Possible involvement of the µ opioid receptor in the antinociception induced by sinomenine on formalin-induced nociceptive behavior in mice.

Sinomenine, an alkaloid originally isolated from the roots and the rhizome of Sinomenium acutum is used as a traditional Chinese herbal medicines for rheumatoid arthritis and neuralgia. The aims of this study were to investigate the effects of oral administration of shinomenine on formalin-induced nociceptive behavior in mice and the opioid receptor subtypes involved in the antinociceptive effects of sinomenine. Our findings showed that a single dose of oral-administrated sinomenine inhibited the formalin induced licking and biting responses in a dose-dependent manner. Intraperitoneal pretreatment with naloxone hydrochloride, an opioid receptor antagonist, and ß-funaltrexamine hydrochloride (ß-FNA), a selective µ-opioid receptor antagonist, significantly attenuated sinomenine induced antinociception, but not by naltrindole, a nonselective δ-opioid receptor antagonist and nor-binaltorphimine, a selective κ-opioid receptor antagonist. Furthermore, in western blot analysis, oral administration of sinomenine resulted in a significant blockage of spinal extracellular signal-regulated protein kinase (ERK1/2) activation induced by formalin. Naloxone hydrochloride and ß-FNA significantly reversed the blockage of spinal ERK1/2 activation induced by sinomenine. These results suggest that sinomenine-induced anti nociceptive effect and blockage of spinal ERK1/2 activation may be triggered by activation of µ-opioid receptors.

Possible involvement of the peripheral Mu-opioid system in antinociception induced by bergamot essential oil to allodynia after peripheral nerve injury.

The essential oil of bergamot (BEO) is one of the most common essential oils and is most familiar to the general public. The aims of this study were to investigate the effect of intraplantar (i.pl.) BEO on neuropathic allodynia induced by partial sciatic nerve ligation (PSNL) in mice and the opioid receptor subtypes involved in the antiallodynic effects of BEO. Our findings showed that a single dose of i.pl. administration of BEO significantly inhibited the PSNL-induced neuropathic pain using the von Frey test. The i.pl pretreatment with naloxone methiodide, a peripherally acting µ-opioid receptor preferring antagonist, ß-funaltrexamine hydrochloride (ß-FNA), a selective µ-opioid receptor antagonist, and ß-endorphin antiserum significantly reversed the antiallodynic effect of BEO in the von Frey test, but not by naltrindole, the nonselective δ-opioid receptor antagonist and nor-binaltorphimine, the selective κ-opioid receptor antagonist. Furthermore, in the western blotting analysis, i.pl. administration of BEO resulted in a significant blockage of spinal extracellular signal-regulated protein kinase (ERK) activation induced by PSNL. Naloxone methiodide and ß-FNA significantly reversed the blockage of spinal ERK activation induced by BEO. These results suggest that i.pl. injection of BEO-induced antiallodynic effect and blockage of spinal ERK activation may be triggered by activation of peripheral µ-opioid receptors.

Cardenolides and bufadienolide glycosides from Kalanchoe tubiflora and evaluation of cytotoxicity.

Two new cardenolides, kalantubolide A (1) and kalantubolide B (2), and two bufadienolide glycosides, kalantuboside A (3) and kalantuboside B (4), as well as eleven known compounds were isolated and characterized from the EtOH extract of Kalanchoe tubiflora. The structures of compounds were assigned based on 1D and 2D NMR spectroscopic analyses including HMQC, HMBC, and NOESY. Biological evaluation indicated that cardenolides (1-2) and bufadienolide glycosides (3-7) showed strong cytotoxicity against four human tumor cell lines (A549, Cal-27, A2058, and HL-60) with IC50 values ranging from 0.01 µM to 10.66 µM. Cardenolides (1-2) also displayed significant cytotoxicity toward HL-60 tumor cell line. In addition, compounds 3, 4, 5, 6, and 7 blocked the cell cycle in the G2/M-phase and induced apoptosis in HL-60 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.

Activations of Both Extrinsic and Intrinsic Pathways in HCT 116 Human Colorectal Cancer Cells Contribute to Apoptosis through p53-Mediated ATM/Fas Signaling by Emilia sonchifolia Extract, a Folklore Medicinal Plant.

Emilia sonchifolia (L.) DC (Compositae), an herbaceous plant found in Taiwan and India, is used as folk medicine. The clinical applications include inflammation, rheumatism, cough, cuts fever, dysentery, analgesic, and antibacteria. The activities of Emilia sonchifolia extract (ESE) on colorectal cancer cell death have not been fully investigated. The purpose of this study explored the induction of apoptosis and its molecular mechanisms in ESE-treated HCT 116 human colorectal cancer cells in vitro. The methanolic ESE was characterized, and γ-humulene was formed as the major constituent (63.86%). ESE induced cell growth inhibition in a concentration- and time-dependent response by MTT assay. Apoptotic cells (DNA fragmentation, an apoptotic catachrestic) were found after ESE treatment by TUNEL assay and DNA gel electrophoresis. Alternatively, ESE stimulated the activities of caspase-3, -8, and -9 and their specific caspase inhibitors protected against ESE-induced cytotoxicity. ESE promoted the mitochondria-dependent and death-receptor-associated protein levels. Also, ESE increased ROS production and upregulated the levels of ATM, p53, and Fas in HCT 116 cells. Strikingly, p53 siRNA reversed ESE-reduced viability involved in p53-mediated ATM/Fas signaling in HCT 116 cells. In summary, our result is the first report suggesting that ESE may be potentially efficacious in the treatment of colorectal cancer.

Cucurbitacin E Induces G(2)/M Phase Arrest through STAT3/p53/p21 Signaling and Provokes Apoptosis via Fas/CD95 and Mitochondria-Dependent Pathways in Human Bladder Cancer T24 Cells.

Cucurbitacin E, a tetracyclic triterpenes compound extracted from cucurbitaceous plants, has been shown to exhibit anticancer and anti-inflammatory activities. The purpose of this study was to elucidate whether cucurbitacin E promotes cell cycle arrest and induces apoptosis in T24 cells and further to explore the underlying molecular mechanisms. The effects of cucurbitacin E on T24 cell's growth and accompanied morphological changes were examined by MTT assay and a phase-contrast microscope. DNA content, mitochondrial membrane potential (ΔΨ(m)) and annexin V/PI staining were determined by flow cytometry. The protein levels were measured by Western blotting. Our results demonstrated that cucurbitacin E-induced G(2)/M arrest was associated with a marked increase in the levels of p53, p21 and a decrease in phospho-signal transducer and activator of transcription 3 (STAT3), cyclin-dependent kinase 1 (CDK1) and cyclin B. Cucurbitacin E-triggered apoptosis was accompanied with up-regulation of Fas/CD95, truncated BID (t-BID) and a loss of ΔΨ(m), resulting in the releases of cytochrome c, apoptotic protease activating factor 1 (Apaf-1) and apoptosis-inducing factor (AIF), and sequential activation of caspase-8, caspase-9, and caspase-3. Our findings provided the first evidence that STAT3/p53/p21 signaling, Fas/CD95 and mitochondria-dependent pathways play critical roles in cucurbitacin E-induced G(2)/M phase arrest and apoptosis of T24 cells.

Houttuynia cordata Thunb extract induces apoptosis through mitochondrial-dependent pathway in HT-29 human colon adenocarcinoma cells.

The Houttuynia cordata Thunb (HCT) extract has been used as a traditional Chinese herb medicine and as well as an effective drug for treating allergic inflammation for thousands of years. In this study, we investigated the anti-cancer activity of HCT and its molecular mechanisms in the human colon adenocarcinoma cell line HT-29. HCT inhibited HT-29 cell viability in a dose- and time-dependent manner by MTT assay. Treatment with 450 microg/ml of HCT for 48 and 72 h led to DNA damage and apoptosis by DAPI staining and comet assay. HCT increased reactive oxygen species production and decreased the levels of mitochondria membrane potential (MMP) in HT-29 cells by flow cytometry analysis. HCT caused the release of cytochrome c, Apaf-1, pro-caspase-9 and AIF from mitochondria via a decrease of the MMP. The decrease of MMP was then associated with a decrease in the ratio of Bax/Bcl-2 and activation of caspase-9 and -3 by Western blotting and caspase activity assay. Caspase-9 and -3 inhibitors almost completely suppressed HCT-induced caspase-9 and -3 activities. Our results demonstrated that the HCT-induced apoptosis in human colon adenocarcinoma cell line HT-29 might be related to a mitochondrial-dependent pathway.

Intrathecal substance P (1-7) prevents morphine-evoked spontaneous pain behavior via spinal NMDA-NO cascade.

Previous research has shown that injection of high-dose of morphine into the spinal lumbar intrathecal (i.t.) space of rats elicits an excitatory behavioral syndrome indicative of severe vocalization and agitation. Substance P N-terminal fragments are known to inhibit nociceptive responses when injected i.t. into animals. In this study, we investigated the effect of i.t. substance P (1-7) on both the nociceptive response and the extracellular concentrations of glutamate and nitric oxide (NO) metabolites (nitrite/nitrate) evoked by high-dose i.t. morphine (500 nmol). The induced behavioral responses were attenuated dose-dependently by i.t. pretreatment with the substance P N-terminal fragment substance P (1-7) (100-400 pmol). The inhibitory effect of substance P (1-7) was reversed significantly by pretreatment with [d-Pro2, d-Phe7]substance P (1-7) (20 and 40 nmol), a d-isomer and antagonist of substance P (1-7). In vivo microdialysis analysis showed a significant elevation of extracellular glutamate and NO metabolites in the spinal cord after i.t. injection of high-dose morphine (500 nmol). Pretreatment with substance P (1-7) (400 pmol) produced a significant reduction on the elevated concentrations of glutamate and NO metabolites evoked by i.t. morphine. The reduced levels of glutamate and NO metabolites were significantly reversed by the substance P (1-7) antagonist (40 nmol). The present results suggest that i.t. substance P (1-7) may attenuate the excitatory behavior (vocalization and agitation) of high-dose i.t. morphine by inhibiting the presynaptic release of glutamate, and reducing NO production in the dorsal spinal cord.

Extracellular signal-regulated kinase (ERK) and nitric oxide synthase mediate intrathecal morphine-induced nociceptive behavior.

Intrathecal (i.t.) administration of morphine at a high dose of 60nmol into the spinal lumbar space in mice produces a severe hindlimb scratching followed by biting and licking. Nitric oxide (NO) is thought to play an important role in signal transduction pathways that enhance nociceptive transmission in the spinal cord. The present study was designed to determine whether high-dose i.t. morphine could influence the activation of the extracellular signal-regulated kinase (ERK), a mitogen-activated protein (MAP) kinase in neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS) activation. Both 7-NI and TRIM, selective inhibitors of nNOS, resulted in a dose-dependent inhibition of high-dose i.t. morphine-induced behavior. The selective iNOS inhibitor W1400 in relatively large doses inhibited in a non dose-dependent manner. The i.t. injection of morphine evoked a definite activation of ERK in the lumbar dorsal spinal cord. Behavioral experiments showed that U0126 (0.5-2.5nmol), a MAP kinase-ERK inhibitor, dose-dependently attenuated the behavioral response to i.t. morphine. In mice treated with high-dose morphine, 7-NI was very effective in blocking ERK activation, whereas W1400 had no effect. Taken together, these results suggest that the behavioral response to high-dose i.t. morphine may be triggered by the nNOS-ERK pathway in the dorsal spinal cord.