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Fitoterapia Sep 2019Veratrum californicum is a rich source of steroidal alkaloids, many of which have proven to be antagonists of the Hedgehog (Hh) signaling pathway that becomes aberrant...
Veratrum californicum is a rich source of steroidal alkaloids, many of which have proven to be antagonists of the Hedgehog (Hh) signaling pathway that becomes aberrant in over twenty types of cancer. These alkaloids first became known in the 1950's due to their teratogenic properties, which resulted in newborn and fetal lambs developing cyclopia as a result of pregnant ewes consuming Veratrum californicum. It was discovered that the alkaloids in V. californicum were concentrated in the root and rhizome of the plant with much lower amounts of the most active alkaloid, cyclopamine, present in the aerial plant, especially in the late growth season. Inspired by the limitations in analytical instrumentation and methods available to researchers at the time of the original investigation, we have used state-of-the-art instrumentation and modern analytical methods to quantitate four steroidal alkaloids based on study parameters including plant part, harvest location, and growth stage. The results of the current inquiry detail differences in alkaloid composition based on the study parameters, provide a detailed assessment for alkaloids that have been characterized previously (cyclopamine, veratramine, muldamine and isorubijervine), and identify at least six alkaloids that have not been previously characterized. This study provides insight into optimal harvest time, plant growth stage, harvest location, and plant part required to isolate, yet to be characterized, alkaloids of interest for exploration as Hh pathway antagonists with desirable medicinal properties.
Topics: Alkaloids; Hedgehog Proteins; Idaho; Molecular Structure; Phytochemicals; Plant Components, Aerial; Rhizome; Seasons; Steroids; Veratrum; Veratrum Alkaloids
PubMed: 31381957
DOI: 10.1016/j.fitote.2019.104281 -
Molecular Pharmaceutics Sep 2019Many oral mucosal conditions cause considerable and prolonged pain that to date has been difficult to alleviate via topical delivery, and the use of injection causes...
Many oral mucosal conditions cause considerable and prolonged pain that to date has been difficult to alleviate via topical delivery, and the use of injection causes many patients dental anxiety and needle-prick pain. Therefore, developing a noninjectable drug delivery system as an alternative administration procedure may vastly improve the health and wellbeing of these patients. Recent advances in the development of mucoadhesive electrospun patches for the direct delivery of therapeutics to the oral mucosa offer a potential solution, but as yet, the release of local anesthetics from this system and their uptake by oral tissue have not been demonstrated. Here, we demonstrate the fabrication of lidocaine-loaded electrospun fiber patches, drug release, and subsequent uptake and permeation through the porcine buccal mucosa. Lidocaine HCl and lidocaine base were incorporated into the electrospun patches to evaluate the difference in drug permeation for the two drug compositions. Lidocaine released from the lidocaine HCl-containing electrospun patches was significantly quicker than from the lidocaine base patches, with double the amount of drug released from the lidocaine HCl patches in the first 15 min (0.16 ± 0.04 mg) compared to that from the lidocaine base patches (0.07 ± 0.01 mg). The permeation of lidocaine from the lidocaine HCl electrospun patches through ex vivo porcine buccal mucosa was also detected in 15 min, whereas permeation of lidocaine from the lidocaine base patch was not detected. Matrix-assisted laser desorption ionization-mass spectrometry imaging was used to investigate localization of lidocaine within the oral tissue. Lidocaine in the solution as well as from the mucoadhesive patch penetrated into the buccal mucosal tissue in a time-dependent manner and was detectable in the lamina propria after only 15 min. Moreover, the lidocaine released from lidocaine HCl electrospun patches retained biological activity, inhibiting veratridine-mediated opening of voltage-gated sodium channels in SH-SY5Y neuroblastoma cells. These data suggest that a mucoadhesive electrospun patch may be used as a vehicle for rapid uptake and sustained anesthetic drug delivery to treat or prevent oral pain.
Topics: Administration, Buccal; Anesthetics; Animals; Cell Line, Tumor; Drug Delivery Systems; Drug Liberation; Facial Pain; Humans; Lidocaine; Mouth Mucosa; Neuroblastoma; Oral Mucosal Absorption; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Swine; Tissue Distribution; Veratridine; Voltage-Gated Sodium Channel Agonists; Voltage-Gated Sodium Channel Blockers
PubMed: 31361498
DOI: 10.1021/acs.molpharmaceut.9b00535 -
Journal of Cellular and Molecular... Sep 2019Liver fibrosis is a wound-healing process of liver featured by the over-deposition of extracellular matrix (ECM) and angiogenesis. However, the effective treatment is...
BACKGROUND
Liver fibrosis is a wound-healing process of liver featured by the over-deposition of extracellular matrix (ECM) and angiogenesis. However, the effective treatment is lacking. Procyanidin B2 (PB2) is a flavonoid extract abundant in grape seeds with anti-oxidant, anti-inflammatory and anti-cancer properties. The present study aimed to determine effects of PB2 on liver fibrosis.
METHOD
The CCl4-induced mouse liver fibrosis model and a human hepatic stellate cell (HSC) line (LX2 cells) were used to study the activation, ECM production and angiogenesis of HSCs through Western blotting analysis, immunohistochemistry, immunofluorescence staining, flow cytometry and tubulogenesis assay. A Hedgehog (Hh) pathway inhibitor (cyclopamine) and Smoothened agonist (SAG) were used to investigate the role of PB2 on Hh pathway.
RESULTS
The results showed that PB2 could inhibit the proliferation and induce apoptosis of HSCs. PB2 could also down-regulate the expressions of VEGF-A, HIF-1α, α-SMA, Col-1 and TGF-β1 of HSCs in vivo and in vitro. The application of SAG and cyclopamine proved that PB2 targets on Hh pathway.
CONCLUSIONS
PB2 inhibited the Hh pathway to suppress the activation, ECM production and angiogenesis of HSCs, therefore reverses the progression of liver fibrosis in vivo and in vitro.
Topics: Animals; Biflavonoids; Carbon Tetrachloride; Catechin; Cell Line; Cell Proliferation; Down-Regulation; Hedgehog Proteins; Hepatic Stellate Cells; Humans; Liver; Liver Cirrhosis; Male; Mice; Mice, Inbred C57BL; Morphogenesis; Neovascularization, Pathologic; Proanthocyanidins; Signal Transduction; Transforming Growth Factor beta1; Veratrum Alkaloids
PubMed: 31328391
DOI: 10.1111/jcmm.14543 -
Biomedicine & Pharmacotherapy =... Sep 2019Jervine is a natural teratogenic compound isolated from Veratrum californicum. In this study, for the first time, we revealed a novel activity of jervine in sensitizing...
Jervine is a natural teratogenic compound isolated from Veratrum californicum. In this study, for the first time, we revealed a novel activity of jervine in sensitizing the anti-proliferation effect of doxorubicin (DOX). We demonstrated that the synergistic mechanism was related to the intracellular accumulation of DOX via modulating ABCB1 transportation. Jervine did not affect the expression of ABCB1 in mRNA nor protein levels. However, jervine increased the ATPase activity of ABCB1 and possibly served as a substrate of ABCB1. The molecular docking results indicated that jervine was bound to a closed ABCB1 conformation and blocked drug entrance to the central binding site at the transmembrane domain. The present study identifies jervine acts as a substrate of ABCB1, and has potential to be developed as a novel and potent chemotherapy sensitizer used for patients developing multidrug resistance.
Topics: ATP Binding Cassette Transporter, Subfamily B; Cell Line, Tumor; Cell Proliferation; Cell Survival; Doxorubicin; Human Umbilical Vein Endothelial Cells; Humans; MCF-7 Cells; Protein Structure, Secondary; Substrate Specificity; Teratogens; Veratrum Alkaloids
PubMed: 31207578
DOI: 10.1016/j.biopha.2019.109059 -
Nature Jul 2019The oncoprotein Smoothened (SMO), a G-protein-coupled receptor (GPCR) of the Frizzled-class (class-F), transduces the Hedgehog signal from the tumour suppressor...
The oncoprotein Smoothened (SMO), a G-protein-coupled receptor (GPCR) of the Frizzled-class (class-F), transduces the Hedgehog signal from the tumour suppressor Patched-1 (PTCH1) to the glioma-associated-oncogene (GLI) transcription factors, which activates the Hedgehog signalling pathway. It has remained unknown how PTCH1 modulates SMO, how SMO is stimulated to form a complex with heterotrimeric G proteins and whether G-protein coupling contributes to the activation of GLI proteins. Here we show that 24,25-epoxycholesterol, which we identify as an endogenous ligand of PTCH1, can stimulate Hedgehog signalling in cells and can trigger G-protein signalling via human SMO in vitro. We present a cryo-electron microscopy structure of human SMO bound to 24(S),25-epoxycholesterol and coupled to a heterotrimeric G protein. The structure reveals a ligand-binding site for 24(S),25-epoxycholesterol in the 7-transmembrane region, as well as a G-coupled activation mechanism of human SMO. Notably, the G protein presents a different arrangement from that of class-A GPCR-G complexes. Our work provides molecular insights into Hedgehog signal transduction and the activation of a class-F GPCR.
Topics: Cryoelectron Microscopy; GTP-Binding Protein alpha Subunits, Gi-Go; Hedgehog Proteins; Humans; Immunoglobulin Fab Fragments; Ligands; Models, Molecular; Oxysterols; Patched-1 Receptor; Protein Conformation; Signal Transduction; Smoothened Receptor; Veratrum Alkaloids
PubMed: 31168089
DOI: 10.1038/s41586-019-1286-0 -
Journal of Enzyme Inhibition and... Dec 2019In this study, we investigated whether jervine (J) could prevent gastrointestinal (GI) side effects of abdominopelvic radiotherapy (RT) in Wistar-Albino female rats....
In this study, we investigated whether jervine (J) could prevent gastrointestinal (GI) side effects of abdominopelvic radiotherapy (RT) in Wistar-Albino female rats. Rats were divided into five groups: control (C), J only (J), J administered at 5 mg/kg/days for 7 days, RT only (RT), J before RT (J + RT), J administered for seven days before RT, J both before and after RT (J + RT + J), and J administered for 7 days before RT and after RT for 3 days. The weights of rats were measured on the 1st, 7th, and 10th days of the study. Rats were sacrificed to obtain tissues from the liver and intestine, which was followed by taking blood samples intracardially. In addition, the tissues were stained with pyruvate dehydrogenase (PDH) immunohistochemically. In our study, J supplementation markedly reduced weight loss, and histopathological, immunohistochemical, biochemical results suggest that J had a protective effect on GI toxicity following RT.
Topics: Animals; Gastrointestinal Agents; Radiation Injuries; Rats; Rats, Wistar; Veratrum Alkaloids
PubMed: 30871382
DOI: 10.1080/14756366.2019.1586681 -
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 -
Medical Science Monitor : International... Feb 2019BACKGROUND Esophageal carcinoma is a common gastrointestinal tumor in humans. Cyclopamine, a Hedgehog (Hh)-pathway-specific inhibitor, is an effective chemotherapeutic...
BACKGROUND Esophageal carcinoma is a common gastrointestinal tumor in humans. Cyclopamine, a Hedgehog (Hh)-pathway-specific inhibitor, is an effective chemotherapeutic drug for suppressing tumor cell differentiation, with unclear mechanisms. We investigated glioma-associated oncogene protein-1 (Gli-1) expression in human esophageal carcinoma tissue and the inhibition of cyclopamine on EC9706 esophageal carcinoma cell growth. MATERIAL AND METHODS Gli-1 in tumor tissue was measured by immunohistochemistry (IHC). EC9706 cells were treated with different concentrations of cyclopamine and incubated for different times. MTT method, flow cytometry, and Acridine orange/ethidium bromide (AO/EB) double-fluorescence staining were applied to detect cell proliferation and apoptosis. Western blot (WB) analysis was performed to assess Gli-1 expression. RESULTS Gli-1 was associated with patient age, gender, lymphatic metastasis, tumor recurrence, and stage, with significantly (P<0.05) positive correlations with age, lymphatic metastasis, tumor recurrence, and stage. At 12 h (F=214.57), 24 h (F=76.832), 48 h (F=236.90), and 72 h (F=164.55), the higher the concentration of cyclopamine, the higher the inhibition rate of suppressing EC9706 proliferation, and this effect was significant (P<0.05). The number of early-apoptosis cells increased as the concentration of cyclopamine increased. Morphology of EC9706 cells appeared as round with rough edges, karyopyknosis, and karyorrhexis. After 48 h, apoptosis rates of EC9706 cells treated with different concentrations of cyclopamine were (7.73±1.25)% at 2.5 μM, (13.37±1.42)% at 5.0 μM, (22.3±2.92)% at 10.0 μM, and (33.57±1.75)% at 20.0 μM, and the effect was dose-dependent. Gli-1 was obviously reduced after cyclopamine treatment and the effect was dose-dependent. CONCLUSIONS Gli-1 is highly expressed in human esophageal carcinoma, and could be a marker for use in assessing tumor stage and the deciding on treatment target.
Topics: Apoptosis; Carcinoma, Squamous Cell; Cell Cycle; Cell Line, Tumor; Cell Proliferation; China; Disease Progression; Down-Regulation; Esophageal Neoplasms; Female; Humans; Lymphatic Metastasis; Male; Neoplasm Recurrence, Local; Signal Transduction; Veratrum Alkaloids; Zinc Finger Protein GLI1
PubMed: 30807555
DOI: 10.12659/MSM.912858 -
Scientific Reports Feb 2019Lung cancer remains the leading cause of cancer-related death, despite the advent of targeted therapies and immunotherapies. Therefore, it is crucial to identify novel...
Lung cancer remains the leading cause of cancer-related death, despite the advent of targeted therapies and immunotherapies. Therefore, it is crucial to identify novel molecular features unique to lung tumors. Here, we show that cyclopamine tartrate (CycT) strongly suppresses the growth of subcutaneously implanted non-small cell lung cancer (NSCLC) xenografts and nearly eradicated orthotopically implanted NSCLC xenografts. CycT reduces heme synthesis and degradation in NSCLC cells and suppresses oxygen consumption in purified mitochondria. In orthotopic tumors, CycT decreases the levels of proteins and enzymes crucial for heme synthesis, uptake, and oxidative phosphorylation (OXPHOS). CycT also decreases the levels of two regulators promoting OXPHOS, MYC and MCL1, and effectively alleviates tumor hypoxia. Evidently, CycT acts via multiple modes to suppress OXPHOS. One mode is to directly inhibit mitochondrial respiration/OXPHOS. Another mode is to inhibit heme synthesis and degradation. Both modes appear to be independent of hedgehog signaling. Addition of heme to NSCLC cells partially reverses the effect of CycT on oxygen consumption, proliferation, and tumorigenic functions. Together, our results strongly suggest that CycT suppress tumor growth in the lung by inhibiting heme metabolism and OXPHOS. Targeting heme metabolism and OXPHOS may be an effective strategy to combat lung cancer.
Topics: A549 Cells; Animals; Carcinogenesis; Carcinoma, Non-Small-Cell Lung; Cell Proliferation; Cell Respiration; Disease Progression; Female; Hedgehog Proteins; Humans; Lung Neoplasms; Mice; Mice, Inbred NOD; Mice, SCID; Mitochondria; Oxidative Phosphorylation; Signal Transduction; Tartrates; Tumor Burden; Veratrum Alkaloids; Xenograft Model Antitumor Assays
PubMed: 30723259
DOI: 10.1038/s41598-018-38345-1 -
PloS One 2019Peiminine is a compound isolated from Bolbostemma paniculatum (Maxim) Franquet (Cucurbitaceae family), which has demonstrated antitumor activities. But its precise...
Peiminine is a compound isolated from Bolbostemma paniculatum (Maxim) Franquet (Cucurbitaceae family), which has demonstrated antitumor activities. But its precise molecular mechanism underlying antitumor activity remain elusive. In this study, peiminine-induced apoptosis towards human hepatocellular carcinoma and its molecular mechanism were investigated. MTT assay was employed to assess anticancer effects of peiminine upon Hela, HepG2, SW480 and MCF-7 cell lines. Nuclear staining and flow cytometry were carried out to detect apoptosis induced by peiminine. Mitochondrial membrane potential evaluation and Western blot analysis were performed to investigate the mechanism of peiminine-induced apoptosis. The results showed peiminine reduced the viability of HepG2 cells in a time- and dose-dependent manner and had an IC50 of 4.58 μg/mL at 24h. Peiminine significantly increased the percentage of apoptotic cells and the mitochondrial membrane potential dose-dependently in HepG2 cells. The results of Western blotting indicated the expressions of Bcl-2, procaspase-3, procaspase-8, procaspase-9, and PARP decreased in HepG2 cells treated with peiminine, while the expressions of Bax, caspase-3, caspase-8, caspase-9, and cleaved PARP1 increased. The result suggests that peiminine can induce apoptosis in human hepatocellular carcinoma HepG2 cells through both extrinsic and intrinsic apoptotic pathways.
Topics: Apoptosis; Carcinoma, Hepatocellular; Cell Proliferation; Cell Survival; Cevanes; Dose-Response Relationship, Drug; Hep G2 Cells; Humans; Liver Neoplasms; Membrane Potential, Mitochondrial; Neoplasm Proteins; Signal Transduction
PubMed: 30615617
DOI: 10.1371/journal.pone.0201864