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Thapsigargin disrupts actin cytoskeletal proteins in A549 cells. The reductions of protein levels of p-Cofilin-1 (Ser3) and p-Paxillin (Tyr118) by thapsigargin treatment (1 μM for 6 and 24 h) in A549 cells were shown by Western blots (a) and histograms (b). Notes showed that the protein level of total Cofilin-1 is not affected by thapsigargin treatment. Results are averages of four independent experiments. Data represent mean ± SEM. **P<0.01, ***P<0.001, N.S, and no statistical difference.
Source publication
The objective of this study was performed to investigate the effects of thapsigargin on apoptosis, actin cytoskeletal dynamics, and actin cytoskeletal proteins in human lung adenocarcinoma cell. Thapsigargin is a specific irreversible inhibitor of ER calcium-ATPase, which may promote ER stress by depletion of lumenal calcium stores and show potenti...
Citations
... It has been widely used to induce ER stress and Ca 2+ homeostasis disturbances in a variety of cells. Previous reports have demonstrated that TG induced apoptosis through activating ER stress and impairing mitochondrial pathway [13,14]. Therefore, this study focused on exploring the molecules that promote cell apoptosis by regulating the ER stress. ...
Background
The incidence of colorectal cancer (CRC) has been increasing in recent years. Thus, the discovery of factors that can assist in alleviating CRC is urgently warranted.
Methods
To identify a potential factor involved in the development of CRC, we screened the upregulated genes in tumor tissues through four datasets from an online database. The expression of reticulocalbin 1 (RCN1), a Ca²⁺-binding protein, was upregulated in the four datasets. Based on loss-of-function experiments, the effect of RCN1 on cell viability was assessed by Cell Counting Kit-8 (CCK-8) assay. The regulatory effect of RCN1 on apoptosis was evaluated through Annexin V-fluorescein 5-isothiocyanate (FITC)/propidium iodide (PI) staining assay and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay in RKO and SW480 cells. Activation of endoplasmic reticulum (ER) stress signaling pathways was confirmed by estimating the phosphorylation and expression of PRKR-like ER kinase (PERK), inositol-requiring kinase-1 (IRE1), transcription factor 6 (ACT6), and CCAAT/enhancer-binding protein-homologous protein (CHOP). The intracellular Ca²⁺ homeostasis regulated by RCN1 was determined through the detection of Ca²⁺ concentration and mitochondrial membrane potential (MMP) measurement. Moreover, whether inositol 1,4,5-trisphosphate receptor type 1 (IP3R1) was involved in the regulation of RCN1 in CRC was verified through the depletion of IP3R1 in RKO cells.
Results
Knockdown of RCN1 reduced cell viability and facilitated apoptosis in RKO and SW480 cells. Phosphorylation of PERK and IRE1, activation of ATF6, and upregulation of CHOP were induced by the absence of RCN1, suggesting that the unfolded protein response (UPR) was activated in CRC cells. The concentration of Ca²⁺ in mitochondria was increased after RCN1 depletion, followed by reduction in the MMP and release of cytochrome c from mitochondria to the cytoplasm in RKO and SW480 cells. Moreover, it was demonstrated that IP3R1 mediates the effect of RCN1 on apoptosis induced by ER stress in CRC cells. The downregulation of IP3R1 restored the RCN1 loss-induced apoptosis and the increased Ca²⁺ concentration.
Conclusion
Taken together, our results confirmed that silencing of RCN1 disrupted intracellular Ca²⁺ homeostasis and promoted cell apoptosis caused by TG-induced ER stress by regulating IP3R1 and activating the UPR signaling pathways.
... TG is a specific inhibitor of the sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) [26]. Additionally, it triggers ectopic calcium influx that has been described to lead to actin depolymerization via cofilin-1 and mTOR-RhoA pathways [27]. BFA on the other hand, is a widely used inhibitor of ER-Golgi transport that blocks the activity of guanine-nucleotide exchange factors (GEF) to activate ADP-ribosylation factors (ARF) [28] including ARF6. ...
... Pertaining actin evaluation, it is worth mentioning that this returned a decrease in the signal upon incubation with TG and BFA in comparison to controls (Fig. 3). Indeed, both compounds can alter the actin cytoskeleton [27,29]. Additionally, this is also compatible with an enlargement of the ER and consequent re-localization of the surrounding cytoskeleton. ...
Bladder cells face a challenging biophysical environment: mechanical cues originating from urine flow and regular contraction to enable the filling voiding of the organ. To ensure functional adaption, bladder cells rely on high biomechanical compliance, nevertheless aging or chronic pathological conditions can modify this plasticity. Obviously the cytoskeletal network plays an essential role, however the contribution of other, closely entangled, intracellular organelles is currently underappreciated. The endoplasmic reticulum (ER) lies at a crucial crossroads, connected to both nucleus and cytoskeleton. Yet, its role in the maintenance of cell mechanical stability is less investigated. To start exploring these aspects, T24 bladder cancer cells were treated with the ER stress inducers brefeldin A (10-40nM BFA, 24 h) and thapsigargin (0.1-100nM TG, 24 h). Without impairment of cell motility and viability, BFA and TG triggered a significant subcellular redistribution of the ER; this was associated with a rearrangement of actin cytoskeleton. Additional inhibition of actin polymerization with cytochalasin D (100nM CytD) contributed to the spread of the ER toward cell periphery, and was accompanied by an increase of cellular stiffness (Young´s modulus) in the cytoplasmic compartment. Shrinking of the ER toward the nucleus (100nM TG, 2 h) was related to an increased stiffness in the nuclear and perinuclear areas. A similar short-term response profile was observed also in normal human primary bladder fibroblasts. In sum, the ER and its subcellular rearrangement seem to contribute to the mechanical properties of bladder cells opening new perspectives in the study of the related stress signaling cascades.
... Another line of evidence linking COFILIN to apoptosis comes from the investigations performed in cell context. Treating A549 cells with thapsigargin, an inhibitor of endoplasmic reticulum calcium ATPase, also induces apoptosis through the inhibition of COFILIN-mediated F-actin reorganization (Wang et al., 2014a). Elevated COFILIN phosphorylation after SSH2 knockdown was previously demonstrated to induce the activation of Caspase-3/7 in a human renal cell carcinoma cell line (Lu et al., 2014), and activation of Caspase-3/7 is known to trigger apoptotic cell death (Saller et al., 2010). ...
The acrosome is a membranous organelle positioned in the anterior portion of the sperm head and is essential for male fertility. Acrosome biogenesis requires the dynamic cytoskeletal shuttling of vesicles towards nascent acrosome which is regulated by a series of accessory proteins. However, much remains unknown about the molecular basis underlying this process. Here, we generated Ssh2 knock-out (KO) mice and HA-tagged Ssh2 knock-in (KI) mice to define the functions of Slingshot phosphatase 2 (SSH2) in spermatogenesis and demonstrated that as a regulator of actin remodeling, SSH2 is essential for acrosome biogenesis and male fertility. In Ssh2 KO males, spermatogenesis was arrested at the early spermatid stage with increased apoptotic index and the impaired acrosome biogenesis was characterized by defective transport/fusion of proacrosomal vesicles. Moreover, disorganized F-actin structures accompanied by excessive phosphorylation of COFILIN were observed in the testes of Ssh2 KO mice. Collectively, our data reveal a modulatory role for SSH2 in acrosome biogenesis through COFILIN-mediated actin remodeling and the indispensability of this phosphatase in male fertility in mice.
... We demonstrated that Vx-809 not only restores the misfolding responsible for cystic fibrosis, but actively participates in the correction of stress-induced misfolding of the ER. ER stress was induced by Thapsigargin, a well-known stress inducer [39], that causes a depletion of calcium from the ER lumen followed by accumulation of unfolded or misfolded proteins in the ER. In our experimental model, Vx-809 counteracts the activation of the UPR mechanism, most likely because it restores protein folding. ...
Correct protein folding is the basis of cellular well-being; thus, accumulation of misfolded proteins within the endoplasmic reticulum (ER) leads to an imbalance of homeostasis that causes stress to the ER. Various studies have shown that protein misfolding is a significant factor in the etiology of many human diseases, including cancer, diabetes, and cystic fibrosis. Misfolded protein accumulation in the ER triggers a sophisticated signal transduction pathway, the unfolded protein response (UPR), which is controlled by three proteins, resident in ER: IRE1α, PERK, and ATF6. Briefly, when ER stress is irreversible, IRE1α induces the activation of pro-inflammatory proteins; PERK phosphorylates eIF2α which induces ATF4 transcription, while ATF6 activates genes encoding ER chaperones. Reticular stress causes an alteration of the calcium homeostasis, which is released from the ER and taken up by the mitochondria, leading to an increase in the oxygen radical species production, and consequently, to oxidative stress. Accumulation of intracellular calcium, in combination with lethal ROS levels, has been associated with an increase of pro-inflammatory protein expression and the initiation of the inflammatory process. Lumacaftor (Vx-809) is a common corrector used in cystic fibrosis treatment which enhances the folding of mutated F508del-CFTR, one of the most prevalent impaired proteins underlying the disease, promoting a higher localization of the mutant protein on the cell membrane. Here, we demonstrate that this drug reduces the ER stress and, consequently, the inflammation that is caused by such events. Thus, this molecule is a promising drug to treat several pathologies that present an etiopathogenesis due to the accumulation of protein aggregates that lead to chronic reticular stress.
... Because of the role of ALMS1 in the centrosome and its relation with endocytic trafficking (Collin et al., 2012), its depletion could probably alter the structure of the membrane, inhibiting the signalling transduction of ligands like THAP. THAP also induces apoptosis impairing cytoskeleton dynamics and disorganization of F-actin via mTORC1 inhibition in the mTORC1-RhoA-Limk-Cofilin-1 axis in A549 cells (Wang et al., 2014). ALMS1 depletion FIGURE 6 (Continued) western-blot of p-SMAD3, SMAD3, p-SMAD2 and SMAD2 levels after 0 and 30 min of stimulation with TGF-β1 (2 ng/μL) ligand and their corresponding loading control (coomassie) in HeLa cells (n = 2). ...
Background: ALMS1 is a ubiquitous gene associated with Alström syndrome (ALMS). The main symptoms of ALMS affect multiple organs and tissues, generating at last, multi-organic fibrosis in the lungs, kidneys and liver. TGF-β is one of the main pathways implicated in fibrosis, controlling the cell cycle, apoptosis, cell migration, cell adhesion and epithelial-mesenchymal transition (EMT). Nevertheless, the role of ALMS1 gene in fibrosis generation and other implicated processes such as cell migration or cell adhesion via the TGF- β pathway has not been elucidated yet.
Methods: Initially, we evaluated how depletion of ALMS1 affects different processes like apoptosis, cell cycle and mitochondrial activity in HeLa cells. Then, we performed proteomic profiling with TGF-β stimuli in HeLa ALMS1 −/− cells and validated the results by examining different EMT biomarkers using qPCR. The expression of these EMT biomarkers were also studied in hTERT-BJ-5ta ALMS1 −/−. Finally, we evaluated the SMAD3 and SMAD2 phosphorylation and cell migration capacity in both models.
Results: Depletion of ALMS1 generated apoptosis resistance to thapsigargin (THAP) and C2-Ceramide (C2-C), and G2/M cell cycle arrest in HeLa cells. For mitochondrial activity, results did not show significant differences between ALMS1 +/+ and ALMS1 −/− . Proteomic results showed inhibition of downstream pathways regulated by TGF-β. The protein-coding genes (PCG) were associated with processes like focal adhesion or cell-substrate adherens junction in HeLa. SNAI1 showed an opposite pattern to what would be expected when activating the EMT in HeLa and BJ-5ta. Finally, in BJ-5ta model a reduced activation of SMAD3 but not SMAD2 were also observed. In HeLa model no alterations in the canonical TGF-β pathway were observed but both cell lines showed a reduction in migration capacity.
Conclusion: ALMS1 has a role in controlling the cell cycle and the apoptosis processes. Moreover, the depletion of ALMS1 affects the signal transduction through the TGF-β and other processes like the cell migration and adhesion capacity.
... Ca 2+ also assists the actin dynamics in brain endothelial cells, by modulating the activity of several related regulators (Mostafavi et al., 2014;Tsai et al., 2015;Wang et al., 2014a). PKC and calmodulin dependent kinases are activated by Ca 2+ . ...
Blood brain barrier (BBB) is formed by the brain microvascular endothelial cells (BMVECs) lining the wall of brain capillaries. Its integrity is regulated by multiple mechanisms, including up/downregulation of tight junction proteins or adhesion molecules, altered Ca2 + homeostasis, remodeling of cytoskeleton, that are confined at the level of BMVECs. Beside the contribution of BMVECs to BBB permeability changes, other cells, such as pericytes, astrocytes, microglia, leukocytes or neurons, etc. are also exerting direct or indirect modulatory effects on BBB. Alterations in BBB integrity play a key role in multiple brain pathologies, including neurological (e.g. epilepsy) and neurodegenerative disorders (e.g. Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis etc.). In this review, the principal Ca2 + signaling pathways in brain microvascular endothelial cells are discussed and their contribution to BBB integrity is emphasized. Improving the knowledge of Ca2 + homeostasis alterations in BMVECa is fundamental to identify new possible drug targets that diminish/prevent BBB permeabilization in neurological and neurodegenerative disorders.
... The robust results of the experimental data analyses demonstrated reduced microglial cell motility through changes in the expression and structure of actin filaments of the cytoskeleton. Ca 2+ also affected the remodeling of actin, which is a crucial element of the cytoskeleton and cellular motility [54,55] that was further confirmed by our ECIS evaluation of BV-2 culture resistance. Although Ca 2+ does not directly bind to actin, it has been shown to affect the activities of multiple actin regulators, including protein kinase C and calmodulin-dependent kinases [56,57]. ...
Background: The ways in which microglia activate and promote neovascularization (NV) are not fully understood.
Recent in vivo evidence supports the theory that calcium is required for the transition of microglia from a
surveillance state to an active one. The objectives of this study were to discover novel L-type voltage-gated channel
(L-VGCC) blockers and investigate their application for the prevention of inflammation and angiogenesis.
Methods: Pharmacophore-based computational modeling methods were used to screen for novel calcium channel
blockers (CCBs) from the ZINC compound library. The effects of CCBs on calcium blockade, microglial proinflammatory activation, and cell toxicity were validated in BV-2 microglial cell and freshly isolated smooth muscle
cell (SMC) cultures. Laser-induced choroidal neovascularization (NV) and the suture-induced inflammatory corneal
NV models of angiogenesis were used for in vivo validation of the novel CCBs. CX3CR1gfp/+ mice were used to
examine the infiltration of GFP-labeled microglial cells.
Results: We identified three compounds from the ZINC database (Zinc20267861, Zinc18204217, and Zinc33254827)
as new blockers of L-type voltage-gated calcium channels (L-VGCC) using a structure-based pharmacophore
approach. The effects of the three CCBs on Ca2+ influx into cells were verified in BV-2 microglial cells using Fura-2
fluorescent dye and in freshly isolated SMCs using the voltage-patch clamp. All three CCBs reduced microglial cell
migration, activation stimulated by lipopolysaccharide (LPS), and reduced the expression of the inflammatory
markers NF-κB (phospho-IκBα) and cyclooxygenase-2 (COX-2) as well as reactive oxygen species. Of the three
compounds, we further examined the in vivo activity of Zinc20267861. Topical treatment with Zinc20267861 in a
rat model of suture-induced inflammatory cornea neovascularization demonstrated efficacy of the compound in
reducing monocyte infiltration and overall corneal NV response. Subconjunctival administration of the compound in
the choroidal NV mouse model effectively prevented CNV and microglial infiltration.
Conclusions: Our findings suggest that the novel CCBs identified here are effective anti-inflammatory agents that
can be further evaluated for treating NV disorders and can be potentially applied in the treatment of ocular
inflammatory and pathological angiogenetic disorders.
... In addition, TG promoted apoptosis in ACC cells (Figures 3, 4A and B). Wang et al 33 revealed that TG can induce apoptosis in human lung adenocarcinoma cells through cofilin-1 and paxillin, which is consistent with our present study. A and B). ...
Objective
Thapsigargin (TG) is a natural product that exists in most parts of the plant Thapsia garganica L. and possesses potential anticancer activities against variety tumor cell lines. TG induces endoplasmic reticulum (ER) stress and apoptosis by inhibiting cancer growth. However, the antineoplastic effect of TG in human adrenocortical carcinoma (ACC) cells is still unknown.
Methods
In this study, two human ACC cell lines including SW-13 and NCI-H295R were employed to explore the potential role of TG in ACC. A mouse xenograft model of SW-13 cells was established to verify the role of TG in vivo. The cell viability was tested using Cell Counting Kit-8 and Transwell assays. Flow cytometry and Hoechst 33,258 staining were employed to analyze cell apoptosis. RT-qPCR and Western blot (WB) were performed to explore the underlying mechanism of TG-induced apoptosis in ACC cells.
Results
The results indicated that TG dose-dependently inhibited proliferation, migration and invasion in human ACC cells. TG significantly increased the mitochondrial rate of apoptosis and ER stress activity in ACC cells and suppressed ACC xenograft growth in vivo. In addition, the expression of Jun N-terminal kinase (JNK) signaling-related genes and proteins was upregulated by the treatment with TG.
Conclusion
Our findings suggest that TG inhibits the viability of ACC cells by inducing apoptosis through the activation of JNK signaling. Thus, TG is expected to be a potential candidate for the treatment of ACC.
... It has been suggested that intervention in cellular skeleton microfilament actin reconstruction may be a functional target of anticancer drugs, and may be regarded as a basis for developing novel antineoplastic drugs (25). Yip et al (26) suggested that thapsigargin modulates osteoclastogenesis through the regulation of F-actin and reactive oxygen species production (27). The present study indicated that thapsigargin inhibits F-actin expression in prostate cancer cells. ...
... The present study suggested that thapsigargin induces cofilin-1 expression in prostate cancer cells. Wang et al (27) demonstrated that thapsigargin induces apoptosis of human lung adenocarcinoma cells through cofilin-1 and paxillin. ...
... The present study identified that thapsigargin significantly inhibited paxillin protein expression in prostate cancer PC3 cells. Wang et al (27) revealed that thapsigargin induces apoptosis in human lung adenocarcinoma cells through cofilin-1 and paxillin. These results of the present study suggest that curcumin inhibits the tumor growth of prostate cancer cells by modulating the F-actin/cofilin-1/paxillin pathway. ...
It is widely considered that endoplasmic reticulum stress may rapidly induce apoptosis. The aim of the present study was to investigate the effect of thapsigargin on the induction of apoptosis in prostate cancer cells, and to explore its possible mechanism. A Cell Counting Kit-8 was selected to determine the effect of thapsigargin (0, 1, 10 and 100 nM) on the proliferation of PC3 cells. Cell proliferation of the prostate cancer cells was effectively inhibited by treatment with thapsigargin, and thapsigargin significantly increased the rate of apoptosis and caspase-3/9 activities in prostate cancer cells. The protein expression of phosphorylated (p)-RAC-α serine threonine-protein kinase, p-mechanistic target of rapamycin, F-actin and paxillin were significantly decreased, and cofilin-1 protein expression was significantly increased by treatment with thapsigargin in prostate cancer cells. Overall, the data of the present study revealed that thapsigargin induced apoptosis in prostate cancer cells through cofilin-1 and paxillin.
... Also, significantly less growth inhibition was observed in Sf9 cells (~20%) as compared to HEK cells (~80%) at 72 h after thapsigargin treatment (Fig. 1c). Since thapsigargin-induced cell death is known to be associated with impaired cytoskeleton dynamics [25], cytoskeletal alterations were observed using phalloidin-FITC, an F-actin probe. Immunofluorescence staining of actin filaments suggests that Sf9 cells do not undergo any noticeable disturbance in their actin network in contrast to the HEK cells which suffered significant filament shortening/contraction (Fig. 1d). ...
