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Biochemical and Biophysical Research... Dec 2020Non-small cell lung cancer (NSCLC) has been identified as a leading cause of tumor-associated death around the world. Presently, it is necessary to find effective and...
Non-small cell lung cancer (NSCLC) has been identified as a leading cause of tumor-associated death around the world. Presently, it is necessary to find effective and safe therapy for its treatment in clinic. Jervine (Jer), a sterodial alkaloid from rhizomes of Veratrum album, exhibits anti-inflammatory and anti-cancer effects. However, its effects on lung cancer progression are still unknown. In this study, we explored if Jer showed any influences on NSCLC development, as well as the underlying molecular mechanisms. The results showed that Jer time- and dose-dependently reduced the proliferation of NSCLC cells, along with inhibited colony formation capacity. Apoptosis was highly induced by Jer in NSCLC cells through promoting the expression of cleaved Caspase-3. Furthermore, Jer treatment led to autophagy in cancer cells, as evidenced by the fluorescence microscopy results and increases of LC3II. Autophagy inhibitor bafilomycinA1 (BafA1) abrogated the inhibitory effects of Jer on cell proliferation and apoptosis induction, showing that Jer triggered autophagy-mediated apoptosis in NSCLC cells. Additionally, AKT and mammalian target of Rapamycin (mTOR) signaling pathway was highly repressed in cancer cells. Importantly, promoting AKT activation greatly rescued the cell survival, while attenuated autophagy and apoptosis in Jer-incubated NSCLC cells, revealing that Jer-modulated autophagic cell death was through the blockage of AKT signaling. Hedgehog signaling pathway was then found to be suppressed by Jer, as proved by the decreased expression of Sonic Hedgehog (Shh), Hedgehog receptor protein patched homolog 1 (PTCH1), smoothened (SMO) and glioma-associated oncogene homolog 1 (Gli1) in NSCLC cells. Of note, enhancing Shh signaling dramatically diminished the stimulative effects of Jer on autophagy-mediated apoptosis in vitro, demonstrating the importance of Hedgehog signaling in Jer-regulated cell death. Moreover, Jer treatment effectively reduced tumor growth in A549-bearing mice with few toxicity. Together, Jer may be a promising and effective therapeutic strategy for NSCLC treatment.
Topics: Animals; Antineoplastic Agents; Apoptosis; Autophagic Cell Death; Autophagy; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Hedgehog Proteins; Humans; Lung Neoplasms; Mice, Nude; Proto-Oncogene Proteins c-akt; Veratrum Alkaloids
PubMed: 32972750
DOI: 10.1016/j.bbrc.2020.08.023 -
Zhongguo Shi Yan Xue Ye Xue Za Zhi Aug 2020To study the effect of SMO inhibitor (Jervine) on proliferation, apoptosis and cell cycle of MDS cell line MUTZ-1, and its mechanism.
OBJECTIVE
To study the effect of SMO inhibitor (Jervine) on proliferation, apoptosis and cell cycle of MDS cell line MUTZ-1, and its mechanism.
METHODS
The effect of different concentrations Jervine on proliferation of MUTZ-1 cells was detected by CCK-8 method. Apoptosis and cell cycle of MUTZ-1 cells were detected by flow cytometry. Western blot was used to detect the changes of Shh signaling pathway effecting proteins BCL2 and CyclinD1. The expression levels of Smo and Gli1 gene were detected by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR).
RESULTS
Jervine inhibited MUTZ-1 cell proliferation in a concentration dependent manner (24 h, r=-0.977), the apoptosis rate of MUTZ-1 cells increased with the enhancement of concentration of Jervine in MUTZ-1 cells (P<0.001), the cell proportion of G phase increased and the cell number of S phase decreased with enhancement of concentration (P<0.001). The result of RT-qPCR and Western blot showed that the expression of Smo, Gli1 mRNA and BCL2, CyclinD1 proteins decreased (P<0.05).
CONCLUSION
SMO inhibitor can effectively inhibit the growth of MDS cell line MUTZ-1 improve the cell apoptosis and induce cell cycle arrest. Its action mechanism may be related with dowm-regulating the expression of BCL2 and CyclinD1.
Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Hedgehog Proteins; Humans; Myelodysplastic Syndromes; Signal Transduction; Veratrum Alkaloids
PubMed: 32798415
DOI: 10.19746/j.cnki.issn.1009-2137.2020.04.036 -
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 -
Anti-cancer Drugs Feb 2021Colorectal cancer (CRC) is a commonly diagnosed type of cancer globally. The molecular mechanism by which peiminine suppressed the progression of CRC is not fully...
Colorectal cancer (CRC) is a commonly diagnosed type of cancer globally. The molecular mechanism by which peiminine suppressed the progression of CRC is not fully addressed. The viability was assessed through cell counting kit 8 assay. Colony formation assay was used to analyze the colony formation ability. The metastasis was evaluated by transwell migration and invasion assays. Quantitative real-time PCR was performed to measure the expression of LINC00659 and miR-760 in CRC cells. The binding sites between miR-760 and LINC00659 were predicted by Starbase software and verified by dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA-pull down assay. The in-vivo function of peiminine in CRC progression was confirmed by murine xenograft model. Peiminine inhibited the viability, colony formation and metastasis of CRC cells. Peiminine notably down-regulated the expression of LINC00659, while the expression of miR-760 was up-regulated by peiminine treatment. MiR-760 was a direct target of LINC00659 in CRC cells. The depletion of miR-760 attenuated the inhibitory effects of LINC00659 intervention on the viability, colony formation and metastasis of CRC cells. Peiminine restrained the progression of CRC through LINC00659 and miR-760. LINC00659 inhibited the growth of CRC tumors through LINC00659/miR-760 axis in vivo. Peiminine suppressed the development of CRC through inhibiting the viability, colony formation and metastasis of CRC cells via LINC00659/miR-760 axis. LINC00659/miR-760 axis might be an underlying target for CRC therapy.
Topics: Animals; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Cevanes; Colorectal Neoplasms; Disease Progression; Down-Regulation; Male; Mice; MicroRNAs; RNA, Long Noncoding; Real-Time Polymerase Chain Reaction; Up-Regulation
PubMed: 32740014
DOI: 10.1097/CAD.0000000000000981 -
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 -
Sudebno-meditsinskaia Ekspertiza 2020The problem of laboratory diagnosis of acute and fatal poisoning by hellebore, which is possible when used in traditional medicine, the erroneous use of hellebore...
The problem of laboratory diagnosis of acute and fatal poisoning by hellebore, which is possible when used in traditional medicine, the erroneous use of hellebore preparations orally or use of various types of this plant for food, remains relevant. Currently, in the practice of chemical-toxicological laboratories and the bureau of forensic medical examination there is no single approach to the laboratory diagnosis of such poisoning. The diagnosis is most often based on anamnesis. In this regard, the development and validation of a legally significant methodology for the determination of hellebore alkaloids in various biological objects seems relevant. The physicochemical and toxic properties of alkaloids of various types of hellebore are characterized. It was shown that for the identification of hellebore alkaloids, it is advisable to use HPLC-MS/MS as the most sensitive and specific instrumental method corresponding to the characteristics of hellebore alkaloids (high molecular weight, high thermal lability, high polarity).
Topics: Helleborus; Poisoning; Tandem Mass Spectrometry; Veratrum; Veratrum Alkaloids
PubMed: 32686389
DOI: 10.17116/sudmed20206304134 -
Frontiers in Immunology 2020Activated fibroblast-like synoviocytes (FLSs) play a central role in the formation of synovial pannus and joint destruction in rheumatoid arthritis (RA). Targeting FLSs...
Activated fibroblast-like synoviocytes (FLSs) play a central role in the formation of synovial pannus and joint destruction in rheumatoid arthritis (RA). Targeting FLSs could be a potential therapeutic strategy. The objective of this study is to explore the role of c-Jun N-terminal kinase (JNK) in proliferation, migration and invasion of FLSs promoted by the sonic hedeghog (SHH) signaling pathway in patients with RA. Activation of SHH signaling was evaluated by real-time PCR and Western Blot. Levels of phosphorylation of JNK and c-Jun were detected by Western Blot. FLSs proliferation was quantified by Cell Counting Kit-8 (CCK-8) assay and flow cytometry. Cell migration and invasion were assessed by wound healing assay and Transwell chamber assay. Invasiveness of FLSs was evaluated using a humanized synovitis animal model. We observed that treatment of SHH agonist (SAG) significantly increased the levels of phosphorylation of JNK and c-Jun, while SHH antagonist (cyclopamine) significantly decreased the expression of phospho-JNK and phospho-c-Jun in FLSs. The elevated level of phospho-c-Jun stimulated by SAG was decreased in the presence of JNK inhibitor (SP600125) ( < 0.001). FLSs proliferation, migration and invasion were promoted by SHH agonist ( < 0.05). However, the enhanced aggressiveness of FLSs was abolished in the presence of JNK inhibitor ( < 0.05). study showed that the invasion of FLSs into cartilage was increased by SHH overexpression and the excessive invasiveness was inhibited by blockade of JNK signaling ( < 0.01). These results suggest that JNK is one of the downstream molecules mediating the effect of SHH signaling in FLSs. These findings indicate that SHH-JNK signaling could be a potential therapeutic target to suppress the aggressiveness of FLSs and prevent articular damage of RA.
Topics: Arthritis, Rheumatoid; Biomarkers; Cell Movement; Cell Proliferation; Cell Survival; Cells, Cultured; Cytokines; Female; Flow Cytometry; Hedgehog Proteins; Humans; Immunohistochemistry; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Male; Matrix Metalloproteinase 1; Middle Aged; Synoviocytes; Veratrum Alkaloids
PubMed: 32670287
DOI: 10.3389/fimmu.2020.01300 -
Life Sciences Sep 2020Vascular smooth muscle cells (VSMCs) play a crucial role in the progression of atherosclerosis. Paired box 9 (Pax9) is a member of the Pax gene family which participates...
AIMS
Vascular smooth muscle cells (VSMCs) play a crucial role in the progression of atherosclerosis. Paired box 9 (Pax9) is a member of the Pax gene family which participates in the development of various tissues and organs. However, the effect of Pax9 on atherosclerosis and VSMCs and the underlying mechanisms remain unclear.
MAIN METHODS
Western blotting was performed to assess Pax9 expression in atherosclerosis and VSMCs. Pax9 siRNA and overexpression plasmid were constructed to explore the biological function. Cell proliferation assay, phalloidin staining, and Transwell assay, accompanied by the sonic hedgehog (Shh) signaling pathway antagonist, cyclopamine (5 μM) and agonist, SAG (100 nM), were used to evaluate the VSMC phenotype, proliferation, and migration, as well as explore the associated mechanisms.
KEY FINDINGS
We first discovered Pax9 to be significantly increased in atherosclerotic mice and platelet-derived growth factor-BB (PDGF-BB)-induced VSMCs. Pax9 knockdown inhibited the phenotypic transformation, proliferation, and migration of VSMCs, whereas the opposite effect was observed when Pax9 was overexpressed. Next, we established that Shh was activated in PDGF-BB-induced VSMCs. Moreover, Pax9 overexpression further activated Shh and exacerbated the phenotypic transformation, proliferation, and migration of PDGF-BB-induced VSMCs. These changes were effectively inhibited by treatment with the Shh signaling pathway antagonist. Consistently, Pax9 knockdown down-regulated Shh expression and inhibited the phenotypic transformation, proliferation, and migration of PDGF-BB-induced VSMCs. Treatment with the Shh signaling pathway agonist prevented these changes.
SIGNIFICANCE
Pax9 regulated VSMC phenotypic transformation, proliferation, and migration via Shh, which may represent a novel target for the treatment of atherosclerosis.
Topics: Animals; Atherosclerosis; Becaplermin; Cell Movement; Cell Proliferation; Cyclohexylamines; Gene Knockdown Techniques; Hedgehog Proteins; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocytes, Smooth Muscle; PAX9 Transcription Factor; Phenotype; Signal Transduction; Thiophenes; Veratrum Alkaloids
PubMed: 32634424
DOI: 10.1016/j.lfs.2020.118053 -
Molecular Medicine Reports Aug 2020The Indian hedgehog (IHH) signaling pathway is an important pathway for bone growth and development. The aim of the present study was to examine the role of the IHH...
The Indian hedgehog (IHH) signaling pathway is an important pathway for bone growth and development. The aim of the present study was to examine the role of the IHH signaling pathway in the development of the ossification of ligamentum flavum (OLF) at the cellular and tissue levels. The expression levels and localization of the osteogenic genes Runt-related transcription factor 2 (RUNX2), Osterix, alkaline phosphatase (ALP), osteocalcin (OCN) and IHH were evaluated in OLF tissues by reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemistry. Non-ossified ligamentum flavum (LF) sections were used as control samples. The tissue explant method was used to obtain cultured LF cells. In addition, OLF cells were subjected to cyclic stretch application for 0, 6, 12 or 24 h. The expression levels of osteogenic genes, and the IHH signaling pathway genes IHH, Smoothened (SMO), GLI family zinc finger 1 (GLI1), GLI2 and GLI3 were evaluated with RT-qPCR and western blotting. Osteogenic differentiation was further evaluated by assessing ALP activity and staining. Moreover, the effect of cyclopamine (Cpn), an IHH signaling inhibitor, on osteogenic differentiation was examined. The RT-qPCR and immunohistochemical results indicated that the mRNA and protein expression levels of RUNX2, Osterix, ALP, OCN and IHH were significantly higher in the OLF group compared with the LF group. Furthermore, application of cyclic stretch to OLF cells resulted in greater ALP activity, and significant increases in mRNA and protein expression levels of RUNX2, Osterix, ALP and OCN in a time-d00ependent manner. Cyclic stretch application also led to significant increases in IHH signaling pathway genes, including IHH, SMO, GLI1 and GLI2, while no significant effect was found on GLI3 expression level. In addition, it was found that Cpn significantly reversed the effect of cyclic stretch on the ALP activity, and the expression levels of RUNX2, Osterix, ALP, OCN, GLI1 and GLI2. Collectively, the present results suggested that the IHH signaling pathway may mediate the effect of cyclic stretch on the OLF cells.
Topics: Adult; Aged; Alkaline Phosphatase; Cell Differentiation; Cells, Cultured; Core Binding Factor Alpha 1 Subunit; Female; Hedgehog Proteins; Humans; Ligamentum Flavum; Male; Middle Aged; Nerve Tissue Proteins; Nuclear Proteins; Ossification, Heterotopic; Osteocalcin; Signal Transduction; Smoothened Receptor; Sp7 Transcription Factor; Stress, Mechanical; Veratrum Alkaloids; Zinc Finger Protein GLI1; Zinc Finger Protein Gli2; Zinc Finger Protein Gli3
PubMed: 32626952
DOI: 10.3892/mmr.2020.11200 -
Life Sciences Sep 2020Glioblastoma is an extremely aggressive glioma, resistant to radio and chemotherapy usually performed with temozolomide. One of the main reasons for glioblastoma...
AIM
Glioblastoma is an extremely aggressive glioma, resistant to radio and chemotherapy usually performed with temozolomide. One of the main reasons for glioblastoma resistance to conventional therapies is due to the presence of cancer stem-like cells. These cells could recapitulate some signaling pathways important for embryonic development, such as Sonic hedgehog. Here, we investigated if the inhibitor of the Sonic hedgehog pathway, cyclopamine, could potentiate the temozolomide effect in cancer stem-like cells and glioblastoma cell lines in vitro.
MAIN METHODS
The viability of glioblastoma cells exposed to cyclopamine and temozolomide treatment was evaluated by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay while the induction of apoptosis was assessed by western blot. The stemness properties of glioma cells were verified by clonogenic and differentiation assay and the expression of stem cell markers were measured by fluorescence microscopy and western blot.
KEY FINDINGS
The glioblastoma viability was reduced by cyclopamine treatment. Cyclopamine potentiated temozolomide treatment in glioblastoma cell lines by inducing apoptosis through activation of caspase-3 cleaved. Conversely, the combined treatment of cyclopamine and temozolomide potentiated the stemness properties of glioblastoma cells by inducing the expression of SOX-2 and OCT-4.
SIGNIFICANCE
Cyclopamine plays an effect on glioblastoma cell lines but also sensibilize them to temozolomide treatment. Thus, first-line treatment with Sonic hedgehog inhibitor followed by temozolomide could be used as a new therapeutic strategy for glioblastoma patients.
Topics: Apoptosis; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Survival; Glioblastoma; Hedgehog Proteins; Humans; Neoplastic Stem Cells; Octamer Transcription Factor-3; SOXB1 Transcription Factors; Signal Transduction; Temozolomide; Veratrum Alkaloids
PubMed: 32622951
DOI: 10.1016/j.lfs.2020.118027