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Cancer Medicine Jul 2021The Sonic Hedgehog (SHH) signaling pathway plays an important role in various types of human cancers including ovarian cancer; however, its function and underlying...
BACKGROUND
The Sonic Hedgehog (SHH) signaling pathway plays an important role in various types of human cancers including ovarian cancer; however, its function and underlying mechanism in ovarian cancer are still not entirely understood.
METHODS
We detected the expressions of SHH and SQSTM1 in borderline ovarian tumor tissues, epithelial ovarian cancer (EOC) tissues and benign ovarian tumor tissues. Cyclopamine (Cyp, a well-known inhibitor of SHH signaling pathway) and chloroquine (CQ, the pharmaceutical inhibitor of autophagy) were used in vivo and in vitro (autophagic flux, CCK-8 assay, wound healing assay, transwell assay, tumor xenograft model). The mechanism of action was explored through Quantitative RT-PCR and Western Blot.
RESULTS
We found up-regulation of SHH and accumulation of SQSTM1/P62 in epithelial ovarian cancer. Cyp induced autophagy through the PI3K/AKT signaling pathway. Moreover, low-dose Cyp and chloroquine (CQ) significantly promoted the migratory ability of SKOV3 cells.
CONCLUSIONS
Our findings suggest that inhibition of the SHH pathway and autophagy may be a potential and effective therapy for the treatment of ovarian cancer.
Topics: Animals; Autophagic Cell Death; Carcinoma, Ovarian Epithelial; Cell Line, Tumor; Cell Movement; Chloroquine; Female; Hedgehog Proteins; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Ovarian Neoplasms; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; RNA-Binding Proteins; Sequestosome-1 Protein; Up-Regulation; Veratrum Alkaloids
PubMed: 34076346
DOI: 10.1002/cam4.4018 -
Harmful Algae Mar 2021Marine biotoxins accumulating in seafood products pose a risk to human health. These toxins are often potent in minute amounts and contained within complex matrices;...
Marine biotoxins accumulating in seafood products pose a risk to human health. These toxins are often potent in minute amounts and contained within complex matrices; requiring sensitive, reliable, and robust methods for their detection. The mouse neuroblastoma (Neuro-2a) cytotoxicity assay (N2a-assay) is a sensitive, high-throughput, in vitro method effective for detecting sodium channel-specific marine biotoxins. The N2a-assay can be conducted to distinguish between specific effects on voltage-gated sodium (Na) channels, caused by toxins that activate (e.g., ciguatoxins (CTXs), brevetoxins (PbTxs)) or block (e.g., tetrodotoxins, saxitoxins) the target Na. The sensitivity and specificity of the assay to compounds activating the Na are achieved through the addition of the pharmaceuticals ouabain (O) and veratridine (V). However, these compounds can be toxic to Neuro-2a cells and their application at insufficient or excessive concentrations can reduce the effectiveness of this assay for marine toxin detection. Therefore, during growth incubation, Neuro-2a cells were exposed to O and V, and surviving cells exhibiting a lower sensitivity to O and V (OV-LS) were propagated. OV-LS Neuro-2a cells were selected for 60-80% survival when exposed to 0.22/0.022 mM O/V during the cytotoxicity assay. At these conditions, OV-LS N2a cells demonstrated a 3.5-fold higher survival rate 71% ± 7.9 SD (n = 232), and lower sensitivity to O/V, compared to the original Neuro-2a cells 20% ± 9.0 SD (n = 16). Additionally, OV-LS N2a cells were 1.3-2.6-fold more sensitive for detecting CTX3C 1.35 pg/ml, CTX1B 2.06 pg/ml, and PbTx-3 3.04 ng/ml compared to Neuro-2a cells using 0.1/0.01 mM O/V. Detection of CTX3C in a complex fish matrix using OV-LS cells was 0.0048 pg CTX3C/mg fish tissue equivalent. This work shows the potential for a significant improvement in sensitivity for CTX3C, CTX1B, and PbTx-3 using the OV-LS N2a-assay.
Topics: Animals; Cell Line, Tumor; Ciguatoxins; Marine Toxins; Neuroblastoma; Ouabain; Oxocins; Veratridine
PubMed: 33980434
DOI: 10.1016/j.hal.2021.101994 -
Pharmaceutical Biology Dec 2021Peimine and paeoniflorin can be combined for the treatment of cough in paediatrics. The interaction during the co-administration could dramatically affect the...
CONTEXT
Peimine and paeoniflorin can be combined for the treatment of cough in paediatrics. The interaction during the co-administration could dramatically affect the bioavailability of drugs.
OBJECTIVE
The interaction between peimine and paeoniflorin was investigated in this study.
MATERIALS AND METHODS
The pharmacokinetics of paeoniflorin (20 mg/kg) with or without the coadministration of peimine (5 mg/kg for 10 days before paeoniflorin) was orally investigated in Sprague-Dawley rats ( = 6). The group without the peimine was set as the control group. The metabolic stability of paeoniflorin was studied in rat liver with microsomes. The effect of peimine on the absorption of paeoniflorin was investigated with Caco-2 cell monolayers.
RESULTS
The (244.98 ± 10.95 vs. 139.18 ± 15.14 μg/L) and AUC (3295.92 ± 263.02 vs. 139.18 ± 15.14 h·μg/L) of paeoniflorin was increased by peimine. The was prolonged from 5.33 ± 1.65 to 14.21 ± 4.97 h and the clearance was decreased from 15.43 ± 1.75 to 4.12 ± 0.57 L/h/kg. Consistently, peimine increased the metabolic stability of paeoniflorin with rat liver microsomes with the increased (56.78 ± 2.62 vs. 26.33 ± 3.15 min) and the decreased intrinsic clearance (24.42 ± 3.78 vs. 52.64 ± 4.47 μL/min/mg protein). Moreover, the transportation of paeoniflorin was also inhibited by peimine as the efflux ratio decreased from 3.06 to 1.63.
DISCUSSION AND CONCLUSIONS
Peimine increased the systemic exposure of paeoniflorin through inhibiting the activity of CYP3A4 and P-gp. These results provide a reference for further studies in a broader population.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; Area Under Curve; Caco-2 Cells; Cevanes; Cytochrome P-450 CYP3A; Drug Interactions; Glucosides; Half-Life; Humans; Male; Microsomes, Liver; Monoterpenes; Rats; Rats, Sprague-Dawley
PubMed: 33721550
DOI: 10.1080/13880209.2021.1875013 -
Cell Death & Disease Mar 2021Chronic myeloid leukemia (CML) patients with complex chromosomal translocations as well as non-compliant CML patients often demonstrate short-lived responses and poor...
Chronic myeloid leukemia (CML) patients with complex chromosomal translocations as well as non-compliant CML patients often demonstrate short-lived responses and poor outcomes on the current therapeutic regimes using Imatinib and its variants. It has been derived so far that leukemic stem cells (LSCs) are responsible for Imatinib resistance and CML progression. Sonic hedgehog (Shh) signaling has been implicated in proliferation of this Imatinib-resistant CD34(+) LSCs. Our work here identifies the molecular mechanism of Shh-mediated mutation-independent Imatinib resistance that is most relevant for treating CML-variants and non-compliant patients. Our results elucidate that while Shh can impart stemness, it also upregulates expression of anti-apoptotic protein-Bcl2. It is the upregulation of Bcl2 that is involved in conferring Imatinib resistance to the CD34(+) LSCs. Sub-toxic doses of Bcl2 inhibitor or Shh inhibitor (<
Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Chromosomes, Human; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Imatinib Mesylate; K562 Cells; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Neoplastic Stem Cells; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Thiazoles; Up-Regulation; Veratrum Alkaloids
PubMed: 33707419
DOI: 10.1038/s41419-021-03542-w -
Journal of Orthopaedic Surgery and... Feb 2021This study was designed to observe the expression of important hedgehog (Hh) signal factors in the bone tissue of rats with chronic fluorosis and cultured osteoblasts in...
OBJECTIVE
This study was designed to observe the expression of important hedgehog (Hh) signal factors in the bone tissue of rats with chronic fluorosis and cultured osteoblasts in order to investigate the role and significance of the Hh signal in fluoride-induced bone injury.
METHODS
Healthy Sprague-Dawley (SD) rats were randomly divided into four groups: the control group, the fluorosis group (F Group), the fluoride + blocker group (F + Cycl group: rats were treated with fluoride + cyclopamine), and the fluoride + blocker control group (F + DMSO group). After 6 months of intervention, the urinary fluoride content of rats in each group was detected. The primary osteoblasts of rats were selected for cell experiment, and the experiment was carried out after the cells were passaged from the second to the fourth generation.
RESULTS
The proliferation rate of primary rat osteoblasts presented time-affected and dose-affected relationships in a short time under treatment with a low dose of sodium fluoride (NaF), but the proliferation of osteoblasts was inhibited by long-term and high-dose NaF exposure. In the F group, the alkaline phosphatase (ALP) activity of osteoblasts increased gradually. The ALP activity was lower in the F + Cycl group than in the F group, and there was no significant difference between the F + DMSO group and F group. With the increase in fluoride exposure, the expression of Hh signal factors and osteogenic-related factor proteins increased gradually. The expressions of Indian hedgehog (Ihh), smoothened (Smo), Glioma-associated oncogene homolog (Gli) 2, and Runt-related transcription factor 2 (Runx2)in the F + Cycl group increased with the dose of fluoride but they were significantly inhibited compared with the F group. Compared with the control group, the content of urinary fluoride in the F group was significantly higher (P < 0.05), but there was no significant change in urinary fluoride content in the F + Cycl group and the F + DMSO group. Compared with the control group, the serum bone alkaline phosphatase (BALP) contents of rats in the other groups increased after 6 months' intake of fluoride water (P < 0.05). After drug blocking, the serum BALP content in the F + Cycl group was lower than that in the F + DMSO group (P < 0.05). The BALP content in the F + DMSO group was similar to that in the F group: it did not decrease. The mRNA expressions of Ihh, Smo, Gli2, and Runx2 in bone tissue of the F group were significantly higher than those in the control group (P < 0.05). After cyclopamine blocking, the expressions decreased (P < 0.05), but the differences between the F + DMSO group and F group were not statistically significant.
CONCLUSION
Hh signal plays an important role in fluoride-induced bone injury. The effective inhibition of cyclopamine is expected to be a new target for the treatment of skeletal damage caused by fluorosis.
Topics: Animals; Bone Diseases; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Gene Expression; Hedgehog Proteins; Osteoblasts; Rats, Sprague-Dawley; Signal Transduction; Smoothened Receptor; Sodium Fluoride; Time Factors; Veratrum Alkaloids; Rats
PubMed: 33637095
DOI: 10.1186/s13018-021-02287-8 -
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 -
Journal of Insect Science (Online) Nov 2020The tea green leafhopper Empoasca onukii Matsuda (Hemiptera: Cicadellidae), the orange spiny whitefly, Aleurocanthus spiniferus (Quaintanca) (Hemiptera: Aleyrodidae),...
Evaluation of Botanicals for Management of Piercing-Sucking Pests and the Effect on Beneficial Arthropod Populations in Tea Trees Camellia sinensis (L.) O. Kuntze (Theaceae).
The tea green leafhopper Empoasca onukii Matsuda (Hemiptera: Cicadellidae), the orange spiny whitefly, Aleurocanthus spiniferus (Quaintanca) (Hemiptera: Aleyrodidae), and the green plant bugs Apolygus lucorum Meyer-Dür (Hemiptera: Miridae) are the important piercing-sucking herbivores in tea trees Camellia sinensis (L.) O. Kuntze (Theaceae). The goal of this study was to evaluate the laboratory toxicities and field control efficacies of botanical insecticides including matrine, azadirachtin, veratrine, and pyrethrin to three tea pests. Via leaf-dip bioassay, toxicity tests with botanical insecticides indicated that there were significant differences between the LC50 values for botanical insecticides within the same insect species. Matrine had the highest toxicity to E. onukii, A. spiniferus, and A. lucorum with the LC50 values of 2.35, 13.10, and 44.88 mg/liter, respectively. Field tests showed that, among four botanical insecticides, matrine at dose of 9 g a.i. ha-1 can significantly reduce the numbers of E. onukii and A. spiniferus and the infestation of A. lucorum on the tea plants. Furthermore, botanical insecticides matrine and azadirachtin had no obvious influence on the coccinellids, spiders, and parasitoids densities in tea plantations. The results of this study indicated that use of botanical insecticides, such as matrine, has the potential to manipulate the population of E. onukii, A. spiniferus, and A. lucorum and will be an effective and environmentally compatible strategy for the control of tea pests.
Topics: Alkaloids; Animals; Camellia sinensis; Hemiptera; Insect Control; Insecticides; Limonins; Pest Control, Biological; Pyrethrins; Quinolizines; Species Specificity; Veratrine; Matrines
PubMed: 33211857
DOI: 10.1093/jisesa/ieaa101 -
Biomedicine & Pharmacotherapy =... Dec 2020Nasopharyngeal carcinoma (NPC) is a malignant tumor originating from the superior mucosal epithelium of the nasopharynx. However, effective therapies for NPC are still...
Nasopharyngeal carcinoma (NPC) is a malignant tumor originating from the superior mucosal epithelium of the nasopharynx. However, effective therapies for NPC are still required. Reducing Hedgehog signaling pathway has been shown to suppress tumor growth. In this study, we attempted to explore whether Jervine (JV), an inhibitor of Hedgehog signaling, had anti-cancer effects on NPC, and the underlying mechanisms. Our findings showed that JV treatments markedly reduced the proliferation of NPC cells in a dose- and time-dependent manner. Cell cycle arrest in G2/M phase was significantly enhanced by JV, along with evident DNA damage. Moreover, JV treatment effectively induced apoptosis in NPC cells through improving Caspase-3 activation. Furthermore, ROS production and mitochondrial impairments were detected in JV-incubated NPC cells with elevated releases of Cyto-c from mitochondria. JV also dramatically triggered autophagy through blocking AKT/mTOR and increasing AMPK signaling pathways. Intriguingly, we showed that JV-induced apoptosis was mainly via an autophagy-dependent manner. In addition, the expression levels of SHH, PTCH1, SMO and GLI1 were markedly suppressed in NPC cells, demonstrating the hindered Hedgehog signaling. Importantly, we found that JV-induced apoptosis and autophagy were closely associated with the blockage of Hedgehog signaling. Our in vivo studies confirmed the anti-cancer effects of JV on NPC through inducing autophagy, as evidenced by the markedly reduced tumor growth rate and weight without side effects and toxicity. Taken together, JV may be a promising and effective agent for human NPC treatment through repressing Hedgehog signaling pathway and inducing autophagic cell death.
Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Cell Line, Tumor; Cell Proliferation; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Hedgehog Proteins; Humans; Male; Mice, Inbred BALB C; Mice, Nude; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Patched-1 Receptor; Signal Transduction; Smoothened Receptor; Veratrum Alkaloids; Xenograft Model Antitumor Assays; Zinc Finger Protein GLI1
PubMed: 33113432
DOI: 10.1016/j.biopha.2020.110898 -
Cells Jul 2020In the liver, energy homeostasis is mainly regulated by mechanistic target of rapamycin (mTOR) signalling, which influences relevant metabolic pathways, including lipid...
In the liver, energy homeostasis is mainly regulated by mechanistic target of rapamycin (mTOR) signalling, which influences relevant metabolic pathways, including lipid metabolism. However, the Hedgehog (Hh) pathway is one of the newly identified drivers of hepatic lipid metabolism. Although the link between mTOR and Hh signalling was previously demonstrated in cancer development and progression, knowledge of their molecular crosstalk in healthy liver is lacking. To close this information gap, we used a transgenic mouse model, which allows hepatocyte-specific deletion of the Hh pathway, and in vitro studies to reveal interactions between Hh and mTOR signalling. The study was conducted in male and female mice to investigate sexual differences in the crosstalk of these signalling pathways. Our results reveal that the conditional Hh knockout reduces mitochondrial adenosine triphosphate (ATP) production in primary hepatocytes from female mice and inhibits autophagy in hepatocytes from both sexes. Furthermore, in vitro studies show a synergistic effect of cyclopamine and rapamycin on the inhibition of mTor signalling and oxidative respiration in primary hepatocytes from male and female C57BL/6N mice. Overall, our results demonstrate that the impairment of Hh signalling influences mTOR signalling and therefore represses oxidative phosphorylation and autophagy.
Topics: Adenosine Triphosphate; Animals; Autophagy; Drug Synergism; Energy Metabolism; Female; Gene Deletion; Hedgehog Proteins; Hepatocytes; Lipid Metabolism; Liver; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Oxidative Phosphorylation; Sex Factors; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Veratrum Alkaloids
PubMed: 32751882
DOI: 10.3390/cells9081817 -
PloS One 2020Endothelial cilia are found in a variety of tissues including the cranial vasculature of zebrafish embryos. Recently, endothelial cells in the developing mouse retina...
Endothelial cilia are found in a variety of tissues including the cranial vasculature of zebrafish embryos. Recently, endothelial cells in the developing mouse retina were reported to also possess primary cilia that are potentially involved in vascular remodeling. Fish carrying mutations in intraflagellar transport (ift) genes have disrupted cilia and have been reported to have an increased rate of spontaneous intracranial hemorrhage (ICH), potentially due to disruption of the sonic hedgehog (shh) signaling pathway. However, it remains unknown whether the endothelial cells forming the retinal microvasculature in zebrafish also possess cilia, and whether endothelial cilia are necessary for development and maintenance of the blood-retinal barrier (BRB). In the present study, we found that the endothelial cells lining the zebrafish hyaloid vasculature possess primary cilia during development. To determine whether endothelial cilia are necessary for BRB integrity, ift57, ift88, and ift172 mutants, which lack cilia, were crossed with the double-transgenic zebrafish strain Tg(l-fabp:DBP-EGFP;flk1:mCherry). This strain expresses a vitamin D-binding protein (DBP) fused to enhanced green fluorescent protein (EGFP) as a tracer in the blood plasma, while the endothelial cells forming the vasculature are tagged by mCherry. The Ift mutant fish develop a functional BRB, indicating that endothelial cilia are not necessary for early BRB integrity. Additionally, although treatment of zebrafish larvae with Shh inhibitor cyclopamine results in BRB breakdown, the Ift mutant fish were not sensitized to cyclopamine-induced BRB breakdown.
Topics: Adaptor Proteins, Signal Transducing; Animals; Animals, Genetically Modified; Blood-Retinal Barrier; Cilia; Endothelial Cells; Hedgehog Proteins; Larva; Mutagenesis; Retinal Vessels; Signal Transduction; Veratrum Alkaloids; Zebrafish; Zebrafish Proteins
PubMed: 32735563
DOI: 10.1371/journal.pone.0225351