Raphanus sativus Linne Protects Human Nucleus Pulposus Cells against H2O2-Induced Damage by Inhibiting TREM2.

Intervertebral disc degeneration (IDD) progresses owing to damage and depletion of nucleus pulposus (NP) cells. Cytoprotection mitigates oxidative stress, nutrient deprivation, and mechanical stress, which lead to cell damage and necrosis. We aimed to examine the protective effect of Raphanus sativus Linne (RSL), common radish, against oxidative stress by H2O2 in human NP cells and whether the RSL extracts can inhibit triggering receptor expressed on myeloid cells 2 (TREM2), an inducer of apoptosis and degeneration in NP cells. We administered hydrogen peroxide (H2O2) to cultured human NP cells treated with RSL extracts. We used immunoblotting and quantitative PCR to investigate expression of the apoptosis-associated proteins in cultured cells. RSL significantly enhanced cell survival by suppressing the activation of cleaved caspase-3 and Bax. In contrast, RSL extract increased Bcl2 concentration to downregulate apoptosis. Additionally, RSL treatment notably enhanced the mRNA levels of ACAN and Col2a1 while significantly reducing those of ADAMTS-4, ADAMTS-5, MMP3, and MMP13, key genes involved in NP degeneration. While H2O2 elevated TREM2 expression, causing disc degeneration, RSL downregulated TREM2 expression. Thus, our findings imply that RSL supports human NP cells under oxidative stress and regulates the pathways underlying disc degeneration, particularly TREM2, and that RSL extracts may potentially prevent IDD.

Repeated epidural delivery of Shinbaro2: effects on neural recovery, inflammation, and pain modulation in a rat model of lumbar spinal stenosis.

The choice of treatment for lumbar spinal stenosis (LSS) depends on symptom severity. When severe motor issues with urinary dysfunction are not present, conservative treatment is often considered to be the priority. One such conservative treatment is epidural injection, which is effective in alleviating inflammation and the pain caused by LSS-affected nerves. In this study, Shinbaro2 (Sh2), pharmacopuncture using natural herbal medicines for patients with disc diseases, is introduced as an epidural to treat LSS in a rat model. The treatment of primary sensory neurons from the rats' dorsal root ganglion (DRG) neurons with Sh2 at various concentrations (0.5, 1, and 2 mg/mL) was found to be safe and non-toxic. Furthermore, it remarkably stimulated axonal outgrowth even under H2O2-treated conditions, indicating its potential for stimulating nerve regeneration. When LSS rats received epidural injections of two different concentrations of Sh2 (1 and 2 mg/kg) once daily for 4 weeks, a significant reduction was seen in ED1+ macrophages surrounding the silicone block used for LSS induction. Moreover, epidural injection of Sh2 in the DRG led to a significant suppression of pain-related factors. Notably, Sh2 treatment resulted in improved locomotor recovery, as evaluated by the Basso, Beattie, and Bresnahan scale and the horizontal ladder test. Additionally, hind paw hypersensitivity, assessed using the Von Frey test, was reduced, and normal gait was restored. Our findings demonstrate that epidural Sh2 injection not only reduced inflammation but also improved locomotor function and pain in LSS model rats. Thus, Sh2 delivery via epidural injection has potential as an effective treatment option for LSS.

Mume Fructus reduces interleukin-1 beta-induced cartilage degradation via MAPK downregulation in rat articular chondrocytes.

Osteoarthritis is the most prevalent type of degenerative arthritis. It is characterized by persistent pain, joint dysfunction, and physical disability. Pain relief and inflammation control are prioritised during osteoarthritis treatment Mume Fructus (Omae), a fumigated product of the Prunus mume fruit, is used as a traditional medicine in several Asian countries. However, its therapeutic mechanism of action and effects on osteoarthritis and articular chondrocytes remain unknown. In this study, we analyzed the anti-osteoarthritis and articular regenerative effects of Mume Fructus extract on rat chondrocytes. Mume Fructus treatment reduced the interleukin-1ß-induced expression of matrix metalloproteinase 3, matrix metalloproteinase 13, and a disintegrin and metalloproteinase with thrombospondin type 1 motifs 5. Additionally, it enhanced collagen type II alpha 1 chain and aggrecan accumulation in rat chondrocytes. Furthermore, Mume Fructus treatment regulated the inflammatory cytokine levels, mitogen-activated protein kinase phosphorylation, and nuclear factor-kappa B activation. Overall, our results demonstrated that Mume Fructus inhibits osteoarthritis progression by inhibiting the nuclear factor-kappa B and mitogen-activated protein kinase pathways to reduce the levels of inflammatory cytokines and prevent cartilage degeneration. Therefore, Mume Fructus may be a potential therapeutic option for osteoarthritis.

Immune-boosting effect of Yookgong-dan against cyclophosphamide-induced immunosuppression in mice.

Immune responses must be strictly regulated to prevent autoimmune and infectious diseases and to protect against infectious agents. As people age, their immunity wanes, leading to a decrease in lymphocyte production in bone marrow and thymus and a decline in the efficacy of mature lymphocytes in secondary lymphoid organs. This study explores the immune-boosting potential of Yookgong-dan (YGD) in enhancing the immune system by activating immune cells. In our in vitro experiments, cyclophosphamide (Cy) treatment led to a significant decrease in primary splenocyte viability. However, subsequent treatment with YGD significantly improved cell viability, with doses ranging between 1 and 25 µg/mL in Cy-treated splenocytes. Flow cytometry analysis demonstrated that the Cy group exhibited reduced positivity of CD3+ T cells and CD45+ leukocytes compared to the blank group. In contrast, treatment with YGD led to a notable, dose-responsive increase in these immune cell types. In our in vivo experiments, YGD was orally administered to Cy-induced immunosuppressed mice at 20 and 100 mg/kg doses for 10 days. The results indicated a dose-dependent elevation in immunoglobulin (Ig)G and IgM levels in the serum, emphasizing the immunostimulatory effect of YGD. Furthermore, the Cy-treated group showed decreased T cells, B (CD19+) cells, and leukocytes in the total splenocyte population. Yet, YGD treatment resulted in a dose-dependent reversal of this pattern, suggesting its ability to counter immunosuppression. Notably, YGD was found to effectively stimulate T (CD4+ and CD8+) lymphocyte subsets and natural killer cells, along with enhancing Th1/Th2 cytokines in immunosuppressed conditions. These outcomes correlated with the modulation of BCL-2 and BAX expression, which are critical for apoptosis. In conclusion, YGD has the potential to bolster immune functionality through the activation of immune cells, thereby enhancing the immune system's capacity to combat diseases and improve overall health and wellness.

Protective effect of Luffa cylindrica Roemer against dexamethasone-induced muscle atrophy in primary rat skeletal muscle cells.

Glucocorticoids (GCs) are commonly used in the treatment of chronic inflammatory conditions. However, the administration of high doses and long-term use of GCs can induce muscle atrophy (MA) in patients, leading to a decline in quality of life and increased mortality. MA leads to protein degradation in skeletal muscle, resulting in a reduction of muscle mass. This process is triggered by GCs like dexamethasone (DEX), which induce the expression of E3 ubiquitin ligases, namely Atrogin-1 and muscle RING-finger protein-1 (MuRF1). In this study, we examined the anti-MA potential of Luffa cylindrica Roemer (LCR) on DEX-treated primary skeletal myotubes. Primary skeletal myotubes stimulated with LCR alone resulted in a significant upregulation of myotube development, characterized by an increase in both the number and diameter of myotubes. Contrastingly, combined treatment with LCR and DEX reduced the expression of Atrogin-1, while treatment with DEX alone induced the expression of MuRF1. Furthermore, LCR treatment successfully restored the number and diameter of myotubes that had been diminished by DEX treatment. These findings suggest that LCR holds potential for treating MA, as an accelerating effect on muscle development and anti-MA effects on primary skeletal muscle cells were observed.

Shinbaro2 enhances axonal extension beyond the glial scar for functional recovery in rats with contusive spinal cord injury.

Spinal cord injury (SCI) is a devastating event that often results in the inflammatory condition of glial scar tissue formation, impeding neural regeneration and recovery. Reducing the inflammatory response and inhibiting glial formation are promising strategies for improving SCI outcomes. Here, we introduce a new role for Shinbaro2 (Sh2), known for its anti-inflammatory and pain-reducing effects, in ameliorating glial scars formed in the damaged spinal cord and promoting axon growth after SCI. Sh2 was applied at various concentrations to cultivate primary spinal cord neurons. Concentrations of 1 and 2 mg/mL effectively enhanced cell viability and axonal outgrowth in spinal cord neurons subjected to hydrogen peroxide or laceration injury. Sh2 helped reduce neuroinflammation by increasing anti-inflammatory M2 macrophages (arginase 1) and decreasing inflammatory cells, ultimately reducing lesion size. In scar formation, Sh2 inhibited the expression of ß-catenin and nestin in reactive astrocytes in the injured spinal cord. Moreover, Sh2 suppressed the expression of chondroitin sulfate proteoglycans and SOX9, which are involved in scar formation. Furthermore, Sh2 promoted the sprouting of serotonergic axons and the growth of neurofibrillary tangles, enhancing motor function recovery in SCI. These findings highlight the potential of Sh2 as an SCI therapeutic intervention, offering hope for neural and functional restoration in individuals with this debilitating condition.

Epidural Injection of Harpagoside for the Recovery of Rats with Lumbar Spinal Stenosis.

Epidural administration is the leading therapeutic option for the management of pain associated with lumbar spinal stenosis (LSS), which is characterized by compression of the nerve root due to narrowing of the spinal canal. Corticosteroids are effective in alleviating LSS-related pain but can lead to complications with long-term use. Recent studies have focused on identifying promising medications administered epidurally to affected spinal regions. In this study, we aimed to investigate the effectiveness of harpagoside (HAS) as an epidural medication in rats with LSS. HAS at various concentrations was effective for neuroprotection against ferrous sulfate damage and consequent promotion of axonal outgrowth in primary spinal cord neurons. When two concentrations of HAS (100 and 200 µg/kg) were administered to the rat LSS model via the epidural space once a day for 4 weeks, the inflammatory responses around the silicone block used for LSS were substantially reduced. Consistently, pain-related factors were significantly suppressed by the epidural administration of HAS. The motor functions of rats with LSS significantly improved. These findings suggest that targeted delivery of HAS directly to the affected area via epidural injection holds promise as a potential treatment option for the recovery of patients with LSS.

Epidural Injection Method for Long-Term Pain Management in Rats with Spinal Stenosis.

Epidural injection is one of the most common nonsurgical treatment options for long-term pain relief in lumbar spinal stenosis. Recently, various nerve block injections have been used for pain management. Among them, nerve block through epidural injection is a safe and effective method for the clinical treatment of low back or lower extremity pain. Although the epidural injection method has a long history, the effectiveness of long-term epidural injections in disc diseases has not been scientifically proven. In particular, to verify the safety and efficacy of drugs in preclinical studies, the route and method of drug administration in terms of the clinical application method and duration of use must be established. However, there is no standardized method for long-term epidural injections in a rat model of stenosis to identify the precise efficacy and safety of epidural injections. Therefore, standardizing the epidural injection method is very important for evaluating the efficacy and safety of drugs used for back or lower extremity pain. We describe the first standardized long-term epidural injection method for evaluating the efficacy and safety of drugs according to their route of administration in rats with lumbar spinal stenosis.

Curcuma aromatica Salisb. Protects from Acetaminophen-Induced Hepatotoxicity by Regulating the Sirt1/HO-1 Signaling Pathway.

Acetaminophen (APAP) overdose-induced hepatotoxicity reduces the activity of sirtuin-1 (Sirt1) along with heme oxygenase 1 (HO-1) and promotes inflammatory responses and oxidative stress. Although the extract of Curcuma aromatica Salisb. (CAS) possesses hepatoprotective properties, scientific evidence on whether CAS prevents hepatotoxicity and the underlying molecular mechanisms are lacking. Here, we hypothesized that CAS ameliorates hepatotoxicity by inhibiting inflammation and oxidative stress via Sirt1/HO-1 signaling. CAS pretreatment at doses of 200 and 400 µg/mL significantly increased cell viability in APAP-treated primary hepatocytes. The expression of inducible nitric oxide synthase (iNOS) substantially increased after APAP treatment; however, this expression significantly decreased in cells pretreated with 100, 200, and 400 µg/mL CAS. CAS increased Sirt1 and HO-1 levels in APAP-treated hepatocytes in a dose-dependent manner. When CAS was orally administered to mice at doses of 20 or 100 mg/kg for 7 days, the APAP-induced increase in serum aspartate aminotransferase and alanine aminotransferase levels was inhibited. Moreover, CAS decreased IL-6, TNF-α, and IL-1ß, increased IL-10, suppressed ROS generation, increased glutathione levels, inhibited iNOS and cyclooxygenase-2, and enhanced Sirt1 and HO-1 in the mouse model of APAP-induced hepatotoxicity. These findings suggest that CAS could be used as a natural hepatoprotective drug to treat APAP-induced injury.

Epigenetic Changes within the Annulus Fibrosus by DNA Methylation in Rat Intervertebral Disc Degeneration Model.

Intervertebral disc degeneration (IDD) is an age-dependent progressive spinal disease that causes chronic back or neck pain. Although aging has long been presented as the main risk factor, the exact cause is not fully known. DNA methylation is associated with chronic pain, suggesting that epigenetic modulation may ameliorate disc degeneration. We examined histological changes in the DNA methylation within the discs and their association with pain-related transient receptor potential vanilloid subtype 1 (TrpV1) expression in rats subjected to IDD. Epigenetic markers (5-hydroxymethylcytosine (5hmC), 5-methylcytosine (5Mc)), DNA methyltransferases (DNMTs), and Ten-eleven translocations (Tets) were analyzed using immunohistochemistry, real-time PCR, and DNA dot-blot following IDD. Results revealed high 5mC levels in the annulus fibrosus (AF) region within the disc after IDD and an association with TrpV1 expression. DNMT1 is mainly involved in 5mC conversion in degenerated discs. However, 5hmC levels did not differ between groups. A degenerated disc can lead to locomotor defects as assessed by ladder and tail suspension tests, no pain signals in the von Frey test, upregulated matrix metalloproteinase-3, and downregulated aggrecan levels within the disc. Thus, we found that the DNA methylation status in the AF region of the disc was mainly changed after IDD and associated with aberrant TrpV1 expression in degenerated discs.

Uwhangchungsimwon Inhibits Oxygen Glucose Deprivation/Re-Oxygenation-Induced Cell Death through Neuronal VEGF and IGF-1 Receptor Signaling and Synaptic Remodeling in Cortical Neurons.

Uwhangchungsimwon (UCW), a multi-component herbal product, has long been used to treat vascular diseases such as headache, dizziness, high blood pressure, and stroke. Though the prophylactic actions of UCW are well known, insufficient experimental evidence exists on its effectiveness against stroke. Here, we investigated the mechanism underlying the efficacy of UCW in oxygen glucose deprivation/re-oxygenation (OGD/R)-injury to the primary cortical neurons using an in vitro ischemia model. Neurons secrete vascular endothelial growth factor (VEGF), which acts as a neurotrophic factor in response to an ischemic injury. VEGF modulates neuroprotection and axonal outgrowth by activating the VEGF receptors and plays a critical role in vascular diseases. In this study, cortical neurons were pretreated with UCW (2, 10, and 50 µg/mL) for 48 h, incubated in oxygen-glucose-deprived conditions for 2 h, and further reoxygenated for 24 h. UCW effectively protected neurons from OGD/R-induced degeneration and cell death. Moreover, the role of UCW in sustaining protection against OGD/R injury is associated with activation of VEGF-VEGFR and insulin-like growth factor 1 receptor expression. Therefore, UCW is a potential herbal supplement for the prevention of hypoxic-ischemic neuronal injury as it may occur after stroke.

Chaenomeles Fructus (CF), the Fruit of Chaenomeles sinensis Alleviates IL-1ß Induced Cartilage Degradation in Rat Articular Chondrocytes.

Osteoarthritis (OA) causes persistent pain, joint dysfunction, and physical disability. It is the most prevalent type of degenerative arthritis, affecting millions of people worldwide. OA is currently treated with a focus on pain relief, inflammation control, and artificial joint surgery. Hence, a therapeutic agent capable of preventing or delaying the progression of OA is needed. OA is strongly associated with the degeneration of the articular cartilage and changes in the ECM, which are primarily associated with a decrease in proteoglycan and collagen. In the progress of articular cartilage degradation, catabolic enzymes, such as matrix metalloproteinases (MMPs), are activated by IL-1ß stimulation. Given the tight relationship between IL-1ß and ECM (extra-cellular matrix) degradation, this study examined the effects of Chaenomeles Fructus (CF) on IL-1ß-induced OA in rat chondrocytes. The CF treatment reduced IL-1ß-induced MMP3/13 and ADAMTS-5 production at the mRNA and protein levels. Similarly, CF enhanced col2a and aggrecan accumulation and chondrocyte proliferation. CF inhibited NF-κB (nuclear factor kappa B) activation, nuclear translocation induced by IL-1ß, reactive oxygen species (ROS) production, and ERK phosphorylation. CF demonstrated anti-OA and articular regeneration effects on rat chondrocytes, thus, suggesting that CF is a viable and fundamental therapeutic option for OA.

Gongjin-Dan Enhances Neurite Outgrowth of Cortical Neuron by Ameliorating H2O2-Induced Oxidative Damage via Sirtuin1 Signaling Pathway.

Gongjin-dan (GJD) is a multiherbal formula produced from 10 medicinal herbs and has been traditonally used as an oriental medicine to treat cardiovascular diseases, alcoholic hepatitis, mild dementia, and anemia. Additionally, increasing evidence suggests that GJD exerts neuroprotective effects by suppressing inflammation and oxidative stress-induced events to prevent neurological diseases. However, the mechanism by which GJD prevents oxidative stress-induced neuronal injury in a mature neuron remains unknown. Here, we examined the preventive effect and mechanism of GJD on primary cortical neurons exposed to hydrogen peroxide (H2O2). In the neuroprotection signaling pathway, Sirtuin1 is involved in neuroprotective action as a therapeutic target for neurological diseases. After pre-treatment with GJD at three concentrations (10, 25, and 50 µg/mL) and stimulation by H2O2 (30 µM) for 24 h, the influence of GJD on Sirtuin1 activation was assessed using immunocytochemistry, real-time PCR, western blotting, and flow cytometry. GJD effectively ameliorated H2O2-induced neuronal death against oxidative damage through Sirtuin1 activation. In addition, GJD-induced Sirtuin1 activation accelerated elongation of new axons and formation of synapses via increased expression of nerve growth factor and brain-derived neurotrophic factor, as well as regeneration-related genes. Thus, GJD shows potential for preventing neurological diseases via Sirtuin1 activation.

Neurotherapeutic Potential of Cervus elaphus Sibericus on Axon Regeneration and Growth Cone Reformation after H2O2-Induced Injury in Rat Primary Cortical Neurons.

Cervus elaphus sibericus (CES), commonly known as deer antler, has been used as a medicinal herb because of its various pharmacological activities, including its anti-infective, anti-arthritic, anti-allergic, and anti-oxidative properties. However, the precise mechanisms by which CES functions as a potent anti-oxidative agent remain unknown; particularly, the effects of CES on cortical neurons and its neurobiological mechanism have not been examined. We used primary cortical neurons from the embryonic rat cerebral cortex and hydrogen peroxide to induce oxidative stress and damage in neurons. After post-treatment of CES at three concentrations (10, 50, and 200 µg/mL), the influence of CES on the neurobiological mechanism was assessed by immunocytochemistry, flow cytometry, and real-time PCR. CES effectively prevented neuronal death caused by hydrogen peroxide-induced damage by regulating oxidative signaling. In addition, CES significantly induced the expression of brain-derived neurotrophic factor and neurotrophin nerve growth factor, as well as regeneration-associated genes. We also observed newly processing elongated axons after CES treatment under oxidative conditions. In addition, filopodia tips generally do not form a retraction bulb, called swollen endings. Thus, CES shows therapeutic potential for treating neurological diseases by stimulating neuron repair and regeneration.

Epigallocatechin-3-Gallate Suppresses Vasculogenic Mimicry through Inhibiting the Twist/VE-Cadherin/AKT Pathway in Human Prostate Cancer PC-3 Cells.

Vasculogenic mimicry (VM) is the alternative process of forming vessel-like networks by aggressive tumor cells, and it has an important role in tumor survival, growth, and metastasis. Epigallocatechin-3-gallate (EGCG) is well known to have diverse bioactivities including anti-cancer effects. However, the efficacy of EGCG on VM is elusive. In this study, we explored whether and how EGCG affects VM in human prostate cancer (PCa) PC-3 cells. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Invasive and VM formation abilities were assessed by an invasion assay and a three-dimensional (3D) culture VM tube formation assay, respectively. Western blots were carried out. An immunofluorescence assay was performed to detect nuclear twist expression. EGCG effectively inhibited the invasive ability, as well as tubular channel formation, without affecting cell viability. EGCG significantly downregulated the expression of vascular endothelial cadherin (VE-cadherin) and its transcription factor, twist, N-cadherin, vimentin, phosphor-AKT, and AKT, but not phospho-erythropoietin-producing hepatocellular receptor A2 (EphA2) and EphA2. In addition, EGCG diminished the nuclear localization of twist. Treatment with SC79, an AKT activator, effectively rescued EGCG-inhibited VM formation. These results demonstrated for the first time that EGCG causes marked suppression of VM through inhibiting the twist/VE-cadherin/AKT pathway in human PCa PC-3 cells.

Serum promotes vasculogenic mimicry through the EphA2/VE-cadherin/AKT pathway in PC-3 human prostate cancer cells.

AIMS: Serum is widely used for in vitro cell culture of eukaryotic cells. Although serum is well known to affect various biological activities in cancer cells, its effect in vasculogenic mimicry (VM) is not yet fully defined. Thus, this study investigated the role of serum in VM in human prostate cancer (PCa) PC-3 cells. MAIN METHODS: Invasion assay and 3D culture VM tube formation assay are performed. VM-related molecules are checked by western blot and reverse transcriptase-polymerase chain reaction. Nuclear twist is detected by confocal after twist-FITC/DAPI double staining. KEY FINDINGS: Serum dramatically induced not only invasion but also VM. Serum increased the phosphorylation of erythropoietin-producing hepatocellular A2 (EphA2) without affecting EphA2 expression. Both the protein and mRNA expression levels of vascular endothelial cadherin (VE-cadherin) are up-regulated by serum. Twist expression was increased in the nucleus by serum. Serum activated AKT through phosphorylation, despite the unchanged AKT expression. Serum caused an increase in matrix metalloproteinase-2 (MMP-2) and laminin subunit 5 gamma-2 (LAMC2) protein expressions. Wortmannin, a phosphoinositide-3-kinase inhibitor, significantly decreased serum-induced invasion and VM. SIGNIFICANCE: These results demonstrated that serum activates EphA2 and up-regulates twist/VE-cadherin, which in turn activate AKT that up-regulates MMP-2 and LAMC2, thereby inducing the invasion and VM of human PCa PC-3 cells.

Luteolin induces caspase-dependent apoptosis via inhibiting the AKT/osteopontin pathway in human hepatocellular carcinoma SK-Hep-1 cells.

AIMS: Luteolin, a naturally occurring flavonoid, possesses anti-cancer effects including induction of apoptosis. This study investigated the involvement of osteopontin (OPN) in luteolin-induced apoptosis in human hepatocellular carcinoma (HCC) SK-Hep-1 cells with high OPN expression. MAIN METHODS: MTT assay was used to determine the cell viability. Cell cycle analysis was performed to identify apoptosis. Apoptosis was confirmed by detecting cytoplasmic histone-associated-DNA-fragments using a cell death detection ELISAPLUS kit. The expression of proteins was evaluated by Western blot. Reverse transcriptase-polymerase chain reaction was employed to detect the expression of mRNA. KEY FINDINGS: Cytotoxic effect of luteolin was higher in cancer cell line SK-Hep-1 cells than in normal cell line AML12 cells. Luteolin led a significantly increase in apoptosis accompanied by activation of caspase 8, -9 and -3 and cleavage of poly (ADP-ribose) polymerase (PARP), which was completely blocked by Z-VAD-FMK, a pan caspase inhibitor. Luteolin significantly downregulated the expression of X-linked inhibitor of apoptosis (XIAP), Mcl-1 and Bid. Furthermore, luteolin effectively decreased OPN expression at both mRNA and protein level. Exogenous OPN markedly blocked apoptosis induction, caspases activation, PARP cleavage, downregulation of XIAP and Mcl-1 in luteolin-treated cells. Luteolin impaired the AKT pathway by inhibiting the phosphorylation of AKT. SC79, an AKT activator, blocked apoptosis induction, caspases activation, PARP cleavage, downregulation of OPN, XIAP, Mcl-1 and Bid in luteolin-treated cells. SIGNIFICANCE: These results demonstrated that luteolin inhibits the AKT/OPN pathway, thereby inducing caspase-dependent apoptosis in human HCC SK-Hep-1 cells with little toxicity.

Dihydroartemisinin induced caspase-dependent apoptosis through inhibiting the specificity protein 1 pathway in hepatocellular carcinoma SK-Hep-1 cells.

AIMS: Dihydroartemisinin (DHA) is a semi-synthetic derivative of artemisinin, well known for a safe and effective first-line antimalarial agent. This study investigated whether and how DHA induces apoptosis focusing on the specificity protein 1 (Sp1) pathway in hepatocellular carcinoma (HCC) SK-Hep-1 cells. MAIN METHODS: The cell viability was evaluated by MTT assay. Cell cycle analysis was performed after PI staining by flow cytometry system. Apoptosis was confirmed by DAPI staining and by detecting cytoplasmic histone-associated-DNA-fragments using a cell death detection ELISAPLUS kit. The expression of proteins involved in apoptosis was evaluated by Western blot. The nuclear localization of Sp1 was evaluated by immunofluorescence assay. KEY FINDINGS: DHA exerted potent cytotoxicity against HCC SK-Hep-1 cells compared with normal hepatocyte AML12 cells. The sub-G1 DNA content and apoptosis index were increased by DHA, which was accompanied by nuclei condensation and fragmentation. DHA activated caspase 3, caspase 8, and caspase 9 and cleaved poly (ADP-ribose) polymerase (PARP). DHA-induced apoptotic cell death, activation of caspases and cleavage of PARP were dramatically inhibited by pan caspase inhibitor Z-VAD-FMK. DHA down-regulated protein expression and nuclear localization of Sp1, which in turn decreased Sp1 downstream target protein, X-linked inhibitor of apoptosis. Decreased Sp1 protein expression by DHA was restored by proteasome inhibitor MG132. DHA led to a down-regulation of phospho-ERK, -p38 and -JNK without affecting their total forms. SIGNIFICANCE: These results demonstrate that DHA induces caspase-dependent apoptosis in HCC SK-Hep-1 cells by proteasome-dependent degradation of Sp1, which is involved in mitogen-activate protein kinase pathway.