scholarly journals 12-Deacetyl-12-epi-Scalaradial, a Scalarane Sesterterpenoid from a Marine Sponge Hippospongia sp., Induces HeLa Cells Apoptosis via MAPK/ERK Pathway and Modulates Nuclear Receptor Nur77

Marine Drugs ◽  
2020 ◽  
Vol 18 (7) ◽  
pp. 375
Author(s):  
Mi Zhou ◽  
Bo-Rong Peng ◽  
Wenjing Tian ◽  
Jui-Hsin Su ◽  
Guanghui Wang ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Western Blotting ◽  
Marine Sponge ◽  
Hela Cells ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Erk Pathway ◽  
Orphan Nuclear Receptors ◽  
Western Blotting Analysis ◽  
Blotting Analysis

12-Deacetyl-12-epi-scalaradial, a scalarane sesterterpenoid from a marine sponge Hippospongia sp, has been reported to possess cytotoxic activity on HepG2, MCF-7, and HCT-116 cells. However, there is no research to indicate that 12-deacetyl-12-epi-scalaradial exhibited anticancer effect on cervical cancer HeLa cells. The aim of this study was to investigate the anticancer activity of 12-deacetyl-12-epi-scalaradial against HeLa cells and to explore the mechanism. The results from a methylthiazolyldiphenyl-tetrazolium (MTT) assay suggested that 12-deacetyl-12-epi-scalaradial suppressed the proliferation of HeLa cells and flow cytometry analysis showed 12-deacetyl-12-epi-scalaradial could induce the apoptosis of HeLa cells in dose- and time-dependent manner. Western blotting analysis demonstrated that 12-deacetyl-12-epi-scalaradial triggered apoptosis via mediating the extrinsic pathway and was found to suppress MAPK/ERK pathway which was associate with cancer cell death. Nur77, a critical number of orphan nuclear receptors, plays diverse roles in tumor development as a transcription factor and has been considered as a promising anticancer drug target. The dual-luciferase reporter assays suggested that 12-deacetyl-12-epi-scalaradial could selectively enhance the trans-activation activity of Nur77. Furthermore, Western blotting analysis and fluorescence quenching showed that 12-deacetyl-12-epi-scalaradial could induce the phosphorylation of Nur77 and interact with the ligand-binding domain (LBD) of Nur77. Our research confirmed 12-deacetyl-12-epi-scalaradial as a potential agent for cervical cancer therapy and provided a view that 12-deacetyl-12-epi-scalaradial may be a modulator of Nur77.

Factor VIII A2 Domain Contains a Binding Site for Factor X

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4226-4226
Author(s):  
Masahiro Takeyama ◽  
Keiji Nogami ◽  
Tomoko Matsumoto ◽  
Midori Shima
Keyword(s):  
Binding Site ◽  
Western Blotting ◽  
Hemophilia A ◽  
Affinity Constant ◽  
Severe Bleeding ◽  
Dependent Manner ◽  
Severe Hemophilia ◽  
Western Blotting Analysis ◽  
Blotting Analysis ◽  
A2 Domain

Abstract Acquired hemophilia A (AHA) is a rare hemorrhagic disease in which autoantibodies against coagulation factor (F) VIII impair the coagulation system. The inhibitors developed in AHA are polyclonal autoantibodies and the majority of FVIII inhibitors bind to the A2, A3, or C2 domains. Depending on the location of the epitope, different mechanisms of action for the anti-FVIII antibodies have been reported. Anti-A3 antibodies neutralize the procoagulant activity of FVIII by preventing its interaction with FIXa. Anti-C2 antibodies inhibit the binding of FVIII to phospholipid membrane and/or von Willebrand factor, whereas A2 and A3 inhibitors block the binding of FVIII to FIXa and FX, respectively, and obstruct the formation of the Xase complex. We have a case of AHA whose inhibitor recognizes only A2 domain and attempted several approaches to determine the mechanism of neutralizing FVIII. Thrombin and plasmin generation assay using patient’s plasma showed that the thrombin and plasmin generation in this AHA patient were decreased compared with that in congenital severe hemophilia A patient. Furthermore, FX generation (Coatest) in this AHA was also decreased compared with that in congenital severe hemophilia A patient (p<0.05). These results indicated that this inhibitor impaired the generation of Xase complex and might cause the severe bleeding disorder in this patient. The IgG subclass of inhibitor in our case was IgG1 and IgG4. Western blotting analysis using FVIIIa revealed that the inhibitor IgG recognized only A2 domain. Furthermore, western blotting analysis using FVIII A2 fragment, digested by activated protein C, showed that the inhibitor IgG bound to FVIII A2N (residue 372-562) fragment. It is known that FVIII A2 domain contains FIXa and thrombin binding sites. Western blotting analysis revealed that the inhibitor IgG inhibited Arg336 cleavage in FVIIIa by FIXa and Arg372 cleavage in FVIII by thrombin. However, the FXa-catalyzed cleavage at Arg372 in FVIII was inhibited by this inhibitor IgG. ELISA-based assay showed that the inhibitor IgG inhibited FX binding to FVIII A2. These results suggest that FX(a) binds to FVIII A2 domain. Therefore, to determine the direct binding of FX and FVIII A2 domain, ELISA-based assay was employed to assess this interaction. ELISA-based assay showed that FVIII A2 fragment bound FX in a dose-dependent manner with moderate affinity (Kd = 338 nM). FX inhibited FVIII A2 fragment binding to immobilized FX up to 70% with an inhibition constant (Ki = 254 nM) similar to the affinity constant. It is known that the residue 484-509 in the A2 domain interacts with FIXa. We hypothesized that FX binding site in the A2 domain might be in the opposite side of FIXa binding site in the A2 domain. According to the 3-D model of FVIII molecule, we prepared synthetic peptides corresponding to FVIII A2 residues 400-409, 409-419, and 420-429. To determine the specificity of these sequences for FX interaction, we examined the effects of these peptides on FVIII A2 binding to FX using ELISA-based assay. The 400-409 peptide inhibited the A2 and FX interaction up to 70%. In contrast, the 410-419 and the 420-429 peptides inhibited the interaction up to 30%. Covalent cross-linking was observed between the 400-409 peptide and FX following reaction with EDC using SDS-PAGE. These results indicate that FVIII A2 domain contains the binding site for FX(a), and the 400-409 region in the FVIII A2 domain contributes to a unique FX(a)-interactive site. Disclosures No relevant conflicts of interest to declare.

scholarly journals Long Non-coding RNA LINC02474 Affects Metastasis and Apoptosis of Colorectal Cancer by Inhibiting the Expression of GZMB

2021 ◽  
Vol 11 ◽  
Author(s):  
Tiantian Du ◽  
Qinglun Gao ◽  
Yinghui Zhao ◽  
Jie Gao ◽  
Juan Li ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Western Blotting ◽  
Noncoding Rna ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Nude Mouse Model ◽  
Western Blotting Analysis ◽  
Blotting Analysis

BackgroundColorectal cancer (CRC) is one of the most frequently diagnosed malignancies. Metastasis is the main event that impedes the therapeutic effect on CRC, and its underlying mechanisms remain largely unclear. LINC02474 is a novel long noncoding RNA (lncRNA) associated with metastasis of CRC, while little is known about how LINC02474 regulates these malignant characteristics.MethodsExpressions of LINC02474 and granzyme B (GZMB) were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) or Western blotting analysis. Cell metastasis was detected by transwell assay and metastatic nude mouse model, and apoptosis was determined by Western blotting analysis and flow cytometry. Besides, the interaction between LINC02474 and GZMB was detected by dual-luciferase reporter assays.ResultsThe expression of LINC02474 was significantly up-regulated in CRC tissues. Moreover, depletion of LINC02474 damaged the metastatic abilities of CRC cells in vivo and in vitro while boosting apoptosis. Besides, up-regulation of LINC02474 could promote migration and invasion, while apoptosis was inhibited in CRC cells. Besides, down-regulation of LINC02474 promoted the expression of GZMB, and interference of GZMB could increase the metastatic abilities of CRC cells while reducing apoptosis. Furthermore, LINC02474 was related to the transcriptional repression of GZMB in CRC cells determined by the dual-luciferase reporter assay.ConclusionsThe findings revealed that a novel lncRNA, LINC02474, as an oncogene, could promote metastasis, but limit apoptosis partly by impeding GZMB expression in CRC. Besides, LINC02474 had the potential to be used as a biomarker in the prognosis of CRC.

scholarly journals miR-18a-3p and Its Target Protein HuR May Regulate Myogenic Differentiation in Immune-Mediated Necrotizing Myopathy

2022 ◽  
Vol 12 ◽  
Author(s):  
Lifang Ye ◽  
Yu Zuo ◽  
Fang Chen ◽  
Qinglin Peng ◽  
Xin Lu ◽  
...  
Keyword(s):  
Western Blotting ◽  
Skeletal Muscles ◽  
Myogenic Differentiation ◽  
Target Protein ◽  
Luciferase Reporter ◽  
Western Blotting Analysis ◽  
Qrt Pcr ◽  
Necrotizing Myopathy ◽  
Blotting Analysis ◽  
Immune Mediated

Immune-mediated necrotizing myopathy (IMNM) is characterized by manifestation of myonecrosis and regeneration of muscle fibers; however, the underlying pathogenesis remains unclear. This study aimed to investigate the role and mechanism of miR-18a-3p and its target RNA-binding protein HuR in IMNM. HuR and miR-18a-3p levels were detected in the skeletal muscles of 18 patients with IMNM using quantitative reverse-transcription real-time polymerase chain reaction (qRT-PCR) and western blotting analysis. Human myoblasts were transfected with small interfering RNA targeting HuR and miR-18a-3p mimic or inhibitor. Myogenic differentiation markers, myogenin and myosin heavy chain, were analyzed by qRT-PCR, western blotting analysis, and immunofluorescence staining. The results showed that miR-18a-3p was upregulated (p=0.0002), whereas HuR was downregulated (p=0.002) in the skeletal muscles of patients with IMNM. The expression of miR-18a-3p in patients with IMNM was negatively correlated with those of HuR (r = -0.512, p = 0.029). We also found that disease activity was positively correlated with HuR expression (r = 0.576, p = 0.012) but muscle activity was negatively correlated with miR-18a-3p expression (r = -0.550, p = 0.017). Besides, bioinformatics analysis and dual-luciferase reporter assays suggested that miR-18a-3p could directly target HuR. Cellular experiments showed that overexpression of miR-18a-3p inhibited myogenic differentiation by targeting HuR, whereas inhibition of miR-18a-3p led to opposite results. Therefore, miR-18a-3p and its target protein HuR may be responsible for modulating the myogenic process in IMNM and can thus be therapeutic targets for the same.

scholarly journals Puerarin Ameliorates Caerulein-Induced Chronic Pancreatitis via Inhibition of MAPK Signaling Pathway

2021 ◽  
Vol 12 ◽  
Author(s):  
Xiang-Peng Zeng ◽  
Jing-Hui Zeng ◽  
Xia Lin ◽  
Yan-Hong Ni ◽  
Chuan-Shen Jiang ◽  
...  
Keyword(s):  
Chronic Pancreatitis ◽  
P38 Mapk ◽  
Western Blotting ◽  
Mapk Signaling ◽  
Pancreatic Fibrosis ◽  
Pancreatic Stellate Cells ◽  
Dependent Manner ◽  
Western Blotting Analysis ◽  
Blotting Analysis ◽  
The Impact

Pancreatic fibrosis is one of the most important pathological features of chronic pancreatitis (CP), and pancreatic stellate cells (PSCs) are considered to be the key cells. Puerarin is the most important flavonoid active component in Chinese herb Radix Puerariae, and it exhibited anti-fibrotic effect in various fibrous diseases recently. However, the impact and molecular mechanism of puerarin on CP and pancreatic fibrosis remain unknown. This study systematically investigated the effect of puerarin on CP and pancreatic fibrosis in vivo and in vitro. H&amp;E staining, Sirius Red staining, qRT-PCR and Western blotting analysis of fibrosis and inflammation related genes of pancreatic tissues showed that puerarin notably ameliorated pancreatic atrophy, inflammation and fibrosis in a model of caerulein-induced murine CP. Western blotting analysis of pancreatic tissues showed the phosphorylation level of MAPK family proteins (JNK1/2, ERK1/2 and p38 MAPK) significantly increased after modeling of cerulein, while puerarin could inhibit their phosphorylation levels to a certain extent. We found that puerarin exerted a marked inhibition on the proliferation, migration and activation of PSCs, determined by CCK-8 assay, transwell migration assay, scratch wound-healing assay and expression levels of α-SMA, Fibronectin, Col1α1 and GFAP. Western blotting result demonstrated that puerarin markedly inhibited the phosphorylation of MAPK family proteins (JNK1/2, ERK1/2 and p38 MAPK) of PSCs in a dose-dependent manner whether or not stimulated by platelet-activating factor. In conclusion, the present study showed that puerarin could be a potential therapeutic candidate in the treatment of CP, and the MAPK pathway might be its important target.

scholarly journals Analysis of the physical activity effects and measurement of pro-inflammatory cytokines in irradiated lungs in rats

2012 ◽  
Vol 27 (3) ◽  
pp. 223-230 ◽  
Author(s):  
Renata Cristiane Gennari Bianchi ◽  
Eduardo Rochete Ropelle ◽  
Carlos Kiyoshi Katashima ◽  
José Barreto Campello Carvalheira ◽  
Luiz Roberto Lopes ◽  
...  
Keyword(s):  
Physical Activity ◽  
Western Blotting ◽  
Inflammatory Cytokines ◽  
Lower Lobe ◽  
Whole Body ◽  
Control Group ◽  
Western Blotting Analysis ◽  
Blotting Analysis ◽  
Tnf Α ◽  
Pro Inflammatory Cytokines

PURPOSE: To study if the pre-radiotherapy physical activity has radio-protective elements, by measuring the radio-induced activation of pro-inflammatory cytokines as interleukin-6 (il-6), transforming growth factor -β (tgf -β), tumor necrosis factor -α (tnf-α) and protein beta kinase β (ikkβ), through western blotting analysis. METHODS: A randomized study with 28 Wistar hannover rats, males, with a mean age of 90 days and weighing about 200 grams. The animals were divided into three groups: (GI, GII and GIII). GIII group were submitted to swimming for eight weeks (zero load, three times a week, about 30 minutes). Then, the groups (except the control group) were submitted to irradiation by cobalt therapy, single dose of 3.5 gray in the whole body. All animals were sacrificed by overdose of pentobarbital, according to the time for analysis of cytokines, and then a fragment of the lower lobe of the right lung went to western blotting analysis. RESULTS: The cytokines IKK β, TNF-α and IL-6 induced by radiation in the lung were lower in the exercised animals. However, exercise did not alter the radiation-induced increase in tgf-β. CONCLUSION: The results show a lower response in relation to inflammatory cytokines in the group that practiced the exercise pre-radiotherapy, showing that exercise can protect tissues from tissue damage due to irradiation.

scholarly journals Baicalein Inhibits the Proliferation of Cervical Cancer Cells Through the GSK3β-Dependent Pathway

2018 ◽  
Vol 26 (4) ◽  
pp. 645-653 ◽  
Author(s):  
Xiaoling Wu ◽  
Zhiqin Yang ◽  
Huimin Dang ◽  
Huixia Peng ◽  
Zhijun Dai
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Cyclin D1 ◽  
Cancer Cells ◽  
Hela Cells ◽  
Glycogen Synthase ◽  
Dependent Manner ◽  
Cervical Cancer Cells ◽  
Dose Dependent ◽  
Dependent Pathway

Baicalein, a flavonoid derived from the root of Scutellaria baicalensis, has been reported to possess multiple pharmacological activities, such as anticancer and anti-inflammatory properties. This study investigated the effect of baicalein in cervical cancer cells. Cell growth curve and MTT assay were performed and revealed that baicalein inhibited the proliferation of SiHa and HeLa cells in a dose-dependent manner. We further found that baicalein arrested the cell cycle of SiHa and HeLa cells at the G0/G1 phase by suppressing the expression of cyclin D1 through the downregulation of phosphorylated protein kinase B (p-AKT) and phosphorylated glycogen synthase kinase 3β (p-GSK3β) according to FACS assays and Western blotting. Moreover, when CHIR-99021, a GSK3β inhibitor, was added to baicalein-treated SiHa cells, the expression of cyclin D1 was recovered, and cell proliferation was promoted. In conclusion, these data indicated that baicalein suspended the cell cycle at the G0/G1 phase via the downregulation of cyclin D1 through the AKT‐GSK3β signaling pathway and further inhibited the proliferation of SiHa and HeLa cervical cancer cells.

scholarly journals Syringin exhibits anticancer effects in HeLa human cervical cancer cells by inducing apoptosis, cell cycle arrest and inhibition of cell migration

2016 ◽  
Vol 11 (4) ◽  
pp. 838 ◽  
Author(s):  
Ning Xia
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Cell Migration ◽  
Cell Cycle Arrest ◽  
Hela Cells ◽  
Dependent Manner ◽  
Cycle Arrest ◽  
Cervical Cancer Cells ◽  
Human Cervical Cancer Cells ◽  
Human Cervical Cancer

<p class="Abstract">The present study was aimed at to demonstrate the antitumor effects of syringin in HeLa human cervical cancer cells. Its effects on apoptosis, cell cycle phase distribution as well as on cell migration were also examined. The effect on cell proliferation was evaluated by MTT assay, while as effects on colony formation were assessed using clonogenic assay. Syringin inhibited cancer cell growth in HeLa cells in a time-dependent as well as in a concentration-dependent manner. Syringin also led to inhibition of colony formation efficacy with complete suppression at 100 µM drug dose. Syringin could induce G2/M cell cycle arrest along with slight sub-G1 cell cycle arrest. HeLa cells began to emit red fluorescence as the dose of syringin increased from 0 µM in vehicle control to 100 µM. Syringin also inhibited cell migration in a dose-dependent manner with 100 µM dose of syringin leading to 100% inhibition of cell migration.</p><p> </p>

scholarly journals Mechanisms Involved in the Anti-Tumor Effects of Toosendanin in Glioma Cells

2021 ◽  
Author(s):  
haiyan huang ◽  
Chaochao Zhang ◽  
Haijun Gao ◽  
Ziqiang Liu ◽  
Jiacheng Lai ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Wound Healing ◽  
Western Blotting ◽  
Colony Formation ◽  
Glioma Cell ◽  
Glioma Cells ◽  
Migration And Invasion ◽  
Antitumor Effects ◽  
Western Blotting Analysis ◽  
Blotting Analysis

Abstract Background: Toosendanin (TSN) is a triterpenoid compound mainly used as an ascaris repellant. Recent studies have shown that it possesses antitumor effects in many types of tumor cells. However, the effects of TSN on glioma cells have rarely been reported. Methods: Different assays were performed to investigate the effects of TSN on the different glioma cell lines including U87MG and LN18. The assays included colony formation, wound healing, and transwell assays. Furthermore, Hoechst 3342 staining, flow cytometry, and western blotting analysis were performed to investigate the apoptotic activities of TSN. Finally, the results were confirmed using a xenograft tumor model that comprised of nude mice. Results: In vitro, the CCK-8 and colony formation assays showed that TSN effectively inhibited glioma cell proliferation. Moreover, the inhibitory effects on glioma cell migration and invasion were demonstrated through the wound healing and transwell assays, respectively. Hoechst 33342 staining, flow cytometry, and western blotting assays demonstrated the significant effect of TSN in the apoptosis induction of glioma cells. Furthermore, the anti-glioma effect of TSN was exerted through the inhibition of the PI3K/Akt/mTOR signaling pathways as demonstrated by western blotting analysis. In addition, the effects of TSN on glioma cell viability, apoptosis, cell cycle arrest, migration, and invasion were reversed by 740Y-P, a PI3K activator. Finally, the mouse xenograft model confirmed the suppressive effect of TSN on tumor growth in vivo. Conclusion: Our results suggest that TSN is a promising chemotherapeutic drug for patients with glioma.

scholarly journals Nucleolin Promotes Cisplatin Resistance in Cervical Cancer by the YB1-MDR1 Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Jing Ke ◽  
Chunming Gu ◽  
Heyan Zhang ◽  
Yang Liu ◽  
Wenhao Zhang ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Drug Resistance ◽  
Hela Cells ◽  
Protein Level ◽  
Molecular Mechanisms ◽  
Cisplatin Resistance ◽  
Mdr1 Gene ◽  
Luciferase Reporter ◽  
Drug Efflux ◽  
Cell Line Hela

Purpose. Cervical cancer is the fourth most common cancer in women worldwide and is the main cause of cancer-related deaths in women. Cisplatin (DDP) is one of the major chemotherapeutic drugs for cervical cancer patients. But, drug resistance limits the effectiveness of cancer therapy. Nucleolin (NCL) is a nucleocytoplasmic multifunctional protein involved in the development of cancer. It has been reported that NCL may be a potential target for modulation of drug resistance. However, the precise molecular mechanisms are poorly understood. Materials and Methods. Human cervical cancer Hela cells and their cisplatin-resistant cell line Hela/DDP were used in this study. The protein level of NCL in cervical cancer cells was measured by western blot analysis. Hela cells and Hela/DDP cells were transfected with NCL overexpression plasmid or NCL siRNA separately. MTT and EdU assay were performed to evaluate the cell viability and sensitivity to cisplatin. The drug efflux function of MDR1 protein was assessed by intracellular rhodamine-123 accumulation assay.The promoter activity of MDR1 was assessed by using a dual-luciferase reporter assay. Results. We found that the protein level of NCL was elevated in Hela/DDP cells. Overexpression of NCL increased cervical cancer cell proliferation and attenuated the sensitivity to cisplatin. Overexpression of NCL increased Multidrug resistance (MDR1) gene expression and drug efflux. Our results demonstrated that NCL was highly related with cisplatin resistance in cervical cancer. NCL played an important role in MDR1 gene transcription through regulation of the transcription factor YB1. Conclusion. Our findings revealed the novel role of NCL in cisplatin-resistant cervical cancer and NCL may be a potential therapeutic target for chemoresistance.

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