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Long-term clinical evaluation of toothpaste and oral rinse containing sanguinaria extract in controlling plaque, gingival inflammation, and sulcular bleeding during orthodontic treatment.

A sanguinaria-containing toothpaste and oral rinse regimen was evaluated during a 6-month period to determine its effectiveness in controlling plaque, gingival inflammation, and sulcular bleeding. Oral hygiene instructions and a 1-month prestudy brushing period failed to produce a significant improvement in health among the 24 subjects as determined by the three evaluation parameters. After the oral hygiene period, the subjects were randomly assigned to the active treatment (marketed Viadent toothpaste and oral rinse) or the placebo treatment (same base formulas without sanguinaria). Treatments were evaluated using the Löe and Silness gingival index, the Silness and Löe plaque index, and the Mühlemann and Son sulcular bleeding index at baseline and monthly through 6 months. The sanguinaria regimen reduced plaque by 57%, gingival inflammation by 60%, and sulcular bleeding by 45% from baseline compared with placebo group reductions of 27% (plaque) and 21% (gingival inflammation), and an increase of 30% in bleeding index. Repeated-measures analysis of covariance with baseline scores used as covariates showed statistically significant differences at 6 months for plaque (active 0.39; placebo 0.68, p less than 0.01), gingival inflammation (active 0.32; placebo 0.76, p less than 0.001), and sulcular bleeding (active 0.34; placebo 0.70, p less than 0.002). Results of this study demonstrated that the combined use of the sanguinaria-containing toothpaste and oral rinse controls and reduces plaque and gingival inflammation in an orthodontic population.

Adolescent↗

Antiplatelet effect of sanguinarine is correlated to calcium mobilization, thromboxane and cAMP production.

Sanguinarine is a plant alkaloid present in the root of Sanguinaria canadensis and Poppy fumaria species. Sanguinarine has been used as an antiseptic mouth rinse and a toothpaste additive to reduce dental plaque and gingival inflammation. In this study, we investigated the antiplatelet effects of sanguinarine, aiming to extend its potential pharmacological applications. Sanguinarine inhibited platelet aggregation induced by arachidonic acid (AA), collagen, U46619 and sub-threshold concentration of thrombin (0.05 U/ml) with IC(50) concentrations of 8.3, 7.7, 8.6 and 4.4 microM, respectively. Sanguinarine (5-10 microM) inhibited 10-31% of platelet TXB(2) production, but not platelet aggregation induced by higher concentration of thrombin (0.1 U/ml). SQ29548, a thromboxane receptor antagonist, inhibited the AA-induced platelet aggregation but not TXB(2) production. Sanguinarine suppressed cyclooxygenase-1 (COX-1) activity (IC(50)=28 microM), whereas its effect on COX-2 activity was minimal. Sanguinarine (8, 10 microM) further inhibited the AA-induced Ca(2+) mobilization by 27-62%. In addition, SQ22536, an adenylate cyclase inhibitor, attenuated the inhibitory effect of sanguinarine toward AA-induced platelet Ca(2+) mobilization and aggregation. These results suggest that sanguinarine is a potent antiplatelet agent, which activates adenylate cyclase, inhibits platelet Ca(2+) mobilization, TXB(2) production as well as suppresses COX-1 enzyme activity. Sanguinarine may have therapeutic potential for treatment of cardiovascular diseases related to platelet aggregation.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

Divergent regulation of Pyk2/CAKbeta phosphorylation by Ca2+ and cAMP in the hippocampus.

Proline-rich tyrosine kinase 2 (Pyk2) is activated in neurones following NMDA receptor stimulation via PKC. Pyk2 is involved in hippocampal LTP and acts to potentiate NMDA receptor function. Elevations of intracellular Ca2+ and cAMP levels are key NMDA receptor-dependent triggering events leading to induction of hippocampal LTP. In this study, we compared the ability of A23187 (Ca2+ ionophore) or forskolin (adenylate cyclase activator) to modulate the phosphorylation of Pyk2 in rat hippocampal slices. Using an immunoprecipitation assay, phosphorylated Pyk2 levels were increased following treatment with A23187, levels peaking at around 10 min. Staurosporine, at concentrations inhibiting conventional and novel isoforms of PKC, and chelerythrine, at concentrations inhibiting the atypical PKC isoform PKMxi, were compared for their ability to attenuate the effect of A23187. Exposure of acute hippocampal slices to either chelerythrine or staurosporine completely blocked enhanced phosphorylation of Pyk2 by A23187, suggesting a possible involvement of PKMxi and typical PKCs in Pyk2 activation by Ca2+. In contrast, application of forskolin reduced phosphorylated Pyk2 below basal levels, suggesting that cAMP inhibits Pyk2. These results implicate Ca2+ and multiple forms of PKC in the activation of Pyk2 downstream of NMDA receptors and suggest that cAMP-dependent processes exert a suppressive action on Pyk2.

Alkaloids↗

Rapid change of glucose concentration promotes mesangial cell proliferation via VEGF: inhibitory effects of thiazolidinedione.

Diabetic nephropathy is a common complication in diabetes mellitus (DM). Thiazolidinedione (TZD) is thought to ameliorate diabetic nephropathy, however, the mechanism has not been elucidated. We hypothesized that VEGF participates in the pathogenesis of diabetic nephropathy and that TZD may be beneficial for the treatment of diabetic nephropathy through its effect on VEGF. Increased VEGF expression was demonstrated in the glomeruli of DM rats and rat mesangial cells (RMC) incubated with high medium glucose. It was also demonstrated that VEGF promoted mesangial cell proliferation, which was inhibited by TZD. It was shown that a rapid fall and rise of ambient glucose concentration induces more VEGF production and cell proliferation in RMC than in cells with continuously high glucose medium, which was also inhibited by TZD. Prostaglandin J2 and protein C kinase inhibitors significantly inhibited [3H]thymidine incorporation in RMC incubated with VEGF, which was inhibited by TZD. These findings indicate that a rapid change of glucose concentration promotes RMC proliferation by the increased production of VEGF. TZD has an inhibitory action through, at least in part, PPAR-gamma.

Alkaloids↗

Identification of a drug target motif: an anti-tumor drug NK109 interacts with a PNxxxxP.

The synthetic compound NK109 shows anti-tumor effects against a number of human cancer cell lines. The mechanism of action is thought to involve the inhibition of DNA topoisomerase II. However, NK109 also exhibits potent anti-tumor activities against doxorubicin-, cisplatin- and etoposide-resistant human cell lines. This paper describes target validation of NK109 using biotinylated NK109 and a T7 phage library screening procedure. Phage particles displaying an affinity for NK109 were isolated and the DNA sequence determined. The amino acid sequences of selected peptides, and the results of mutation experiments by alanine scanning, confirmed that the binding target motif of NK109 is PNxxxxP. In silico analysis of the interaction between NK109 and the peptide, by docking simulation and molecular dynamics, supported this conclusion. The PNxxxxP motif exists in the C2 domain of protein kinase Calpha. NK109 was confirmed to bind the C2 domain from surface plasmon resonance analysis. Furthermore, NK109 moderately inhibited protein kinase C activity in vitro. Our results show that the anti-tumor activity of NK109 stems from interactions with multiple protein targets.

Amino Acid Motifs↗

Synthesis and cytotoxic activity of benzo[c][1,7] and [1,8]phenanthrolines analogues of nitidine and fagaronine.

Fagaronine and nitidine are natural benzo[c] phenanthridinium alkaloids, which display antileukemic activity. Both act as topoisomerase I and topoisomerase II inhibitors. The objective of the present study was to prepare noncharged isosters of these compounds, with replacement of the aromatic A ring by a pyridine ring, present in other topoisomerase I inhibitors. Various 7,8- and 8,9-dimethoxy and metylenedioxy benzo[c][1,7] and [1,8]phenanthrolines were readily synthesized by benzyne-mediated cyclization of the corresponding substituted N-(2-halobenzyl)-5-quinolinamines or 5-isoquinolinamines. In both series, compounds bearing oxygenated substituents at positions 8 and 9 exhibited cytotoxic properties towards L1210 murine leukemia cells, which may result from their capacities to intercalate into DNA. Topoisomerase I inhibition was observed for all active compounds.

Alkaloids↗

Effect of protein kinase C blockade on phosphorylation of NR1 in dorsal horn and spinothalamic tract cells caused by intradermal capsaicin injection in rats.

We have previously reported that protein kinase A (PKA) is involved in the phosphorylation of NR1 subunits of N-methyl-d-aspartate (NMDA) receptors in dorsal horn neurons after intradermal injection of capsaicin (CAP). To see if protein kinase C (PKC) also participates in the phosphorylation of NR1, we used electron microscopic techniques to determine further where the phosphorylated NR1 subunits (pNR1) are expressed in the spinothalamic tract (STT) cells and immunohistochemistry to examine whether a PKC inhibitor, chelerythrine chloride, blocks the enhanced phosphorylation of NR1 on serine 896. The pNR1 subunits were in the soma and dendrites of STT cells and in presynaptic endings. Western blots showed that pretreatment with the PKC inhibitor caused a decrease in CAP-induced phosphorylation of NR1 protein. In immunofluorescence staining, the number of pNR1-like immunoreactive neurons was significantly decreased on the side ipsilateral to the injection when chelerythrine chloride was administered intrathecally before CAP injection. In addition, when STT cells were labeled by microinjection of the retrograde tracer, fluorogold (FG), into the thalamus, we found that the proportion of p-NR1-LI STT cells was markedly reduced after PKC inhibition. Combined with our previous findings, these results strongly suggest that NR1 subunits in spinal dorsal horn neurons are phosphorylated following CAP injection, and this phosphorylation is catalyzed by PKC, as well as by PKA.

Alkaloids↗

Protective effect of bioantioxidants on argemone oil/sanguinarine alkaloid induced genotoxicity in mice.

Our prior studies have shown that argemone oil (AO) and its alkaloid sanguinarine causes DNA damage in mice and Epidemic Dropsy patients. Since some of the bioantioxidants including riboflavin and alpha-tocopherol offered protection to Epidemic Dropsy patients, a combination of riboflavin and alpha-tocopherol was evaluated on AO and sanguinarine induced genotoxicity using alkaline comet assay. Single administration of combination of riboflavin (50mg/kg) and alpha-tocopherol (150mg/kg) to mice, 24h prior to or immediately after AO (2.0ml/kg) exposure showed significant decrease in tail moment (70-72%), tail length (37-44%), and tail DNA (49-53%) in bone marrow cells. Single or multiple doses of antioxidants given after 24h of AO exposure resulted in substantial (P<0.05) decrease in all the parameters of comet assay in bone marrow cells. Single dose of antioxidants given either 24h prior to or immediately after sanguinarine (21.6mg/kg) exposure caused significant decrease in tail moment (56-62%), tail length (69%) and tail DNA (34-42%) in bone marrow cells of mice. Single or multiple doses of antioxidants given after 24h of sanguinarine treated resulted in decrease in tail moment (50-71%), tail length (54-63%) and tail DNA (29-43%) in bone marrow cells. Similar protective response of combination of antioxidants was observed in blood cells of mice treated either with AO or sanguinarine alkaloid. Further, the frequency of bone marrow and blood cells in Olive tail moment category of 8 and onwards were found to be substantially reduced in antioxidants treated animals as compared to respective AO or sanguinarine exposed mice. Based on these results, it can be suggested that a combination of riboflavin and alpha-tocopherol provides protection against AO and sanguinarine induced genotoxicity.

Alkaloids↗

Sanguinarine induces apoptosis of human pancreatic carcinoma AsPC-1 and BxPC-3 cells via modulations in Bcl-2 family proteins.

Pancreatic cancer is associated with low responsiveness to conventional chemotherapies and its incidence nearly equals its death rate. This warrants the development of novel mechanism-based approaches for the management of pancreatic cancer. This study was designed to determine the potential of sanguinarine, a plant alkaloid known to possess strong antimicrobial, anti-inflammatory, and antioxidant activities, against human pancreatic carcinoma cells. Employing human pancreatic carcinoma AsPC-1 and BxPC-3 cells, we specifically evaluated the pro-apoptotic and cell cycle deregulatory effects of sanguinarine and evaluated the involvement of Bcl-2 family proteins and p53 as the mechanism of the biological effects of sanguinarine. Our data demonstrated that sanguinarine (at low concentrations of 0.1-10 microM; for 24 h) treatment to AsPC-1 and BxPC-3 cells resulted in a dose dependent (i) inhibition of viability and growth, (ii) colony formation ability, (iii) induction of apoptosis, and (iv) G0-G1 phase cell cycle arrest. Further, sanguinarine-treatment to AsPC-1 and BxPC-3 cells resulted in a dose dependent (i) increase in pro-apoptotic Bax, Bid and Bak proteins; (ii) decrease in anti-apoptotic Bcl-2 and Bcl-X(L) proteins; and (iii) decrease in p53 with an increase in its phosphorylation. Based on our study, we suggest that sanguinarine may be developed as an agent for the management of pancreatic cancer. Indeed, more in depth studies both in vitro as well as in vivo in appropriate relevant animal models are needed to strengthen this suggestion.

Adenocarcinoma↗

Delayed preconditioning of the human myocardium: signal transduction and clinical implications.

OBJECTIVE: Ischemic preconditioning confers cardioprotection in early and delayed phases. We investigated the delayed window of pharmacological and ischemic preconditioning in human myocardium, and the involvement of mitoKATP, PKC and p38MAPK. METHODS: These studies were carried out using human right atrial tissue in a cell necrosis model. The tissue was obtained from patients undergoing coronary artery surgery. RESULTS: The second window triggered by ischemia, phenylephrine or adenosine resulted in similar cardioprotection between 24 and 72 h following the intervention. Atrial tissue taken from patients with a single episode of angina between 24 and 72 h prior to surgery were already protected and preconditioning with ischemia, phenylephrine or adenosine did not add to the protection. The trigger of preconditioning with mitoKATP channel opener diazoxide, PKC activator PMA and p38MAPK activator anisomycin produced similar delayed protection to that of ischemia or phenylephrine. Cardioprotection was lost when mitoKATP channels were blocked by 5HD, PKC by chelerythrine and p38MAPK by SB203580 24 h after the trigger of preconditioning. CONCLUSIONS: Ischemic and pharmacological preconditioning induce similar delayed cardioprotection of the human heart. This second window of protection that is seen between 24 and 72 h occurs in vitro and in vivo and requires opening of mitoKATP channels and activation of PKC and p38MAPK.

Acetylcholine↗

Effects of alpha1-adrenergic stimulation on normal and hypertrophied mouse hearts. Relation to caveolin-3 expression.

BACKGROUND: Modulation of the transduction efficiency through G-protein coupled receptors, caused by external stimulation, is essential in designing antihypertrophic treatment strategies in the dysfunctional heart. We compared protein-kinase C (PKC)-dependent regulation of positive inotropic effect via alpha1-adrenoreceptor (ADR)/Gq protein in hyperdynamic versus hypertrophied myocardium. METHODS: Inotropic (work performing isolated heart) and cellular effects of alpha1-adrenoreceptor stimulation were studied in nontransgenic (Ntg) and transgenic (Tg) mice with cardiac specific overexpression of L-type voltage-dependent calcium channels (L-type VDCC). RESULTS: Transgenic hyperdynamic and hypertrophic myocardium (due to overexpression of the L-type VDCC alpha1 subunit) were characterized by a lack of positive inotropic effect (PIE) to alpha1-ADR stimulation with phenylephrine (PE), as compared to a positive response in Ntg hearts. This was partially restored by PKC inhibition with chelerythrine and staurosporine only at the hyperdynamic stage. The inability of PKC inhibition to increase positive inotropy was associated with markedly decreased cardiac-specific caveolin-3 expression, and no changes in Galphaq, PLC-beta1, caveolin-1 and alpha1-adrenoreceptor expression. CONCLUSION: In the hyperdynamic myocardium, PKC activation may be one of the switches responsible for an impaired alpha1-adrenergic positive inotropic response. In the hypertrophied myocardium, the interruption of the transduction from Galphaq-protein coupled receptors to downstream effectors may be due to the down-regulation of caveolin-3 expression.

Action Potentials↗

Investigation of signaling pathways that mediate the inotropic effect of urotensin-II in human heart.

OBJECTIVE: This study investigated signaling pathways that may contribute to the potent positive inotropic effect of human urotensin-II (hU-II) in human isolated right atrial trabeculae obtained from patients with coronary artery disease. METHODS: Trabeculae were set up in tissue baths and stimulated to contract at 1 Hz. Tissues were incubated with 20 nM hU-II with or without phorbol 12-myristate 13-acetate (PMA, 10 microM) to desensitize PKC, the PKC inhibitor chelerythrine (10 microM), 10 microM 4alpha-phorbol that does not desensitize PKC, the myosin light chain kinase inhibitor wortmannin (50 nM, 10 microM), or the Rho kinase inhibitor Y-27632 (0.1-10 microM). Activated RhoA was determined by affinity immunoprecipitation, and phosphorylation of signaling proteins was determined by SDS-PAGE. RESULTS: hU-II caused a potent positive inotropic response in atrial trabeculae, and this was concomitant with increased phosphorylation of regulatory myosin light chain (MLC-2, 1.8+/-0.4-fold, P<0.05, n=6) and PKCalpha/betaII (1.4+/-0.2-fold compared to non-stimulated controls, P<0.05, n=7). Pretreatment of tissues with PMA caused a marked reduction in the inotropic effect of hU-II, but did not affect hU-II-mediated phosphorylation of MLC-2. The inotropic response was inhibited by chelerythrine, but not 4alpha-phorbol or wortmannin. Although Y-27632 also reduced the positive inotropic response to hU-II, this was associated with a marked reduction in basal force of contraction. RhoA.GTP was immunoprecipitated in tissues pretreated with or without hU-II, with findings showing no detectable activation of RhoA in the agonist stimulated tissues. CONCLUSIONS: The findings indicated that hU-II increased force of contraction in human heart via a PKC-dependent mechanism and increased phosphorylation of MLC-2, although this was independent of PKC. The positive inotropic effect was independent of myosin light chain kinase and RhoA-Rho kinase signaling pathways.

Alkaloids↗

alpha1A- but not alpha1B-adrenergic receptors precondition the ischemic heart by a staurosporine-sensitive, chelerythrine-insensitive mechanism.

OBJECTIVE: Brief periods of ischemia stimulate an endogenous mechanism in the heart that protects the myocardium from subsequent ischemic injury. alpha1-Adrenergic receptors (ARs) have been implicated in this process. However, the lack of sufficiently selective antagonists has made it difficult to determine which alpha1-AR subtype protects the heart from ischemic injury. The goal of this study was to identify the alpha1-AR subtype that is involved in ischemic preconditioning. METHODS: We developed transgenic mice that express constitutively active mutant (CAM) forms of the alpha1A-AR or the alpha1B-AR regulated by their endogenous promoters. Hearts isolated from transgenic and non-transgenic mice were perfused by the Langendorff method using an ischemic preconditioning perfusion protocol or a non-preconditioning perfusion protocol prior to 30-min ischemia and 40-min reperfusion. Contractile function was continuously monitored through an intraventricular balloon. RESULTS: The contractile function of non-transgenic hearts perfused according to the ischemic preconditioning protocol completely recovered from 30-min ischemia. However, non-transgenic hearts perfused according to the non-preconditioning protocol recovered only 60% of their contractile function. The contractile function of CAM alpha1A-AR hearts, but not CAM alpha1B-AR hearts, completely recovered from 30-min ischemia even though they were perfused according to the non-preconditioning protocol. Thus, CAM alpha1A-AR hearts, but not CAM alpha1B-AR hearts, were inherently preconditioned against ischemic injury. Staurosporine, but not chelerythrine, completely reversed the preconditioning effect of CAM alpha1A-ARs. CONCLUSIONS: These data demonstrate that alpha1A-ARs protect the heart from ischemic injury through a staurosporine-sensitive signaling pathway that is independent of protein kinase C.

Alkaloids↗

Infarct-sparing effect of myocardial postconditioning is dependent on protein kinase C signalling.

OBJECTIVE: Using non-selective and selective protein kinase C (PKC) epsilon and delta isoform inhibitors, we tested the hypothesis that the cardioprotective phenotype invoked by postconditioning (postcon) is dependent on PKC signalling. Furthermore, we determined whether postconditioning alters pPKCepsilon and/or pPKCdelta in cytosolic and mitochondrial fractions. METHODS: Male Sprague-Dawley rats underwent 30 min left coronary artery (LCA) occlusion followed by 3 h of reperfusion. Rats were randomised to the following groups: Untreated, no intervention either before or after LCA occlusion; Postcon, 3 cycles of 10-s full reperfusion and 10-s re-occlusion, initiated immediately at the onset of reperfusion; Chelerythrine (non-selective PKC inhibitor, 5 mg/kg)+/-postcon; Rottlerin (PKCdelta inhibitor, 0.3 mg/kg)+/-postcon; KIE1-1 (PKCepsilon inhibitor, 3.8 mg/kg)+/-postcon. A subset of rats was employed to assess pPKCepsilon and/or pPKCdelta in sham, Isch/RP (30-min LCA occlusion followed by 30-min reperfusion), and postcon-treated hearts. RESULTS: Infarct size, expressed as area of necrosis as a percentage of the area at risk, AN/AAR (%), was significantly reduced by postcon compared to control (untreated) rats (39+/-2% vs. 53+/-1% in control, P<0.001). Treatment with chelerythrine alone or the PKCepsilon antagonist KIE1-1 alone at reperfusion had no effect on infarct size compared to control. In contrast, the infarct-sparing effect of postcon was abrogated by non-selective PKC inhibition and PKCepsilon antagonism (50+/-2% and 50+/-1%, respectively, P<0.002). Inhibition of PKCdelta reduced infarct size to values comparable to that in postcon group (36+/-3% vs. 39+/-2%). However, postcon in the presence of PKCdelta inhibitor did not enhance the infarct-sparing effects (38+/-2%). In addition, pPKCepsilon in postcon hearts was significantly higher in the total cell homogenate (10338+/-1627 vs. 4165+/-608 in Isch/RP, arbitrary units), and pPKCdelta translocation to mitochondria was significantly less (>2-fold decrease) compared to Isch/RP. CONCLUSION: These data suggest that postcon modulates PKC during early reperfusion by increasing PKCepsilon expression and translocation to a site other than the outer mitochondrial membrane, and limits translocation of PKCdelta to mitochondria and associated deleterious signalling.

Acetophenones↗

Postconditioning protects rabbit hearts through a protein kinase C-adenosine A2b receptor cascade.

OBJECTIVE: Ischemic postconditioning protects the reperfused heart from infarction, and this protection is dependent on the occupancy of adenosine receptors. We further explored the role of adenosine receptors in this salvage. METHODS: In situ rabbit hearts underwent 30 min of regional ischemia and 3 h of reperfusion, and postconditioning was effected with four cycles of 30-s reperfusion/30-s coronary artery occlusion at the end of ischemia. RESULTS: Postconditioning reduced infarct size from 40.2+/-3.4% of the risk zone in untreated hearts to 15.5+/-2.5%. Protection by postconditioning was blocked by either the non-selective adenosine receptor blocker 8-p-(sulfophenyl)theophylline or the A2b-selective antagonist MRS 1754, injected intravenously 5 min before reperfusion. The protein kinase C (PKC) antagonist chelerythrine also aborted postconditioning's salvage, indicating a PKC-dependent mechanism. Neither the A1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine nor the A2a-selective antagonist 8-(13-chlorostyryl)caffeine had an effect on protection. The non-selective but A2b-potent adenosine agonist 5'-(N-ethylcarboxamido)adenosine (NECA) infused from 5 min before to 1h after reperfusion mimicked postconditioning's effect on infarct size (17.2+/-2.7% infarction) and MRS 1754 blocked the NECA-induced cardioprotection, confirming that A2b activation was protective. The PKC activator phorbol 12-myristate 13-acetate delivered just before reperfusion also duplicated the protective effect of postconditioning (16.3+/-4.1% infarction), and co-administration of the PKC antagonist chelerythrine aborted PMA's protection, confirming that the protection was the result of PKC activation. NECA's protective effect was not affected by chelerythrine, but rather MRS 1754 blocked PMA's salutary effect (42.8+/-1.0% infarction), suggesting that the A2b receptor's effect is under control of PKC. Finally, wortmannin, a blocker of phosphatidylinositol 3-kinase, also abrogated protection by PMA. CONCLUSIONS: Salvage of ischemic myocardium by postconditioning is dependent on activation of A2b receptors, which in turn depends on activation of PKC. It is still unclear why PKC activation is required to make the heart's adenosine become protective.

Acetamides↗

Sanguinarine is a potent inhibitor of oxidative burst in DMSO-differentiated HL-60 cells by a non-redox mechanism.

Sanguinarine (SA), a member of the benzo[c]phenanthridine isoquinoline alkaloids, has been shown to possess antimicrobial, anti-inflammatory, and antioxidant properties. We examined the effects of SA on oxidative burst in DMSO-differentiated HL-60 cells, an excellent model for studying oxidative burst. SA inhibited both N-formyl-Met-Leu-Phe (fMLP) and phorbol 12-myristate 13-acetate (PMA)-induced oxidative burst with half-maximal concentration for inhibition (IC(50)) of 1.5 and 1.8 microM, respectively. Despite suggestions of SA antioxidant activity this inhibition cannot be ascribed to radical scavenging property of SA because the IC(50) for superoxide dismutase-like activity in a non-cellular system was 60 microM. TROLOX, a water-soluble vitamin E analog, had IC(50) of 3 microM in the same system. Moreover, cyclic voltammetry measurements show that SA is not an easily oxidizable species, with a peak anodic potential at 700 mV, as compared to TROLOX with peak anodic potential at 200 mV. On the other hand, TROLOX, when used in cell suspension, was much poorer inhibitor of oxidative burst than SA. When testing direct effect of SA on NADPH oxidase in the post-granular fraction of disrupted cells, the IC(50) was found to be 8.3 microM. It is higher than that observed in whole cells, however, the shift may be ascribed to SDS effect on SA activity. We conclude the SA inhibition of oxidative burst is not caused by SA redox activity but most likely is a result of SA affecting the activity of NADPH oxidase directly and in part by preventing the formation of NADPH oxidase protein complex.

Alkaloids↗

Protein kinase C delta is not activated by caspase-3 and its inhibition is sufficient to induce apoptosis in the colon cancer line, COLO 205.

Activation of protein kinase C delta (PKCdelta) is believed to be pro-apoptotic. PKCdelta is reported to be reduced in colon cancers. Using a colon cancer cell line, COLO 205, we have examined the roles of PKCdelta in apoptosis and of caspase-3 in the activation and inhibition of PKCdelta. PKCdelta activation with bistratene A and its inhibition with rottlerin induced apoptosis. Effects of PKC activators and inhibitors were additive, suggesting that PKCdelta down-regulation was responsible for the effects on apoptosis. Different apoptotic pathways induced PKCdelta cleavage, but the fragment produced was inactive in kinase assays. Caspase-3 inhibition did not block DNA fragmentation or PKCdelta proteolysis despite blocking intracellular caspase-3 activity. Calpain inhibition with calpeptin did not prevent TPA-induced PKCdelta cleavage. We conclude that in colonocytes, inhibition of PKCdelta is sufficient to lead to caspase-3-independent apoptosis. Caspase-3 does not cleave PKCdelta to an active form, nor does caspase-3 inhibition block apoptosis.

Acetamides↗

Capillary electrophoretic studies of acid-base properties of sanguinarine and chelerythrine alkaloids.

Capillary zone electrophoresis with UV detection was used for determination of dissociation constants of alkaloids sanguinarine and chelerythrine. Despite the limited solubility of the uncharged forms of the alkaloids resulting in insufficient analytical signal at higher pH the reliable dissociation constants were obtained when acidified samples containing low amount of the alkaloid were injected into the capillary. The precipitation of the alkaloid in the capillary induced by injecting sample of low pH into the background electrolyte of higher pH does not affect the mobility of the alkaloid if its concentration injected exceeds the solubility only to a small extent. Dissociation constants (pK(R+)) of sanguinarine and chelerythrine calculated to 8.3 +/- 0.1 and 9.2 +/- 0.1, respectively, are relevant to Good buffers of ionic strength of 30 mM.

Alkaloids↗