Therapeutic Potential and Mechanisms of Novel Simple O-Substituted Isoflavones against Cerebral Ischemia Reperfusion.

Isoflavones have been widely studied and have attracted extensive attention in fields ranging from chemotaxonomy and plant physiology to human nutrition and medicine. Isoflavones are often divided into three subgroups: simple O-substituted derivatives, prenylated derivatives, and glycosides. Simple O-substituted isoflavones and their glycosides, such as daidzein (daidzin), genistein (genistin), glycitein (glycitin), biochanin A (astroside), and formononetin (ononin), are the most common ingredients in legumes and are considered as phytoestrogens for daily dietary hormone replacement therapy due to their structural similarity to 17-ß-estradiol. On the basis of the known estrogen-like potency, these above isoflavones possess multiple pharmacological activities such as antioxidant, anti-inflammatory, anticancer, anti-angiogenetic, hepatoprotective, antidiabetic, antilipidemic, anti-osteoporotic, and neuroprotective activities. However, there are very few review studies on the protective effects of these novel isoflavones and their related compounds in cerebral ischemia reperfusion. This review primarily focuses on the biosynthesis, metabolism, and neuroprotective mechanism of these aforementioned novel isoflavones in cerebral ischemia reperfusion. From these published works in in vitro and in vivo studies, simple O-substituted isoflavones could serve as promising therapeutic compounds for the prevention and treatment of cerebral ischemia reperfusion via their estrogenic receptor properties and neuron-modulatory, antioxidant, anti-inflammatory, and anti-apoptotic effects. The detailed mechanism of the protective effects of simple O-substituted isoflavones against cerebral ischemia reperfusion might be related to the PI3K/AKT/ERK/mTOR or GSK-3ß pathway, eNOS/Keap1/Nrf-2/HO-1 pathway, TLRs/TIRAP/MyD88/NFκ-B pathway, and Bcl-2-regulated anti-apoptotic pathway. However, clinical trials are needed to verify their potential on cerebral ischemia reperfusion because past studies were conducted with rodents and prophylactic administration.

Antioxidant Effects and Phytochemical Properties of Seven Taiwanese Cirsium Species Extracts.

In the present investigation, we compared the radical-scavenging activities and phenolic contents of seven Taiwanese Cirsium species with a spectrophotometric method. We further analyzed their phytochemical profiles with high-performance liquid chromatography-photodiode array detection (HPLC-DAD). We found that the flower part of Cirsium japonicum var. australe (CJF) showed the best radical-scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and the hypochlorite ion, for which the equivalents were 6.44 ± 0.17 mg catechin/g, 54.85 ± 0.66 mmol Trolox/g and 418.69 ± 10.52 mmol Trolox/g respectively. CJF also had the highest contents of total phenolics (5.23 ± 0.20 mg catechin/g) and phenylpropanoids (29.73 ± 0.72 mg verbascoside/g). According to the Pearson's correlation coefficient, there was a positive correlation between the total phenylpropanoid content and ABTS radical-scavenging activities (r = 0.979). The radical-scavenging activities of the phenylpropanoids are closely related to their reducing power (r = 0.986). HPLC chromatograms obtained in validated HPLC conditions confirm that they have different phytochemical profiles by which they can be distinguished. Only CJF contained silicristin (0.66 ± 0.03 mg/g) and silydianin (9.13 ± 0.30 mg/g). CJF contained the highest contents of apigenin (5.56 ± 0.09 mg/g) and diosmetin (2.82 ± 0.10 mg/g). Among the major constituents, silicristin had the best radical-scavenging activities against DPPH (71.68 ± 0.66 mg catechin/g) and ABTS (3.01 ± 0.01 mmol Trolox/g). However, diosmetin had the best reducing power and radical-scavenging activity against the hypochlorite anion (41.57 ± 1.14 mg mmol Trolox/g). Finally, we found that flavonolignans (especial silicristin and silydianin) and diosmetin acted synergistically in scavenging radicals.

Fisetin Inhibits Cell Proliferation through the Induction of G0/G1 Phase Arrest and Caspase-3-Mediated Apoptosis in Mouse Leukemia Cells.

Fisetin, a naturally occurring flavonoid, is found in common fruits and vegetables and has been shown to induce cytotoxic effects in many human cancer cell lines. No information has shown that fisetin induced cell cycle arrest and apoptosis in mouse leukemia WEHI-3 cells. We found that fisetin decreased total viable cells through G0/G1 phase arrest and induced sub-G1 phase (apoptosis). We have confirmed fisetin induced cell apoptosis by the formation of DNA fragmentation and induction of apoptotic cell death. Results indicated that fisetin induced intracellular Ca 2+ increase but decreased the ROS production and the levels of ΔΨ m in WEHI-3 cells. Fisetin increased the activities of caspase-3, -8 and -9. Cells were pre-treated with inhibitors of caspase-3, -8 and -9 and then treated with fisetin and results showed increased viable cell number when compared to fisetin treated only. Fisetin reduced expressions of cdc25a but increased p-p53, Chk1, p21 and p27 that may lead to G0/G1 phase arrest. Fisetin inhibited anti-apoptotic protein Bcl-2 and Bcl-xL and increased pro-apoptotic protein Bax and Bak. Furthermore, fisetin increased the protein expression of cytochrome c and AIF. Fisetin decreased cell number through G0/G1 phase arrest via the inhibition of cdc25c and induction of apoptosis through caspase-dependent and mitochondria-dependent pathways. Therefore, fisetin may be useful as a potential therapeutic agent for leukemia.

Deguelin Impairs Cell Adhesion, Migration and Invasion of Human Lung Cancer Cells through the NF-[Formula: see text]B Signaling Pathways.

Deguelin, a rotenoid, is isolated from a natural plant species, and has biological activities including antitumor function. In the present study, we investigated the effect of deguelin on the cell adhesion, migration and invasion of NCI-H292 human lung cancer cells in vitro. Cell viability was analyzed by using flow cytometer. Cell adhesion was determined by using the cell-matrix adhesion assay. Wound healing assay was used to examine cell migration. Cell migration and invasion were investigated using a Boyden chamber assay. The protein expression was measured by Western blotting and confocal laser microscopy. The electrophoretic mobility shift assay was used to measure NF-[Formula: see text]B p65 binding to DNA.We selected the concentrations of deguelin at 0, 0.5, 1.0, 1.5, 2.0 and 2.5[Formula: see text][Formula: see text]M and we found that those concentrations of deguelin did not induce significant cytotoxic effects on NCI-H292 cells. Thus, we selected those concentrations of deguelin for metastasis assay. We found that deguelin inhibited cell adhesion, migration and invasion in dose-dependent manners that was assayed by wound healing and transwell methods, respectively. Deguelin decreased the expression of MMP-2/-9, SOS 1, Rho A, p-AKT (Thr308), p-ERK1/2, p-p38, p-JNK, NF-[Formula: see text]B (p65) and uPA in NCI-H292 cells. Deguelin suppressed the expression of PI3K, SOS 1, NF-[Formula: see text]B (p65), but did not significantly affect PKC and Ras in the nuclei of NCI-H292 cells that were confirmed by confocal laser microscopy. We suggest that deguelin may be used as a novel anticancer metastasis of lung cancer in the future.

Anticancer effects of cantharidin in A431 human skin cancer (Epidermoid carcinoma) cells in vitro and in vivo.

Cantharidin (CTD), a potential anticancer agent of Traditional Chinese Medicine has cytotxic effects in different human cancer cell lines. The cytotoxic effects of CTD on A431 human skin cancer (epidermoid carcinoma) cells in vitro and in A431 cell xenograft mouse model were examined. In vitro, A431 human skin cell were treated with CTD for 24 and 48 h. Cell phase distribution, ROS production, Ca2+ release, Caspase activity and the level of apoptosis associated proteins were measured. In vivo, A431 cell xenograft mouse model were examined. CTD-induced cell morphological changes and decreased percentage of viable A431 cells via G0/G1 phase arrest and induced apoptosis. CTD-induced G0/G1 phase arrest through the reduction of protein levels of cyclin E, CDK6, and cyclin D in A431 cells. CTD-induced cell apoptosis of A431 cells also was confirm by DNA gel electrophoresis showed CTD-induced DNA fragmentation. CTD reduced the mitochondrial membrane potential and stimulated release of cytochrome c, AIF and Endo G in A431 cells. Flow cytometry demonstrated that CTD increased activity of caspase-8, -9 and -3. However, when cells were pretreated with specific caspase inhibitors activity was reduced and cell viability increased. CTD increased protein levels of death receptors such as DR4, DR5, TRAIL and levels of the active form of caspase-8, -9 and -3 in A431 cells. AIF and Endo G proteins levels were also enhanced by CTD. In vivo studies showed that CTD significantly inhibited A431 cell xenograft tumors in mice. Taken together, these in vitro and in vivo results provide insight into the mechanisms of CTD on cell growth and tumor production. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 723-738, 2017.

Allyl Isothiocyanate Induces Cell Toxicity by Multiple Pathways in Human Breast Cancer Cells.

Isothiocyanates (ITCs) occur in many cruciferous vegetables. These compounds, which have significant anticancer actions, can induce apoptosis in different human cancer cell lines. In the present study, we investigated if allyl isothiocyanate (AITC) would induce toxicity in human breast cancer MCF-7 (estrogen receptor positive) and MDA-MB-231 (estrogen receptor negative) cells. We found that AITC stimulated reactive oxygen species and Ca[Formula: see text] production, and decreased the mitochondrial membrane potential. Activity of caspase-8, -9 and -3 was increased by AITC in both cell lines. AITC also induced mitochondrial-mediated apoptosis, as shown by cytochrome c, AIF and Endo G release from mitochondria, activation of caspase-9 and caspase-3, and formation of DAPI-positive cells. There was a significant reduction in the levels of the anti-apoptotic protein Bcl-2 along with a marked increase in the pro-apoptotic protein Bax in both cell lines. AITC induced apoptosis in human breast cancer MCF-7 cells via AIF and Endo G signaling pathways, but in MDA-MB-231 cells apoptosis occurred via the GADD153 pathway. This study has revealed novel anti-cancer mechanisms of AITC, a compound that is ordinarily present in human diets and may have potential therapeutic effects in various cancers.

Suppression of the TNF-alpha level is mediated by Gan-Lu-Yin (traditional Chinese medicine) in human oral cancer cells through the NF-kappa B, AKT, and ERK-dependent pathways.

Oral cancer is one of the major causes of deaths in the male population of Taiwan. Gan-Lu-Yin (GLY) is used for an adjuvant treatment of Traditional Chinese Medicine in clinical patients. In this study, we investigated the molecular mechanisms in oral cancer cell lines after exposure to GLY. The cytometric bead-based array (CBA) method was used for the examining and analyzing of tumor necrosis factor-alpha (TNF-α) secretion level. TNF-α mRNA expression was determined by real-time PCR analysis. Nuclear factor kappa B (NF-κB) activity and other relative proteins were determined by NF-κB promoter assay, Western blotting, electrophoretic mobility shift assay (EMSA), and immuno-staining analyses. GLY decreased the secretion of TNF-α from the oral cancer CAL 27 cells. Furthermore, 2000 µg/mL of GLY significantly suppressed TNF-α mRNA expression of CAL 27 cells in a time-dependent manner. GLY reduced the levels of proteins, including nuclear NF-κB (p65 and p50), p-IKK (ser176), p-IκB, p-AKT, p-ERK, and nuclear Egr-1 in a time and dose-dependent manner. GLY also suppressed the NF-κB activity and translocation in CAL 27 cells. We suggest that GLY might promote the cure of oral cancer through decreasing the level of TNF-α cytokine, and these actions were mediated partially through the NF-κB, AKT, and ERK-dependent pathways in vitro. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1196-1205, 2016.

Crude extract of Euphorbia formosana induces apoptosis of DU145 human prostate cancer cells acts through the caspase-dependent and independent signaling pathway.

Prostate cancer is the most frequently diagnosed malignancy in men and the second highest contributor of male cancer mortality. The crude extract of Euphorbia formosana (CEEF) has been used for treatment of different diseases but the cytotoxic effects of CEEF on human cancer cells have not been reported. The purpose of the present experiments was to determine effects of CEEF on cell cycle distribution and induction of apoptosis in DU145 human prostate cancer cells in vitro. Contrast-phase microscope was used for examining cell morphological changes. Flow cytometric assays were used for cell viability, cell cycle, apoptosis, reactive oxygen species, and Ca2+ production and mitochondria membrane potential (ΔΨm ). Western blotting was used for examining protein expression of cell cycle and apoptosis associated proteins. Real-time PCR was used for examining mRNA levels of caspase-3, -8, and -9, AIF, and Endo G. Confocal laser microscope was used to examine the translocation of AIF, Endo G, and cytochrome in DU145 cells after CEEF exposure. CEEF-induced cell morphological changes, decreased the percentage of viable cells, and induced S phase arrest and apoptosis in DU145 cells. Furthermore, CEEF promoted RAS and Ca2+ production and reduced ΔΨm levels. Real-time QPCR confirmed that CEEF promoted the mRNA expression of caspase-3 and -9, AIF and Endo G and we found that AIF and Endo G and cytochrome c were released from mitochondria. Taken together, CEEF-induced cytotoxic effects via ROS production, induced S phase arrest and induction of apoptosis through caspase-dependent and independent and mitochondria-dependent pathways in DU245 cancer cells. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1600-1611, 2016.

Crude extract of Polygonum cuspidatum promotes immune responses in leukemic mice through enhancing phagocytosis of macrophage and natural killer cell activities in vivo.

Polygonum cuspidatum is a traditional Chinese herbal medicine used in the treatment of various diseases. In the present study, we investigated whether the crude extract of Polygonum cuspidatum (CEPC) could affect immune responses of murine leukemia cells in vivo. Normal BALB/c mice were i.p. injected with WEHI-3 cells to generate leukemic mice and then were treated orally with CEPC at 0, 50, 100 and 200 mg/kg for three weeks. Animals were weighed and blood, liver, spleen samples were collected for further analyses. Results indicated that CEPC did not significantly affect the body and liver weight of animals, but reduced the weight of spleen when compared to control groups. Flow cytometric assay demonstrated that CEPC increased the percentage of CD3- (T-cell marker) and CD19- (B-cell marker) positive cells, but reduced that of CD11b-positive ones (monocytes). However, it did not significantly affect the proportion of Mac-3-positive cells (macrophages), compared to control groups. Results indicated that CEPC promoted phagocytosis by macrophages from blood samples at all examined doses but did not affect that of macrophages from the peritoneal cavity. CEPC also promoted natural killer cell activity of splenocytes at 200 mg/kg of CEPC. CEPC promoted B-cell proliferation at 200 mg/kg treatment when cells were stimulated with lipopolysaccharides but did not promote T-cell proliferation at three doses of CEPC treatment on concanavalin A stimulation.

The crude extract of Corni Fructus inhibits the migration and invasion of U-2 OS human osteosarcoma cells through the inhibition of matrix metalloproteinase-2/-9 by MAPK signaling.

Osteosarcoma is the most common primary malignancy of the bone cancers. In the Chinese population, the crude extract of Corni Fructus (CECF) has been used as Traditional Chinese medicine to treat several different diseases for hundreds of years. In the present study, effects of CECF on inhibition of migration and invasion in U-2 OS human osteosarcoma cells were examined. CECF significantly inhibited migration and invasion of U-2 OS human osteosarcoma cells. We also found that CECF inhibited activities of matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-9 (MMP-9). CECF decreased protein levels of FAK, PKC, SOS1, MKK7, MEKK3, GRB2, NF-κB p65, COX-2, HIF-1α, PI3K, Rho A, ROCK-1, IRE-1α, p-JNK1/2, p-ERK1/2, p-p38, Ras, p-PERK, MMP-2, MMP-9, and VEGF in U-2 OS cells. Results of this study indicate that CECF may have potential as a novel anticancer agent for the treatment of osteosarcoma by inhibiting migration and invasion of cancer cells.

Solanum lyratum Extracts Induce Extrinsic and Intrinsic Pathways of Apoptosis in WEHI-3 Murine Leukemia Cells and Inhibit Allograft Tumor.

We investigated the molecular mechanisms of cell cycle arrest and apoptotic death induced by Solanum lyratum extracts (SLE) or diosgenin in WEHI-3 murine leukemia cells in vitro and antitumor activity in vivo. Diosgenin is one of the components of SLE. Our study showed that SLE and diosgenin decreased the viable WEHI-3 cells and induced G(0)/G(1) phase arrest and apoptosis in concentration- or time-dependent manners. Both reagents increased the levels of ROS production and decreased the mitochondrial membrane potential (ΔΨ(m)). SLE- and diosgenin-triggered apoptosis is mediated through modulating the extrinsic and intrinsic signaling pathways. Intriguingly, the p53 inhibitor (pifithrin-α), anti-Fas ligand (FasL) mAb, and specific inhibitors of caspase-8 (z-IETD-fmk), caspase-9 (z-LEHD-fmk), and caspase-3 (z-DEVD-fmk) blocked SLE- and diosgenin-reduced cell viability of WEHI-3 cells. The in vivo study demonstrated that SLE has marked antitumor efficacy against tumors in the WEHI-3 cell allograft model. In conclusion, SLE- and diosgenin-induced G(0)/G(1) phase arrest and triggered extrinsic and intrinsic apoptotic pathways via p53 activation in WEHI-3 cells. SLE also exhibited antitumor activity in vivo. Our findings showed that SLE may be potentially efficacious in the treatment of leukemia in the future.

Evaluation of the antioxidant activity of five endemic Ligustrum species leaves from Taiwan flora in vitro.

Leaves from the plant species belonging to the genus Ligustrum are widely used as tea or herbal medicine in Europe, China, and Japan. The antioxidant properties of five Ligustrum species from Taiwan were compared using in vitro antioxidant methods such as DPPH radical scavenging, TEAC, and FRAP assays. Cell-based antioxidant methods were used, including Fe(2+)/ascorbate-induced lipid peroxidation on brain homogenate and AAPH-induced erythrocyte haemolysis. The amounts of major phenolic compounds from the Ligustrum species, including phenylpropanoids, flavonoids, and iridoids, were determined by spectrophotometric methods. The results showed that all Ligustrum species exhibited antioxidant, radical-scavenging, anti-haemolytic, and lipid peroxidation-inhibiting activities at different magnitudes of potency. A significant correlation was found between antioxidant activity and the amount of antioxidant components, in particular, total phenolics and phenylpropanoids. Among all Ligustrum species from Taiwan, Ligustrum morrisonense is presented as potential source of natural antioxidants.

Triterpenoid contents and anti-inflammatory properties of the methanol extracts of ligustrum species leaves.

Ligustrum (privet) plants are used by Chinese physicians to prevent and cure hepatitis and chronic bronchitis. Three common Ligustrum plant spp., namely Ligustrum lucidum Ait. (LL), L. pricei Hayata (LP) and L. sinensis Lour. (LS) were collected to assess their analgesic/anti-inflammatory properties on chemical-induced nociception and carrageenan-induced inflammation in rodents. The methanol extracts from Ligustrum plants leaves effectively inhibited nociceptive responses induced by 1% acetic acid and 1% formalin. LP and LL reduced the edema induced by 1% carrageenan. LP exhibited the best potency of the Ligustrum plants. Furthermore, LP reduced the abdominal Evan's blue extravasations caused by lipopolysaccharide, lipoteichoic acid, autocrines and sodium nitroprusside. The triterpenoid content of the three Ligustrum spp. was measured by high performance liquid chromatography using a photodiode array detector. LP contained the highest content of amyrin, betulinic acid and lupeol. LL had the highest content of oleanolic acid and ursolic acid. The various degrees of analgesic/anti-inflammatory effects among three Ligustrum plants may be related to their different triterpenoid contents. LP is a potential analgesic and anti-inflammatory Ligustrum plant. The effects of LP are partially related to the inhibition of cyclooxygenase-2 activity and a decrease in microvascular permeability via the actions of autocrines and kinins.

Protective effect of Broussonetia papyrifera against hydrogen peroxide-induced oxidative stress in SH-SY5Y cells.

This study compared the antioxidant activity of various parts of Broussonetia papyrifera (BP), and further evaluated the protective effects of BP against hydrogen peroxide (H(2)O(2))-induced neuronal injury in human neuroblastoma SH-SY5Y cells. Among four BP parts, BP radix and leaf possessed the best scavenging activities for 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical, and H(2)O(2). These two BP parts also had higher phenolic and phenylpropanoid contents. Following exposure of cells to H(2)O(2), there was a marked decrease in cell survival, intracellular glutathione levels, and antioxidant enzymes, as well as an increase in intracellular oxidative stress. DNA fragmentation was also observed, but pretreatment of the cells with BP radix but not leaf prior to H(2)O(2) exposure blocked these H(2)O(2)-induced cellular events. The present findings indicate that BP radix exerts protective effects against H(2)O(2) toxicity via its free radical scavenging activity, which might be due to its total phenolic compounds.