Prenatal inflammation exposure accelerates lung cancer tumorigenesis in offspring mouse: possible links to IRE1α/XBP1-mediated M2-like polarization of TAMs and PD-L1 up-expression.

BACKGROUND: Prenatal inflammation exposure (PIE) can increase the disease susceptibility in offspring such as lung cancer. Our purpose was to investigate the mechanisms of PIE on lung cancer. METHODS: Prenatal BALB/c mice were exposed to lipopolysaccharide (LPS), and then, their offspring were intraperitoneally instilled with urethane to establish the two-stage lung cancer carcinogenesis model. At the 48 weeks of age, the offspring mice were killed and lung tissues were collected for HE, immunohistochemistry, immunofluorescence, and Luminex MAGPIX®-based assays. CD11b + F4/80 + tumor-associated macrophages (TAMs) were sorted out from lung tumor tissues by cell sorting technique. Flow cytometry was employed to evaluate the extent of M2-like polarization of TAMs and PD-L1 expression. RESULTS: The offspring of PIE mice revealed more lung lesion changes, including atypical hyperplasia and intrapulmonary metastases. The number of lung nodules, lung organ index, and PCNA, MMP-9 and Vimentin positive cells in lung tissue of PIE group were higher than those of Control group. The increases of mRNA encoding M2 macrophage markers and cytokines in offspring of prenatal LPS-treated mice confirmed the induced effect of PIE on macrophage polarization. Additionally, PIE treatment increased the percentage of CD163 + CD206 + cells in the sorted TAMs. Importantly, endoplasmic reticulum (ER) stress-markers like GRP78/BIP and CHOP, p-IRE1α and XBP1s, and PD-L1 were up-regulated in TAMs from PIE group. Besides, we also observed that IRE1α inhibitor (KIRA6) reversed the M2-like TAMs polarization and metastasis induced by PIE. CONCLUSIONS: IRE1α/XBP1-mediated M2-like TAMs polarization releases the pro-tumorigenic cytokines and PD-L1 expression, which may be the regulatory mechanism of accelerating lung cancer in offspring of mice undergoing PIE.

Cyclophosphamide stimulates endoplasmic reticulum stress and induces apoptotic cell death in human glioblastoma cell lines.

Cyclophosphamide (CP) is an alkylating chemotherapeutic agent commonly used in cancer treatments. In this study, we aimed to investigate the effects of 4-Hydroperoxy cyclophosphamide (4-HC), which is active form of CP, on glucose-regulated protein 78 (GRP78), activating transcription factor 6 (ATF6), phospho-protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (p-PERK), phospho-inositol-requiring enzyme 1 alpha (p-IRE1α), eukaryotic translation initiation factor 2 alpha (eIF2α), and caspase-3 messenger ribonucleic acids (mRNAs) and proteins that play roles in the ER stress pathway and apoptosis in U87 and T98 human glioblastoma cell lines. U87 and T98 human glioblastoma cell lines were divided into control and 4-HC-treated groups. Cell viability assay was used to detect the half maximal inhibitory concentration (IC50) for 24 hours of 4-HC. Immunocytochemistry and quantitative polymerase chain reaction (qPCR) methods were used to evaluate the levels of proteins and their mRNAs. The IC50 values of U87 and T98 cells were calculated as 15.67±0.58 µM and 19.92±1 µM, respectively. The levels of GRP78, ATF6, p-PERK, p-IRE1α, eIF2α, and caspase-3 protein expressions in the 4-HC-treated group compared to that in the control group. These increased protein expressions also were correlated with the mRNA levels. The ER stress signal pathway could be active in 4-HC-induced cell death. Further studies of ER-related stress mechanisms in anticancer treatment would be important for effective therapeutic strategies.

Licochalcone A induces endoplasmic reticulum stress-mediated apoptosis of endometrial cancer cells via upregulation of GRP78 expression.

Licochalcone A (LicA), a natural compound extracted from licorice root, has been shown to exert a variety of anticancer activities. Whether LicA has such effects on endometrial cancer (EMC) is unclear. This study aims to investigate the antitumor effects of LicA on EMC. Our results show that LicA significantly reduced the viability and induced apoptosis of EMC cells and EMC-7 cells from EMC patients. LicA was also found to induce endoplasmic reticulum (ER) stress, leading to increased expression of ER-related proteins (GRP78/PERK/IRE1α/CHOP) in EMC cell lines. Suppression of GRP78 expression in human EMC cells treated with LicA significantly attenuated the effects of LicA, resulting in reduced ER-stress mediated cell apoptosis and decreased expression of ER- and apoptosis-related proteins. Our findings demonstrate that LicA induces apoptosis in EMC cells through the GRP78-mediated ER-stress pathway, emphasizing the potential of LicA as an anticancer therapy for EMC.

PBA alleviates cadmium-induced mouse spermatogonia apoptosis by suppressing endoplasmic reticulum stress.

OBJECTIVE: Endoplasmic reticulum (ER) stress mediates Cd-caused germ cell apoptosis in testis. The effects of 4-phenylbutyric acid (PBA), a classical chaperone, were investigated on Cd-induced apoptosis in mouse GC-1 spermatogonia cells. METHODS: The cells were pretreated with PBA before Cd exposure. TUNEL and flow cytometry assays were applied to determine apoptosis. Some key biomarkers of ER stress were analyzed using RT-PCR and western blot. RESULTS: as expected, the apoptotic cells exposed to Cd apparently increased. The mRNA and protein expression levels of GRP78 and ATF6α, were elevated in the Cd groups. Additional experiments displayed that Cd notably increased IRE1α and JNK phosphorylation, and upregulated XBP-1 mRNA and protein expression. Moreover, p-eIF2α and CHOP expressions were clearly elevated in the Cd groups. Interestingly, PBA almost completely inhibited ER stress and protected spermatogonia against apoptosis induced by Cd. CONCLUSION: PBA alleviated Cd-induced ER stress and spermatogonia apoptosis, and may have the therapeutic role in Cd-induced male reproductive toxicity.

Polystyrene nanoplastics-induced lung apoptosis and ferroptosis via ROS-dependent endoplasmic reticulum stress.

It has been shown that exposure to nanoplastics (MNPs) through inhalation can induce pulmonary toxicity, but the toxicological mechanism of MNPs on the respiratory system remains unclear. Therefore, we explored the toxicological mechanism of exposure to polystyrene nanoplastics (PS-NPs) (0.05, 0.15, 0.2 mg/mL) on BEAS-2B cells. Results revealed that PS-NPs induce oxidative stress, increased apoptosis rate measured by flow cytometry, the key ferroptosis protein (GPX4 and FTH1) reduction, increased iron content, mitochondrial alterations, and increased malondialdehyde (MDA) level. Besides, consistent results were observed in mice exposed to PS-NPs (5 mg/kg/2d, 10 mg/kg/2d). Thus, we proved that PS-NPs induced cell death and lung damage through apoptosis and ferroptosis. In terms of mechanism, the elevation of the endoplasmic reticulum (ER) stress protein expression (IRE1α, PERK, XBP1S, and CHOP) revealed that PS-NPs induce lung damage by activating the two main ER stress pathways. Furthermore, the toxicological effects of PS-NPs observed in this study are attenuated by the ROS inhibitor N-acetylcysteine (NAC). Collectively, NPs-induced apoptosis and ferroptosis are attenuated by NAC via inhibiting the ROS-dependent ER stress in vitro and in vivo. This improves our understanding of the mechanism by which PS-NPs exposure leads to pulmonary injury and the potential protective effects of NAC.

Expression of GRP78 and its copartners in HEK293 and pancreatic cancer cell lines (BxPC-3/PANC-1) exposed to MRI and CT contrast agents.

Endoplasmic reticulum (ER) stress-associated chaperones trigger a defense mechanism called as unfolded protein response (UPR) which can manage apoptosis and be determinative in cell fate. Both anticancer drug effects and potential toxicity effects of magnetic resonance imaging (MRI) and computed tomography (CT) contrast agents were aimed to be evaluated. For this purpose, we investigated expression profiles of endoplasmic reticulum stress-associated chaperone molecules in human pancreatic tumor lines BxPC-3 and PANC-1 and control human embryonic kidney cells 293 (HEK293) induced with a variety of gadolinium and iohexol contrast agents. Protein expression levels of ER stress-associated chaperones (master regulator: GRP78/Bip and its copartners: Calnexin, Ero1, PDI, CHOP, IRE1α and PERK) were evaluated with Western blotting. Expression levels at mRNA level were also assessed for GRP78/Bip and CHOP with real-time PCR. Induction of cells was carried out with four different Gd-based contrast agents (GBCAs): (Dotarem, Optimark, Primovist and Gadovist) and two different iohexol agents (Omnipol, Omnipaque). CT contrast agents tested in the study did not result in significant ER stress in HEK293 cells. However, they do not seem to have theranostic potential in pancreas cancer through ER pathway. The potential efficiency of macrocyclic MRI contrast agents to provoke apoptosis via ER stress-associated chaperones in BxPC-3 cells lends credibility for their future theranostic use in pancreas cancer as long as undesired toxicity effects were carefully considered. ER stress markers and/or contrast agents seem to have promising potential to be translated into the clinical practice to manage pancreas cancer progression.

Identification of the unfolded protein response pathway as target for radiosensitization in pancreatic cancer.

BACKGROUND AND PURPOSE: Due to the high intrinsic radioresistance of pancreatic ductal adenocarcinoma (PDAC), radiotherapy (RT) is only beneficial in 30% of patients. Therefore, this study aimed to identify targets to improve the efficacy of RT in PDAC. MATERIALS AND METHODS: Alamar Blue proliferation and colony formation assay (CFA) were used to determine the radioresponse of a cohort of 38 murine PDAC cell lines. A gene set enrichment analysis was performed to reveal differentially expressed pathways. CFA, cell cycle distribution, γH2AX FACS analysis, and Caspase 3/7 SYTOX assay were used to examine the effect of a combination treatment using KIRA8 as an IRE1α-inhibitor and Ceapin-A7 as an inhibitor against ATF6. RESULTS: The unfolded protein response (UPR) was identified as a pathway highly expressed in radioresistant cell lines. Using the IRE1α-inhibitor KIRA8 or the ATF6-inhibitor Ceapin-A7 in combination with radiation, a radiosensitizing effect was observed in radioresistant cell lines, but no substantial alteration of the radioresponse in radiosensitive cell lines. Mechanistically, increased apoptosis by KIRA8 in combination with radiation and a cell cycle arrest in the G1 phase after ATF6 inhibition and radiation have been observed in radioresistant cell lines. CONCLUSION: So, our data show evidence that the UPR is involved in radioresistance of PDAC. Increased apoptosis and a G1 cell cycle arrest seem to be responsible for the radiosensitizing effect of UPR inhibition. These findings are supportive for developing novel combination treatment concepts in PDAC to overcome radioresistance.

Experimental periodontitis induced hypoadiponectinemia by IRE1α-mediated endoplasmic reticulum stress in adipocytes.

BACKGROUD: Hypoadiponectinemia is the important cause of insulin resistance. Recent studies have shown that periodontitis is associated with hypoadiponectinemia. The purpose of this study was to investigate the effect of periodontitis-induced endoplasmic reticulum stress (ERS) in visceral adipocytes on hypoadiponectinemia. METHODS: Rat periodontitis models were established by local ligation with silk around the bilateral maxillary second molars. Porphyromonas gingivalis-lipopolysaccharid (P.g-LPS) was also used to stimulate the visceral adipocytes in vitro. The protein expression levels of glucose regulated protein 78 (GRP78), inositol-requiring protein 1α (IRE1α), protein kinase RNA-like ER kinase (PERK), activating transcription factor 6 (ATF6) and adiponectin were detected. IRE1α lentiviruses were transfected into visceral adipocytes in vitro, and an IRE1α inhibitor (KIRA6) was injected in epididymal adipose tissue of rats to detect and verify the effect of ERS on adiponectin expression in visceral adipocytes in vivo. RESULTS: Hypoadiponectinemia was observed in periodontitis rat, and the expression levels of ERS key proteins GRP78 and the phosphorylation levels of IRE1α (p-IRE1α)/IRE1α in visceral adipocytes were increased, while the expression levels of adiponectin protein were decreased. After KIRA6 injection into epididymal adipose tissue of rats with periodontitis, adiponectin levels in visceral adipocytes increased, and serum adiponectin levels recovered to a certain extent. The protein expression levels of GRP78 and p-IRE1α/IRE1α were increased and adiponectin protein expression was decreased in P.g-LPS-induced visceral adipocytes. Overexpression of IRE1α further inhibited adiponectin expression in P.g-LPS-stimulated visceral adipocytes, and conversely, IRE1α inhibition restored adiponectin expression. CONCLUSIONS: Our findings suggest that periodontitis induces ERS in visceral adipocytes leading to hypoadiponectinemia. IRE1α is a key protein regulating adiponectin expression in visceral adipocytes.

Quercetin alleviates cadmium-induced BRL-3A cell apoptosis by inhibiting oxidative stress and the PERK/IRE1α/ATF6 signaling pathway.

Cadmium (Cd) is a highly toxic environmental pollutant. The liver is an important metabolic organ in the body and is susceptible to Cd toxicity attacks. Quercetin (Que) is a flavonoid compound with pharmacological activities of scavenging free radicals and antioxidant activity. Previous studies have shown that Que can alleviate Cd caused hepatocyte apoptosis in rats, but the specific mechanism remains unclear. To explore the specific mechanism, we established a model of Cd toxicity and Que rescue in BRL-3A cells and used 4-phenylbutyrate (4-PBA), an endoplasmic reticulum stress (ERS) inhibitor, as positive control. Set up a control group, Cd treatment group, Cd and Que co treatment group, Que treatment group, Cd and 4-PBA co treatment group, and 4-PBA treatment group. Cell Counting Kit-8 (CCK-8) method was employed to measure cell viability. Fluorescence staining was applied to observe cell apoptosis. Flow cytometry was performed to detect reactive oxygen species levels. Real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot method was adopted to detect the mRNA and protein expression levels of ERS and apoptosis-related genes. The results showed that compared with the control group, the Cd treated group showed a significant decrease in cell viability (P < 0.01), an increase in intracellular ROS levels, and apoptosis. The mRNA and protein expression levels of ERS and apoptosis related factors such as GRP78, IRE1α, XBP1, ATF6, Caspase-12, Caspase-3 and Bax in the cells were significantly increased (P < 0.01), while the mRNA and protein expression levels of Bcl-2 were significantly reduced (P < 0.01). Compared with the Cd treatment group, the Cd and Que co treatment group and the Cd and 4-PBA co treatment group showed a significant increase in cell viability (P < 0.01), a decrease in intracellular ROS levels, a decrease in cell apoptosis, and a significant decrease in the expression levels of ERS and apoptosis related factors mRNA and protein (P < 0.01), as well as a significant increase in Bcl-2 mRNA and protein expression (P < 0.01). We confirmed that Que could alleviate the apoptosis caused by Cd in BRL-3A cells, and the effects of Que were similar to those of ERS inhibitor.

Effects of 3021 meal replacement powder protect NAFLD via suppressing the ERS, oxidative stress and inflammatory responses.

Objective: To explore the specific protective mechanism of 3021 meal replacement powder (MRP) against non-alcoholic fatty liver disease (NAFLD). Materials and Methods: C57BL/6J male mice were divided into four groups: control group, 3021 MRP group, model group and test group. The lipid accumulation and endoplasmic reticulum stress (ERS)-related proteins in hepatocytes of mice were detected by hematoxylin-eosin (HE) staining, oil red O staining and Western blotting. Results: The expressions of GRP78, GRP94, p-PERK and p-IRE1α were significantly inhibited in test group compared with those in model group. The protein expressions of p-NF-κB, p-JNK, IL-1ß, IL-18 and NOX4 in test group were also significantly lower than those in model group. In vivo and in vitro experiments revealed that the body weight and lipid droplet content, and the expressions of ERS-related proteins (including BIP and XBP-1) in liver tissues all significantly declined in model group compared with those in 3021 MRP group. Conclusion: In conclusion, 3021 MRP can greatly reduce lipid accumulation by inhibiting ERS, oxidative stress and inflammatory response in NAFLD.

Inhibition of SK2 and ER stress ameliorated inflammation and apoptosis in liver ischemia-reperfusion injury.

Ischemia-reperfusion injury (IRI) remains a major cause of mortality and morbidity after liver surgery. Endoplasmic reticulum (ER) stress is a critical mechanism of inflammatory injury during hepatic IRI. In this study, we investigated the effect of sphingosine kinases 2 (SK2) on ER stress and hepatic IRI. We established hepatic IRI mice and hepatocellular hypoxia/reoxygenation in vitro model. We observed the SK2 and ER stress protein IRE1α expression. Then, we used an SK2 inhibitor and knocked down IRE1α/SK2, to observe the effect of SK2 during IRI. Our results showed that the expression of ER stress and SK2 was significantly elevated during hepatic IRI. Inhibition of SK2 ameliorated liver inflammation and reduced cell apoptosis in hepatic IRI mice. Consistently, we found that the inhibition of IRE1α also downregulated SK2 expression and reduced mitochondrial membrane permeability. Furthermore, the knockdown of SK2 could also reduce cell damage and reduce the expression of inflammatory factors but did not influence ER stress-related signaling pathway. Taken together, our results suggested that ER stress and SK2 played important and regulatory roles in hepatic IRI. Inhibition of ER stress and SK2 could significantly improve liver function after hepatic IRI.

Endoplasmic reticulum stress mediates environmental particle-induced inflammatory response in bronchial epithelium.

While the detailed mechanisms for how particulate matter (PM) causes adverse health effects in the lungs remain largely unknown, endoplasmic reticulum (ER) stress has been implicated in PM-induced lung injury. The present study was undertaken to examine how/if ER stress might regulate PM-induced inflammation, and to begin to define potential underlying molecular mechanisms. Here, ER stress hallmarks were examined in human bronchial epithelial (HBE) cells exposed to PM. To confirm roles of certain pathways, siRNA targeting ER stress genes and an ER stress inhibitor were employed. Expression of select inflammatory cytokines and related signaling pathway components by the cells were assessed as well. The results showed that PM exposure induced elevations in two ER stress hallmarks, i.e. GRP78 and IRE1α, in time-and/or dose-related manners in the HBE cells. Inhibition of ER stress by siRNA for GRP78 or IRE1α significantly alleviated the PM-induced effects. Further, ER stress appeared to regulate PM-induced inflammation - likely through downstream autophagy and NF-κB pathways - as implied by studies showing that inhibition of ER stress by siRNA of GRP78 or IRE1α caused significant amelioration of PM-induced autophagy and subsequent activation of NF-κB pathways. Moreover, the ER stress inhibitor 4-PBA were used to confirm the protective effects against PM-induced outcomes. Together, the results suggest ER stress plays a deleterious role in PM-induced airway inflammation, possibly through activation of autophagy and NF-κB signaling. Accordingly, protocols/treatments that could lead to inhibited ER stress could potentially be effective for treatment of PM-related airway disorders.

STF-083010 an inhibitor of IRE1α endonuclease activity affects mitochondrial respiration and generation of mitochondrial membrane potential.

STF-083010 is an inhibitor of endonuclease activity of inositol requiring-enzyme 1α (IRE1α) that is involved in activation of IRE1α-XBP1 axis of the unfolded protein response after ER stress. STF-083010 was tested as a possible antitumor agent in some previous studies exhibiting the ability either to induce death of tumour cells or to increase sensitivity of tumours cells to other neoplastic agents. STF-083010 exhibits also hepatoprotective effects in different models of liver injury and hepatic steatohepatitis. We have shown that STF-083010 has significant impact on mitochondrial functions that is not dependent on the way of STF-083010 application. We have observed that STF-083010 decrease of both maximal respiration (representing maximal electron transfer capacity of mitochondrial respiratory chain) and spare respiratory capacity after either incubation of the SH-SY5Y cells with STF-083010 or direct addition of STF-083010 to the respiration medium. In addition, we have documented impact of STF-083010 on generation of mitochondrial membrane potential (ΔΨm) that could be a result of decreased mitochondrial substrate level phosphorylation. Finally, increased sensitivity of ΔΨm to uncoupler in the presence of STF-083010 was documented. Our results indicate that STF-083010 has important impact on mitochondrial functions independently of its ability to inhibit endonuclease activity of IRE1α that is involved in activation of IRE1α-XBP1 axis of the unfolded protein response after ER stress. The impact of STF-083010 on mitochondrial functions could be associated with its possible off-target effect.

TSG-6 alleviates cerebral ischemia/reperfusion injury and blood-brain barrier disruption by suppressing ER stress-mediated inflammation.

Tumor necrosis factor-stimulated gene-6 (TSG-6) exhibits promising neuroprotective activity, but how it influences cerebral ischemia/reperfusion (CIR) injury remains to be established. Here, the impact of TSG-6 on the CIR-induced disturbance in the blood-brain barrier (BBB) and associated neurological degeneration was assessed, and the related molecular processes were explored. In this study, TSG-6 markedly reduced CIR-mediated increases in neurological deficit scores, decreased infarct volume, and protected against the apoptotic death of neurons in MCAO/R model rats. Similarly, TSG-6 pretreatment protected cultured neurons against OGD/R-associated neuronal death. TSG-6 also restored BBB integrity, suppressing PERK-eIF2α and IRE1α-TRAF2 pathway activation in CIR model systems, thereby inhibiting NF-κB, TNF-α, IL-1ß, and IL-6. The further use of specific inhibitors of ER stress, 4-phenyl butyric acid (4-PBA), PERK (GSK2656157), and IRE1α (STF083010) demonstrated the ability of ER stress to drive inflammatory activity in the context of CIR injury i the PERK-eIF2α-NF-κB and IRE1α-TRAF2-NF-κB pathways. Consistently, the activation of ER stress using tunicamycin resulted in reversing the beneficial effects of TSG-6 on CIR-associated BBB disruption and neurological damage in vitro and in vivo. Treatment with TSG-6 can protect against CIR injury via the inhibition of ER stress-related inflammatory activity induced through the PERK-eIF2α-NF-κB and IRE1α-TRAF2-NF-κB pathways.

Modulation of the autophagy-lysosomal pathway and endoplasmic reticulum stress by ketone bodies in experimental models of stroke.

Ischemic stroke is a leading cause of disability worldwide. There is no simple treatment to alleviate ischemic brain injury, as thrombolytic therapy is applicable within a narrow time window. During the last years, the ketogenic diet (KD) and the exogenous administration of the ketone body ß-hydroxybutyrate (BHB) have been proposed as therapeutic tools for acute neurological disorders and both can reduce ischemic brain injury. However, the mechanisms involved are not completely clear. We have previously shown that the D enantiomer of BHB stimulates the autophagic flux in cultured neurons exposed to glucose deprivation (GD) and in the brain of hypoglycemic rats. Here, we have investigated the effect of the systemic administration of D-BHB, followed by its continuous infusion after middle cerebral artery occlusion (MCAO), on the autophagy-lysosomal pathway and the activation of the unfolded protein response (UPR). Results show for the first time that the protective effect of BHB against MCAO injury is enantiomer selective as only D-BHB, the physiologic enantiomer of BHB, significantly reduced brain injury. D-BHB treatment prevented the cleavage of the lysosomal membrane protein LAMP2 and stimulated the autophagic flux in the ischemic core and the penumbra. In addition, D-BHB notably reduced the activation of the PERK/eIF2α/ATF4 pathway of the UPR and inhibited IRE1α phosphorylation. L-BHB showed no significant effect relative to ischemic animals. In cortical cultures under GD, D-BHB prevented LAMP2 cleavage and decreased lysosomal number. It also abated the activation of the PERK/eIF2α/ATF4 pathway, partially sustained protein synthesis, and reduced pIRE1α. In contrast, L-BHB showed no significant effects. Results suggest that protection elicited by D-BHB treatment post-ischemia prevents lysosomal rupture allowing functional autophagy, preventing the loss of proteostasis and UPR activation.

XBP1s gene of endoplasmic reticulum stress enhances proliferation and osteogenesis of human periodontal ligament cells.

BACKGROUND: The endoplasmic reticulum stress (ERS) pathway, inositol-requiring enzyme-1 alpha-X-box binding protein-1 (IRE1α-XBP1), has been considered as a critical factor of human periodontal ligament cells (hPDLCs) in proliferation and osteogenesis. This study aimed to explore the effect and mechanism of XBP1s, which was cleaved by IRE1α on the proliferation and osteogenesis of hPDLCs. METHODS: ERS model was induced by tunicamycin (TM); cell proliferation was assessed by CCK-8 assay; pLVX-XBP1s-hPDLCs cell line was established by lentivirus infaction; expression of ERS-related protein including eIF2α, GRP78, ATF4 and XBP1s, autophagy-related P62 and LC3, and apoptosis-related Bcl-2 and Caspase-3 were detected by Western Blot; expression of osteogenic genes was detected by RT-qPCR, and senescence of hPDLCs was explored by ß-galactosidase staining. Furthermore, the interaction between XBP1s and human bone morphogenetic protein 2 (BMP2) was examined by immunofluorescence antibody test (IFAT). RESULTS: The results showed an increase in proliferation of hPDLCs from 0 to 24 h when ERS was induced by TM treatment (P < 0.05). XBP1s overexpression induced hPDLCs proliferation, upgraded autophagy and degraded apoptosis significantly (P < 0.05). In pLVX-XBP1s-hPDLCs, the ratio of senescent cells was markedly decreased after several passages (P < 0.05); After infection with pLVX-BMP2 lentiviral supernatant, IFAT result showed that XBP1s and BMP2 well co-located in the cytoplasm of pLVX-XBP1s-hPDLCs and PERK-ATF4 ERS branch was activated, meanwhile, there were obviously more mineralized nodules and mRNA expression of osteogenesis-related genes was continually up-regulated (P < 0.05). CONCLUSIONS: XBP1s promotes the proliferation via regulating the autophagy and apoptosis, and enhances expression of osteogenic genes in hPDLCs. The mechanisms in this regard need exploring further for periodontal tissue regeneration, functionalization and clinical applications.

Glutamine inhibits inflammation, oxidative stress, and apoptosis and ameliorates hyperoxic lung injury.

Glutamine (Gln) is the most widely acting and abundant amino acid in the body and has anti-inflammatory properties, regulates body metabolism, and improves immune function. However, the mechanism of Gln's effect on hyperoxic lung injury in neonatal rats is unclear. Therefore, this work focused on examining Gln's function in lung injury of newborn rats mediated by hyperoxia and the underlying mechanism. We examined body mass and ratio of wet-to-dry lung tissue weights of neonatal rats. Hematoxylin and eosin (HE) staining was performed to examine histopathological alterations of lung tissues. In addition, enzyme-linked immunoassay (ELISA) was conducted to measure pro-inflammatory cytokine levels within bronchoalveolar lavage fluid (BALF). Apoptosis of lung tissues was observed using TUNEL assay. Western blotting was performed for detecting endoplasmic reticulum stress (ERS)-associated protein levels. The results showed that Gln promoted body weight gain, significantly reduced pathological damage and oxidative stress in lung tissue, and improved lung function in neonatal rats. Gln reduced pro-inflammatory cytokine release as well as inflammatory cell production in BALF and inhibited apoptosis in lung tissue cells. Furthermore, we found that Gln could downregulate ERS-associated protein levels (GRP78, Caspase-12, CHOP) and inhibit c-Jun N-terminal kinase (JNK) and inositol-requiring enzyme 1 alpha (IRE1α) phosphorylation. These results in an animal model of bronchopulmonary dysplasia (BPD) suggest that Gln may have a therapeutic effect on BPD by reducing lung inflammation, oxidative stress, and apoptosis and improving lung function; its mechanism of action may be related to the inhibition of the IRE1α/JNK pathway.

Cryptotanshinone induces apoptosis of activated hepatic stellate cells via modulating endoplasmic reticulum stress.

BACKGROUND: Cryptotanshinone (CPT) has wide biological functions, including anti-oxidative, antifibrosis, and anti-inflammatory properties. However, the effect of CPT on hepatic fibrosis is unknown. AIM: To investigate the effects of CPT treatment on hepatic fibrosis and its underlying mechanism of action. METHODS: Hepatic stellate cells (HSCs) and normal hepatocytes were treated with different concentrations of CPT and salubrinal. The CCK-8 assay was used to determine cell viability. Flow cytometry was used to measure apoptosis and cell cycle arrest. Reverse transcription polymerase chain reaction (RT-PCR) and Western blot analyses were used to measure mRNA levels and protein expression of endoplasmic reticulum stress (ERS) signaling pathway related molecules, respectively. Carbon tetrachloride (CCL4) was used to induce in vivo hepatic fibrosis in mice. Mice were treated with CPT and salubrinal, and blood and liver samples were collected for histopathological examination. RESULTS: We found that CPT treatment significantly reduced fibrogenesis by modulating the synthesis and degradation of the extracellular matrix in vitro. CPT inhibited cell proliferation and induced cell cycle arrest at the G2/M phase in cultured HSCs. Furthermore, we found that CPT promoted apoptosis of activated HSCs by upregulating expression of ERS markers (CHOP and GRP78) and activating ERS pathway molecules (PERK, IRE1α, and ATF4), which were inhibited by salubrinal. Inhibition of ERS by salubrinal partially eliminated the therapeutic effect of CPT in our CCL4-induced hepatic fibrosis mouse model. CONCLUSION: CPT can promote apoptosis of HSCs and alleviate hepatic fibrosis through modulating the ERS pathway, which represents a promising strategy for treating hepatic fibrosis.

Induction of immunogenic cell death effect of licoricidin in cervical cancer cells by enhancing endoplasmic reticulum stress-mediated high mobility group box 1 expression.

Licoricidin (LCD) is an activity compound of the roots of Glycyrrhiza uralensis, which has therapeutic efficacy, including anti-virus, anti-cancer, and enhanced immunity in Traditional Chinese Medicine. Herein, this study aimed to clarify the effect of LCD on cervical cancer cells. In the present study, we found that LCD significantly inhibited cell viability via inducing cell apoptosis and companies with cleaved-PARP protein expression and caspase-3/-9 activity. Cell viability was markedly reversed these effects by pan-caspase inhibitor Z-VAD-FMK treatment. Furthermore, we showed that LCD-induced ER (endoplasmic reticulum) stress triggers upregulating the protein level of GRP78 (Bip), CHOP, and IRE1α, and subsequently confirmed the mRNA level by quantitative real-time polymerase chain reaction. In addition, LCD exhibited the release of danger-associated molecular patterns from cervical cancer cells, such as the release of high-mobility group box 1 (HMGB1), secretion of ATP, and exposure of calreticulin (CRT) on the cell surface, which led to immunogenic cell death (ICD). These results provide a novel foundation that LCD induces ICD via triggering ER stress in human cervical cancer cells. LCD might be an ICD inducer of immunotherapy in progressive cervical cancer.

Three tyrosine kinase inhibitors cause cardiotoxicity by inducing endoplasmic reticulum stress and inflammation in cardiomyocytes.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) are anti-cancer therapeutics often prescribed for long-term treatment. Many of these treatments cause cardiotoxicity with limited cure. We aim to clarify molecular mechanisms of TKI-induced cardiotoxicity so as to find potential targets for treating the adverse cardiac complications. METHODS: Eight TKIs with different levels of cardiotoxicity reported are selected. Phenotypic and transcriptomic responses of human cardiomyocytes to TKIs at varying doses and times are profiled and analyzed. Stress responses and signaling pathways that modulate cardiotoxicity induced by three TKIs are validated in cardiomyocytes and rat hearts. RESULTS: Toxicity rank of the eight TKIs determined by measuring their effects on cell viability, contractility, and respiration is largely consistent with that derived from database or literature, indicating that human cardiomyocytes are a good cellular model for studying cardiotoxicity. When transcriptomes are measured for selected TKI treatments with different levels of toxicity in human cardiomyocytes, the data are classified into 7 clusters with mainly single-drug clusters. Drug-specific effects on the transcriptome dominate over dose-, time- or toxicity-dependent effects. Two clusters with three TKIs (afatinib, ponatinib, and sorafenib) have the top enriched pathway as the endoplasmic reticulum stress (ERS). All three TKIs induce ERS in rat primary cardiomyocytes and ponatinib activates the IRE1α-XBP1s axis downstream of ERS in the hearts of rats underwent a 7-day course of drug treatment. To look for potential triggers of ERS, we find that the three TKIs induce transient reactive oxygen species followed by lipid peroxidation. Inhibiting either PERK or IRE1α downstream of ERS blocks TKI-induced cardiac damages, represented by the induction of cardiac fetal and pro-inflammatory genes without causing more cell death. CONCLUSIONS: Our data contain rich information about phenotypic and transcriptional responses of human cardiomyocytes to eight TKIs, uncovering potential molecular mechanisms in modulating cardiotoxicity. ER stress is activated by multiple TKIs and leads to cardiotoxicity through promoting expression of pro-inflammatory factors and cardiac fetal genes. ER stress-induced inflammation is a promising therapeutic target to mitigate ponatinib- and sorafenib-induced cardiotoxicity.