs using 3D cellular cancer models.

5-Fluorouracil-Immobilized Hyaluronic Acid Hydrogel Arrays on an Electrospun Bilayer Membrane as a Drug Patch.

The hyaluronic acid (HA) hydrogel array was employed for immobilization of 5-fluorouracil (5-FU), and the electrospun bilayer (hydrophilic: polyurethane/pluronic F-127 and hydrophobic: polyurethane) membrane was used to support the HA hydrogel array as a patch. To visualize the drug propagating phenomenon into tissues, we experimentally investigated how FITC-BSA diffused into the tissue by applying hydrogel patches to porcine tissue samples. The diffusive phenomenon basically depends on the FITC-BSA diffusion coefficient in the hydrogel, and the degree of diffusion of FITC-BSA may be affected by the concentration of HA hydrogel, which demonstrates that the high density of HA hydrogel inhibits the diffusive FITC-BSA migration toward the low concentration region. YD-10B cells were employed to investigate the release of 5-FU from the HA array on the bilayer membrane. In the control group, YD-10B cell viability was over 98% after 3 days. However, in the 5-FU-immobilized HA hydrogel array, most of the YD-10B cells were not attached to the bilayer membrane used as a scaffold. These results suggest that 5-FU was locally released and initiated the death of the YD-10B cells. Our results show that 5-FU immobilized on HA arrays significantly reduces YD-10B cell adhesion and proliferation, affecting cells even early in the cell culture. Our results suggest that when 5-FU is immobilized in the HA hydrogel array on the bilayer membrane as a drug patch, it is possible to control the drug concentration, to release it continuously, and that the patch can be applied locally to the targeted tumor site and administer the drug in a time-stable manner. Therefore, the developed bilayer membrane-based HA hydrogel array patch can be considered for sustained release of the drug in biomedical applications.

Microfluidic Electroceuticals Platform for Therapeutic Strategies of Intervertebral Disc Degeneration: Effects of Electrical Stimulation on Human Nucleus Pulposus Cells under Inflammatory Conditions.

The degeneration of an intervertebral disc (IVD) is a major cause of lower back pain. IVD degeneration is characterized by the abnormal expression of inflammatory cytokines and matrix degradation enzymes secreted by IVD cells. In addition, macrophage-mediated inflammation is strongly associated with IVD degeneration. However, the precise pathomechanisms of macrophage-mediated inflammation in IVD are still unknown. In this study, we developed a microfluidic platform integrated with an electrical stimulation (ES) array to investigate macrophage-mediated inflammation in human nucleus pulposus (NP). This platform provides multiple cocultures of different cell types with ES. We observed macrophage-mediated inflammation and considerable migration properties via upregulated expression of interleukin (IL)-6 (p < 0.001), IL-8 (p < 0.05), matrix metalloproteinase (MMP)-1 (p < 0.05), and MMP-3 (p < 0.05) in human NP cells cocultured with macrophages. We also confirmed the inhibitory effects of ES at 10 µA due to the production of IL-6 (p < 0.05) and IL-8 (p < 0.01) under these conditions. Our findings indicate that ES positively affects degenerative inflammation in diverse diseases. Accordingly, the microfluidic electroceutical platform can serve as a degenerative IVD inflammation in vitro model and provide a therapeutic strategy for electroceuticals.

DNA methylation dynamics and dysregulation delineated by high-throughput profiling in the mouse.

We have developed a mouse DNA methylation array that contains 296,070 probes representing the diversity of mouse DNA methylation biology. We present a mouse methylation atlas as a rich reference resource of 1,239 DNA samples encompassing distinct tissues, strains, ages, sexes, and pathologies. We describe applications for comparative epigenomics, genomic imprinting, epigenetic inhibitors, patient-derived xenograft assessment, backcross tracing, and epigenetic clocks. We dissect DNA methylation processes associated with differentiation, aging, and tumorigenesis. Notably, we find that tissue-specific methylation signatures localize to binding sites for transcription factors controlling the corresponding tissue development. Age-associated hypermethylation is enriched at regions of Polycomb repression, while hypomethylation is enhanced at regions bound by cohesin complex members. Apc Min/+ polyp-associated hypermethylation affects enhancers regulating intestinal differentiation, while hypomethylation targets AP-1 binding sites. This Infinium Mouse Methylation BeadChip (version MM285) is widely accessible to the research community and will accelerate high-sample-throughput studies in this important model organism.

Effect of intramuscular midazolam premedication on patient satisfaction in women undergoing general anaesthesia: a randomised control trial.

OBJECTIVE: To determine the effect of premedication with intramuscular midazolam on patient satisfaction in women undergoing general anaesthesia. TRIAL DESIGN, SETTING AND PARTICIPANTS: Double-blind, parallel randomised control trial at a tertiary care medical centre in South Korea. Initially, 140 women aged 20-65 years who underwent general anaesthesia and had an American Society of Anesthesiology physical status classification of I or II were randomly assigned to the intervention group or the control group, and 134 patients (intervention n=65; control n=69) completed the study. INTERVENTION: Intramuscular administration of midazolam (0.05 mg/kg) or placebo (normal saline 0.01 mL/kg) on arrival at the preoperative holding area. MAIN OUTCOMES: The primary outcome was the patient's overall satisfaction with the anaesthesia experience as determined by questionnaire responses on the day after surgery. Satisfaction was defined as a response of 3 or 4 on a five-point scale (0-4). The secondary outcomes included blood pressure, heart rate, oxygen desaturation, recovery duration and postoperative pain. RESULTS: Patients who received midazolam were more satisfied than those who received placebo (percentage difference: 21.0%, OR 3.56, 95% CI 1.46 to 8.70). A subgroup analysis revealed that this difference was greater in patients with anxiety, defined as those whose Amsterdam Preoperative Anxiety and Information Scale anxiety score was ≥11, than that for the whole sample population (percentage difference: 24.0%, OR 4.33, 95% CI 1.25 to 14.96). Both groups had similar heart rates, blood pressure and oxygen desaturation. CONCLUSION: Intramuscular administration of midazolam in women before general anaesthesia in the preoperative holding area improved self-reported satisfaction with the anaesthesia experience, with an acceptable safety profile. TRIAL REGISTRATION NUMBER: KCT0006002.

Recent Advances in Multicellular Tumor Spheroid Generation for Drug Screening.

Multicellular tumor spheroids (MCTs) have been employed in biomedical fields owing to their advantage in designing a three-dimensional (3D) solid tumor model. For controlling multicellular cancer spheroids, mimicking the tumor extracellular matrix (ECM) microenvironment is important to understand cell-cell and cell-matrix interactions. In drug cytotoxicity assessments, MCTs provide better mimicry of conventional solid tumors that can precisely represent anticancer drug candidates' effects. To generate incubate multicellular spheroids, researchers have developed several 3D multicellular spheroid culture technologies to establish a research background and a platform using tumor modelingvia advanced materials science, and biosensing techniques for drug-screening. In application, drug screening was performed in both invasive and non-invasive manners, according to their impact on the spheroids. Here, we review the trend of 3D spheroid culture technology and culture platforms, and their combination with various biosensing techniques for drug screening in the biomedical field.

Efficacy of 0.4 mg tamsulosin monotherapy in patients with moderate-to-severe lower urinary tract symptoms.

PURPOSE: To evaluate efficacy of 0.4 mg tamsulosin monotherapy in patients with benign prostatic hyperplasia with moderate-to-severe International Prostate Symptom Score. MATERIAL AND METHODS: From May 2015 to May 2017, 102 patients were analyzed, retrospectively. The patients were classified into three groups according to the combination of medication (tamsulosin 0.4 mg vs tamsulosin 0.4 mg + solifenacin 5 mg vs tamsulosin 0.4 mg + mirabegron 50 mg). Baseline characteristics (e.g. age, body weight, height, and underlying medical disease) were collected. International Prostate Symptom Score, prostate specific antigen, prostate volume, peak urinary flow rate (Qmax), voided volume, and post-voided volume before after treatment were evaluated. RESULTS: We classified and analyzed the patients into three groups depending on the medication. And there were no significant differences between all parameters among the groups. Voided volume at 3 months after treatment in each group was 170.54 ± 125.83, 121.55 ± 46.19, and 274.63 ± 132.30 (p = 0.019). Differences of voiding symptom score and difference of post-voided volume among the groups before after treatment was 5.00 ± 5.42, 1.92 ± 3.92, and 0.11 ± 5.11 and 8.37 ± 34.32, 0.78 ± 14.86, -33.63 ± 28.58 (p = 0.037, p = 0.007). CONCLUSION: We think tamsulosin monotherapy will be feasible as a first-line therapy for the patients with benign prostatic hyperplasia who has struggled with moderate-to-severe lower urinary tract symptoms.

Ginkgonitroside, a new nitrophenyl glycoside and bioactive compounds from Ginkgo biloba leaves controlling adipocyte and osteoblast differentiation.

Eight compounds (1-8) including one novel nitrophenyl glycoside, ginkgonitroside (1) were isolated from the leaves of Ginkgo biloba, a popular medicinal plant. The structure of the new compound was characterized using extensive spectroscopic analyses via 1D and 2D NMR data interpretations, HR-ESIMS, and chemical transformation. To the best of our knowledge, the present study is the first to report the presence of nitrophenyl glycosides, which are relatively unique phytochemicals in natural products, in G. biloba. The isolated compounds (1-8) were examined for their effects on the regulation of mesenchymal stem cell (MSC) differentiation. Compounds 1-3 and 8 were able to suppress MSC differentiation toward adipocytes. In contrast, compounds 5 and 8 showed activity promoting osteogenic differentiation of MSCs. These findings demonstrate that the active compounds showed regulatory activity on MSC differentiation between adipocytes and osteocytes.

A case report of arthroscopic surgery under extended spinal anesthesia for 402 minutes, assisted by monitored anesthesia care.

INTRODUCTION: Surgical spinal anesthesia is usually maintained for approximately 3 h with bupivacaine, but it is difficult to accurately predict the duration of surgery for each case. When an operation continues for an extended duration, regression of spinal anesthesia often leads to general anesthesia. Here we present a case of extended spinal anesthesia assisted by monitored anesthesia care. CASE PRESENTATION: A 32-year-old male who suffered from persistent pain of the right knee was diagnosed with rupture of the right anterior cruciate ligament. Arthroscopic surgery of the right knee was conducted with spinal anesthesia. A local anesthetic mixture of 0.5% hyperbaric bupivacaine 12 mg with 50 µg of epinephrine was used. The surgery took longer than expected with a total anesthesia time of 402 minutes. In the final 30 minutes of surgery, spinal anesthesia regressed and the procedure was completed under monitored anesthesia care (MAC). CLINICAL DISCUSSION: When spinal anesthesia is on regression during the final stage of surgery, the application of MAC safely secures additional operation time. By adopting MAC, the patient avoided general anesthesia and had minimal physiological distress and a rapid recovery. Another benefit of MAC is the reduced consumption of resources. Further, by avoiding endotracheal intubation and mechanical ventilation, the risk of transmission of infectious agents is minimized. CONCLUSION: In situations where spinal anesthesia is regressing close to the end of a surgical procedure, the application of MAC has potential benefits over general anesthesia. These benefits are particularly relevant during the current COVID-19 pandemic.

Immunostimulatory Activity of Polysaccharides Extracted from Celosia cristata Flowers.

Macrophages play a major role in innate immune responses by producing a variety of immune mediators and cytokines. The stimulation of macrophages by natural products may lead to an enhanced innate immune system. This study evaluated the immunostimulatory effects of a polysaccharide-rich crude fraction of Celosia cristata L. flowers (CCP) on murine macrophages. CCP treatment induced the production of inducible nitric oxide synthase, cyclooxygenase-2, and cytokines by macrophages. Mechanistically, the activation of mitogen-activated protein kinases, NF-κB and toll-like receptor 4 were found to be associated with the stimulatory functions of CCP. CCP was found to be primarily composed of galacturonic acid and glucose in addition to small amounts of arabinose and galactose. This study demonstrated that CCP may enhance the innate immune responses and potentially improve the immune functions in the body.

Species Prioritization Based on Spectral Dissimilarity: A Case Study of Polyporoid Fungal Species.

Biological species collections are critical for natural product drug discovery programs. However, prioritization of target species in massive collections remains difficult. Here, we introduce an untargeted metabolomics-based prioritization workflow that uses MS/MS molecular networking to estimate scaffold-level distribution. As a demonstration, we applied the workflow to 40 polyporoid fungal species. Nine species were prioritized as candidates based on the chemical structural and compositional similarity (CSCS) metric. Most of the selected species showed relatively higher richness and uniqueness of metabolites than those of the others. Cryptoporus volvatus, one of the prioritized species, was investigated further. The chemical profiles of the extracts of C. volvatus culture and fruiting bodies were compared, and it was shown that derivative-level diversity was higher in the fruiting bodies; meanwhile, scaffold-level diversity was similar. This showed that the compounds found from a cultured fungus can also be isolated in wild mushrooms. Targeted isolation of the fruiting body extract yielded three unknown (1-3) and six known (4-9) cryptoporic acid derivatives, which are drimane-type sesquiterpenes with isocitric acid moieties that have been reported in this species. Cryptoporic acid T (1) is a trimeric cryptoporic acid reported for the first time. Compounds 2 and 5 exhibited cytotoxicity against HCT-116 cell lines with IC50 values of 4.3 and 3.6 µM, respectively.

A Spheroid-Forming Hybrid Gold Nanostructure Platform That Electrochemically Detects Anticancer Effects of Curcumin in a Multicellular Brain Cancer Model.

In this study, a multifunctional platform that enables the highly efficient formation of 3D multicellular cancer spheroids and precise real-time assessments of the anticancer effects of curcumin in a brain tumor coculture model is reported. A highly conductive gold nanostructure (HCGN) is fabricated to facilitate cancer spheroid formation without using anti-cell adhesion molecules. A neuroblastoma (SH-SY5Y) and glioblastoma (U-87MG) coculture model is generated on HCGN with a specific cell-to-cell ratio (SH-SY5Y: U-87MG = 1:1), and their redox behaviors are successfully measured without destroying the distinct 3D structure of the multicellular spheroids. Using electrochemical signals as an indicator of spheroid viability, the effects of potential anticancer compounds on cocultured spheroids are further assessed. Remarkably, decreased cell viability in 3D spheroids caused by a low concentration of curcumin (30 µM) is detectable using the electrochemical method (29.4%) but not with a conventional colorimetric assay (CCK-8). The detection is repeated more than ten times for both short- (63 h) and long-term cultivation (144 h) without damaging the spheroids, enabling real-time, non-destructive pharmacokinetic analysis of various drug candidates. Therefore, it can be concluded that the hybrid platform is a highly promising, precise, and high-throughput drug screening tool based on 3D cell cultivation.

Complications of fluid overload during hysteroscopic surgery: cardiomyopathy and epistaxis - A case report.

BACKGROUND: Hysteroscopic surgery has been used in various gynecological fields. However, massive fluid overload can occur as a complication due to persistent infusion of media for uterine cavity distension. We present the case of a woman who developed cardiomyopathy with pulmonary edema and epistaxis during hysteroscopic surgery. CASE: A 76-year-old female underwent hysteroscopic septectomy. She manifested abrupt, active nasal bleeding and regurgitation in the intravenous line. Heart rate, SpO2, and PETCO2 decreased from 55 beats/min to 29 beats/min, from 100% to 56%, and from 31 mmHg to 9 mmHg, respectively. After the operation, brain CT showed bilateral prominent superior ophthalmic vein dilation. Echocardiography showed left ventricle apical ballooning and global hypokinesia. The patient recovered after two days of conservative management, with no sequelae. CONCLUSIONS: Although hysteroscopic surgery is a simple procedure, careful monitoring is necessary to prevent complications from absorption of fluid distending media during the procedure.

Anti-Proliferative Effects of Standardized Cornus officinalis on Benign Prostatic Epithelial Cells via the PCNA/E2F1-Dependent Cell Cycle Pathway.

Cornus officinalis, widely used in traditional Chinese medicine, exhibits pharmacological effects against erectile dysfunction and pollakisuria, which are pathological symptoms of benign prostatic hyperplasia (BPH). Although traditional usage and a study on BPH have been reported, to our knowledge, no study has investigated the exact molecular mechanism(s) underlying the anti-proliferative effects of standardized C. officinalis on prostatic cells. We standardized C. officinalis 30% ethanol extract (COFE) and demonstrated the therapeutic effects of COFE on human BPH epithelial cells and testosterone-induced BPH in rats. In vitro studies using BPH-1 cells demonstrated an upregulation of BPH-related and E2F Transcription Factor 1(E2F1)-dependent cell cycle markers, whereas treatment with COFE clearly inhibited the proliferation of BPH epithelial cells and reduced the overexpression of G1 and S checkpoint genes. Additionally, COFE administration alleviated the androgen-dependent prostatic enlargement in a testosterone-induced BPH animal model. COFE exerted these anti-BPH effects by the inhibition of anti-apoptotic markers, suppression of PCNA expression, and regulation of E2F1/pRB-dependent cell cycle markers in rats with BPH. These results suggest that COFE exerts anti-proliferative effect by regulating PCNA/E2F1-dependent cell cycle signaling pathway both in vivo and in vitro. These findings reveal the therapeutic potential of COFE, which could be used as a substitute for BPH treatment.

Locally Controlled Diffusive Release of Bone Morphogenetic Protein-2 Using Micropatterned Gelatin Methacrylate Hydrogel Carriers.

In this work, a novel and simple bone morphogenetic protein (BMP)-2 carrier is developed, which enables localized and controlled release of BMP-2 and facilitates bone regeneration. BMP-2 is localized in the gelatin methacrylate (GelMA) micropatterns on hydrophilic semi-permeable membrane (SNM), and its controlled release is regulated by the concentration of GelMA hydrogel and BMP-2. The controlled release of BMP-2 is verified using computational analysis and quantified using fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) diffusion model. The osteogenic differentiation of osteosarcoma MG-63 cells is manipulated by localized and controlled BMP-2 release. The calcium deposits are significantly higher and the actin skeletal networks are denser in MG-63 cells cultured in the BMP-2-immobilized GelMA micropattern than in the absence of BMP-2. The proposed BMP-2 carrier is expected to not only act as a barrier membrane that can prevent invasion of connective tissue during bone regeneration, but also as a carrier capable of localizing and controlling the release of BMP-2 due to GelMA micropatterning on SNM. This approach can be extensively applied to tissue engineering, including the localization and encapsulation of cells or drugs.

Ginkgobilol, a new diarylpentanoid and an osteogenic diarylpentanoid analog from Ginkgo biloba leaves.

Phytochemical analysis of methanol extracts of Ginkgo biloba leaves resulted in the isolation of a novel diarylpentanoid, ginkgobilol (1) and a known diarylpentanoid analog (2). The structure of the new compound was elucidated by analyzing NMR spectroscopic data and HR-ESIMS, and the absolute configuration was determined using gauge-including atomic orbital NMR chemical shift calculations, followed by DP4+ analysis and specific rotation value. Diarylpentanoids comprise two aromatic rings linked by a five-carbon bridge; these are relatively unique examples in natural products. To the best of our knowledge, the present study is the first to report the presence of diarylpentanoids in G. biloba. Compound 2 increased alkaline phosphatase (ALP) production in C3H10T1/2, a murine mesenchymal stem cell line, in a dose-dependent manner. The promotion of osteogenic differentiation by the active compound 2 mediated by induction of transcriptional ALP and osteopontin (OPN) gene expression was confirmed using quantitative real time polymerase chain reaction, thus indicating its remarkable bone formation activity.

Aviculin Isolated from Lespedeza cuneata Induce Apoptosis in Breast Cancer Cells through Mitochondria-Mediated Caspase Activation Pathway.

The global incidence of breast cancer has increased. However, there are many impediments to the development of safe and effective anticancer drugs. The aim of the present study was to evaluate the effect of aviculin isolated from Lespedeza cuneata (Dum. Cours.) G. Don. (Fabaceae) on MCF-7 human breast cancer cells and determine the underlying mechanism. Using the bioassay-guided isolation by water soluble tetrazolium salt (WST-1)-based Ez-Cytox assay, nine compounds (four lignan glycosides (1-4), three flavonoid glycosides (5-7), and two phenolic compounds (8 and 9)) were isolated from the ethyl acetate (EA) fraction of the L. cuneata methanolic extract. Of these, aviculin (2), a lignan glycoside, was the only compound that reduced metabolic activity on MCF-7 cells below 50% (IC50: 75.47 ± 2.23 µM). The underlying mechanism was analyzed using the annexin V Alexa Fluor 488 binding assay and Western blotting. Aviculin (2) was found to induce apoptotic cell death through the intrinsic apoptosis pathway, as indicated by the increased expression of initiator caspase-9, executioner caspase-7, and poly (ADP-ribose) polymerase (PARP). Aviculin (2)-induced apoptotic cell death was accompanied by an increase in the Bax/Bcl-2 ratio. These findings demonstrated that aviculin (2) could induce breast cancer cell apoptosis through the intrinsic apoptosis pathway, and it can therefore be considered an excellent candidate for herbal treatment of breast cancer.

Inhibitory Effect of 1,5-Dimethyl Citrate from Sea Buckthorn (Hippophae rhamnoides) on Lipopolysaccharide-Induced Inflammatory Response in RAW 264.7 Mouse Macrophages.

Hippophae rhamnoides L. (Elaeagnaceae; commonly known as "sea buckthorn" and "vitamin tree"), is a spiny deciduous shrub whose fruit is used in foods and traditional medicines. The H. rhamnoides fruit (berry) is rich in vitamin C, with a level exceeding that found in lemons and oranges. H. rhamnoides berries are usually washed and pressed to create pomace and juice. Today, the powder of the aqueous extract of H. rhamnoides berries are sold as a functional food in many countries. As part of our ongoing effort to identify bioactive constituents from natural resources, we aimed to isolate and identify those from the fruits of H. rhamnoides. Phytochemical analysis of the extract of H. rhamnoides fruits led to the isolation and identification of six compounds, namely, a citric acid derivative (1), a phenolic (2), flavonoids (3 and 4), and megastigmane compounds (5 and 6). Treatment with compounds 1-6 did not have any impact on the cell viability of RAW 264.7 mouse macrophages. However, pretreatment with these compounds suppressed lipopolysaccharide (LPS)-induced NO production in RAW 264.7 mouse macrophages in a concentration-dependent manner. Among the isolated compounds, compound 1 was identified as the most active, with an IC50 of 39.76 ± 0.16 µM. This value was comparable to that of the NG-methyl-L-arginine acetate salt, a nitric oxide synthase inhibitor with an IC50 of 28.48 ± 0.05 µM. Western blot analysis demonstrated that compound 1 inhibited the LPS-induced expression of IKKα/ß (IκB kinase alpha/beta), I-κBα (inhibitor of kappa B alpha), nuclear factor kappa-B (NF-κB) p65, iNOS (inducible nitric oxide synthase), and COX-2 (cyclooxygenase-2) in RAW 264.7 cells. Furthermore, LPS-stimulated cytokine production was detected using a sandwich enzyme-linked immunosorbent assay. Compound 1 decreased interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) production in LPS-stimulated RAW 264.7 cells. In summary, the mechanism of action of 1 included the suppression of LPS-induced NO production in RAW 264.7 cells by inhibiting IKKα/ß, I-κBα, NF-κB p65, iNOS, and COX-2, and the activities of IL-6 and TNF-α.