A sesquiterpene lactone, thapsigargin, is a phytochemical found in the roots and fruits of Mediterranean plants from Thapsia L. species that have been used for centuries in folk medicine to treat rheumatic pain, lung diseases, and female infertility. More recently thapsigargin was found to be a potent cytotoxin that induces apoptosis by inhibiting the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) pump, which is necessary for cellular viability. This biological activity encouraged studies on the use of thapsigargin as a novel antineoplastic agent, which were, however, hampered due to high toxicity of this compound to normal cells. In this review, we summarized the recent knowledge on the biological activity and molecular mechanisms of thapsigargin action and advances in the synthesis of less-toxic thapsigargin derivatives that are being developed as novel anticancer drugs.

In glioma C6 cells, extracellular ATP generates inositol 1,4,5-trisphosphate (InsP3), indicating the presence of purinergic receptors coupled to phosphoinositide turnover. To identify the effect of ATP (acting via InsP3) and thapsigargin (acting without InsP3 production as a specific inhibitor of the endoplasmic reticulum Ca(2+)-ATPase) on intracellular Ca2+ pools we used video imaging of Fura-2 loaded into single, intact glioma C6 cells. It has been shown that ATP and thapsigargin initiate Ca2+ response consistent with the capacitative model of Ca2+ influx. When the cells were stimulated by increasing concentrations of ATP (1, 10, 50 and 100 microM) the graded, quantal Ca2+ response was observed. In the absence of extracellular Ca2+ thapsigargin and ionomycin-releasable Ca2+ pools are overlapping, demonstrating that Ca2+ stores are located mainly in the endoplasmic reticulum. After maximal Ca2+ mobilization by ATP, thapsigargin causes further increase in cytosolic Ca2+ concentration, whereas emptying of thapsigargin-sensitive intracellular stores prevents any further Ca2+ release by ATP. Thus, the thapsigargin-sensitive intracellular pool of Ca2+ in glioma C6 cells seems to be larger than that sensitive to InsP3. Two hypothesis to explain this result are proposed. One postulates a presence of two different Ca2+ pools, sensitive and insensitive to InsP3 and both discharged by thapsigargin, and the other, the same intracellular pool of Ca2+ completely emptying by thapsigargin and only partially by InsP3. These results may contribute to understanding the mechanism of Ca2+ signalling mediated by ATP, the most potent intracellular Ca2+ mobilizing agonist in all types of glial cells.

Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR) signaling via the accumulation of unfolded and misfolded proteins. ER stress leads to the production of reactive oxygen species (ROS), which are necessary to maintain redox homeostasis in the ER. Although peroxiredoxin 1 (Prx1) is an antioxidant enzyme that regulates intracellular ROS levels, the link between Prx1 and ER stress remains unclear. In this study, we investigated the role of Prx1 in X-box binding protein 1 (XBP-1) activation, the C/EBP homologous protein (CHOP) pathway, and apoptosis in response to ER stress. We observed that Prx1 overexpression inhibited the nuclear localization of XBP-1 and the expression of XBP-1 target genes and CHOP after thapsigargin (Tg) treatment to induce ER stress. In addition, Prx1 inhibited apoptosis and ROS production during ER stress. The ROS scavenger inhibited ER stress-induced apoptosis but did not affect XBP-1 activation and CHOP expression. Therefore, the biological role of Prx1 in ER stress may have important implications for ER stress-related diseases.

Colorectal cancer (CRC) is the third most common tumor in the world; however, the role and mechanism of endoplasmic reticulum (ER) stress in CRC metastasis remains largely unclear. Metastasis‑associated lung adenocarcinoma transcript 1 (MALAT1) is a long non‑coding RNA (lncRNA), which has previously been associated with CRC metastasis. It has been suggested that ER stress pathways regulate lncRNA expression; however, the effect of ER stress on MALAT1 expression in cancer is unknown. The present study aimed to investigate the relationship between ER stress pathways, MALAT1 expression and cell migration in CRC cells. ER stress was induced by thapsigargin (TG); low dose TG induced the migration of HT29 and HCT116 cells, but not SW1116 and SW620 cells. This effect was associated with increased expression levels of MALAT1, as the knockdown of MALAT1 prevented TG‑induced cell migration. TG‑induced MALAT1 expression was associated with inositol‑requiring enzyme 1 (IRE1) expression and activation of the protein kinase R (PKR)‑like ER kinase (PERK) signaling pathway. X‑box‑binding protein 1 (XBP1) and activating transcription factor 4 (ATF4) binding sites were predicted to be located in the MALAT1 gene promoter regions and the expression of MALAT1 was positively associated with XBP1 and ATF4 expression levels in CRC tissue samples. Thus, these findings indicated that ER stress may promote the migration of CRC cells and contribute to the progression of CRC through the activation of the IRE1/XBP1 and PERK/eIF2α/ATF4 signaling pathways. In conclusion, to the best of our knowledge, this study is the first report that lncRNA MALAT1 expression is regulated by the IRE1/XBP1 pathway in CRC.

We previously reported that the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) down-regulates TLR4 signaling and lipopolysaccharide-induced apoptosis of hepatocytes. There have been several reports regarding the association between HCV infection and endoplasmic reticulum (ER) stress. Here, we examined the regulation of HCV NS5A on the apoptosis of hepatocytes induced by thapsigargin, an inducer of ER stress.

This study investigates the effect of astragalus polysaccharides (APS) in protecting against thapsigargin-induced endoplasmic reticulum (ER) stress in HT29 cells by suppressing the PERK-eIF2a signaling pathway.

Thapsigargin (TGN) is a potent and selective inhibitor of sarco-endoplasmic Ca2+-ATPase, leading to rapid elevation of cytoplasmic Ca2+ concentration. Previous reports have shown that TGN increases the production of various cytokines from macrophages and dendritic cells. Here, we examine the effects of TGN on murine T cells. Nanomolar concentrations of TGN are a significant inducer of IL-2 production with full activity at 50 nM. Micromolar concentrations of TGN, however, are inhibitory to IL-2 production and T cell proliferation. The IL-2 production-inducing activity of TGN is much more prominent when T cells are primed with concanavalin A or anti-CD3 mAb, and is due to the increase of cytoplasmic Ca2+ concentration. TGN at 50 nM does not affect interferon-gamma or IL-4 production from T cells. Thus, the present study shows that low nanomolar concentrations of TGN could be useful in potentiating IL-2 production from antigen-primed T cells.

Changes in ionic concentration have a fundamental effect on numerous physiological processes. For example, IP3-gated thapsigargin sensitive intracellular calcium (Ca2+) storage provides a source of the ion for many cellular signaling events. Less is known about the dynamics of other intracellular ions. The present study investigated the intracellular source of zinc (Zn2+) that has been reported to play a role in cell signaling.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent for esophageal squamous cell carcinoma (ESCC). Forced expression of CHOP, one of the key downstream transcription factors during endoplasmic reticulum (ER) stress, upregulates the death receptor 5 (DR5) levels and promotes oxidative stress and cell death. In this study, we show that ER stress mediated by thapsigargin promoted CHOP and DR5 synthesis thus sensitizing TRAIL treatment, which induced ESCC cells apoptosis. These effects were reversed by DR5 siRNA in vitro and CHOP siRNA both in vitro and in vivo. Besides, chemically inhibition of AMPK by Compound C and AMPK siRNA weakened the anti-cancer effect of thapsigargin and TRAIL co-treatment. Therefore, our findings suggest ER stress effectively sensitizes human ESCC to TRAIL-mediated apoptosis via the TRAIL-DR5-AMPK signaling pathway, and that activation of ER stress may be beneficial for improving the efficacy of TRAIL-based anti-cancer therapy.

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 potential to induce cell death. The effects of thapsigargin on the apoptosis in A549 cells were assayed by Hoechst staining. Moreover, the F-actin staining by Rhodamine-phalloidin and RhoA antibody for cytoskeleton organizations were applied to A549 cells. To confirm the impairment of cytoskeletal dynamics treated with thapsigargin, western blots were applied to analyze the protein levels of p-Cofilin-1 (Ser3), Cofilin-1, and pPaxillin (Tyr118), as well as RhoA and pS6 (S240/244). Results suggest that thapsigargin may induce cell death in A549 cells with a time- and dose-dependent manner. The F-actin fibers and RhoA signals are also reduced with a time- and dose-dependent manner by thapsigargin treatment. The phosphorylation forms of Cofilin-1 and paxillin are attenuated by 1 μM thapsigargin treatment for 24 h. These alternations may be caused by the inhibition of of mTORC1 activities (indicated by pS6 (Ser240/244)) and RhoA pathways after thapsigargin treatment. The present findings highlight important roles of calcium entry in cytoskeleton organization and apoptosis in human lung adenocarcinoma cells and will help to set a stage to the clinical treatment of cancer cell metastasis.

Chronic endoplasmic reticulum (ER) stress has deleterious effects on pancreatic β-cell function and survival in type 2 diabetes (T2D). Cyclin-dependent kinase 5 (CDK5) plays a critical role in β-cell failure under diabetic milieu conditions. However, little information is available on CDK5's ability to impair the function of β-cells via a chemical ER stress inducer thapsigargin. Myricetin, a natural flavonoid, has therapeutic potential for the treatment of type 2 diabetes mellitus. Therefore, we examined the effect of CDK5 on thapsigargin-induced β-cell apoptosis, and explored the relationship between myricetin and CDK5. Exposure of beta cells with thapsigargin, induced a Src-mediated redox signaling (VAV2-Rac1-NOX) formation and CDK5 activation. Activated CDK5 induced antiapoptotic protein myeloid cell leukemia sequence 1 (Mcl-1) degradation which was associated with p66Shc serine 36 phosphorylation, causing beta cell apoptosis via mitochondrial dysfunction. Exposure of beta cells to myricetin resulted in an acute inhibition of Src-mediated redox signaling (VAV2-Rac1-NOX) formation and CDK5 activation. Myricetin inhibited CDK5 activation by directly binding to its ATP-binding pocket. Treatment with myricetin also enhanced the stability of Mcl-1 after thapsigargin treatment. Inhibition of CDK5 with myricetin or roscovitine, a CDK5 inhibitor attenuates thapsigargin induced p66Shc serine 36 phosphorylation and also reduced mitochondrial dysfunction by decreasing mitochondrial ROS and caspase-3 activation. In addition, myricetin was observed to enhance PDX-1 and insulin mRNA expression and potentiate glucose stimulated insulin secretion (GSIS). Taken together, these findings indicate that thapsigargin-induced early molecular events lead to CDK5-p66Shc signalosome contributes to thapsigargin-induced pancreatic β-cell dysfunction. Myricetin blocked thapsigargin induced CDK5-p66Shc signalosome formation and prevented pancreatic beta cell dysfunction. In this study, we demonstrated for the first time that thapsigargin initiated CDK5-p66Shc signalosome mediates the pancreatic beta cell dysfunction and myricetin protects the pancreatic beta cells through the inhibition of CDK5-p66Shc signalosome.

Biological effects of electromagnetic fields (EMFs) have previously been identified for cellular proliferation and changes in expression and conduction of diverse types of ion channels. The major effect elicited by EMFs seems to be directed toward Ca2+ homeostasis. This is particularly remarkable since Ca2+ acts as a central modulator in various signaling pathways, including, but not limited to, cell differentiation and survival. Despite this, the mechanisms underlying this modulation have yet to be unraveled. Here, we assessed the effect of EMFs on intracellular [Ca2+ ], by exposing HEK 293 cells to both radio-frequency electromagnetic fields (RF-EMFs) and static magnetic fields (SMFs). We detected a constant and significant increase in [Ca2+ ] subsequent to exposure to both types of fields. Strikingly, the increase was nulled by administration of 10 μM Thapsigargin, a blocker of sarco/endoplasmic reticulum Ca2+ -ATPases (SERCAs), indicating the involvement of the endoplasmic reticulum (ER) in EMF-related modulation of Ca2+ homeostasis.

Previous studies have shown that pretreatment with thapsigargin (TG), a cellular stress inducer, produced potent protective actions against various pathologic injuries. So far there is no information on the effects of TG on the development of bacterial sepsis. Using lipopolysaccharides- and cecal ligation/puncture-induced sepsis models in mice, we demonstrated that preconditioning with a single bolus administration of TG conferred significant improvements in survival. The beneficial effects of TG were not mediated by ER stress induction or changes in Toll-like receptor 4 signaling. In vivo and in cultured macrophages, we identified that TG reduced the protein production of pro-inflammatory cytokines, but exhibited no significant effects on steady state levels of their transcriptions. Direct measurement on the fraction of polysome-bound mRNAs revealed that TG reduced the translational efficiency of pro-inflammatory cytokines in macrophages. Moreover, we provided evidence suggesting that repression of the mTOR (the mammalian target of rapamycin) signaling pathway, but not activation of the PERK (protein kinase R-like endoplasmic reticulum kinase)-eIF2α (eukaryotic initiation factor 2α) pathway, might be involved in mediating the TG effects on cytokine production. In summary, our results support that pharmacological preconditioning with TG may represent a novel strategy to prevent sepsis-induced mortality and organ injuries.

Estivation (aerobic dormancy) is characterized by sustained metabolic rate depression, which is crucial to survival in the face of unfavorable environmental conditions and enables the preservation of endogenous fuel reserves. Ion pumping is one of the most energetically taxing physiological processes in cells, and ion motive ATPases are likely loci to be differentially regulated in models of metabolic arrest. We proposed that the sarcoendoplasmic reticulum (SER) calcium-ATPase (SERCA) would be deactivated in the estivating desert snail Otala lactea, potentially contributing to the overall suppression of metabolism. SERCA kinetic parameters [decreased maximal velocities, increased substrate K (m) values, increased Arrhenius activation energy (E (a))] were indicative of a less active enzyme in the estivated state. Interestingly, the less active SERCA population in dormant snails featured greater kinetic (K (m) Mg.ATP versus temperature) and conformational (resistance to urea denaturation) stability than that in active snails. Western blotting confirmed that SERCA protein content did not change during estivation. In light of this observation, we proposed that estivation-dependent changes in SERCA activity was due to changes in SERCA phosphorylation state. In vitro studies promoting specific kinase or phosphatase action indicated that decreased SERCA activity in estivation was linked with endogenous kinase activity whereas reactivation of SERCA was facilitated by endogenous protein phosphatases (PP).

In neutrophils, intracellular Ca2+ levels are regulated by several transporters and pathways, namely SERCA [sarco(endo)plasmic reticulum Ca2+-ATPase], SOCE (store-operated calcium entry), and ROCE (receptor-operated calcium entry). However, the exact mechanisms involved in the communication among these transporters are still unclear. In the present study, thapsigargin, an irreversible inhibitor of SERCA, and ML-9, a broadly used SOCE inhibitor, were applied in human neutrophils to better understand their effects on Ca2+ pathways in these important cells of the immune system. The thapsigargin and ML-9 effects in the intracellular free Ca2+ flux were evaluated in freshly isolated human neutrophils, using a microplate reader for monitoring fluorimetric kinetic readings. The obtained results corroborate the general thapsigargin-induced intracellular pattern of Ca2+ fluctuation, but it was also observed a much more extended effect in time and a clear sustained increase of Ca2+ levels due to its influx by SOCE. Moreover, it was obvious that ML-9 enhanced the thapsigargin-induced emptying of the internal stores. Indeed, ML-9 does not have this effect by itself, which indicates that, in neutrophils, thapsigargin does not act only on the influx by SOCE, but also by other Ca2+ pathways, that, in the future, should be further explored.

Endoplasmic reticulum (ER) stress-related apoptosis is involved in the pathophysiology of many cardiovascular diseases, and Panax quinquefolium saponin (PQS) is able to inhibit excessive ER stress-related apoptosis of cardiomyocytes following hypoxia/reoxygenation and myocardial infarction. However, the pathway by which PQS inhibits the ER stress-related apoptosis is not well understood. To further investigate the protective effect of PQS against ER stress-related apoptosis, primary cultured cardiomyocytes were stimulated with thapsigargin (TG), which is widely used to model cellular ER stress, and it could induce apoptotic cell death in sufficient concentration.

To investigate the effect of TAP7f, a penicillin derivative previously characterized as a potent antitumor agent that promotes ER stress and apoptosis, in combination with thapsigargin, an ER stress inducer, on melanoma cells.

The struggle to control the COVID-19 pandemic is made challenging by the emergence of virulent SARS-CoV-2 variants. To gain insight into their replication dynamics, emergent Alpha (A), Beta (B) and Delta (D) SARS-CoV-2 variants were assessed for their infection performance in single variant- and co-infections. The effectiveness of thapsigargin (TG), a recently discovered broad-spectrum antiviral, against these variants was also examined. Of the 3 viruses, the D variant exhibited the highest replication rate and was most able to spread to in-contact cells; its replication rate at 24 h post-infection (hpi) based on progeny viral RNA production was over 4 times that of variant A and 9 times more than the B variant. In co-infections, the D variant boosted the replication of its co-infected partners at the expense of its own initial performance. Furthermore, co-infection with AD or AB combination conferred replication synergy where total progeny (RNA) output was greater than the sum of corresponding single-variant infections. All variants were highly sensitive to TG inhibition. A single pre-infection priming dose of TG effectively blocked all single-variant infections and every combination (AB, AD, BD variants) of co-infection at greater than 95% (relative to controls) at 72 hpi. Likewise, TG was effective in inhibiting each variant in active preexisting infection. In conclusion, against the current backdrop of the dominant D variant that could be further complicated by co-infection synergy with new variants, the growing list of viruses susceptible to TG, a promising host-centric antiviral, now includes a spectrum of contemporary SARS-CoV-2 viruses.

Disruption of the endoplasmic reticulum (ER) homeostasis is the cause of ER stress. We performed microRNA (miRNA) analysis (deep sequencing) to search for coping responses (including signaling pathways) induced by disrupted ER Ca(2 +) homeostasis. Our focus was on a specific branch of UPR namely the bi-functional protein kinase/endoribonuclease inositol-requiring element 1α (IRE1α). Activated IRE1α undergoes autophosphorylation and oligomerization, leading to the activation of the endoribonuclease domain and splicing of the mRNA encoding XBP1 specific transcription factor. This processing changes the coding reading frame, producing a potent transcription factor termed XBP1s. We utilized the XBP1 splicing luciferase reporter to screen for modulators of the IRE1α branch of the unfolded protein response (UPR). Here, we describe a detailed experimental design and bioinformatics analysis of ER Ca(2 +) depletion (thapsigargin treated)-induced microRNA (deep sequencing) profile. The data can be access at the Gene Expression Omnibus (GEO), the National Center for Biotechnology Information (NCBI), reference number GSE57138.

1. Thapsigargin (TPG, 3 microM) and cyclopiazonic acid (CPA, 10 microM) slowly increased muscle tone in the guinea-pig isolated tracheal muscle. A large sustained contraction was produced when 2.4 mM Ca2+ was readmitted after 10 min exposure to Ca2+-free solution following 30 min treatment with TPG or CPA. 2. The sustained contraction after Ca2+ readmission was partially inhibited by nifedipine (3 microM) and highly dependent on external Ca2+. The TPG- and CPA-induced sustained contractions were 75% and 67%, respectively, of the sustained contraction produced by carbachol (Cch, 1 microM, EC80) in the presence of nifedipine. 3. The contractions produced by Cch, TPG and CPA were all inhibited by isoprenaline (ISO) and sodium nitroprusside (SNP). In the presence of nifedipine, the IC50 of ISO was 11, 17, and 23 nM and that of SNP was 0.5, 1, 0.8 microM for Cch-, TPG-, and CPA-induced contractions, respectively. The contraction produced by 60 mM K+ was only weakly inhibited by ISO and SNP. As with ISO and SNP, the Cch-, TPG- and CPA-induced contractions were also similarly inhibited by SKF 96365 (100 microM) and cadmium (Cd2+, 100 microM). 4. It was concluded that TPG and CPA increased Ca2+ influx probably via a mechanism activated by Ca2+ depletion of the sarcoplasmic reticulum. The susceptibility of the contraction produced by TPG, CPA and Cch to inhibition by ISO and SNP and also by SKF-96365 and Cd2+ suggests that the contractions use common pathways for increasing intracellular Ca2+, and that the contractions produced by K+ involve a different mechanism.