Resveratrol 3-O-D-glucuronide and resveratrol 4'-O-D-glucuronide inhibit colon cancer cell growth: evidence for a role of A3 adenosine receptors, cyclin D1 depletion, and G1 cell cycle arrest.

SCOPE: Resveratrol is a plant-derived polyphenol with chemotherapeutic properties in animal cancer models and many biochemical effects in vitro. Its bioavailability is low and raises the possibility that the metabolites of resveratrol have biological effects. Here we investigate the actions of resveratrol 3-O-D-glucuronide, resveratrol 4-O-D-glucuronide, and resveratrol 3-O-Dsulfate on the growth of colon cancer cells in vitro. METHODS AND RESULTS: The growth of Caco-2, HCT-116, and CCL-228 cells was measured using the neutral red and MTT assays. Resveratrol and each metabolite inhibited cell growth with IC50 values of 9.8­31 µM. Resveratrol caused S phase arrest in all three cell lines. Resveratrol 3-O-D-glucuronide and resveratrol 4-O-D-glucuronide caused G1 arrest in CCL-228 and Caco-2 cells. Resveratrol 3-O-D-sulfate had no effect on cell cycle. Growth inhibition was reversed by an inhibitor of AMP-activated protein kinase (compound C) or an adenosine A3 receptor antagonist (MRS1191). The A3 receptor agonist 2Cl-IB-MECA inhibited growth and A3 receptors were detected in all cell lines. The resveratrol glucuronides also reduced cyclin D1 levels but at higher concentrations than in growth experiments and generally did not increase phosphorylated AMP-activated protein kinase. CONCLUSION: Resveratrol glucuronides inhibit cell growth by G1 arrest and cyclin D1 depletion, and our results strongly suggest a role for A3 adenosine receptors in this inhibition.

A novel steroidal saponin glycoside from Fagonia indica induces cell-selective apoptosis or necrosis in cancer cells.

Fagonia indica is a small spiny shrub of great ethnopharmacological importance in folk medicine. The aqueous decoction of aerial parts is a popular remedy against various skin lesions, including cancer. We used a biological activity-guided fractionation approach to isolate the most potent fraction of the crude extract on three cancer cell lines: MCF-7 oestrogen-dependent breast cancer, MDA-MB-468 oestrogen-independent breast cancer, and Caco-2 colon cancer cells. A series of chromatographic and spectroscopic procedures were utilised on the EtOAc fraction, which resulted in the isolation of a new steroidal saponin glycoside. The cytotoxic activity of the saponin glycoside was determined in cancer cells using the MTT and neutral red uptake assays. After 24h treatment, the observed IC(50) values of the saponin glycoside were 12.5 µM on MDA-MB-468 and Caco-2 cells, but 100 µM on MCF-7 cells. Several lines of evidence: PARP cleavage, caspase-3 cleavage, DNA ladder assays, and reversal of growth inhibition with the pan-caspase inhibitor Z-VAD-fmk, suggested stimulation of apoptosis in MDA-MB-468 and Caco-2 cells, but not in MCF-7 cells, which do not express caspase-3. The haemolytic activity of the saponin glycoside was confirmed in sheep red blood cells, with cell lysis observed at >100 µM, suggesting that, at this concentration, the saponin glycoside caused necrosis through cell lysis in MCF-7 cells. Using the DNA ladder assay, the saponin glycoside (12.5 µM) was not toxic to HUVEC (human umbilical vein endothelial cells) or U937 cells, indicating some selectivity between malignant and normal cells. We conclude that the steroidal saponin glycoside isolated from F. indica is able to induce apoptosis or necrosis in cancer cells depending on the cell type.

Inhibition of stretching-evoked ATP release from bladder mucosa by anticholinergic agents.

OBJECTIVE: To determine whether muscarinic receptor antagonism affects stretching-induced release of ATP. MATERIALS AND METHODS: Mucosal strips, dissected from guinea pig (male, 450g; n = 10) urinary bladders, were placed in horizontal organ baths and superfused with Ca(2+) -free Tyrode's solution. Superfusate samples were taken pre- and post- intervention (rapid stretching or relaxation) and ATP concentration was quantified using a luciferin-luciferase assay. The effect of muscarinic acetylcholine receptor antagonism on ATP release was assessed by addition of methoctramine (1 µM) and 4-DAMP (10 nM). RESULTS: Rapid stretching (0 to 13.3 ± 1.2 mN; no. strips = 20) increased ATP in the superfusate to a median threefold increase over basal levels. After a period of equilibration, tension in the mucosal strips relaxed until it had reached a new steady-state after 60 min and stretching was repeated. In the presence of 4-DAMP (10 nM) or methoctramine (1 µM), ATP concentrations after stretching reduced to 61% or 20%, respectively. By contrast, ATP concentrations in mucosa-matched controls, perfused with vehicle, increased in response to stretching by 391% and 1500%, respectively. Rapid relaxation also stimulated ATP release. This release did not appear to be sensitive to 4-DAMP or methoctramine. CONCLUSIONS: An alteration of resting mucosal tension is the key determinant of ATP release, as ATP is released from the mucosa in response to both stretching and relaxation. Muscarinic receptor antagonism inhibits stretching-evoked ATP release from bladder mucosa, suggesting that anticholinergic agents used to treat human lower urinary tract pathologies act on urothelial muscarinic receptors.

Novel acylated steroidal glycosides from Caralluma tuberculata induce caspase-dependent apoptosis in cancer cells.

AIM OF THE STUDY: Pregnane glycosides are potent cytotoxic agents which may represent new leads in the development of anti-tumour drugs, particularly in the treatment of breast cancer, because of the structural similarity to estrogenic agonists. Caralluma species are natural sources of a wide variety of pregnane glycosides. The aim of the study was to isolate, using an activity-guided fractionation approach, novel pregnane glycosides for testing on breast cancer and other tumour lines. MATERIALS AND METHODS: The effect of crude extracts, specific organic fractions and isolated compounds from Caralluma tuberculata was tested on the growth and viability of MCF-7 estrogen-dependent, and MDA-MB-468 estrogen-independent breast cancer cells, Caco-2 human colonic cells, HUVECs and U937 cells. Neutral red uptake and MTT assays were used. Apoptosis was detected by Western blot of poly-(ADP ribose) polymerase (PARP) as were other markers of nuclear fragmentation (DNA ladder assay, staining of cells with nuclear dye DAPI). The involvement of caspases was investigated using the pan-caspase inhibitor Z-VAD-FMK. RESULTS: The ethyl acetate fraction of Caralluma tuberculata was found to be the most potent anti-proliferative fraction against all three cancer cell lines. Two novel steroidal glycosides were isolated from the active fraction after a series of chromatographic experiments. The structure of the isolated compounds was elucidated solely based on 2D-NMR (HMBC, HETCOR, DQF-COSY) and MS spectral analysis as compound 1: 12-O-benzoyl-20-O-acetyl-3ß,12ß,14ß,20ß-tetrahydroxy-pregnan-3-ylO-ß-D-glucopyranosyl-(1→4)-ß-D-glucopyranosyl-(1 → 4)-3-methoxy-ß-D-ribopyranoside, and as compound 2: 7-O-acetyl-12-O-benzoyl-3ß,7ß,12ß,14ß-tetrahydroxy-17ß-(3-methylbutyl-O-acetyl-1-yl)-androstan-3-ylO-ß-D-glucopyranosyl-(1 → 4)-6-deoxy-ß-D-allopyranosyl-(1 → 4)-ß-D-cymaropyranosyl-(1 → 4)-ß-D-cymapyranosyl-(1→ 4)-ß-D-cymaropyranoside. Compound 1 (pregnane glycoside) and compound 2 (androstan glycoside) induced apoptosis at <25 µM after 48 h as assessed by cell shrinkage, PARP cleavage, DNA fragmentation, and reversal with the caspase inhibitor. CONCLUSIONS: Two novel steroid glycosides isolated from Caralluma tuberculata possess moderate, micromolar cytotoxic activity on breast cancer and other cells in vitro, which may indicate a source of activity in vivo of interest to future drug design.

Cystic fibrosis airway epithelial Ca2+ i signaling: the mechanism for the larger agonist-mediated Ca2+ i signals in human cystic fibrosis airway epithelia.

In cystic fibrosis (CF) airways, abnormal epithelial ion transport likely initiates mucus stasis, resulting in persistent airway infections and chronic inflammation. Mucus clearance is regulated, in part, by activation of apical membrane receptors coupled to intracellular calcium (Ca(2+)(i)) mobilization. We have shown that Ca(2+)(i) signals resulting from apical purinoceptor (P2Y(2)-R) activation are increased in CF compared with normal human airway epithelia. The present study addressed the mechanism for the larger apical P2Y(2)-R-dependent Ca(2+)(i) signals in CF human airway epithelia. We show that the increased Ca(2+)(i) mobilization in CF was not specific to P2Y(2)-Rs because it was mimicked by apical bradykinin receptor activation, and it did not result from a greater number of P2Y(2)-R or a more efficient coupling between P2Y(2)-Rs and phospholipase C-generated inositol 1,4,5-trisphosphate. Rather, the larger apical P2Y(2)-R activation-promoted Ca(2+)(i) signals in CF epithelia resulted from an increased density and Ca(2+) storage capacity of apically confined endoplasmic reticulum (ER) Ca(2+) stores. To address whether the ER up-regulation resulted from ER retention of misfolded DeltaF508 CFTR or was an acquired response to chronic luminal airway infection/inflammation, three approaches were used. First, ER density was studied in normal and CF sweat duct human epithelia expressing high levels of DeltaF508 CFTR, and it was found to be the same in normal and CF epithelia. Second, apical ER density was morphometrically analyzed in airway epithelia from normal subjects, DeltaF508 homozygous CF patients, and a disease control, primary ciliary dyskinesia; it was found to be greater in both CF and primary ciliary dyskinesia. Third, apical ER density and P2Y(2)-R activation-mobilized Ca(2+)(i), which were investigated in airway epithelia in a long term culture in the absence of luminal infection, were similar in normal and CF epithelia. To directly test whether luminal infection/inflammation triggers an up-regulation of the apically confined ER Ca(2+) stores, normal airway epithelia were chronically exposed to supernatant from mucopurulent material from CF airways. Supernatant treatment expanded the apically confined ER, resulting in larger apical P2Y(2)-R activation-dependent Ca(2+)(i) responses, which reproduced the increased Ca(2+)(i) signals observed in CF epithelia. In conclusion, the mechanism for the larger Ca(2+)(i) signals elicited by apical P2Y(2)-R activation in CF airway epithelia is an expansion of the apical ER Ca(2+) stores triggered by chronic luminal airway infection/inflammation. Greater ER-derived Ca(2+)(i) signals may provide a compensatory mechanism to restore, at least acutely, mucus clearance in CF airways.

Regulation of Ins(3,4,5,6)P(4) signaling by a reversible kinase/phosphatase.

Regulation of Cl(-) channel conductance by Ins(3,4,5,6)P(4) provides receptor-dependent control over salt and fluid secretion, cell volume homeostasis, and electrical excitability of neurones and smooth muscle. Ignorance of how Ins(3,4,5,6)P(4) is synthesized has long hindered our understanding of this signaling pathway. We now show Ins(3,4,5,6)P(4) synthesis by Ins(1,3,4,5,6)P(5) 1-phosphatase activity by an enzyme previously characterized as an Ins(3,4,5,6)P(4) 1-kinase. Rationalization of these phenomena with a ligand binding model unveils Ins(1,3,4)P(3) as not simply an alternative kinase substrate, but also an activator of Ins(1,3,4,5,6)P(5) 1-phosphatase. Stable overexpression of the enzyme in epithelial monolayers verifies its physiological role in elevating Ins(3,4,5,6)P(4) levels and inhibiting secretion. It is exceptional for a single enzyme to catalyze two opposing signaling reactions (1-kinase/1-phosphatase) under physiological conditions. Reciprocal coordination of these opposing reactions offers an alternative to general doctrine that intracellular signals are regulated by integrating multiple, distinct phosphatases and kinases.