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The Journal of Organic Chemistry May 2020The synthesis of the stereotriad core in the eastern portion of the alkaloids jervine (), cyclopamine (), and veratramine () is reported. Starting from a known...
The synthesis of the stereotriad core in the eastern portion of the alkaloids jervine (), cyclopamine (), and veratramine () is reported. Starting from a known β-methyltyrosine derivative (), the route utilizes a diastereoselective substrate-controlled 1,2-reduction to establish the stereochemistry of the vicinal amino alcohol motif embedded within the targets. Oxidative dearomatization is demonstrated to be a viable approach for the synthesis of the spirocyclic DE ring junction found in jervine and cyclopamine.
Topics: Veratrum; Veratrum Alkaloids
PubMed: 32352768
DOI: 10.1021/acs.joc.0c00685 -
Curcumin- and Cyclopamine-Loaded Liposomes to Enhance Therapeutic Efficacy Against Hepatic Fibrosis.Drug Design, Development and Therapy 2020Hepatic fibrosis is a public health problem characterized by activation of hepatic stellate cells (HSCs), which triggers excessive production of extracellular matrix...
BACKGROUND AND PURPOSE
Hepatic fibrosis is a public health problem characterized by activation of hepatic stellate cells (HSCs), which triggers excessive production of extracellular matrix (ECM). Inhibition of HSC activation may be an effective treatment. Since various pathways control HSC activation, a combination of drugs with different mechanisms may be more effective than monotherapy.
METHODS
Here, we prepared liposomes loaded with curcumin and cyclopamine to inhibit HSC activation. We systematically analyzed the physicochemical characteristics of liposomes loaded with the two drugs, as well as their effects on HSC proliferation, activation and collagen production on gene, protein and cellular levels.
RESULTS
The prepared liposomes helped solubilize both drugs, contributing to their uptake by cells. Liposomes loaded with both drugs inhibited cell proliferation, migration and invasion, as well as induced more apoptosis and perturbed the cell cycle more than the free combination of both drugs in solution or liposomes loaded with either drug alone. Liposomes loaded with both drugs strongly suppressed HSC activation and collagen secretion.
CONCLUSION
Our results suggest that liposome encapsulation can increase the uptake of curcumin and cyclopamine as well as the synergism between them in anti-fibrosis. This approach shows potential for treating hepatic fibrosis.
Topics: Animals; Apoptosis; Cell Proliferation; Cells, Cultured; Collagen; Curcumin; Liposomes; Liver Cirrhosis; Rats; Veratrum Alkaloids
PubMed: 33380787
DOI: 10.2147/DDDT.S287442 -
PloS One 2019Voltage-gated sodium channels (NaVs) are key therapeutic targets for pain, epilepsy and cardiac arrhythmias. Here we describe the development of a no-wash fluorescent...
Voltage-gated sodium channels (NaVs) are key therapeutic targets for pain, epilepsy and cardiac arrhythmias. Here we describe the development of a no-wash fluorescent sodium influx assay suitable for high-throughput screening and characterization of novel drug leads. Addition of red-violet food dyes (peak absorbance range 495-575 nm) to assays in HEK293 cells heterologously expressing hNaV1.1-1.8 effectively quenched background fluorescence of the sodium indicator dye Asante NaTRIUM Green-2 (ANG-2; peak emission 540 nm), negating the need for a wash step. Ponceau 4R (1 mM) was identified as a suitable quencher, which had no direct effect on NaV channels as assessed by patch-clamp experiments, and did not alter the pharmacology of the NaV1.1-1.7 activator veratridine (EC50 10-29 μM) or the NaV1.1-1.8 inhibitor tetracaine (IC50's 6-66 μM). In addition, we also identified that the food dyes Ponceau 4R, Brilliant Black BN, Allura Red and Amaranth are effective at quenching the background fluorescence of the calcium indicator dyes fluo-4, fura-2 and fura-5F, identifying them as potential inexpensive alternatives to no-wash calcium ion indicator kits. In summary, we have developed a no-wash fluorescent sodium influx assay suitable for high-throughput screening based on the sodium indicator dye ANG-2 and the quencher Ponceau 4R.
Topics: Fluorescent Dyes; HEK293 Cells; High-Throughput Screening Assays; Humans; Patch-Clamp Techniques; Sodium; Spectrometry, Fluorescence; Tetracaine; Veratridine; Voltage-Gated Sodium Channel Agonists; Voltage-Gated Sodium Channel Blockers; Voltage-Gated Sodium Channels
PubMed: 30856233
DOI: 10.1371/journal.pone.0213751 -
Bioscience Reports May 2020Precartilaginous stem cells (PCSCs) are adult stem cells that can initiate chondrocytes and bone development. In the present study, we explored whether miR-132/212 was...
Precartilaginous stem cells (PCSCs) are adult stem cells that can initiate chondrocytes and bone development. In the present study, we explored whether miR-132/212 was involved in the proliferation of PCSCs via Hedgehog signaling pathway. PCSCs were isolated and purified with the fibroblast growth factor receptor-3 (FGFR-3) antibody. Cell viability, DNA synthesis and apoptosis were measured using MTT, BrdU and flow cytometric analysis. The mRNA and protein expression were detected by real-time PCR and Western blot, respectively. The target gene for miR-132/212 was validated by luciferase reporter assay. Results showed that transfection with miR-132/212 mimic significantly increased cell viability and DNA synthesis, and inhibited apoptosis of PCSCs. By contrast, miR-132/212 inhibitor could suppress growth and promote apoptosis of PCSCs. Luciferase reporter assays indicated that transfection of miR-132/212 led to a marked reduction of luciferase activity, but had no effect on PTCH1 3'-UTR mutated fragment, suggesting that Patched1 (PTCH1) is a target of miR-132/212. Furthermore, treatment with miR-132/212 mimics obviously increased the protein expression of Indian hedgehog (Ihh) and parathyroid hormone related protein (PTHrP), which was decreased after treatment with Hedgehog signaling inhibitor, cyclopamine. We also found that inhibition of Ihh/PTHrP signaling by cyclopamine significantly suppressed growth and DNA synthesis, and induced apoptosis in PCSCs. These findings demonstrate that miR-132/212 promotes growth and inhibits apoptosis in PCSCs by regulating PTCH1-mediated Ihh/PTHrP pathway, suggesting that miR-132/212 cluster might serve as a novel target for bone diseases.
Topics: Adult Stem Cells; Animals; Apoptosis; Cartilage, Articular; Cell Proliferation; Cells, Cultured; Chondrocytes; Hedgehog Proteins; MicroRNAs; Multigene Family; Parathyroid Hormone-Related Protein; Patched-1 Receptor; Primary Cell Culture; Rabbits; Veratrum Alkaloids
PubMed: 32319512
DOI: 10.1042/BSR20191654 -
Developmental Biology Feb 2003From time of embryonic emergence, the gustatory papilla types on the mammalian tongue have stereotypic anterior and posterior tongue locations. Furthermore, on anterior...
Cyclopamine and jervine in embryonic rat tongue cultures demonstrate a role for Shh signaling in taste papilla development and patterning: fungiform papillae double in number and form in novel locations in dorsal lingual epithelium.
From time of embryonic emergence, the gustatory papilla types on the mammalian tongue have stereotypic anterior and posterior tongue locations. Furthermore, on anterior tongue, the fungiform papillae are patterned in rows. Among the many molecules that have potential roles in regulating papilla location and pattern, Sonic hedgehog (Shh) has been localized within early tongue and developing papillae. We used an embryonic, tongue organ culture system that retains temporal, spatial, and molecular characteristics of in vivo taste papilla morphogenesis and patterning to study the role of Shh in taste papilla development. Tongues from gestational day 14 rat embryos, when papillae are just beginning to emerge on dorsal tongue, were maintained in organ culture for 2 days. The steroidal alkaloids, cyclopamine and jervine, that specifically disrupt the Shh signaling pathway, or a Shh-blocking antibody were added to the standard culture medium. Controls included tongues cultured in the standard medium alone, and with addition of solanidine, an alkaloid that resembles cyclopamine structurally but that does not disrupt Shh signaling. In cultures with cyclopamine, jervine, or blocking antibody, fungiform papilla numbers doubled on the dorsal tongue with a distribution that essentially eliminated inter-papilla regions, compared with tongues in standard medium or solanidine. In addition, fungiform papillae developed on posterior oral tongue, just in front of and beside the single circumvallate papilla, regions where fungiform papillae do not typically develop. The Shh protein was in all fungiform papillae in embryonic tongues, and tongue cultures with standard medium or cyclopamine, and was conspicuously localized in the basement membrane region of the papillae. Ptc protein had a similar distribution to Shh, although the immunoproduct was more diffuse. Fungiform papillae did not develop on pharyngeal or ventral tongue in cyclopamine and jervine cultures, or in the tongue midline furrow, nor was development of the single circumvallate papilla altered. The results demonstrate a prominent role for Shh in fungiform papilla induction and patterning and indicate differences in morphogenetic control of fungiform and circumvallate papilla development and numbers. Furthermore, a previously unknown, broad competence of dorsal lingual epithelium to form fungiform papillae on both anterior and posterior oral tongue is revealed.
Topics: Animals; Body Patterning; Epithelium; Female; Hedgehog Proteins; Immunohistochemistry; Microscopy, Electron, Scanning; Organ Culture Techniques; Pregnancy; Rats; Rats, Sprague-Dawley; Signal Transduction; Taste Buds; Tongue; Trans-Activators; Veratrum Alkaloids
PubMed: 12606278
DOI: 10.1016/s0012-1606(02)00014-3 -
The Journal of General Physiology Jul 1987Macroscopic currents in Na channels were recorded from adult frog skeletal muscle under voltage clamp as various toxins were added to the bathing medium. Veratridine,... (Comparative Study)
Comparative Study
Macroscopic currents in Na channels were recorded from adult frog skeletal muscle under voltage clamp as various toxins were added to the bathing medium. Veratridine, cevadine, and 3-(4-ethoxybenzoyl)-veracevine modified the Na channels in a use-dependent manner during depolarizations and held them open for 3, 2.4, and 1.2 s, respectively, at -90 mV. The three alkaloids modified channels in the same way. Activation gating was shifted about -100 mV by the modification, and reversible closing of the channels by strong hyperpolarizations slowed reversal of the modification. The synthetic insecticides deltamethrin, EDO, GH739, and GH414 also modified channels during depolarizations that opened channels. The modification lasted 3 s with deltamethrin, but only 3-5 ms with the others. Hyperpolarization speeded the shutting off of current in insecticide-modified channels, but no reversible activation gating could be demonstrated. The ionic selectivity, PNa/PNH4, of channels was decreased by all of the toxins. This ratio was 0.11 in normal channels, 0.26 in insecticide-modified channels, and 0.7-1.6 in veratrum-alkaloid-modified channels. During use-dependent modification, the veratrum alkaloids reduced the total Na current markedly, while deltamethrin did not. Thus, alkaloid and insecticide modifications share many features but differ in how much the conducting properties of the pore are changed and whether the channel can close reversibly while the toxin remains bound.
Topics: Animals; In Vitro Techniques; Insecticides; Ion Channels; Membrane Potentials; Models, Biological; Muscles; Rana pipiens; Sodium; Veratrum Alkaloids
PubMed: 2442297
DOI: 10.1085/jgp.90.1.75 -
Anesthesiology Feb 1991
Topics: Anesthetics, Local; Humans; Nerve Block; Nerve Fibers; Veratridine
PubMed: 1990892
DOI: 10.1097/00000542-199102000-00001 -
Pharmaceutical Biology Dec 2021Eriodictyol (EDT) is a flavonoid with strong anti-inflammatory, anti-apoptotic, and antioxidant properties.
CONTEXT
Eriodictyol (EDT) is a flavonoid with strong anti-inflammatory, anti-apoptotic, and antioxidant properties.
OBJECTIVE
To investigate the protective effect and mechanism of EDT in ulcerative colitis (UC).
MATERIALS AND METHODS
UC model was induced by 3% dextran sulphate sodium (DSS) solution for 7 days, meanwhile, EDT and Smoothened (Smo) inhibitor cyclopamine (Cyc) were intraperitoneally injected. In the first experiment, C57BL/6 mice divided into blank control, DSS, DSS + EDT (20 or 40 mg/kg) groups. In second experiment, added Cyc (5 mg/kg) and EDT + Cyc groups. All mice were sacrificed on day 8. Disease activity index (DAI), colon length and colon histology as well as MDA levels, SOD, and GSH-Px activities were measured. The expression of Sonic hedgehog (Shh), Patched, Smo, glioblastoma-1, zonula occludens-1 (ZO-1), occludin, cleaved caspase 3, Bax and Bcl-2 in colon was detected using RT-PCR and Western blotting.
RESULTS
After EDT treatment, compared with the DSS group, DAI (2.33 ± 0.516 vs. 3.67 ± 0.516), colon shortening (5.27 ± 0.476 vs. 4.53 ± 0.528 cm) and histological score (6.67 ± 1.211 vs. 12 ± 1.265) was significantly decreased. EDT also reduced inflammation, oxidative stress and apoptosis in colon. Additionally, EDT increased the expression of the tight junction proteins ZO-1 (35%) and occludin (66.3%). Mechanistically, EDT upregulated the Shh signalling pathway. However, Cyc-mediated inhibition of the Shh pathway partially abolished the effects of EDT.
DISCUSSION AND CONCLUSIONS
These results indicate EDT attenuates DSS-induced colitis by activating the Shh pathway. Further clinical trials are needed to demonstrate its efficacy on UC.
Topics: Animals; Colitis, Ulcerative; Colon; Cytokines; Dextran Sulfate; Flavanones; Hedgehog Proteins; Inflammation; Male; Mice; Mice, Inbred C57BL; Models, Animal; Oxidative Stress; Signal Transduction; Veratrum Alkaloids
PubMed: 34348563
DOI: 10.1080/13880209.2021.1948066 -
Anticancer Research Nov 2009Hedgehog (Hh) and Wnt signaling pathways are involved in the stimulation of growth of leukemia and lymphoma cells. In the present study, whether or not the Hh inhibitor,...
BACKGROUND
Hedgehog (Hh) and Wnt signaling pathways are involved in the stimulation of growth of leukemia and lymphoma cells. In the present study, whether or not the Hh inhibitor, cyclopamine, and the Wnt inhibitor, quercetin, suppress cell growth was investigated.
MATERIALS AND METHODS
The effects of cyclopamine and quercetin on the in vitro growth and protein expression of ten acute leukemia and B-cell lymphoma cell lines were examined.
RESULTS
Cyclopamine and quercetin suppressed cell growth and induced apoptosis in seven and eight cell lines respectively. Cyclopamine decreased the level of Gli1 protein, a target gene product of Hh signaling. Quercetin decreased the level of Notch1 protein and its active fragment in the DND-41 T-lymphoblastic leukemia cell line with constitutive Notch activation.
CONCLUSION
Cyclopamine and quercetin suppress the growth of a number of leukemia and lymphoma cells. This finding suggests the potential use of these compounds in molecularly-targeted therapy for leukemia and lymphoma.
Topics: Apoptosis; Cell Growth Processes; Cell Line, Tumor; Dose-Response Relationship, Drug; HL-60 Cells; Humans; Jurkat Cells; Leukemia; Lymphoma; Protein Biosynthesis; Quercetin; Receptor, Notch1; Transcription Factors; Veratrum Alkaloids; Zinc Finger Protein GLI1
PubMed: 20032413
DOI: No ID Found -
Cellular Signalling Mar 2015Cholesterol modification of Hedgehog (Hh) ligands is fundamental for the activity of Hh signaling, and cholesterol biosynthesis is also required for intracellular Hh...
Cholesterol modification of Hedgehog (Hh) ligands is fundamental for the activity of Hh signaling, and cholesterol biosynthesis is also required for intracellular Hh signaling transduction. Here, we investigated the roles and underlying mechanism of Hh signaling in metabolism of cholesterol. The main components of the Hh pathway are abundantly expressed in both human cytotrophoblasts and trophoblast-like cells. Activation of Hh signaling induces the conversion of cholesterol to progesterone (P4) and estradiol (E2) through up-regulating the expression of steroidogenic enzymes including P450 cholesterol side chain cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase type 1 (3β-HSD1), and aromatase. Moreover, inhibition of Hh signaling attenuates not only Hh-induced expression of steroidogenic enzymes but also the conversion of cholesterol to P4 and E2. Whereas Gli3 is required for Hh-induced P450scc expression, Gli2 mediates the induction of 3β-HSD1 and aromatase. Finally, in ovariectomized nude mice, systemic inhibition of Hh signaling by cyclopamine suppresses circulating P4 and E2 levels derived from a trophoblast-like choricarcinoma xenograft, and attenuates uterine response to P4 and E2. Together these results uncover a hitherto uncharacterized role of Hh signaling in metabolism of cholesterol.
Topics: 17-Hydroxysteroid Dehydrogenases; Animals; Aromatase; Cells, Cultured; Cholesterol; Estradiol; Female; Hedgehog Proteins; Humans; Kruppel-Like Transcription Factors; Mice; Mice, Inbred BALB C; Mice, Nude; Nerve Tissue Proteins; Oncogene Proteins; Progesterone; RNA, Small Interfering; Signal Transduction; Trans-Activators; Transplantation, Heterologous; Trophoblasts; Veratrum Alkaloids; Zinc Finger Protein GLI1; Zinc Finger Protein Gli2; Zinc Finger Protein Gli3
PubMed: 25582983
DOI: 10.1016/j.cellsig.2015.01.004