scholarly journals Tanshinone IIA suppresses the progression of lung adenocarcinoma through regulating CCNA2-CDK2 complex and AURKA/PLK1 pathway

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ziheng Li ◽  
Ying Zhang ◽  
Yuan Zhou ◽  
Fuqian Wang ◽  
Chao Yin ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Lung Adenocarcinoma ◽  
Annexin V ◽  
Tanshinone Iia ◽  
Dependent Manner ◽  
Information Analysis ◽  
Cdk2 Complex ◽  
Dual Staining

AbstractLung adenocarcinoma (LUAD) belongs to a subgroup of non-small cell lung cancer (NSCLC) with an increasing incidence all over the world. Tanshinone IIA (TSA), an active compound of Salvia miltiorrhiza Bunge., has been found to have anti-tumor effects on many tumors, but its anti-LUAD effect and its mechanism have not been reported yet. In this study, bio-information analysis was applied to characterize the potential mechanism of TSA on LUA, biological experiments were used to verify the mechanisms involved. TCGA, Pubchem, SwissTargetPrediction, Venny2.1.0, STRING, DAVID, Cytoscape 3.7.2, Omicshare, GEPIA, RSCBPDB, Chem Draw, AutoDockTools, and PyMOL were utilized for analysis in the bio-information analysis and network pharmacology. Our experiments in vitro focused on the anti-LUAD effects and mechanisms of TSA on LUAD cells (A549 and NCI-H1975 cells) via MTT, plate cloning, Annexin V-FITC and PI dual staining, flow cytometry, and western blot assays. A total of 64 differentially expressed genes (DEGs) of TSA for treatment of LUAD were screened out. Gene ontology and pathway analysis revealed characteristic of the DEGs network. After GEPIA-based DEGs confirmation, 46 genes were considered having significant differences. Further, 10 key DEGs (BTK, HSD11B1, ADAM33, TNNC1, THRA, CCNA2, AURKA, MIF, PLK1, and SORD) were identified as the most likely relevant genes from overall survival analysis. Molecular Docking results showed that CCNA2, CDK2 and PLK1 had the lowest docking energy. MTT and plate cloning assays results showed that TSA inhibited the proliferation of LUAD cells in a concentration-dependent manner. Annexin V-FITC and PI dual staining and flow cytometry assays results told that TSA promoted the apoptosis of the two LUAD cells in different degrees, and induced cycle arrest in the G1/S phase. Western blot results showed that TSA significantly down-regulated the expression of CCNA2, CDK2, AURKA, PLK1, and p-ERK. In summary, TSA could suppress the progression of LUAD by inducing cell apoptosis and arresting cell cycle, and these were done by regulating CCNA2-CDK2 complex and AURKA/PLK1 pathway. These findings are the first to demonstrate the molecular mechanism of TSA in treatment of LUAD combination of network bio-information analysis and biological experiments in vitro.

MDM2 Inhibitor Nutlin-3a Triggers Autophagic Cell Death In Addition to Apoptosis In Leukemia Cell Lines with Wild-Type p53

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3300-3300
Author(s):  
Seshagiri Duvvuri ◽  
Vivian Ruvolo ◽  
Duncan H. Mak ◽  
Kensuke Kojima ◽  
Marina Konopleva ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Western Blot ◽  
Cell Lines ◽  
Research Funding ◽  
Leukemia Cell ◽  
Annexin V ◽  
Dependent Manner ◽  
Autophagic Vacuoles ◽  
Leukemia Cell Lines

Abstract Abstract 3300 Background: Nutlin-3a is a small molecule inhibitor of MDM2 and has been shown to induce apoptosis and cell cycle arrest in various cancer models in a p53 dependent manner. Autophagy is a programmed cell death that can occur concurrently with apoptosis or in its absence. There is significant debate whether autophagy is a protective mechanism or a bona fide mechanism of cell death. While autophagy can function as tumor cell defense mechanism against cellular stress induced death, mutation/loss of alleles of certain genes regulating autophagy have been associated with development of cancer (e.g. Beclin-1 in breast cancer [Nature, 1999, 402: 672–676]). Multiple proteins involved in autophagy are transcriptional targets of p53 but Nutlin-3a has not been evaluated for its role in inducing autophagy. Here we present data suggesting that low dose Nutlin-3a induces autophagy in addition to apoptosis in leukemia cell lines in a p53 dependent manner. Methods and results: OCI-AML-3 cells (p53-WT) treated with Nutlin-3a (2.5 and 5.0μM for 48, 72 and 96 hrs) were stained with mono-dansyl-cadaverine (MDC), a dye that accumulates in acidic autophagic vacuoles. OCI-AML-3 cells showed increasing staining with MDC in a dose and time dependent fashion by both flow cytometry (54%, 57% and 51% MDC positive after treatment with Nutlin-3a 5.0μM for 48, 72 and 96 hrs) and by confocal microscopy. Nutlin-3a treated cells also were positive for Annexin-V (flow cytometry 22%, 26% and 36% at 48, 72 and 96 hrs time points), and some of the cells were double-positive for Annexin-V and MDC (9.2%, 5% and 7% at 48, 72 and 96 hrs) suggesting that both apoptosis and autophagy can occur simultaneously. Autophagy induction was confirmed by Transmission Electron Microscopy (TEM). Large, multiple autophagic vacuoles were observed in OCI-AML-3 cells treated with Nutlin-3a. OCI-AML-3 cells with stable p53 knockdown by shRNA or HL-60 cells (p53-null) did not show increased MDC staining by flow cytometry (both cell lines) or autophagic vacuoles by TEM (HL-60) after similar treatment. Western blot analysis showed increases in LC3-II and in conjugation of Atg5/12, early and late autophagy markers respectively, in OCI-AML-3 cells after treatment with Nutlin-3a. Increased expression of the autophagy markers (LC3-II and Atg 5/12 conjugate) were also seen by Western blot analysis in the ALL cell lines REH and NALM-6 (both p53-WT) after treatment with Nutlin-3a. Western blot and/or RT-PCR analysis showed upregulation of other p53 related proteins involved in autophagy e.g. DRAM, AMPK-β, LKB1, pLKB1 in OCI-AML-3 cells treated with Nutlin-3a. As mTOR/Akt pathway inhibits autophagy, analysis of mTOR targets showed downregulation of the total and phospho-ribosomal-S6-protein levels, whereas there was no change in total or phospho-4-EBP-1 levels. Knockdown of Beclin-1 (ATG6), one of the proteins required for initiation of the formation of autophagic vacuoles, caused reduction in autophagic vacuoles (MDC staining by confocal microscopy) in OCI-AML-3 and REH cells without affecting apoptosis induction (Annexin V by flow cytometry). Pharmacologic inhibition of late autophagy by Bafilomycin (10nM for 2 hours) reduced MDC staining in OCI-AML-3 cells treated with Nutlin-3a for 48 hrs (32% without and 9% with Bafilomycin) while having limited inhibition of apoptosis (Annexin V positive 42% without and 33% with Bafilomycin). Conclusion: Nutlin-3a induces autophagy in leukemia cells by a p53 dependent manner. We also demonstrate that autophagy could go hand-in-hand with apoptosis and in a fraction of cells both processes may occur concomitantly. Inhibition of autophagy does not necessarily enhance apoptosis. Disclosures: Andreeff: Roche: Research Funding. Borthakur:ASCO: Research Funding.

Arsenic Trioxide Induces Platelet Apoptosis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5149-5149
Author(s):  
Yicun Wu ◽  
Jin Dai ◽  
Weilin Zhang ◽  
Rong Yan ◽  
Changgeng Ruan ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Western Blot ◽  
Arsenic Trioxide ◽  
Caspase 3 ◽  
Conflicts Of Interest ◽  
Pulmonary Haemorrhage ◽  
Tumour Cells ◽  
Dependent Manner ◽  
Platelet Apoptosis

Abstract Abstract 5149 Objective: Arsenic trioxide, a component of Traditional Chinese Medicine, is known as an effective anticancer drug especially in the treatment of acute promyelocytic leukaemia (APL). APL has emerged as the most curable subtype of acute myeloid leukaemia since the widely use of arsenic trioxide-based chemotherapy. However, recent researches show that thrombocytopenia occurred in 79% of the relapsed or refractory APL patients treated with arsenic trioxide, and part of the APL patients had to be stopped treatment because of catastrophic bleeding, such as intracranial and pulmonary haemorrhage. Thrombocytopenia also occurred in 43% of the myelodysplastic syndrome patients treated with arsenic trioxide. Recently, arsenic trioxide has been proved to have a pro-apoptotic effect on various kinds of nucleated tumour cells or non-tumour cells. The effect of arsenic trioxide on enucleated platelet, however, still remains unclear. The aim of current study is to investigate whether arsenic trioxide induces platelet apoptosis. Methods: Washed platelets (3 × 108/ml) were incubated with different concentrations of arsenic trioxide or vehicle at 37°C for 4 hours. Then, mitochondrial inner transmembrane potential (ΔΨm) and phosphatidylserine (PS) exposure were tested by flow cytometry. In the mean time, the treated platelets were analyzed by western blot for the expression levels of pro-apoptotic protein (Bax), and anti-apoptotic proteins (Bcl-2 and Bcl-XL). Activation of caspase-3 was also examined by western blot using an anti-caspase-3 antibody. Results: ΔΨm depolarization and PS exposure were dose-dependently induced in platelets incubated with different concentrations (2 uM, 4 uM, 8 uM, 16 uM) of arsenic trioxide as detected by flow cytometry, and the lowest concentration of arsenic trioxide incurring ΔΨm depolarization and PS exposure was 4 umol/L. Simultaneously, arsenic trioxide induced up-regulation of Bax and down-regulation of Bcl-2 and Bcl-XL in a dose-dependent manner. Furthermore, 17 kD cleaved caspase-3 fragments were dose-dependently induced in platelets treated with different concentrations of arsenic trioxide indicating that caspase-3 was activated by arsenic trioxide. Conclusions: Taken together, the data indicate that arsenic trioxide induces platelet apoptosis in vitro, which might suggest a novel pathogenic mechanism of thrombocytopenia in the patients who treated with arsenic trioxide. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mitofusin-2 Triggers Cervical Carcinoma Cell Hela Apoptosis via Mitochondrial Pathway in Mouse Model

2018 ◽  
Vol 46 (1) ◽  
pp. 69-81 ◽  
Author(s):  
Weiqiong Wang ◽  
Xiaowen Liu ◽  
Xiaomei Guo ◽  
Huanhuan Quan
Keyword(s):  
Flow Cytometry ◽  
Mouse Model ◽  
Western Blot ◽  
Cervical Carcinoma ◽  
Hela Cells ◽  
Mitochondrial Pathway ◽  
Dependent Manner ◽  
Caspase 9 ◽  
Mitofusin 2

Background/Aims: Cervical carcinoma continues to be one of the most dangerous cancer types, and more effective therapies are urgently needed for cervical carcinoma treatment. Mitochondria-associated Mitofusin 2 has influence on the progression of many cancers. In the current study, we aimed to focus on the cell apoptotic effects of Mfn2 on cervical carcinoma HeLa cells in vitro and to try to explore its underlying mechanisms. Moreover, we investigated the anticancer potential of Mfn2 in a cervical carcinoma mouse model. Methods: Adenovirus-Mfn2 (Adv-Mfn2) was used to deliver mfn2 into HeLa cells and tumour tissues in a nude mouse model. CCK-8, TUNEL assay, Western blot and immunohistochemical staining were performed to detect the effects of Mfn2. The mRNA level of Mfn2 was determined by quantitative realtime PCR (qRT-PCR) analysis. The effect of Mfn2 on cell apoptosis was investigated by flow cytometry. Flow cytometry was used to assess the change of the mitochondrial membrane potential of the cells treated with JC-1 assay. Mfn2, Bax, Bcl-2, cytochrome c, cleaved caspase-3, and cleaved caspase-9 protein levels were analysed by Western blot. Results: Data from CCK-8 and flow cytometry showed that Mfn2 could inhibit proliferation and induce apoptosis in a dose- and time-dependent manner in HeLa cells. JC-1 test results revealed that the membrane potential of the mitochondrial decreased in a dose-dependent manner in HeLa cells after Adv-Mfn2 treatment. The data from Western blot confirmed that higher cytosolic amounts of cytochrome c with increasing doses of Adv-Mfn2 signified the onset of the intrinsic apoptotic pathway. Levels of cleaved caspase-3 and cleaved caspase-9 increased in HeLa cells with Adv-Mfn2 treatment. We also found significant increases in the Bax level and a decreased Bcl-2 level with Adv-Mfn2 treatment. We further confirmed that Mfn2 could significantly inhibit the growth of the cervical tumour in the xenografted cervical carcinoma mouse model. After a 9-day-treatment, the tumours of the Adv-mfn2 group were inhibited and induced into apoptosis. The results demonstrated that the overexpression of Mfn2 could not only increase the levels of Bax and Bid in cervical tumour cells but also decrease the phosphorylation of Bad and the expression of Bcl-2. Conclusion: These studies suggested that the overexpression of Mfn2 could trigger cervical tumour apoptosis in vitro and in vivo, which was related to the mitochondrial pathway, and may provide a new treatment target for cervical carcinoma.

scholarly journals Involvement of Na+/H+ exchanger in desmopressin-induced platelet procoagulant response.

2004 ◽  
Vol 51 (3) ◽  
pp. 773-788 ◽  
Author(s):  
Maria M Tomasiak ◽  
Halina Stelmach ◽  
Anna Bodzenta-Łukaszyk ◽  
Marian Tomasiak
Keyword(s):  
Flow Cytometry ◽  
Thrombin Generation ◽  
Annexin V ◽  
Underlying Mechanism ◽  
Dependent Manner ◽  
Platelet Volume ◽  
Cell Sizing ◽  
Dose Dependent ◽  
In Vitro Treatment

Desmopressin (DDAVP) action on platelets is associated with the development of procoagulant response but the underlying mechanism of this phenomenon is not known. We investigated whether this effect of DDAVP might be due to activation of plasma membrane Na+/H+ exchanger. The DDAVP-induced platelet procoagulant response, measured as phospholipid-dependent thrombin generation, was dose dependent and significantly weaker than that produced by collagen or monensin (mimics Na+/H+ antiport). Both the DDAVP- and collagen-produced procoagulant responses were less pronounced in the presence of EIPA, an Na+/H+ exchanger inhibitor. Flow cytometry studies revealed that in vitro treatment of platelets with DDAVP or collagen was associated with the appearance of both degranulated (and fragmented) and swollen cells. The DDAVP-evoked rise in size and granularity heterogeneity was similar to that produced by collagen or monensin and was not observed in the presence of EIPA. Using flow cytometry and annexin V-FITC as a probe for phosphatidylserine (PS) we demonstrated increased and uniform binding of this marker to all subsets of DDAVP-treated platelet population. The DDAVP-evoked PS expression was dose dependent, strongly reduced by EIPA and weaker than that caused by monensin or collagen. As judged by optical swelling assay, DDAVP in a dose dependent manner produced a rise in platelet volume. The swelling was inhibited by EIPA and its kinetics was similar to that observed in the presence of monensin. Electronic cell-sizing measurements showed an increase in mean platelet volume and a decrease in platelet count and platelet crit upon treatment with DDAVP. DDAVP elicited a slow (much slower than collagen) alkalinization of platelet cytosol. Altogether the data indicate an involvement of Na+/H+ exchanger in the generation of procoagulant activity in DDAVP-treated platelets.

VDAC1 Mediated Anticancer Activity of Gallic Acid in Human Lung Adenocarcinoma A549 Cells

2018 ◽  
Vol 18 (2) ◽  
pp. 255-262 ◽  
Author(s):  
Aikebaier Maimaiti ◽  
Amier Aili ◽  
Hureshitanmu Kuerban ◽  
Xuejun Li
Keyword(s):  
Western Blot ◽  
Cell Viability ◽  
Gallic Acid ◽  
Molecular Mechanisms ◽  
A549 Cells ◽  
Dependent Manner ◽  
Lung Carcinoma Cells ◽  
Induced Apoptosis ◽  
The Impact

Aims: Gallic acid (GA) is generally distributed in a variety of plants and foods, and possesses cell growth-inhibiting activities in cancer cell lines. In the present study, the impact of GA on cell viability, apoptosis induction and possible molecular mechanisms in cultured A549 lung carcinoma cells was investigated. Methods: In vitro experiments showed that treating A549 cells with various concentrations of GA inhibited cell viability and induced apoptosis in a dose-dependent manner. In order to understand the mechanism by which GA inhibits cell viability, comparative proteomic analysis was applied. The changed proteins were identified by Western blot and siRNA methods. Results: Two-dimensional electrophoresis revealed changes that occurred to the cells when treated with or without GA. Four up-regulated protein spots were clearly identified as malate dehydrogenase (MDH), voltagedependent, anion-selective channel protein 1(VDAC1), calreticulin (CRT) and brain acid soluble protein 1(BASP1). VDAC1 in A549 cells was reconfirmed by western blot. Transfection with VDAC1 siRNA significantly increased cell viability after the treatment of GA. Further investigation showed that GA down regulated PI3K/Akt signaling pathways. These data strongly suggest that up-regulation of VDAC1 by GA may play an important role in GA-induced, inhibitory effects on A549 cell viability.

scholarly journals The Olive Leaves Extract Has Anti-Tumor Effects against Neuroblastoma through Inhibition of Cell Proliferation and Induction of Apoptosis

Nutrients ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 2178
Author(s):  
Fabio Morandi ◽  
Veronica Bensa ◽  
Enzo Calarco ◽  
Fabio Pastorino ◽  
Patrizia Perri ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Cell Viability ◽  
Cell Cycle Progression ◽  
Treatment Strategies ◽  
Annexin V ◽  
Dependent Manner ◽  
Induction Of Apoptosis ◽  
Dose Dependent

Neuroblastoma (NB) is the most common extra-cranial solid tumor of pediatric age. The prognosis for high-risk NB patients remains poor, and new treatment strategies are desirable. The olive leaf extract (OLE) is constituted by phenolic compounds, whose health beneficial effects were reported. Here, the anti-tumor effects of OLE were investigated in vitro on a panel of NB cell lines in terms of (i) reduction of cell viability; (ii) inhibition of cell proliferation through cell cycle arrest; (iii) induction of apoptosis; and (iv) inhibition of cell migration. Furthermore, cytotoxicity experiments, by combining OLE with the chemotherapeutic topotecan, were also performed. OLE reduced the cell viability of NB cells in a time- and dose-dependent manner in 2D and 3D models. NB cells exposed to OLE underwent inhibition of cell proliferation, which was characterized by an arrest of the cell cycle progression in G0/G1 phase and by the accumulation of cells in the sub-G0 phase, which is peculiar of apoptotic death. This was confirmed by a dose-dependent increase of Annexin V+ cells (peculiar of apoptosis) and upregulation of caspases 3 and 7 protein levels. Moreover, OLE inhibited the migration of NB cells. Finally, the anti-tumor efficacy of the chemotherapeutic topotecan, in terms of cell viability reduction, was greatly enhanced by its combination with OLE. In conclusion, OLE has anti-tumor activity against NB by inhibiting cell proliferation and migration and by inducing apoptosis.

scholarly journals SAT0363 DELPHINIDIN DOSE-DEPENDENTLY DIMINISHES PERIPHERAL IL-17 AND IFN-Γ PRODUCING LYMPHOCYTES IN PSORIATIC ARTHRITIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1128.1-1129
Author(s):  
A. Mavropoulos ◽  
S. Tsiogkas ◽  
D. Skyvalidas ◽  
C. Liaskos ◽  
A. Roussaki-Schulze ◽  
...  
Keyword(s):  
Psoriatic Arthritis ◽  
Cell Viability ◽  
Annexin V ◽  
Nkt Cells ◽  
Inhibitory Effect ◽  
Dependent Manner ◽  
Research Support ◽  
Research Activities ◽  
Ifn Γ

Background:Delphinidin, a dietary anthocyanidin and powerful anti-oxidant from pigmented fruits and vegetables, has broad anti-inflammatory properties. In a human skin model of psoriasis, delphinidin reduced expression of proliferative and inflammatory markers (1).Objectives:The rationale of our study was to assess whether delphinidin can in vitro suppress IL-17 and IFN-γ production in peripheral blood mononuclear cell (PBMC) subsets from patients with psoriatic arthritis (PsA).Methods:PBMCs were obtained from 24 patients with PsA attending the outpatient clinic of the Department of Rheumatology/clinical Immunology at the University General Hospital of Larissa, Greece. 16 age- and sex-matched healthy volunteers were also included in the study. Delphinidin was supplemented at a concentration ranging from 1 to 50μg/ml, one hour prior to cell stimulation. Cell viability (Annexin V staining) and innate/adaptive lymphocyte subpopulations were assessed by flow cytometry with a panel of fluorochrome-conjugated antibodies against CD56, CD3, CD4 and CD8. Intracellular expression of IL-17 and IFN-γ was measured following PMA/ionomycin stimulation for 5 hours using standard cell permeabilization protocols and monoclonal antibodies against IL-17 and IFN-γResults:Delphinidin at concentration ≥10 μg/ml sharply diminished IL-17-production by CD4(+) T cells (Th17) and CD56(+)CD3(+) (NKT) cells from patients with psoriatic arthritis and normal controls (p≤0.05). IFN-γ producing T (CD4 and CD8) cells, as well as NK and NKT cells were also dose-dependently suppressed following delphinidin pre-incubation in both patients and healthy controls. Inhibition of IFN-γ(+) cells ranged from 27 to 69% and peaked at delphinidin concentration 20-50μg/ml. The inhibitory effect of delphinidin on IL-17 and IFN-γ producing lymphocytes was not due to compromised cell viability, as assessed by annexin V binding.Conclusion:Delphinidin exerts, in a dose-dependent manner, a profound in vitro inhibitory effect on T cell and NKT cell IL-17 and IFN-γ production in PsA, and therefore, it may be used as a dietary immunosuppressant, complementary to standard treatment.References:[1]Chamcheu JC Skin Pharmacol Physiol. 2015;28(4):177-88. doi: 10.1159/000368445Disclosure of Interests:ATHANASIOS MAVROPOULOS: None declared, Sotirios Tsiogkas: None declared, Dimitrios Skyvalidas: None declared, Christos Liaskos: None declared, Aggeliki Roussaki-Schulze Grant/research support from: Received a grant to support the educational and research activities of the department from Genesis Pharma (2018), Speakers bureau: Received honoraria from Genesis Pharma and Janssen(2017) and from Roche and Pharmaserve Lilly(2018), Efterpi Zafiriou Speakers bureau: Received honoraria from Genesis Pharma, Abbvie, Novartis, Roche, Jansses(2017) and Novartis, Abbvie(2018), Dimitrios Bogdanos: None declared, Lazaros Sakkas Grant/research support from: Received a grant to support the educational and research activities of the department from Bristol-Meyers Squib, Speakers bureau: Received honoraria from Actellion(2018), Janssen(2017), Novartis(2017), Sanofi-Aventis(2018), Abbvie(2017) and Roche(2017)

scholarly journals Geigerin-induced cytotoxicity in a murine myoblast cell line (C2C12)

2017 ◽  
Vol 84 (1) ◽  
Author(s):  
Christo J. Botha ◽  
Sarah J. Clift ◽  
Gezina C.H. Ferreira ◽  
Mxolisi G. Masango
Keyword(s):  
Flow Cytometry ◽  
Cell Line ◽  
Ethical Issues ◽  
Sesquiterpene Lactones ◽  
Annexin V ◽  
Metabolic Activation ◽  
Suitable Model ◽  
Myoblast Cell Line ◽  
Myoblast Cell

Geigeria poisoning in sheep, locally known as ‘vermeersiekte’, is an economically important plant poisoning in southern Africa. The toxic principles contained by the toxic plants are believed to be several sesquiterpene lactones, such as geigerin, vermeeric acid and vermeerin, which cause striated muscle lesions in small stock. Because of ethical issues surrounding the use of live animals in toxicity studies, there is currently a dire need to establish an in vitro model that can be used to replace traditional animal experimentation. The objective of this study was to determine the cytotoxicity of geigerin in a murine myoblast cell line (C2C12) using methyl-thiazol-tetrazolium (MTT) and lactate dehydrogenase (LDH) assays, annexin V and propidium iodide (PI) flow cytometry and transmission electron microscopy (TEM). Mouse myoblasts were exposed to 2.0 mM, 2.5 mM and 5.0 mM geigerin for 24, 48 and 72 h. A concentration-dependent cytotoxic response was observed. Apoptosis was detected by means of annexin V flow cytometry during the first 24 h and apoptotic bodies were also visible on TEM. According to the LDH and PI flow cytometry results, myoblast cell membranes were not injured. We concluded that the murine myoblast cell line (C2C12) is a suitable model for future studies planned to evaluate the cytotoxicity of other and combinations of sesquiterpene lactones, with and without metabolic activation, implicated in ‘vermeersiekte’ and to elucidate the subcellular effects of these myotoxins on cultured myoblasts.

scholarly journals Tanshinone IIA Attenuates Chronic PancreatitisInduced Pain in Rats via Downregulation of HMGB1 and TRL4 Expression in the Spinal Cord

2015 ◽  
Vol 18;4 (4;18) ◽  
pp. E615-E628
Author(s):  
Lei Chen
Keyword(s):  
Chronic Pancreatitis ◽  
Western Blot ◽  
Proinflammatory Cytokine ◽  
Mechanical Allodynia ◽  
Tanshinone Iia ◽  
Trinitrobenzene Sulfonic Acid ◽  
Dependent Manner ◽  
Toll Like Receptor ◽  
Tnf Α ◽  
Dose Dependent

Background: Chronic pancreatitis (CP) is a long-standing inflammation of the exocrine pancreas, which typically results in severe and constant abdominal pain. Previous studies on the mechanisms underlying CP-induced pain have primarily focused on the peripheral nociceptive system. A role for a central mechanism in the mediation or modulation of abdominal pain is largely unknown. Tanshinone IIA (TSN IIA), an active component of the traditional Chinese medicine Danshen, exhibits anti-inflammatory properties via downregulation of the expression of high-mobility group protein B1 (HMGB1), a late proinflammatory cytokine. HMGB1 binds and activates toll-like receptor 4 (TLR4) to induce spinal astrocyte activation and proinflammatory cytokine release in neuropathic pain. Objective: In this study, we investigated the effect of TSN IIA on pain responses in rats with trinitrobenzene sulfonic acid (TNBS)-induced CP. The roles of central mechanisms in the mediation or modulation of CP were also investigated. Study Design: A randomized, double-blind, placebo-controlled animal trial. Methods: CP was induced in rats by intrapancreatic infusion of trinitrobenzene sulfonic acid (TNBS). Pancreatic histopathological changes were characterized with semi-quantitative scores. The abdomen nociceptive behaviors were assessed with von Frey filaments. The effects of intraperitoneally administered TSN IIA on CP-induced mechanical allodynia were tested. The spinal protein expression of HMGB1 was determined by western blot. The spinal mRNA and protein expression of proinflammatory cytokines IL-1β, TNF-α, and IL-6 were determined by RT-PCR and western blot, respectively. The spinal expression of the HMGB1 receptor TRL4 and the astrocyte activation marker glial fibrillary acidic protein (GFAP) were determined by western blot or immunohistological staining after intraperitoneal injection of TSN IIA or intrathecal administration of a neutralizing anti-HMGB1 antibody. Results: TNBS infusion resulted in pancreatic histopathological changes of chronic pancreatitis and mechanical allodynia in rats. TSN IIA significantly attenuated TNBS-induced mechanical allodynia in a dose-dependent manner. TNBS significantly increased the spinal expression of HMGB1 and proinflammatory cytokines IL-1β, TNF-α, and IL-6. These TNBS-induced changes were significantly inhibited by TSN IIA in a dose-dependent manner. Furthermore, TSN IIA, but not the neutralizing anti-HMGB1 antibody, significantly inhibited TNBS-induced spinal TLR4 and GFAP expression. Limitations: In addition to TLR4, HMGB1 can also bind to toll-like receptor-2 (TLR2) and the receptor for advanced glycation end products (RAGE). Additional studies are warranted to ascertain whether HMGB1 contributes to CP-induced pain through activation of these receptors. Conclusions: Our results suggest that spinal HMGB1 contributes to the development of CPinduced pain and can potentially be a therapeutic target. TSN IIA attenuates CP-induced pain via downregulation of spinal HMGB1 and TRL4 expression. Therefore, TSN IIA may be a potential anti-nociceptive drug for the treatment of CP-induced pain. Key words: Chronic pancreatitis, HMGB1, proinflammatory cytokine, Tanshinone IIA, spinal cord, astrocyte, TLR4

scholarly journals Ascorbic acid attenuates activation and cytokine production in sepsis-like monocytes

2021 ◽  
Author(s):  
Tobias Schmidt ◽  
Robin Kahn ◽  
Fredrik Kahn
Keyword(s):  
Flow Cytometry ◽  
Ascorbic Acid ◽  
Cytokine Production ◽  
In Vitro Study ◽  
Surface Expression ◽  
High Dose ◽  
Functional Assay ◽  
Dependent Manner ◽  
Dose Dependent

Objective To investigate the effects of high dose ascorbic acid (AA) on monocyte polarization and cytokine production in vitro Design Experimental in vitro study of cells from healthy subjects and patients with sepsis Setting University research laboratory and academic hospital Subjects Six healthy controls and three patients with sepsis Interventions Monocytes were isolated from whole blood of healthy donors (n=6) and polarized in vitro for 48hrs using LPS or LTA. Polarization was confirmed by surface marker expression using flow cytometry. As a comparison, monocytes were also isolated from septic patients (n=3) and analyzed for polarization markers. The effect of AA on monocyte polarization was evaluated. As a functional assay, AA-treated monocytes were analyzed for cytokine production of TNF and IL-8 by intracellular staining and flow cytometry following activation with LPS or LTA. Measurements and Main Results Both LPS and LTA induced polarization in healthy monocytes in vitro, with increased expression of both pro- (CD40 and PDL1, p<0.05) and anti-inflammatory (CD16 and CD163, p<0.05) polarization markers, with non-significant effects on CD86 and CD206. This pattern resembled, at least partly, that of monocytes from septic patients. Treatment with AA significantly inhibited the upregulation of surface expression of CD16 and CD163 (p<0.05) in a dose dependent manner, but not CD40 or PDL-1. Finally, AA attenuated LPS or LTA-induced cytokine production of IL-8 and TNF in a dose-dependent manner (both p<0.05). Conclusions AA inhibits upregulation of anti-, but not pro-inflammatory related markers in LPS or LTA polarized monocytes. Additionally, AA attenuates cytokine production from in vitro polarized monocytes, displaying functional involvement. This study provides important insight into the immunological effects of high dose AA on monocytes, and potential implications in sepsis.

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