Hibiscus Anthocyanins Extracts Induce Apoptosis by Activating AMP-Activated Protein Kinase in Human Colorectal Cancer Cells.

Apoptosis, a programmed cell death process preventing cancer development, can be evaded by cancer cells. AMP-activated protein kinase (AMPK) regulates energy levels and is a key research topic in cancer prevention and treatment. Some bioactive components of Hibiscus sabdariffa L. (HAs), including anthocyanins, have potential anticancer properties. Our study investigated the in vitro cytotoxic potential and mode of action of HAs extracts containing anthocyanins in colorectal cancer cells. The results showed that Hibiscus anthocyanin-rich extracts induced apoptosis in human colorectal cancer cells through the activation of multiple signaling pathways of AMPK. We observed the dose-response and time-dependent induction of apoptosis with HAs. Subsequently, the activation of Fas-mediated proteins triggered apoptotic pathways associated with Fas-mediated apoptosis-related proteins, including caspase-8/tBid. This caused the release of cytochrome C from the mitochondria, resulting in caspase-3 cleavage and apoptosis activation in intestinal cancer cells. These data elucidate the relationship between Has' regulation of apoptosis-related proteins in colorectal cancer cells and apoptotic pathways.

2-(4-Benzyloxy-3-methoxyphenyl)-5-(carbethoxyethylene)-7-methoxy-benzofuran, a Benzofuran Derivative, Suppresses Metastasis Effects in P53-Mutant Hepatocellular Carcinoma Cells.

2-(4-Benzyloxy-3-methoxyphenyl)-5-(carbethoxyethylene)-7-methoxy-benzofuran (BMBF), a benzofuran derivative, is an intermediate found in the process of total synthesis of ailanthoidol. Benzofuran derivatives are a class of compounds that possess various biological and pharmacological activities. The present study explored the anti-metastasis effects of BMBF in hepatocellular carcinoma (HCC). Our preliminary findings indicate that BMBF suppresses the proliferation and changes the morphology of Huh7-an HCC cell line with a mutated p53 gene (Y220C). According to a scratching motility assay, non-cytotoxic concentrations of BMBF significantly inhibited the motility and migration in Huh7 cells. BMBF upregulated the expression of E-cadherin and downregulated the expression of vimentin, Slug, and MMP9, which are associated with epithelial-mesenchymal transition (EMT) and metastasis in Huh7 cells. BMBF decreased the expression of integrin α7, deactivated its downstream signal FAK/AKT, and inhibited p53 protein levels. Cell transfection with p53 siRNA resulted in the prevention of cell invasion because of the reduction in integrin α7, Slug, and MMP-9 in Huh7 cells. BMBF had anti-metastatic effects in PLC/PRF/5-an HCC cell line with R249S, a mutated p53 gene. Our findings indicate that BMBF has anti-metastatic effects in downregulating p53 and mediating the suppression of integrin α7, EMT, and MMP-9 in HCC cells with a mutated p53 gene.

Inhibition of MZF1/c-MYC Axis by Cantharidin Impairs Cell Proliferation in Glioblastoma.

Myeloid zinc finger 1 (MZF1), also known as zinc finger protein 42, is a zinc finger transcription factor, belonging to the Krüppel-like family that has been implicated in several types of malignancies, including glioblastoma multiforme (GBM). MZF1 is reportedly an oncogenic gene that promotes tumor progression. Moreover, higher expression of MZF1 has been associated with a worse overall survival rate among patients with GBM. Thus, MZF1 may be a promising target for therapeutic interventions. Cantharidin (CTD) has been traditionally used in Chinese medicine to induce apoptosis and inhibit cancer cell proliferation; however, the mechanism by which CTD inhibits cell proliferation remains unclear. In this study, we found that the expression of MZF1 was higher in GBM tissues than in adjacent normal tissues and low-grade gliomas. Additionally, the patient-derived GBM cells and GBM cell lines presented higher levels of MZF1 than normal human astrocytes. We demonstrated that CTD had greater anti-proliferative effects on GBM than a derivative of CTD, norcantharidin (NCTD). MZF1 expression was strongly suppressed by CTD treatment. Furthermore, MZF1 enhanced the proliferation of GBM cells and upregulated the expression of c-MYC, whereas these effects were reversed by CTD treatment. The results of our study suggest that CTD may be a promising therapeutic agent for patients with GBM and suggest a promising direction for further investigation.

Suppression of the Proliferation of Huh7 Hepatoma Cells Involving the Downregulation of Mutant p53 Protein and Inactivation of the STAT 3 Pathway with Ailanthoidol.

Ailanthoidol (ATD) has been isolated from the barks of Zanthoxylum ailanthoides and displays anti-inflammatory, antioxidant, antiadipogenic, and antitumor promotion activities. Recently, we found that ATD suppressed TGF-ß1-induced migration and invasion of HepG2 cells. In this report, we found that ATD exhibited more potent cytotoxicity in Huh7 hepatoma cells (mutant p53: Y220C) than in HepG2 cells (wild-type p53). A trypan blue dye exclusion assay and colony assay showed ATD inhibited the growth of Huh7 cells. ATD also induced G1 arrest and reduced the expression of cyclin D1 and CDK2. Flow cytometry analysis with Annexin-V/PI staining demonstrated that ATD induced significant apoptosis in Huh7 cells. Moreover, ATD increased the expression of cleaved PARP and Bax and decreased the expression of procaspase 3/8 and Bcl-xL/Bcl-2. In addition, ATD decreased the expression of mutant p53 protein (mutp53), which is associated with cell proliferation with the exploration of p53 siRNA transfection. Furthermore, ATD suppressed the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) and the expression of mevalonate kinase (MVK). Consistent with ATD, the administration of S3I201 (STAT 3 inhibitor) reduced the expression of Bcl-2/Bcl-xL, cyclin D1, mutp53, and MVK. These results demonstrated ATD's selectivity against mutp53 hepatoma cells involving the downregulation of mutp53 and inactivation of STAT3.

Neochlorogenic Acid Attenuates Hepatic Lipid Accumulation and Inflammation via Regulating miR-34a In Vitro.

Neochlorogenic acid (5-Caffeoylquinic acid; 5-CQA), a major phenolic compound isolated from mulberry leaves, possesses anti-oxidative and anti-inflammatory effects. Although it modulates lipid metabolism, the molecular mechanism is unknown. Using an in-vitro model of nonalcoholic fatty liver disease (NAFLD) in which oleic acid (OA) induced lipid accumulation in HepG2 cells, we evaluated the alleviation effect of 5-CQA. We observed that 5-CQA improved OA-induced intracellular lipid accumulation by downregulating sterol regulatory element-binding protein 1 (SREBP1) and fatty acid synthase (FASN) expression, which regulates the fatty acid synthesis, as well as SREBP2 and HMG-CoA reductases (HMG-CoR) expressions, which regulate cholesterol synthesis. Treatment with 5-CQA also increased the expression of fatty acid ß-oxidation enzymes. Remarkably, 5-CQA attenuated OA-induced miR-34a expression. A transfection assay with an miR-34a mimic or miR-34a inhibitor revealed that miR-34a suppressed Moreover, Sirtuin 1 (SIRT1) expression and inactivated 5' adenosine monophosphate-activated protein kinase (AMPK). Our results suggest that 5-CQA alleviates lipid accumulation by downregulating miR-34a, leading to activation of the SIRT1/AMPK pathway.

Ailanthoidol, a Neolignan, Suppresses TGF-ß1-Induced HepG2 Hepatoblastoma Cell Progression.

Ailanthoidol (ATD), a neolignan, possessed an antitumor promotion effect in the mouse skin model in our previous investigation. However, other antitumor properties remain to be elucidated. Liver cancer is a major cause of death in the world, and its prognosis and survival rate are poor. Therefore, the prevention and therapy of liver cancer have received much attention. TGF (transforming growth factor)-ß1, a cytokine, plays a critical role in the progression of liver cancer. This study determined the inhibitory effects of ATD on the migration and invasion induced by TGF-ß1 in HepG2 hepatoblastoma cells. Furthermore, ATD reduced the TGF-ß1-promoted colony number of HepG2 hepatoblastoma cells. In addition to reversing TGF-ß1-induced cell scattering, ATD suppressed TGF-ß1-induced expression of integrin α3, vimentin, N-cadherin, and matrix metalloproteinase 2 (MMP2). Finally, this study found that ATD significantly inhibited TGF-ß1-promoted phosphorylation of p-38 mitogen-activated protein kinase (MAPK) and Smad 2. Furthermore, the administration of SB203580 (p38MAPK inhibitor) suppressed TGF-ß1-induced expression of integrin α3, N-cadherin, and MMP2. These results demonstrate a novel mechanism of ATD against progression of liver cancer.

Inhibitory Effect of Nelumbo nucifera Leaf Extract on 2-Acetylaminofluorene-induced Hepatocarcinogenesis Through Enhancing Antioxidative Potential and Alleviating Inflammation in Rats.

Leaf extract of Nelumbo nucifera (NLE) has been demonstrated to possess anti-atherosclerosis, improve alcohol-induced steatohepatitis, prevent high-fat diet-induced obesity, and inhibit the proliferation and metastasis of human breast cancer cells. This study determines the chemopreventive role of NLE against 2-acetylaminofluorene (AAF)-induced hepatocellular carcinoma (HCC) in rats. AAF was used to induce hepatocarcinogenesis in rats through genetic and nongenetic effects. After administration for 12 weeks, NLE (0.5-2%) supplementation orally inhibited AAF (0.03%)-induced hepatic fibrosis which appears during the development of premalignant lesions in rats. After the 6-month experiment, NLE supplementation resulted in decreasing AAF-induced serum parameters of hepatic injury, including the level of triglycerides, total cholesterol, alpha-fetoprotein (AFP), and inflammatory mediator interleukin (IL)-6 and tumor necrosis factor (TNF)-α as well as the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (γGT). NLE supplementation also reduced AAF-induced lipid peroxidation and 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation in the rat liver. Hepatic histopathological investigation revealed that NLE supplementation attenuated the AAF-induced HCC and glutathione S-transferase-Pi (GST-Pi) expression. Furthermore, NLE supplementation increased the expression of transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream targets, including catalase, glutathion peroxidase (GPx), and superoxide dismutase 1 (SOD-1) in the rat liver. Our findings indicate that NLE supplementation inhibited AAF-induced hepatocarcinogenesis by enhancing antioxidative potential and alleviating inflammation in rats.

Protective Effects of Morus Root Extract (MRE) Against Lipopolysaccharide-Activated RAW264.7 Cells and CCl4-Induced Mouse Hepatic Damage.

BACKGROUND/AIMS: Inflammation is one of the main contributors to chronic diseases such as cancer. It is of great value to identify the potential activity of various medicinal plants for regulating or blocking uncontrolled chronic inflammation. We investigated whether the root extract of Morus australis possesses antiinflammatory and antioxidative stress potential and hepatic protective activity. METHODS: The microwave-assisted extractionwere was used to prepare the ethanol extract from the dried root of Morus australis (MRE), including polyphenolic and flavonoid contents. Lipopolysaccharide (LPS)-stimulated RAW264.7 cells was examined the anti-inflammatory and anti-oxidative potential of MRE. CCl4-induced mouse hepatic damage were performed to detect the hepatic protective potential in vivo. Immunohistochemistry (IHC) and western blot assays were used to detect target proteins. RESULTS: MRE contained approximately 23% phenolic compounds and 3% flavonoids. The major flavonoid component of MRE was morusin. MRE and morusin inhibited lipopolysaccharide-induced production of nitrite and prostaglandin E2 in RAW264.7 cells. MRE and morusin also suppressed the formation of intracellular reactive oxygen species and the expression of iNOS and COX-2. In an in vivo study, a thiobarbituric acid reactive substances assay showed that MRE inhibited CCl4-induced oxidative stress and expression of nitrotyrosine. MRE also decreased CCl4-induced hepatic iNOS and COX-2 expression, as well as CCl4-induced hepatic inflammation and necrosis in mice. CONCLUSION: MRE exhibited antiinflammatory and hepatic protective activity.

Mulberry leaf extract inhibit hepatocellular carcinoma cell proliferation via depressing IL-6 and TNF-α derived from adipocyte.

Epidemiological studies have revealed that obesity and being overweight are associated with increased cancer risk. Adipose tissue is regarded as an endocrine organ that secretes proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), which are related to the progression of hepatocellular carcinoma (HCC). In this study, adipocytes from 3T3-L1 cells were induced and stained with Oil Red O, which revealed marked intracellular lipid accumulation. Adding 15% conditioned medium (CM) from adipogenic -differentiated 3T3-L1 cells, which contained adipocyte-derived factors, to a culture medium of HepG2 cells was discovered to promote cell proliferation by a factor of up to 1.3 compared with the control. Mulberry leaf extract (MLE), with major components including chlorogenic acid and neochlorogenic acid, was revealed to inhibit CM-promoted HepG2 cell proliferation. The inhibitory effect of MLE on the proliferation of the signal network was evaluated. Expression of the CM-activated IκB/NFκB, STAT3, and Akt/mTOR pathways were reduced when MLE was administered. Although adipocyte-derived factors are complex, administrating anti-TNF-α and anti-IL-6 revealed that MLE blocks signal activation promoted by TNF-α and IL-6. Taken together, these results demonstrated that MLE targets the proliferation signal pathway of the inflammatory response of adipocytes in HCC and could be to prevent obesity-mediated liver cancer.

Inhibition of the Proliferation and Invasion of C6 Glioma Cells by Tricin via the Upregulation of Focal-Adhesion-Kinase-Targeting MicroRNA-7.

Tricin, a natural flavonoid present in large amounts in rice bran, was investigated for the mechanisms by which it exhibited antiproliferation and anti-invasion in C6 glioma cells. The results indicated that treatment with 5, 10, 25, and 50 µM tricin for 48 h significantly ( p < 0.05) inhibited cell numbers and colony numbers with values of 134.3 ± 5.5, 114.6 ± 2.5, 106.3 ± 3.2, and 57.3 ± 10.2, respectively. Tricin also inhibited C6-cell motility, migration, and invasion. Tricin changed the cytoskeletal organization, reduced matrix-metalloproteinase (MMP) expression, and upregulated E-cadherin. Tricin decreased FAK protein levels and suppressed focal-adhesion-kinase (FAK)-downstream-signal activation. Most importantly, tricin dose-dependently upregulated microRNA-7 (miR-7). Transfection with an miR-7 inhibitor suppressed miR-7 expression, increased FAK expression, and promoted the proliferation and invasion in C6 cells. The data support a novel anticancer mechanism of tricin that involves upregulation of FAK-targeting miR-7 in C6 glioma cells.

Stimulation of Fas/FasL-mediated apoptosis by luteolin through enhancement of histone H3 acetylation and c-Jun activation in HL-60 leukemia cells.

Luteolin (3',4',5,7-tetrahydroxyflavone), which exists in fruits, vegetables, and medicinal herbs, is used in Chinese traditional medicine for treating various diseases, such as hypertension, inflammatory disorders, and cancer. However, the gene-regulatory role of luteolin in cancer prevention and therapy has not been clarified. Herein, we demonstrated that treatment with luteolin resulted in a significant decrease in the viability of human leukemia cells. In the present study, by evaluating fragmentation of DNA and poly (ADP-ribose) polymerase (PARP), we found that luteolin was able to induce PARP cleavage and nuclear fragmentation as well as an increase in the sub-G0 /G1 fraction. In addition, luteolin also induced Fas and Fas ligand (FasL) expressions and subsequent activation of caspases-8 and -3, which can trigger the extrinsic apoptosis pathway, while knocking down Fas-associated protein with death domain (FADD) prevented luteolin-induced PARP cleavage. Immunoblot and chromatin immunoprecipitation (ChIP) analyses revealed that luteolin increased acetylation of histone H3, which is involved in the upregulation of Fas and FasL. Moreover, both the extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways are involved in luteolin-induced histone H3 acetylation. Finally, luteolin also activated the c-Jun signaling pathway, which contributes to FasL, but not Fas, gene expression and downregulation of c-Jun expression by small interfering RNA transfection which resulted in a significant decrease in luteolin-induced PARP cleavage. Thus, our results demonstrate that luteolin induced apoptosis of HL-60 cells, and this was associated with c-Jun activation and histone H3 acetylation-mediated Fas/FasL expressions.

Involvement of the antioxidative property of morusin in blocking phorbol ester-induced malignant transformation of JB6 P+ mouse epidermal cells.

Chemoprevention has been acknowledged as an important and practical strategy for managing cancer. We have previously synthesized morusin, a prenylated flavonoid that exhibits anti-cancer progression activity. In the present study, we evaluated the anti-cancer promotion potential of morusin by using the mouse epidermal JB6 P+ cell model. Extensive evidence shows that tumor promotion by phorbol esters is due to the stimulation of reactive oxygen species (ROS). Therefore, the effect of morusin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ROS production was assessed. Noncytotoxic concentrations of morusin were found to dose-dependently reduce TPA-induced ROS production. Moreover, morusin inhibited TPA-induced activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB) activation, which can mediate cell proliferation and malignant transformation. Furthermore, morusin inhibited the TPA upregulation of cyclooxygenase 2 (COX-2), which may be regulated by AP-1 and NF-κB. In addition, noncytotoxic concentrations of morusin reduced the TPA-promoted cell growth of JB6 P+ cells and inhibited TPA-induced malignant properties, such as cytoskeletal rearrangement and cell migration of JB6 P+ cells. Similar to the effects of glutathione (GSH) pretreatment, morusin inhibited TPA-induced expression of N-cadeherin and vimentin, which are malignant cell surface proteins. Finally, morusin treatment dose-dependently suppressed the TPA-induced anchorage-independent cell transformation of JB6 P+ cells. In conclusion, our results evidence that morusin possesses anti-cancer promotion potential because of its antioxidant property, which mediates multiple transformation-associated gene expression.

Inhibition of MDA-MB-231 breast cancer cell proliferation and tumor growth by apigenin through induction of G2/M arrest and histone H3 acetylation-mediated p21WAF1/CIP1 expression.

Apigenin (4',5,7-trihydroxyflavone), a flavonoid commonly found in fruits and vegetables, has anticancer properties in various malignant cancer cells. However, the molecular basis of the anticancer effect remains to be elucidated. In this study, we investigated the cellular mechanisms underlying the induction of cell cycle arrest by apigenin. Our results showed that apigenin at the nonapoptotic induction concentration inhibited cell proliferation and induced cell cycle arrest at the G2/M phase in the MDA-MB-231 breast cancer cell line. Immunoblot analysis indicated that apigenin suppressed the expression of cyclin A, cyclin B, and cyclin-dependent kinase-1 (CDK1), which control the G2-to-M phase transition in the cell cycle. In addition, apigenin upregulated p21WAF1/CIP1 and increased the interaction of p21WAF1/CIP1 with proliferating cell nuclear antigen (PCNA), which inhibits cell cycle progression. Furthermore, apigenin significantly inhibited histone deacetylase (HDAC) activity and induced histone H3 acetylation. The subsequent chromatin immunoprecipitation (ChIP) assay indicated that apigenin increased acetylation of histone H3 in the p21WAF1/CIP1 promoter region, resulting in the increase of p21WAF1/CIP1 transcription. In a tumor xenograft model, apigenin effectively delayed tumor growth. In these apigenin-treated tumors, we also observed reductions in the levels of cyclin A and cyclin B and increases in the levels of p21WAF1/CIP1 and acetylated histone H3. These findings demonstrate for the first time that apigenin can be used in breast cancer prevention and treatment through epigenetic regulation. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 434-444, 2017.

Moniliformediquinone as a potential therapeutic agent, inactivation of hepatic stellate cell and inhibition of liver fibrosis in vivo.

BACKGROUND: Moniliformediquinone (MFD), a phenanthradiquinone in Dendrobium moniliforme, was synthesized in our laboratory. Beyond its in vitro inhibitory effects on cancer cells, other biological activity of MFD is unknown. The purpose of the present study was to investigate the effects of MFD on hepatic fibrogenesis in vitro and in vivo. METHODS: Hepatic stellate HSC-T6 was cultured. Cell viability assay and western blot analyses were performed. Male ICR mice were evaluated on CCl4-induced hepatotoxicity using both histological examination and immunohistochemical staining. RESULTS: First, in vitro study showed that the synthesized MFD effectively attenuated the expression of transforming growth factor-ß1 (TGF-ß1), connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), and type I collagen (COL-1), which modulated the hepatic fibrogenesis. Furthermore, MFD reduced the phosphorylation of p65 NFκB in HSC-T6 cells. In vivo, liver fibrosis was induced by CCl4 twice a week for 10 weeks in mice. The administration of the MFD was started after 1 week of CCl4 thrice-weekly; the MFD significantly reduced plasma aspartate transaminase (AST) and lactose dehydrogenase (LDH) as well as hepatic hydroxy-proline, α-SMA, and COL-1 expression in CCl4-treated mice. Pathological analysis showed that the MFD alleviated CCl4-induced hepatic inflammation, necrosis and fibrosis. These results suggest that MFD possesses therapeutic potential for liver fibrosis. CONCLUSIONS: The synthesized MFD exhibits anti-fibrotic potential by inactivation of HSCs in vitro and decreases mouse hepatic fibrosis in vivo. Further investigation into their clinical therapeutic potential is required.

Solanum nigrum Suppress Angiogenesis-Mediated Tumor Growth Through Inhibition of the AKT/mTOR Pathway.

Solanum nigrum L., an edible plant and local dish, has been assigned anticancer activities. However, the anticancer mechanisms of S. nigrum are poorly understood. Here, we investigated whether the water or polyphenol extracts of S. nigrum (SNWE or SNPE) could inhibit angiogenesis-mediated tumor growth. In nude mice bearing tumor xenografts, SNWE or SNPE significantly reduced the volume and weight of the tumors, and decreased the expression of CD31, a marker for angiogenesis. SNWE or SNPE was found to inhibit the VEGF-induced capillary structure formation of endothelial cells. The chicken egg chorioallantoic membrane (CAM) and matrigel plug assays showed further that SNWE or SNPE inhibited tumor angiogenesis. In human umbilical vascular endothelial cells (HUVECs), SNWE or SNPE suppressed the VEGF-induced activation of AKT and mTOR. Moreover, SNWE or SNPE inhibited the viability of human hepatoma HepG2 cells, and these effects were correlated with the extent of inhibition of the AKT/mTOR pathway. Taken together, our data imply that SNWE or SNPE downregulated the AKT/mTOR pathway in HUVECs and HepG2 cells, which lead to reduced tumor growth and angiogenesis.

Antitumor progression potential of morusin suppressing STAT3 and NFκB in human hepatoma SK-Hep1 cells.

Morusin is a prenylated flavonoid that has been isolated from the root bark of the mulberry tree (Morus species, Moraceae), a Chinese traditional medicine. It has been synthesized by our laboratory from commercially available phloroglucinol, and has demonstrated to possess antitumor effects of cell lines including A549, MCF-7, and MDA-MB-231. In this study, at non-cytotoxic concentrations, morusin altered invasive morphology and suppressed cell-matrix adhesion, cell motility and cell invasion in SK-Hep1 cells. Morusin also increased the expression of E-cadherin, an epithelial cell junction protein, decreased the expression of vimentin, a mesecnchymal marker, and α2-, α6-, ß1- integrin, which regulated cancer attachment and migration. In addition, morusin reduced the activity of matrix metalloproteinase-2 and 9 (MMP-2 and MMP-9), which were involved in extracellular matrix (ECM) degradation and promoting cancer cell invasion. Furthermore, morusin suppressed the signal transducer and activator of transcription 3 (STAT3) and nuclear factor-κB (NFκB) signaling pathways, which modulate the protein expression involved in the invasion process. Finally, morusin decreased the lung colonization of the SK-Hep1 cells in the nude mice. These results indicate morusin possesses antitumor progression potential through suppressing STAT3 and NFκB.

Quercetin induces mitochondrial-derived apoptosis via reactive oxygen species-mediated ERK activation in HL-60 leukemia cells and xenograft.

Quercetin is a plant-derived bioflavonoid that was recently shown to have multiple anticancer activities in various solid tumors. Here, novel molecular mechanisms through which quercetin exerts its anticancer effects in acute myeloid leukemia (AML) cells were investigated. Results from Western blot and flow cytometric assays revealed that quercetin significantly induced caspase-8, caspase-9, and caspase-3 activation, poly ADP-ribose polymerase (PARP) cleavage, and mitochondrial membrane depolarization in HL-60 AML cells. The induction of PARP cleavage by quercetin was also observed in other AML cell lines: THP-1, MV4-11, and U937. Moreover, treatment of HL-60 cells with quercetin induced sustained activation of extracellular signal-regulated kinase (ERK), and inhibition of ERK by an ERK inhibitor significantly abolished quercetin-induced cell apoptosis. MitoSOX red and 2',7'-dichlorofluorescin fluorescence, respectively, showed that mitochondrial superoxide and intracellular peroxide levels were higher in quercetin-treated HL-60 cells compared with the control group. Moreover, both N-acetylcysteine and the superoxide dismutase mimetic, MnTBAP, reversed quercetin-induced intracellular reactive oxygen species production, ERK activation, and subsequent cell death. The in vivo xenograft mice experiments revealed that quercetin significantly reduced tumor growth through inducing intratumoral oxidative stress while activating the ERK pathway and subsequent cell apoptosis in mice with HL-60 tumor xenografts. In conclusions, our results indicated that quercetin induced cell death of HL-60 cells in vitro and in vivo through induction of intracellular oxidative stress following activation of an ERK-mediated apoptosis pathway.

Ethyl acetate extract of Wedelia chinensis inhibits tert-butyl hydroperoxide-induced damage in PC12 cells and D-galactose-induced neuronal cell loss in mice.

BACKGROUND: Wedelia chinensis is traditionally used as a hepatoprotective herb in Taiwan. The aim of this study was to evaluate the neuroprotective potential of W. chinensis. METHODS: An ethyl acetate extract of W. chinensis (EAW) was prepared and analyzed by HPLC. The neuroprotective potential of EAW was assessed by tert-butylhydroperoxide (t-BHP)-induced damage in PC12 cells and D-galactose-induced damage in mouse cortex. RESULTS: EAW exhibited potent radical scavenging property and highly contained luteolin and wedelolactone. EAW decreased t-BHP-induced reactive oxygen species (ROS) accumulation, cytotoxicity and apoptosis in PC12 cells. EAW and its major constituents blocked t-BHP-induced cytochrome C release and Bcl-2 family protein ratio change. EAW and its major constituents increased the endogenous antioxidant capacity evaluated by the binding activity assay of nuclear factor E2-related factor 2 (Nrf2) to antioxidant response element (ARE) and nuclear translocation of Nrf2 respectively in PC12 cells. Finally, EAW inhibited D-galactose-induced lipid peroxidation, apoptosis and neuron loss in the cerebral cortex of mice. CONCLUSION: These results demonstrate that W. chinensis has neuroprotective potential through blocking oxidative stress-induced damage and that luteolin and wedelolactone contribute to the protective action.

Activation of neutral-sphingomyelinase, MAPKs, and p75 NTR-mediating caffeic acid phenethyl ester-induced apoptosis in C6 glioma cells.

BACKGROUND: Caffeic acid phenethyl ester (CAPE), a component of propolis, is reported to possess anti-inflammatory, anti-bacterial, anti-viral, and anti-tumor activities. Previously, our laboratory demonstrated the in vitro and in vivo bioactivity of CAPE and addressed the role of p53 and the p38 mitogen-activated protein kinase (MAPK) pathway in regulating CAPE-induced apoptosis in C6 glioma cells. RESULTS: C6 cancer cell lines were exposed to doses of CAPE; DNA fragmentation and MAPKs and NGF/P75NTR levels were then determined. SMase activity and ceramide content measurement as well as western blotting analyses were performed to clarify molecular changes. The present study showed that CAPE activated neutral sphingomyelinase (N-SMase), which led to the ceramide-mediated activation of MAPKs, including extracellular signal-regulated kinase (ERK), Jun N-terminus kinase (JNK), and p38 MAPK. In addition, CAPE increased the expression of nerve growth factor (NGF) and p75 neurotrophin receptor (p75NTR). The addition of an N-SMase inhibitor, GW4869, established that NGF/p75NTR was the downstream target of N-SMase/ceramide. Pretreatment with MAPK inhibitors demonstrated that MEK/ERK and JNK acted upstream and downstream, respectively, of NGF/p75NTR. Additionally, CAPE-induced caspase 3 activation and poly [ADP-ribose] polymerase cleavage were reduced by pretreatment with MAPK inhibitors, a p75NTR peptide antagonist, or GW4869. CONCLUSIONS: Taken together, N-SMase activation played a pivotal role in CAPE-induced apoptosis by activation of the p38 MAPK pathway and NGF/p75NTR may explain a new role of CAPE induced apoptosis in C6 glioma.

Luteolin enhances paclitaxel-induced apoptosis in human breast cancer MDA-MB-231 cells by blocking STAT3.

The potential use of low-dose chemotherapy has been appealing because lower dosages are more attainable during cancer therapy and cause less toxicity in patients. Combination therapy of paclitaxel, a promising frontline chemotherapy agent, with natural anti-tumor agents that are considerably less toxic and possess the capability of activating additional apoptotic signals may provide a rational molecular basis for novel chemotherapeutic strategies. Luteolin, a natural flavone, possesses multiple biological activities, including anti-tumor potential. In the present study, the effects of concomitant administration of luteolin and paclitaxel were investigated in human breast cancer MDA-MB-231 cells. Luteolin alone demonstrated an anti-proliferative effect. Co-administration of luteolin and paclitaxel resulted in an increase in apoptosis compared with the treatment of paclitaxel alone as evidenced by the results of a diamidino-2-phenylindole (DAPI) stain and Annexin-V-based assay. Moreover, immunoblotting analysis also showed that the co-administration of luteolin and paclitaxel activated caspase-8 and caspase-3 and increased the expression of Fas. Furthermore, the increased expression of Fas due to co-administration was shown to be due to the blocking of signal transducer and activator of transcription 3 (STAT3). Finally, combination therapy with luteolin and paclitaxel significantly reduced tumor size and tumor weight in an orthotopic tumor model of MDA-MB-231 cells in nude mice. These results suggest that the luteolin-paclitaxel combination could be a novel strategy for the treatment of breast cancer.