Himachalol induces apoptosis in B16-F10 murine melanoma cells and protects against skin carcinogenesis.

ETHNOPHARMACOLOGICAL RELEVANCE: Cedrus libani A. Rich (C. libani) is majestic evergreen Mediterranean conifer growing in the mountains of Lebanon. The ethnobotanical and traditional uses of cedar wood oil traces back to ancient times for the treatment of various ailments including cancer. Previous work in our laboratories revealed that himachalol (7-HC), a major sesquiterpene isolated from C. libani, possesses potent cytotoxic activity against various human cancer cell lines as well as promising anti-inflammatory effect in isolated rat monocytes. AIM OF THE STUDY: The present study aims to elucidate the mechanism of action behind the cytotoxic activity of 7-HC against murine melanoma cells (B16F-10) and evaluates its chemopreventive effect against chemically-induced skin carcinogenesis in mice. MATERIALS AND METHODS: 7-HC was extracted and purified from Cedrus libani wood. Cell viability was evaluated using WST-1 kit. Cell cycle analysis and apoptosis were assessed by Flow cytometry using propidium iodide (PI) and fluorescein Isothiocyanate (FITC)-conjugated Annexin V/PI staining respectively. Apoptosis related protein were quantified using western blot. The chemopreventive activity of 7-HC was evaluated for 20 weeks using a DMBA/TPA induced skin carcinogenesis model in Balb/c mice. RESULTS: 7-HC displayed a potent anti-proliferative activity against the melanoma cells with an IC50 of 8.8 µg/ml and 7.3 µg/ml at 24 and 48 h, respectively. Co-treatment with Cisplatin did not show any synergistic or additive effect on cell viability. Flow cytometry analysis using PI revealed that 7-HC treatment (5 and 10 µg/ml) induces the accumulation of cells in the sub-G1 phase and causes a decline in cell populations in the S and G2/M phases. Annexin/PI staining also reveals that 7-HC treatment significantly increases the percentage of cells undergoing early and late apoptosis. Western blot analysis shows that 7-HC treatment decreases the level of the anti-apoptotic protein Bcl-2 and increases the level of the pro-apoptotic protein Bax. A reduction in the level of phosphorylated Erk and Akt was also observed. 7-HC via topical (2.5%), intraperitoneal (10, 25 and 50 mg/kg) or gavage (50 mg/kg) treatment revealed a significant decrease in papilloma volume with no adverse effect on liver and kidney function. CONCLUSIONS: The present study demonstrates that 7-HC treatment protects against chemically-induced skin carcinogenesis, promotes cell cycle arrest and induces apoptosis partially through an inhibition of both the MAPK/Erk and PI3K/Akt pathways.

Purpurogallin is a novel mitogen-activated protein kinase kinase 1/2 inhibitor that suppresses esophageal squamous cell carcinoma growth in vitro and in vivo.

Purpurogallin is a natural compound that is extracted from nutgalls and oak bark and it possesses antioxidant, anticancer, and anti-inflammatory properties. However, the anticancer capacity of purpurogallin and its molecular target have not been investigated in esophageal squamous cell carcinoma (ESCC). Herein, we report that purpurogallin suppresses ESCC cell growth by directly targeting the mitogen-activated protein kinase kinase 1/2 (MEK1/2) signaling pathway. We found that purpurogallin inhibits anchorage-dependent and -independent ESCC growth. The results of in vitro kinase assays and cell-based assays indicated that purpurogallin also strongly attenuates the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and also directly binds to and inhibits MEK1 and MEK2 activity. Furthermore, purpurogallin contributed to S and G2 phase cell cycle arrest by reducing cyclin A2 and cyclin B1 expression and also induced apoptosis by activating poly (ADP ribose) polymerase (PARP). Notably, purpurogallin suppressed patient-derived ESCC tumor growth in an in vivo mouse model. These findings indicated that purpurogallin is a novel MEK1/2 inhibitor that could be useful for treating ESCC.

Widdrol, a sesquiterpene isolated from Juniperus chinensis, inhibits angiogenesis by targeting vascular endothelial growth factor receptor 2 signaling.

Widdrol is an odorous compound derived from Juniperus chinensis that is widely used in traditional medicine to treat fever, inflammation and cancer. It was previously reported that widdrol has antitumor activity by apoptosis induction in cancer cells in vitro. However, its anti-angiogenic activity remains elusive. In the present study, we investigated the anti­angiogenic activity of widdrol and the molecular mechanisms involved. Widdrol inhibited cell proliferation via G1 phase arrest induction in human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Additionally, it was associated with a decreased expression of cyclin-dependent kinase 2 (CDK2) and an increased expression of p21, a CDK inhibitor. Widdrol significantly inhibited the cell migration and tube formation of HUVECs using an in vitro angiogenesis assay. The results showed that widdrol suppressed phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and its downstream proteins, such as AKT, focal adhesion kinase (FAK) and endothelial nitric oxide synthase (eNOS). Moreover, widdrol effectively reduced tumor growth and blood vessel formation in colon tumor xenograft mice. Collectively, these results suggested that widdrol may act as a potential anti-angiogenic agent by inhibiting vessel sprouting and growth, which may have implications for angioprevention.

The Axl receptor tyrosine kinase is an adverse prognostic factor and a therapeutic target in esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) arises in the backdrop of reflux-induced metaplastic phenomenon known as Barrett esophagus. The prognosis of advanced EAC is dismal, and there is an urgent need for identifying molecular targets for therapy. Serial Analysis of Gene Expression (SAGE) was performed on metachronous mucosal biopsies from a patient who underwent progression to EAC during endoscopic surveillance. SAGE confirmed significant upregulation of Axl "tags" during the multistep progression of Barrett esophagus to EAC. In a cohort of 92 surgically resected EACs, Axl overexpression was associated with shortened median survival on both univariate (p < 0.004) and multivariate (p < 0.036) analysis. Genetic knockdown of Axl receptor tyrosine kinase (RTK) function was enabled in two EAC lines (OE33 and JH-EsoAd1) using lentiviral short hairpin RNA (shRNA). Genetic knockdown of Axl in EAC cell lines inhibited invasion, migration, and in vivo engraftment, which was accompanied by downregulation in the activity of the Ral GTPase proteins (RalA and RalB). Restoration of Ral activation rescued the transformed phenotype of EAC cell lines, suggesting a novel effector mechanism for Axl in cancer cells. Pharmacological inhibition of Axl was enabled using a small molecule antagonist, R428 (Rigel Pharmaceuticals). Pharmacological inhibition of Axl with R428 in EAC cell lines significantly reduced anchorage-independent growth, invasion and migration. Blockade of Axl function abrogated phosphorylation of ERBB2 (Her-2/neu) at the Tyr877 residue, indicative of receptor crosstalk. Axl RTK is an adverse prognostic factor in EAC. The availability of small molecule inhibitors of Axl function provides a tractable strategy for molecular therapy of established EAC.

Widdrol induces cell cycle arrest, associated with MCM down-regulation, in human colon adenocarcinoma cells.

Widdrol, an odorous compound extracted from Juniperus chinensis, has been shown to inhibit the in vitro growth of in human cancer cells. This study was conducted on cultured human colon adenocarcinoma HT29 cells to elucidate the possible mechanisms by which widdrol exerts its anti-proliferative activity, which until now has remained poorly understood. It was found that widdrol induces accumulation of sub-G1 phase and arrests in the G1 phase of the cell cycle. Induction of G1 arrest by widdrol was correlated with induction of Chk2, p53 phosphorylation and CDK inhibitor p21 expression as well as inhibition of cyclin E, cyclin-dependent kinase (CDK2) and retinoblastoma protein (pRB). Moreover, mini-chromosome maintenance (MCM) proteins were markedly down-regulated in HT29 cells treated with widdrol. Altogether, these results show widdrol possesses potential anti-cancer activity against colon adenocarcinoma cells by inhibiting their proliferation and inducing cell cycle G1 arrest.

Pathway-specific action of gamma-hydroxybutyric acid in sensory thalamus and its relevance to absence seizures.

The systemic injection of gamma-hydroxybutyric acid (GHB) elicits spike and wave discharges (SWDs), the EEG hallmark of absence seizures, and represents a well established, widely used pharmacological model of this nonconvulsive epilepsy. Despite this experimental use of GHB, as well as its therapeutic use in narcolepsy and its increasing abuse, however, the precise cellular mechanisms underlying the different pharmacological actions of this drug are still unclear. Because sensory thalamic nuclei play a key role in the generation of SWDs and sleep rhythms, and because direct injection of GHB in the ventrobasal (VB) thalamus elicits SWDs, we investigated GHB effects on corticothalamic EPSCs and GABAergic IPSCs in VB thalamocortical (TC) neurons. GHB (250 microm-10 mm) reversibly decreased the amplitude of electrically evoked EPSCs and GABAA IPSCs via activation of GABAB receptors; however, approximately 60% of the IPSCs were insensitive to low (250 microm-1.0 mm) GHB concentrations. The putative GHB receptor antagonist NSC 382 applied alone had a number of unspecific effects, whereas it either had no action on, or further increased, the GHB-elicited effects on synaptic currents. Low GHB concentrations (250 microm) were also effective in increasing absence-like intrathalamic oscillations evoked by cortical afferent stimulation. These results indicate that low concentrations of GHB, similar to the brain concentrations that evoke SWDs in vivo, differentially affect excitatory and inhibitory synaptic currents in TC neurons and promote absence-like intrathalamic oscillations. Furthermore, the present data strengthen previous suggestions on the GHB mechanism of sleep promotion and will help focus future studies on the cellular mechanisms underlying its abuse.

Inhibitory effect of green tea polyphenols on membrane-type 1 matrix metalloproteinase, MT1-MMP.

Matrix metalloproteinases (MMPs), especially membrane-type 1 matrix metalloproteinase (MT1-MMP), which generates an active form of MMP-2 from proMMP-2, are deeply involved in angiogenesis as well as in tumor cell migration and metastasis. To obtain a specific inhibitor for MT1-MMP, we screened a number of natural and synthetic compounds using recombinant human MMP-2, MMP-7, and soluble MT1-MMP in a fluorogenic peptide cleavage assay. (-)-Epigallocatechin 3-O-gallate (EGCG) followed by (-)-epigallocatechin 3,5-di-O-gallate and epitheaflagallin 3-O-gallate, was found to have potent and distinct inhibitory activity against MT1-MMP. Therefore, we investigated the effect of EGCG on the suppression of MMP-2 activation as determined by gelatin zymography, and observed that the active form of MMP-2 in the conditioned medium of human umbilical vein endothelial cells was decreased in the presence of EGCG. The results suggest the possibility that tea polyphenols suppress tumor growth through the suppression of angiogenesis.

Therapeutic intervention in mice deficient for succinate semialdehyde dehydrogenase (gamma-hydroxybutyric aciduria).

Therapeutic intervention for human succinic semialdehyde dehydrogenase (SSADH) deficiency (gamma-hydroxybutyric aciduria) has been limited to vigabatrin (VGB). Pharmacologically, VGB should be highly effective due to 4-aminobutyrate-transaminase (GABA-transaminase) inhibition, lowering succinic semialdehyde and, thereby, gamma-hydroxybutyric acid (GHB) levels. Unfortunately, clinical efficacy has been limited. Because GHB possesses a number of potential receptor interactions, we addressed the hypothesis that antagonism of these interactions in mice with SSADH deficiency could lead to the development of novel treatment strategies for human patients. SSADH-deficient mice have significantly elevated tissue GHB levels, are neurologically impaired, and die within 4 weeks postnatally. In the current report, we compared oral versus intraperitoneal administration of VGB, CGP 35348 [3-aminopropyl(diethoxymethyl)phosphinic acid, a GABA(B) receptor antagonist], and the nonprotein amino acid taurine in rescue of SSADH-deficient mice from early death. In addition, we assessed the efficacy of the specific GHB receptor antagonist NCS-382 (6,7,8,9-tetrahydro-5-[H]benzocycloheptene-5-ol-6-ylideneacetic acid) using i.p. administration. All interventions led to significant lifespan extension (22-61%), with NCS-382 being most effective (50-61% survival). To explore the limited human clinical efficacy of VGB, we measured brain GHB and gamma-aminobutyric acid (GABA) levels in SSADH-deficient mice receiving VGB. Whereas high-dose VGB led to the expected elevation of brain GABA, we found no parallel decrease in GHB levels. Our data indicate that, at a minimum, GHB and GABA(B) receptors are involved in the pathophysiology of SSADH deficiency. We conclude that taurine and NCS-382 may have therapeutic relevance in human SSADH deficiency and that the poor clinical efficacy of VGB in this disease may relate to an inability to decrease brain GHB concentrations.

Inhibition of oncogene product enzyme activity as an approach to cancer chemoprevention. Tyrosine-specific protein kinase inhibition by purpurogallin from Quercus sp. nutgall.

Inhibitors of oncogene product enzyme activity were sought as a prescreen for potential cancer chemopreventive agents. Purpurogallin, a polyphenol from Quercus sp. nutgall, was found to inhibit the tyrosine-specific protein kinase of the human erb-b oncogene product (epidermal growth factor receptor) for both autophosphorylation (IC50 = 27.5 microM) and phosphorylation of an exogenous substrate (IC50 = 45.3 microM). An examination of enzyme kinetics indicated that purpurogallin is a competitive inhibitor of both ATP (Ki = 54.9 microM for autophosphorylation, Ki = 33.9 microM for phosphorylation of exogenous substrate) and the tyrosine-containing acceptor substrate poly(glutamate, alanine, tyrosine) 6:3:1 (Ki = 83.7 microM).

gamma-Hydroxybutyrate modulates synthesis and extracellular concentration of gamma-aminobutyric acid in discrete rat brain regions in vivo.

gamma-Hydroxybutyrate possesses most of the properties of a neurotransmitter/neuromodulator that acts via specific pathways and receptors in brain. Beside its regulatory effects on dopaminergic transmission, gamma-hydroxybutyrate was thought for many years to interfere with gamma-aminobutyric acid (GABA)ergic processes in the brain. The present study demonstrates that in the rat frontal cortex in vivo, gamma-hydroxybutyrate or its agonist NCS-356 administered systemically at a high dose (500 mg/kg) increases GABA contents in dialysates via a mechanism blocked by the peripheral administration of the gamma-hydroxybutyrate antagonist NCS-382. Under the same conditions, the extracellular concentration of this amino acid was not modified in the hippocampus. However, when administered at a low dose (250 mg/kg), gamma-hydroxybutyrate decreases GABA content of the dialysates of the frontal cortex by an NCS-382-sensitive mechanism. Spontaneous [3H]GABA release was observed in the frontal cortex of rats at 160 min after i.p. [3H]-gamma-hydroxybutyrate administration. This result indicates that gamma-hydroxybutyrate in vivo could be the precursor of an extracellular GABA pool in the frontal cortex. After i.p. [3H]-gamma-hydroxybutyrate administration in the rat, the amino acid contents of several brain regions were quantified 160 min later, and the radioactivity in each region was measured. [3H]GABA, [3H]glutamate, and [3H]glycine were detected in most, but not all, of the brain regions studied. In particular, radioactive GABA was not detected in the hippocampus. The other amino acids were not labeled. These results show that gamma-hydroxybutyrate modulates the synthesis and the extracellular concentrations of GABA in specific regions of the rat brain. Identification of these GABA pools and determination of their functional role remain to be defined.

Evaluation of himachalol in murine invasive aspergillosis.

Himachalol (a sesquiterpene alcohol) showed low in vitro minimum inhibitory concentrations against Aspergillus fumigatus by the macro- and microbroth dilution techniques (genometric mean 250 micrograms ml-1 and 46.4 micrograms ml-1 respectively) compared with saperconazole. Swiss mice treated with the phytoproduct (200 mg kg-1, p.o.) once daily for 7 days exhibited a significant (P < 0.001) protection (60%) together with an increase in the mean survival time (15 days) and a reduced CFU (mean log10) burden of A. fumigatus in the kidneys.

Intrathalamic injections of gamma-hydroxybutyric acid increase genetic absence seizures in rats.

The effects of intrathalamic injections of gamma-hydroxybutyric acid (GHB) and of NCS 382 85, a specific antagonist of GHB receptors, were evaluated in rats with spontaneous generalized absence epilepsy. Bilateral injections of GHB (25 and 50 micrograms/side) into the mediolateral thalamus increased spontaneous spike and wave discharges (SWD) in a dose-dependent fashion. This effect was suppressed by administration of NCS 382 85 (50 micrograms/side). Bilateral injection of NCS 382 85 alone (50 and 100 micrograms/side) into the same sites had no effect on SWD duration. Injection of GHB or NCS 382 85 into the midline thalamus and the area of reticular nuclei did not modify the SWD. These data suggest that GHB receptors in the mediolateral thalamus may be involved in the control of spontaneous SWD in this rat model of petit mal epilepsy.

Spasmolytic constituents of Cedrus deodara (Roxb.) Loud: pharmacological evaluation of himachalol.

Himachalol has been identified as the major antispasmodic constituent in the wood of Cedrus deodara. The pharmacological studies of himachalol on various isolated smooth muscles (guinea pig ileum, rabbit jejunum, rat uterus, and guinea pig seminal vesicle) and against different agonists (acetylcholine, histamine, serotonin, nicotine, and barium chloride) indicated spasmolytic activity similar to that of papaverine. It was a more potent antagonist of barium chloride-induced spasm of guinea pig ileum than papaverine but less effective in reverting a similar spasm of rabbit jejunum and had no relaxing effect alone. In the conscious immobilized cat, intragastric administration of himachalol or papaverine (100 mg/kg) produced equal inhibition of carbachol-induced spasm of the intestine, lasting about 2 hr, but himachalol had a faster onset of action. Himachalol was devoid of spasmolytic effect on the bronchial musculature of guinea pig but was 3.3 times more potent than papaverine in antagonizing epinephrine-induced contraction of the guinea pig seminal vesicle. Intravenous injection of himachalol (3-10 mg/kg) in the cat produced a dose-dependent fall in blood pressure and an increased femoral blood flow.

Possible role of microtubules in thyroid secretion.

Colchicine and other microtubule-active agents have been found to block the release, stimulated by either thyroid-stimulating hormone or by dibutyryl cyclic adenosine 3':5'-monophosphate, of (131)I from previously (131)I-labeled mouse thyroid glands in vitro. The time and concentration characteristics of these inhibitors are consistent with their actions on microtubules in other systems. [(3)H]colchicine was also shown to be bound to a soluble 6S protein of bovine thyroid slices similar to the protein identified in other systems as a microtubular subunit. The demonstrated inhibition of colloid droplet formation and absence of an effect on thyroidal adenyl cyclase or cyclic 3':5'-phosphodiesterase suggests a colchicine-sensitive role for microtubules in colloid endocytosis in the thyroid gland.