Ascochlorin induces caspase-independent necroptosis in LPS-stimulated RAW 264.7 macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Plant-specific fungus of natural compound of Ascochyta viciae has traditionally been used in the treatment of sleeping sickness and tumors. The anti-tumor activities of the compounds obtained from Pisum sativum L were evaluated in this study. AIM OF THE STUDY: In this study, during the prolonged incubation, treatment of the LPS-stimulated tumor-like macrophage RAW 264.7 cells with ASC exhibited the shift of anti-inflammatory behavior to a type of necroptotic cell death named necroptosis. MATERIALS AND METHODS: Ascochlorin (ASC) purified from plant-specific fungus Ascochyta viciae is a natural compound with the trimethyl oxocyclohexyl structure and an anti-cancer and antibiotic agent. The fungus contributes to the Ascochyta blight disease complex of pea (Pisum sativum L). RAW 264.7 cells have been stimulated with LPS and treated with ASC. Cell viability of the LPS-treated RAW 264.7 cells and bone marrow-derived macrophage (BMDM) cells were examined. Flow cytometry analysis with 7AAD and Annexin V was examined for the apoptotic or necroptosis/late-apoptosis. Cleaved caspase-3, -7 and -8 as well as cleaved PARP were assessed with a caspase inhibitor, z-VAD-fmk. LPS-responsible human leukemic U937 and colon cancer SW480 and HT-29 cells were also examined for the cell viabilities. RESULTS: Flow cytometry analysis after Annexin V and 7AAD double staining showed that ASC alone induces apoptosis in RAW 264.7 cells, while it induces necroptosis/late-apoptosis in LPS-treated RAW 264.7 cells. 7AAD and Annexin V positive populations were increased in the LPS-treated cells with ASC. Although viability of LPS-treated cells with ASC was decreased, the amounts of cleaved caspase-3, -7 and -8 as well as cleaved PARP were reduced when compared with ASC-treated cells. Upon ASC treatment, the cleaved caspase-8 level was not changed, however, cleaved caspase-3, -7, and PARP were reduced in LPS-stimulated RAW 264.7 cells treated with ASC, claiming a caspase-8 independent necroptosis of ASC. Furthermore, ASC and LPS-cotreated cells which a caspase inhibitor, z-VAD-fmk, was pretreated, showed the decreased cell viability compared with control cells without the inhibitor. Cell viability of RAW 264.7 cells co-treated with ASC and LPS when treated with z-VAD was decreased. In the LPS-responsible human leukemic U937 and colon cancer SW480 and HT-29 cells, cell viabilities were decreased by 10 µM ASC. CONCLUSION: Prolonged stimulation of ASC with LPS induces the necroptosis in RAW cells. Activated immune cells may share the susceptibility of antitumor agents with the cancer cells.

Esculentoside B inhibits inflammatory response through JNK and downstream NF-κB signaling pathway in LPS-triggered murine macrophage RAW 264.7 cells.

Natural compound esculentoside B (EsB), (2S,4aR,6aR,6aS,6bR,8aR,9R,10R,11S,12aR,14bS)-11-hydroxy-9-(hydroxymethyl)-2 methoxycarbonyl-2,6a,6b,9,12a-pentamethyl-10-[(2S,3R,4S,5R)-3,4,5-trihydroxyoxan-2-yl]oxy-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylic acid with molecular weight of 664.833, isolated from roots of Phytolacca acinosa Roxb has been widely used as a constituent of traditional Chinese medicine (TCM). However, the anti-inflammatory capacity of EsB has not been reported yet. Therefore, the objective of this study was to investigate anti-inflammatory activities of EsB in LPS-treated macrophage RAW 264.7 cells. EsB could inhibit nitric oxide (NO) production. EsB also suppressed gene and protein expression levels of inducible isoform of NO synthase (NOS) and cyclooxygenase-2 in a dose-dependent manner. In addition, EsB decreased gene expression and protein secretion levels of pro-inflammatory cytokines such as IL-1ß, TNF-α, and IL-6. EsB remarkably suppressed nuclear translocation of nuclear factor kappa-B (NF-κB) from cytosolic space. Phosphorylation of IκB was also inhibited by EsB. Moreover, EsB specifically down-regulated phospho-c-Jun N-terminal kinase (p-JNK), but not p-p38 or phospho-extracellular signal-regulated kinase 1/2 (p-ERK1/2). Taken together, these results suggest that EsB has inhibitory effect on inflammatory response by inactivating NF-κB and p-JNK. It could be used as a new modulatory drug for effective treatment of inflammation-related diseases.

Oldenlandia diffusa suppresses metastatic potential through inhibiting matrix metalloproteinase-9 and intercellular adhesion molecule-1 expression via p38 and ERK1/2 MAPK pathways and induces apoptosis in human breast cancer MCF-7 cells.

ETHNOPHARMACOLOGY RELEVANCE: Oldenlandia diffusa (OD) has long been known as an apoptotic inducer in breast tumors in ethnomedicine. AIM OF THE STUDY: To scientifically confirm the anti-breast cancer effects of water, methanol (MeOH) and butanol (BuOH) extracts of O. diffusa on cell apoptosis, matrix metalloproteinases (MMPs), intercellular adhesion molecule (ICAM)-1 and intracellular signaling in MCF-7 breast cancer cells. MATERIALS AND METHODS: MeOH extracts (MOD) and BuOH extracts (BOD) were prepared and examined for their ability to inhibit phorbol myristate acetate (PMA)-induced matrix metalloproteinase (MMP)-9 and intercellular adhesion molecule (ICAM)-1 expressions in MCF-7 human breast cancer cells. Additionally, transwell migration, invasion and transcriptional activity were assessed. Results of immunofluorescence confocal microscopy for translocation of NF-κB and p-ERK and p-p38 were also checked. Finally, apoptotic signals including processed caspase-8, caspase-7, poly ADP-ribose polymerase, Bax and Bcl-2 were examined. RESULTS: MOD and BOD specifically inhibited PMA-induced MMP-9 expression as well as invasive and migration potential via ICAM-1. The inhibitory activity was also based on the suppressed transcriptional activity in MCF-7 breast cancer cells. Results of immunofluorescence confocal microscopy showed that translocation of NF-κB decreased upon BOD and MOD treatments, with a decreased level of p-ERK and p-p38 phosphorylation. In addition, treatment of MCF-7 cells with MOD and BOD activated apoptosis-linked proteins including enzymatically active forms of processed caspase-8, caspase-7 and poly ADP-ribose polymerase, together with increased expression of mitochondrial apoptotic protein, Bax and decreased expression of Bcl-2. CONCLUSION: The results indicate that OD as an anti-metastatic agent suppresses the metastatic response by targeting p-ERK, p-38 and NF-κB, thus reducing the invasion capacity of MCF-7 breast cancer cells through inhibition of MMP-9 and ICAM-1 expression and plays an important role in the regulation of breast cancer cell apoptosis.

Cytochrome 2C19 polymorphism and response to adjunctive cilostazol versus high maintenance-dose clopidogrel in patients undergoing percutaneous coronary intervention.

BACKGROUND: Among patients treated with clopidogrel, carriers of the cytochrome P450 (CYP) 2C19 loss-of-function allele have shown increased platelet reactivity and higher rates of ischemic events. Although adjunctive cilostazol to dual antiplatelet therapy (or "triple antiplatelet therapy") intensifies platelet inhibition, it remains unknown whether triple antiplatelet therapy after percutaneous coronary intervention can achieve adequate platelet inhibition in patients with the CYP2C19 mutant allele. METHODS AND RESULTS: CYP2C19 genotyping for *1, *2, and *3 was performed in 134 high-risk patients undergoing elective percutaneous coronary intervention. After measurement of preprocedural platelet reactivity, patients were randomly assigned to receive either adjunctive cilostazol 100 mg twice daily (triple group; n=69) or high maintenance-dose (MD) clopidogrel of 150 mg daily (high-MD group; n=65). Using light transmittance aggregometry and the VerifyNow P2Y(12) assay, platelet reactivity was assessed before the index procedure and at 30-day follow-up. The primary end point was absolute change in maximal platelet aggregation (ΔAgg(max)) according to CYP2C19 genotyping. High posttreatment platelet reactivity was defined as 5 µmol/L ADP-induced maximal platelet aggregation >50%. In noncarriers of the CYP2C19*2/*3 mutant allele, ΔAgg(max) values after 5 and 20 µmol/L ADP stimuli did not differ significantly between the triple (n=22) versus the high-MD group (n=22) (23.6±21.6% versus 16.6±15.4%, P=0.224 and 26.4±22.2% versus 18.6±14.9%, P=0.174, respectively). Absolute changes in late platelet aggregation and P2Y(12) reaction unit were not different between the groups. The rate of high posttreatment platelet reactivity at 30-day follow-up also was comparable between the triple versus the high-MD group (4.5% versus 13.6%, P=0.607). In carriers of at least 1 CYP2C19*2/*3 mutant allele, the triple group (n=47) showed greater values of ΔAgg(max) after addition of 5 µmol/L (25.8±16.8% versus 11.1±19.8%, P<0.001) and 20 µmol/L ADP (26.3±16.0% versus 11.5±16.3%, P<0.001) compared with the high-MD group (n=43). Likewise, absolute changes in late platelet aggregation and P2Y(12) reaction unit were consistently greater in the triple versus the high-MD group. Fewer patients in the triple group met the criteria of high posttreatment platelet reactivity at 30-day follow-up compared with the high-MD group (6.4% versus 37.2%, P<0.001). CONCLUSIONS: Among high-risk patients undergoing elective percutaneous coronary intervention, adjunctive cilostazol can achieve consistently intensified platelet inhibition and reduce the risk of high posttreatment platelet reactivity irrespective of CYP2C19 genotyping. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01012193.

Adding cilostazol to dual antiplatelet therapy achieves greater platelet inhibition than high maintenance dose clopidogrel in patients with acute myocardial infarction: Results of the adjunctive cilostazol versus high maintenance dose clopidogrel in patients with AMI (ACCEL-AMI) study.

BACKGROUND: Optimal platelet inhibition is an important therapeutic adjunct in patients acute myocardial infarction (AMI) undergoing coronary stenting. Whether adjunctive cilostazol to dual antiplatelet therapy (triple antiplatelet therapy) can inhibit enhanced platelet reactivity in patients with AMI yet has not been determined. The aim of this study was to assess the degree of platelet inhibition by triple antiplatelet therapy in patients with AMI. METHODS AND RESULTS: Immediately after emergency room arrival, patients with AMI received clopidogrel (600-mg loading dose, followed by 75 mg daily) and aspirin (300-mg loading dose and 200 mg daily throughout the study period). After patients underwent coronary stenting (n=90), they were randomly assigned to 1 of 3 groups before discharge: standard group, clopidogrel of 75 mg daily (n=30); high maintenance dose (MD) group, clopidogrel of 150 mg daily (n=30); and triple group, adjunctive cilostazol of 100 mg twice daily to clopidogrel of 75 mg daily (n=30). Platelet reactivity was assessed at predischarge and 30-day follow-up by conventional aggregometry and the VerifyNow P2Y12 assay. Predischarge platelet reactivities were similar in the 3 groups. At 30-day follow-up, inhibition of maximal aggregation with 20 microM ADP stimuli was 6.0% in the standard group, 19.1% in the high-MD group, and 42.4% in the triple group (P<0.001), whereas inhibition of late aggregation with 20microM ADP stimuli was 10.8%, 38.1%, and 66.4%, respectively (P<0.001). Similar results were demonstrated when 5 microM ADP was used. Furthermore, percent changes of P2Y12 reaction unit were significantly different among regimens (10.6% in the standard group, 30.7% in the high-MD group, and 43.0% in the triple group; P<0.001). With respect to high-postclopidogrel platelet reactivity (prespecified as 20 microM ADP-induced maximal aggregation >50% of light transmission), fewer patients in the triple group (13.3%) met the criteria as compared with those in the standard (76.7%) and high-MD groups (56.7%) at 30-day follow-up (P<0.001). In the triple group, there were more potent and consistent platelet inhibitions by all parameters as compared with the high-MD group except for percent changes of P2Y12 reaction unit (P=0.071). CONCLUSIONS: Among patients with AMI undergoing coronary stenting, triple antiplatelet therapy results in a greater antiplatelet effect at 30 days as compared with a high-MD clopidogrel or standard dual antiplatelet therapy.