2,3,5,4'-Tetrahydroxystilbene-2-O-ß-glucoside Attenuates Reactive Oxygen Species-Dependent Inflammation and Apoptosis in Porphyromonas gingivalis-Infected Brain Endothelial Cells.

We recently reported that the periodontopathic bacteria Porphyromonas gingivalis (P. gingivalis) initiates an inflammatory cascade that disrupts the balance of reactive oxygen species (ROS), resulting in apoptotic cell death in brain endothelial cells. An extract from Polygonum multiflorum Thunb., 2,3,5,4'-Tetrahydroxystilbene-2-O-ß-glucoside (THSG) has been well-reported to diminish the inflammation in many disease models. However, the effects of THSG in the area of the brain-oral axis is unknown. In this study, we examined the effects of THSG in P. gingivalis-stimulated inflammatory response and apoptotic cell death in brain endothelial cells. THSG treatment remarkably lessened the upregulation of IL-1ß and TNF-α proteins in bEnd.3 cells infected with P. gingivalis. Treatment of THSG further ameliorated brain endothelial cell death, including apoptosis caused by P. gingivalis. Moreover, the present study showed that the inhibitory effects on NF-κB p65 and antiapoptotic properties of THSG is through inhibiting the ROS pathway. Importantly, the ROS inhibitory potency of THSG is similar to a ROS scavenger N-Acetyl-L-Cysteine (NAC) and NADPH oxidase inhibitor apocynin. Furthermore, the protective effect of THSG from P. gingivalis infection was further confirmed in primary mouse brain endothelial cells. Taken together, this study indicates that THSG attenuates an ROS-dependent inflammatory response and cell apoptosis in P. gingivalis-infected brain endothelial cells. Our results also suggest that THSG could be a potential herbal medicine to prevent the risk of developing cerebrovascular diseases from infection of periodontal bacteria.

Comparison of the Phytochemical Properties, Antioxidant Activity and Cytotoxic Effect on HepG2 Cells in Mongolian and Taiwanese Rhubarb Species.

The Mongolian rhubarb-Rheum undulatum L. (RU)-and Rumex crispus L. (RC)-a Taiwanese local rhubarb belonging to the family of Polygonaceae-are principal therapeutic materials in integrative medicine due to their rich quantities of bioactive compounds; however, their phytochemical and antioxidant properties, and anti-cancer activity is poorly investigated. Furthermore, the phytochemical characteristics of both species may be affected by their different geographical distribution and climatic variance. The current study aimed to compare RU with RC extracts in different polarity solvents (n-hexane, ethyl acetate, acetone, ethanol, and water) for their phytochemical contents including the total phenolic content (TPC), total anthraquinone content (TAC), total flavonoid content (TFC), antioxidant and free radical scavenging capacities, and anticancer ability on the HepG2 cell. Except for the n-hexane extract, all of the RU extracts had considerably higher TPCs than RC extracts, ranging from 8.39 to 11.16 mg gallic acid equivalent (GAE) per gram of dry weight, and the TPCs of each extract were also significantly correlated with their antioxidant capacities by ABTS, DPPH, and FRAP assays (p < 0.05). Moreover, there was no remarkable association between the antioxidant capacities and either TACs or TFCs in both the RU and RC extracts. Besides, high-performance liquid chromatography (HPLC) analysis revealed that both the RU and RC extracts contained chrysophanol, emodin, and physcion, and those bioactive compounds were relatively higher in the n-hexane solvent extracts. Additionally, we observed different levels of dose-dependent cytotoxic effects in all the extracts by cell viability assay. Notably, the ethanol extract of RU had a compelling cytotoxic effect with the lowest half-maximum inhibition concentration (IC50-171.94 ± 6.56 µg/mL at 48 h) among the RU extracts than the ethanol extract of RC. Interestingly, the ethanol extract of RU but not RC significantly induced apoptosis in the human liver cancer cell line, HepG2, with a distinct pattern in caspase-3 activation, resulting in increased PARP cleavage and DNA damage. In summary, Mongolian Rhubarb, RU, showed more phytochemical contents, as well as a higher antioxidant capacity and apoptotic effect to HepG2 than RC; thus, it can be exploited for the proper source of natural antioxidants and liver cancer treatment in further investigation.

Hispolon Suppresses LPS- or LTA-Induced iNOS/NO Production and Apoptosis in BV-2 Microglial Cells.

Hispolon (HIS) is an active polyphenol compound derived from Phellinus linteus (Berkeley & Curtis), and our previous study showed that HIS effectively inhibited inflammatory responses in macrophages [Yang, L.Y., S.C. Shen, K.T. Cheng, G.V. Subbaraju, C.C. Chien and Y.C. Chen. Hispolon inhibition of inflammatory apoptosis through reduction of iNOS/NO production via HO-1 induction in macrophages. J. Ethnopharmacol. 156: 61-72, 2014]; however, its effect on neuronal inflammation is still undefined. In this study, HIS concentration- and time-dependently inhibited lipopolysaccharide (LPS)- and lipoteichoic acid (LTA)-induced inducible nitric oxide (NO) synthase (iNOS)/NO production with increased heme oxygenase (HO)-1 proteins in BV-2 microglial cells. Accordingly, HIS protected BV-2 cells from LPS- or LTA-induced apoptosis, characterized by decreased DNA ladder formation, and caspase-3 and poly(ADP ribose) polymerase (PARP) protein cleavage in BV-2 cells. Similarly, the NOS inhibitor, N-nitro-L-arginine methyl ester (NAME), inhibited LPS- or LTA-induced apoptosis of BV-2 cells, but neither NAME nor HIS showed any inhibition of NO production or cell death induced by the NO donor, sodium nitroprusside (SNP), indicating the involvement of NO in the inflammatory apoptosis of microglial cells. Activation of c-Jun N-terminal kinase (JNK) and nuclear factor (NF)-[Formula: see text]B contributed to LPS- or LTA-induced iNOS/NO production and apoptosis of BV-2 cells, and that was suppressed by HIS. Additionally, HIS possesses activity to induce HO-1 protein expression via activation of extracellular signal-regulated kinase (ERK) in BV-2 cells, and application of the HO inhibitor, tin protoporphyrin (SnPP), or knockdown of HO-1 protein by HO-1 small interfering (si)RNA significantly reversed HIS inhibition of NO production and cell death in BV-2 cells stimulated by LPS. Results of an analysis of the effects of HIS and two structurally related chemicals, i.e. dehydroxy-HIS (D-HIS) and HIS-methyl ester (HIS-ME), showed that HIS expressed the most potent inhibitory effects on iNOS/NO production, JNK activation, and apoptosis in BV-2 microglial cells activated by LPS with increased HO-1 protein expression. Overall these results suggested that HIS possesses inhibitory activity against LPS- or LTA-induced inflammatory responses including iNOS/NO production and apoptosis in BV-2 microglial cells and that the mechanisms involve upregulation of the HO-1 protein and downregulation of JNK/NF-[Formula: see text]B activation. A critical role of hydroxyl at position C3 in the anti-inflammatory actions of HIS against activated BV-2 microglial cells was suggested.

Evodiamine Prevents Glioma Growth, Induces Glioblastoma Cell Apoptosis and Cell Cycle Arrest through JNK Activation.

Evodiamine (EVO) is an active medicinal compound derived from the traditional herbal medicine Evodia rutaecarpa. It has been reported that evodiamine has several beneficial biological properties, including anticancer and anti-inflammatory activities. However, the in vitro and in vivo anticancer activities of EVO against the growth of glioblastoma cells remain undefined. EVO induced significant decreases in the viability of U87 and C6 glioma cells, but not of primary astrocytes, according with the occurrence of apoptotic characteristics including DNA ladders, caspase-3 and poly(ADP ribose) polymerase (PARP) protein cleavage, and hypodiploid cells. The disruption of the mitochondrial membrane potential (MMP) was detected, and it was found that the peptidyl caspase-9 inhibitor, Z-LEHD-FMK, significantly prevented glioma cells from EVO-induced apoptosis. Increased c-Jun N-terminal kinase (JNK) protein phosphorylation by EVO was observed, and the addition of JNK inhibitors, SP600125 and JNKI inhibited the EVO-induced apoptosis was inhibited. Additionally, EVO treatment induced G2/M arrest with increased polymerized tubulin protein expression in U87 and C6 cells. Elevated expressions of the cyclin B1, p53, and phosphorylated (p)-p53 proteins were detected in EVO-treated glioma cells, and these were inhibited by JNK inhibitors. An in vivo study showed that EVO significantly reduced the growth of gliomas elicited by the subcutaneous injection of U87 cells with increases in cyclin B1, p53, and p-p53 protein expressions in tumors. An analysis of eight EVO-related chemicals showed that alkyl groups at position 14 in EVO are important for its anti-glioma effects which involve both apoptosis and G2/M arrest. Evidence is provided that supports EVO induction of apoptosis and G2/M arrest via the activation of JNK-mediated gene expression and disruption of MMP in glioblastoma cells. EVO was shown to penetrate the blood-brain barrier; EVO is therefore predicted to be a promising compound for the chemotherapy of glioblastomas and deserves further investigations.

Evodiamine from Evodia rutaecarpa induces apoptosis via activation of JNK and PERK in human ovarian cancer cells.

BACKGROUND: Evodiamine (EVO; 8,13,13b,14-tetrahydro-14-methylindolo[2'3'-3,4]pyrido[2,1-b]quinazolin-5-[7H]-one derived from the traditional herbal medicine Evodia rutaecarpa was reported to possess anticancer activity; however, the anticancer mechanism of EVO against the viability of human ovarian cancer cells is still unclear. PURPOSE: A number of studies showed that chemotherapeutic benefits may result from targeting the endoplasmic reticular (ER) stress signaling pathway. The objective of the study is to investigate the mechanism by which ER stress protein PERK plays in EVO-induced apoptosis of human ovarian cancer cells. METHODS: Cell death analysis was performed by MTT assay, DNA fragmentation assay, and Giemsa staining. DiOC6 staining was used for mitochondrial membrane potential measurement. Protein levels were analyzed by Western blotting. Pharmacological studies using MAPK inhibitors and PERK inhibitor GSK2606414 were involved. RESULTS: The viability of human ovarian cancer cells A2780, A2780CP, ES-2, and SKOV-3 was inhibited by EVO at various concentrations in accordance with increases in the percentage of apoptotic cells, DNA ladders, and cleavage of caspase 3 and poly(ADP ribose) polymerase (PARP) proteins. Decreased viability of cells was reversed by adding caspase inhibitors VAD and DEVD in SKOV-3 and A2780CP cells, and incubation of cells with JNK inhibitor SP600125 (SP) and JNKI, but not other MAPK and AKT inhibitors including PD98059, SB203580, significantly prevented the apoptosis elicited by EVO in human ovarian cancer cells. Furthermore, increased expression of phospho-eIF2α (peIF2α) and phospho-PERK (pPERK) proteins was detected in EVO-treated human ovarian cancer cells, and that was inhibited by adding JNK inhibitors SP600125 and JNKI. Application of a PERK inhibitor GSK2606414 showed a significant protection of human ovarian cancer cells A2780 and A2780CP from EVO-induced apoptosis. EVO disruption of mitochondrial membrane potential (MMP) was also inhibited by adding JNK or PERK inhibitors. The structure-activity relationship study indicated that the alkyl group at position 14 in EVO is important for apoptosis induction via activation of JNK and PERK in human ovarian cancer cells. CONCLUSION: Evidence supporting EVO induction of apoptosis via activation of JNK and PERK to disrupt MMP in human ovarian cancer cells is provided, and the alkyl at position 14 is a critical substitution for the apoptotic actions of EVO.

Early decline in serum phospho-CSE1L levels in vemurafenib/sunitinib-treated melanoma and sorafenib/lapatinib-treated colorectal tumor xenografts.

BACKGROUND: Although targeted therapies have improved the clinical outcomes of cancer treatment, tumors resistance to targeted drug are often detected too late and cause mortality. CSE1L is secreted from tumor and its phosphorylation is regulated by ERK1/2. ERK1/2 is located downstream of various growth factor receptors and kinases, the targets of most targeted drugs. Serum phospho-CSE1L may be a marker for monitoring the efficacy of targeted therapy. METHODS: We used mice tumor xenograft model to study the assay of serum phosphorylated CSE1L for early detecting the efficacy of targeted drugs. The phosphorylation status of CSE1L in vemurafenib and sorafenib treated tumor cells were assayed by immunoblotting with antibody against phosphorylated CSE1L. RESULTS: Ras activation increased phospho-CSE1L expression in B16F10 melanoma cells. Vemurafenib and sorafenib treatment did not significantly reduce the total CSE1L levels; however, they inhibited ERK1/2 and CSE1L phosphorylation in A375 melanoma cells and HT-29 colorectal cancer cells. In the melanoma xenograft model, serum phospho-CSE1L level declined 5 days after vemurafenib/sunitinib treatment and 3 days after sorafenib/lapatinib treatment in the HT-29 colon cancer xenograft model. Vemurafenib/sunitinib and sorafenib/lapatinib treatments resulted in tumor regression. CONCLUSIONS: Our results indicated that serum phospho-CSE1L is useful for early detecting the efficacy of targeted therapy in initial treatment and for monitoring emerging secondary drug resistance to facilitate timely therapeutic decision making.

Hispolon inhibition of inflammatory apoptosis through reduction of iNOS/NO production via HO-1 induction in macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Phellinus linteus (Berkeley & Curtis), a well-known medical fungus, has long been used as a traditional medicine in Oriental countries to treat various diseases, and hispolon (HIS) is one of its bioactive components. HIS is known to possess potent antineoplastic and antiviral properties; however, its effect on inflammatory apoptosis is still undefined. MATERIALS AND METHODS: RAW264.7 macrophages were incubated with HIS for 30 min followed by LPS, LTA, or PGN stimulation for 12h. The expression of indicated proteins AP-1 and NF-κB transcriptional activities was examined by Western blotting using specific antibodies. Levels of NO and ROS were examined by Griess reaction, and DCHF-DA staining via flow cytometric analysis, respectively. AP-1 and NF-κB transcriptional activities were detected by luciferase reporter assay. Knockdown of HO-1 protein expression was performed by transfection of macrophages with HO-1 siRNA. Pharmacological inhibitors including ROS scavenger NAC, JNK inhibitor SP600125, NF-κB inhibitor BAY117082 were applied for mechanism study. RESULTS: HIS showed concentration-dependent inhibition of LPS, LTA, and PGN-induced iNOS protein expressions and NO production by RAW264.7 macrophages. Accordingly, HIS protected RAW264.7 cells from LPS-, LTA-, and PGN-induced apoptosis. Increased HO-1 by HIS was detected at both protein and mRNA levels along with an increase in intracellular peroxide, and this was inhibited by the translational inhibitor, cycloheximide (CHX), the transcriptional inhibitor, actinomycin D (Act D), and the reactive oxygen species scavenger, N-acetylcysteine (NAC). A mechanistic study indicated that inhibition of c-Jun N-terminal kinase (JNK) protein phosphorylation, and activator protein (AP)-1 and nuclear factor (NF)-κB activation were involved in the anti-inflammatory actions of HIS in macrophages. A structure-activity relationship analysis showed that HIS expressed the most potent effect of inhibiting iNOS and apoptosis elicited by LPS, LTA, and PGN with a significant increase in HO-1 protein in macrophages. CONCLUSIONS: Evidence supporting HIS prevention of inflammatory apoptosis via blocking NO production and inducing HO-1 protein expression in macrophages is provided, and the hydroxyl at position C3 is a critical substitution for the anti-inflammatory actions of HIS.

Activation of JNK contributes to evodiamine-induced apoptosis and G2/M arrest in human colorectal carcinoma cells: a structure-activity study of evodiamine.

Evodiamine (EVO; 8,13,13b,14-tetrahydro-14-methylindolo[2'3'-3,4]pyrido[2,1-b]quinazolin-5-[7H]-one derived from the traditional herbal medicine Evodia rutaecarpa was reported to possess anticancer activity; however, the anticancer mechanism is still unclear. In this study, we investigated the anticancer effects of EVO on human colon COLO205 and HT-29 cells and their potential mechanisms. MTT and lactate dehydrogenase (LDH) release assays showed that the viability of COLOL205 and HT-29 cells was inhibited by EVO at various concentrations in accordance with increases in the percentage of apoptotic cells and cleavage of caspase-3 and poly(ADP ribose) polymerase (PARP) proteins. Disruption of the mitochondrial membrane potential by EVO was accompanied by increased Bax, caspase-9 protein cleavage, and cytochrome (Cyt) c protein translocation in COLO205 and HT-29 cells. Application of the antioxidant N-acetyl-L-cysteine (NAC) inhibited H2O2-induced reactive oxygen species (ROS) production and apoptosis, but did not affect EVO-induced apoptosis of COLO205 or HT-29 cells. Significant increases in the G2/M ratio and cyclinB1/cdc25c protein expression by EVO were respectively identified in colon carcinoma cells via a flow cytometric analysis and Western blotting. Induction of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) protein phosphorylation was detected in EVO-treated cells, and the JNK inhibitor, SP600125, but not the ERK inhibitor, U0126, inhibited EVO-induced phosphorylated JNK protein expression, apoptosis, and G2/M arrest of colon carcinoma cells. Data of the structure-activity analysis showed that EVO-related chemicals containing an alkyl group at position 14 were able to induce apoptosis, G2/M arrest associated with increased DNA ladder formation, cleavage of caspase-3 and PARP, and elevated cycB1 and cdc25c protein expressions in COLO205 and HT-29 cells. Evidence supporting JNK activation leading to EVO-induced apoptosis and G2/M arrest in colon carcinoma cells is provided, and alkylation at position 14 of EVO is a critical substitution for treatment of colonic cancer.

HSP90 Inhibitors, Geldanamycin and Radicicol, Enhance Fisetin-Induced Cytotoxicity via Induction of Apoptosis in Human Colonic Cancer Cells.

We revealed the cytotoxic effect of the flavonoid, fisetin (FIS), on human COLO205 colon cancer cells in the presence and absence of the HSP90 inhibitors, geldanamycin (GA) and radicicol (RAD). Compared to FIS treatment alone of COLO205 cells, GA and RAD significantly enhanced FIS-induced cytotoxicity, increased expression of cleaved caspase-3 and the PAPR protein, and produced a greater density of DNA ladder formation. GA and RAD also reduced the MMPs with induction of caspase-9 protein cleavage in FIS-treated COLO205 cells. Increased caspase-3 and -9 activities were detected in COLO205 cells treated with FIS+GA or FIS+RAD, and the intensity of DNA ladder formation induced by FIS+GA was reduced by adding the caspase-3 inhibitor, DEVD-FMK. A decrease in Bcl-2 but not Bcl-XL or Bax protein by FIS+GA or FIS+RAD was identified in COLO205 cells by Western blotting. A reduction in p53 protein with increased ubiquitin-tagged proteins was observed in COLO205 cells treated with FIS+GA or FIS+RAD. Furthermore, GA and RAD reduced the stability of the p53 protein in COLO205 cells under FIS stimulation. The evidence supports HSP90 inhibitors possibly sensitizing human colon cancer cells to FIS-induced apoptosis, and treating colon cancer by combining HSP90 inhibitors with FIS deserves further in vivo study.

Screening of flavonoids for effective osteoclastogenesis suppression.

Flavonoids are natural compounds derived from plants and some of them have been shown to inhibit osteoclast formation, implicating their potential use for the treatment of osteoporosis. Conventionally, the screening of antiosteoclastic agents is a tedious process that requires visual counting of the number of osteoclasts produced. The purpose of this study was to establish an easier and faster method for screening the antiosteoclastogenic flavonoids by using an enzyme assay. Tartrate-resistant acid phosphatase (TRAP) is a marker enzyme of the osteoclast. Results obtained demonstrated that cellular TRAP activity tended to correlate with the number of osteoclasts formed. However, the secreted TRAP activity was actually responsible for the resorption activities of the functional osteoclasts. Consequently, the effectiveness of antiosteoclastogenic agents was screened for by assessing their inhibition on receptor activator of NF-κB ligand (RANKL)-induced TRAP secretion. The half-inhibitory concentrations of flavonoids on TRAP secretion were employed as indices to compare the effectiveness of various flavonoids. The effective flavonoids also exhibited similar inhibitory potencies in the pit-formation analysis. This protocol provides a rapid analysis to screen for effective antiosteoclastogenic agents.

Wogonin but not Nor-wogonin inhibits lipopolysaccharide and lipoteichoic acid-induced iNOS gene expression and NO production in macrophages.

Wogonin (Wog; 5,7-dihydroxy-8-methoxy flavone) has been shown to effectively inhibit lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) gene expression and nitric oxide production in our previous study. In the present study, we found that Nor-wogonin (N-Wog; 5,7,8-trihydroxyl flavone), a structural analogue of Wog with an OH substitution at C8, performed different effect on LPS- or lipoteichoic acid (LTA)-induced iNOS gene expression and nitric oxide (NO) production in macrophages. Wog, but not N-Wog, significantly inhibits LPS- or LTA-induced NO production through suppressing iNOS gene expression at both protein and mRNA without affecting NO donor sodium nitroprusside-induced NO production, NOS enzyme activity, and cells viability. Activation of JNKs (not ERKs) via phosphorylation induction, and an increase in c-Jun (not c-Fos) protein expression were involved in LPS- and LTA-treated RAW264.7 cells, and those events were blocked by Wog, but not N-Wog, addition. Furthermore, 5,7-diOH flavone, but not 5-OH flavone, 7-OH flavone, 5-OH-7-OCH(3) flavone, significantly inhibits LPS-induced iNOS protein expression and NO production, and 7,8-diOCH(3) flavone performs more effective inhibitory activity on LPS-induced NO production and iNOS protein expression than 7-OCH(3)-8-OH flavone. These data suggest that OHs at both C5 and C7 are essential for NO inhibition of flavonoids, and OCH(3) at C8 may contribute to this activity, and suppression of JNKs-c-Jun activation is involved.