This study aims to investigate the anti-tumor effect and mechanism of Guiqi Yiyuan Ointment combined with cisplatin on Lewis lung carcinoma-bearing mice via the protein kinase RNA-like endoplasmic reticulum kinase(PERK)/eukaryotic translation initiation factor 2α(eIF2α)/activated transcription factor 4(ATF4)/C/EBP homologous protein(CHOP) signaling pathway. Sixty SPF-grade male C57BL/6 mice were selected and assigned into a blank group and a modeling group by the random number table method. After modeling of the Lewis lung carcinoma, the mice in the modeling group were randomized into model, cisplatin(5 mg·kg~(-1), once a week), and low-, medium-, and high-dose(1.7, 3.5, and 7.05 g·kg~(-1), respectively, once a day) Guiqi Yiyuan Ointment+cisplatin(5 mg·kg~(-1)) groups(n=10). After 14 days of continuous intervention, the spleen, thymus, and tumor samples of the mice were collected, weighed, and recorded, and the spleen index, thymus index, and tumor suppression rate were calculated. Hematoxylin-eosin(HE) staining was employed to observe the pathological changes in the tumor tissue. The morphological changes of the endoplasmic reticulum of tumor cells were observed by transmission electron microscopy. The positive expression of phosphorylated eIF2α(p-eIF2α) and ATF4 in the tumor tissue was detected by immunofluorescence. Western blot was employed to determine the protein levels of phosphorylated PERK(p-PERK), p-eIF2α, ATF4, CHOP, B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), cyclin-dependent kinase inhibitor 1A(p21), and cyclinD1 in the tumor tissue. Real-time fluorescent quantitative PCR was employed to determine the mRNA levels of PERK, eIF2α, ATF4, CHOP, Bax, Bcl-2, p21, and cyclinD1 in the tumor tissue. Compared with the blank group, the model group showed decreases in spleen index and thymus index(P<0.05). Compared with the model group, the cisplatin group showed decreases in spleen index and thymus index(P<0.05), and the medium-and high-dose Guiqi Yiyuan Ointment+cisplatin groups presented increases in spleen index and thymus index(P<0.05). In addition, the treatment groups all showed decreased tumor mass(P<0.05), increased tumor cell lysis and nuclear rupture, widened gap between rough endoplasmic reticulum, enhanced average fluorescence intensity of p-eIF2α and ATF4(P<0.05), up-regulated protein levels of p-PERK/PERK, p-eIF2α/eIF2α, ATF4, CHOP, Bax, and p21(P<0.05), down-regulated protein and mRNA levels of Bcl-2 and cyclinD1(P<0.05), and up-regulated mRNA levels of PERK, eIF2α, ATF4, CHOP, Bax, and p21(P<0.05). Compared with the cisplatin group, the combination groups showed increases in spleen index and thymus index(P<0.05) as well as mean optical density(P<0.05), and the high-dose Guiqi Yiyuan Ointment+cisplatin group showed decreased tumor mass(P<0.05). In addition, the medium-and high-dose Guiqi Yiyuan Ointment+cisplatin groups showcased enhanced average fluorescence intensity of p-eIF2α and ATF4(P<0.05), up-regulated protein levels of p-PERK/PERK, p-eIF2α/eIF2α, ATF4, CHOP, Bax, and p21(P<0.05), down-regulated protein and mRNA levels of Bcl-2 and cyclinD1(P<0.05), and up-regulated mRNA levels of PERK, eIF2α, ATF4, CHOP, Bax, and p21(P<0.05). In conclusion, Guiqi Yiyuan Ointment combined with cisplatin can effectively inhibit the growth of Lewis lung carcinoma in mice by regulating the expression of proteins related to the PERK/eIF2α/ATF4/CHOP signaling pathway and promoting cell cycle arrest and apoptosis.
Animals ; Cisplatin/administration & dosage* ; Activating Transcription Factor 4/genetics* ; Eukaryotic Initiation Factor-2/genetics* ; eIF-2 Kinase/genetics* ; Carcinoma, Lewis Lung/pathology* ; Drugs, Chinese Herbal/administration & dosage* ; Male ; Mice ; Signal Transduction/drug effects* ; Mice, Inbred C57BL ; Transcription Factor CHOP/genetics* ; Ointments/administration & dosage* ; Humans ; Cell Proliferation/drug effects* ; Antineoplastic Agents/administration & dosage*

Study on the mechanism of Fangxia Dihuang Formula(FXDH) in treating breast cancer complicated with depression through the regulation of M1/M2 microglial polarization via the PERK/eIF2α axis. In addition to control group and 4T1 group, a mouse model of breast cancer complicated with depression was established using 4T1 cells combined with corticosterone. The mice were divided into model group, PERK/eIF2α signaling axis agonist(CCT020312, 2 mg·kg~(-1)·d~(-1)) group, CCT020312(2 mg·kg~(-1)·d~(-1)) + FXDH(13.65 g·kg~(-1)·d~(-1)) group, FXDH(13.65 g·kg~(-1)·d~(-1)) group, FXDH(13.65 g·kg~(-1)·d~(-1)) + Capecitabine Tablets(CAP, 390 mg·kg~(-1)·d~(-1)) group, and Fluoxetine Hydrochloride Capsules(FXT, 2.6 mg·kg~(-1)·d~(-1)) + CAP(390 mg·kg~(-1)·d~(-1)) group, with continuous intervention for 21 d. Depression-like behaviors in mice were assessed through sugar preference test and open field test. Hematoxylin-eosin(HE) staining was used to evaluate the morphology of tumor and hippocampal DG region neurons. Nissl staining was employed to detect changes in Nissl bodies in the hippocampal CA3 region. Immunofluorescence was used to observe cluster of differentiation 86(CD86)/ionized calcium-binding adapter molecule 1(Iba-1) and cluster of differentiation 206(CD206)/Iba-1 in hippocampal tissue. Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) was used to detect the mRNA expression of M1-type microglia [interleukin-6(IL-6), tumor necrosis factor-α(TNF-α)] and M2-type [arginase-1(Arg-1), IL-10] in hippocampal tissue. Western blot was used to detect the protein expression of key factors in the PERK/eIF2α axis, including PERK, eIF2α, activating transcription factor 4(ATF4), and C/EBP homologous protein(CHOP) in hippocampal tissue. The results showed that compared to model group/CCT020312 + FXDH group, FXDH group increased sugar preference index, total movement distance, central zone distance, and central zone entries; reduced tumor mass and volume; tumor cells were sparsely arranged, with a smaller nuclear-to-cytoplasmic ratio and reduced nuclear division figures, increased Nissl body count, and alleviated neuronal nuclear pyknosis; increased CD206-positive M2-type microglia expression, decreased CD86/Iba-1-positive M1-type microglia expression; reduced IL-6 and TNF-α mRNA expression, and increased Arg-1 and IL-10 mRNA expression; downregulated PERK, eIF2α, ATF4, and CHOP protein expression levels. The results indicate that the mechanism of FXDH in treating breast cancer complicated with depression may be related to inhibiting the activity of the PERK/eIF2α axis, reducing the proportion of M1-type microglia, increasing the proportion of M2-type microglia, thereby suppressing neuronal immune inflammation, improving depressive symptoms, and subsequently delaying the progression of breast cancer.
Animals ; Drugs, Chinese Herbal/administration & dosage* ; Female ; Microglia/cytology* ; Mice ; Depression/complications* ; eIF-2 Kinase/genetics* ; Humans ; Breast Neoplasms/psychology* ; Eukaryotic Initiation Factor-2/genetics* ; Mice, Inbred BALB C ; Signal Transduction/drug effects* ; Cell Line, Tumor

OBJECTIVES: To explore the effects of cannabidiol on endoplasmic reticulum stress and neuronal apoptosis in rats with multiple concussions (MCC). METHODS: SD rats were randomized into sham group, MCC group, 1% tween20 (TW) treatment group, and low-dose (10 mg/kg) and high-dose (40 mg/kg) cannabidiol treatment groups. In all but the sham group, MCC models were established using a metal pendulum percussion device, after which the rats received daily intraperitoneal injections of the corresponding agents for 2 weeks. The expressions of PERK, eIF2α, ATF4, CHOP, TRIB3, p-Akt and pro-caspase-3 in the brain tissue of the rats were detected with qRT-PCR, Western blotting and immunofluorescence staining. The core targets of cannabidiol in treatment of traumatic brain injury (TBI) were identified by network pharmacology analysis, and molecular docking was carried out to simulate the interaction of cannabidiol with the factors related to endoplasmic reticulum stress and apoptosis. RESULTS: Compared with the sham-operated rats, the rat models of MCC showed significantly increased mRNA expressions of PERK, eIF2α and CHOP and protein expressions of PERK, eIF2α, ATF4, CHOP, TRIB3, p-AKT and pro-caspase-3 in the cerebral cortex. CBD treatment, especially at the high dose, obviously increased the expression of p-Akt and lowered the expression levels of the other factors tested in the rat models. Network pharmacology analysis indicated interactions of the core targets of CBD with the factors related to endoplasmic reticulum stress and TBI, and molecular docking study showed a high binding energy of CBD with multiple factors pertaining to endoplasmic reticulum stress and apoptosis. CONCLUSIONS MCC induce endoplasmic reticulum stress and apoptosis in rat brain tissues, for which CBD, especially at a high dose, provides neuroprotective effects by inhibiting endoplasmic reticulum stress and cell apoptosis.
Animals ; Endoplasmic Reticulum Stress/drug effects* ; Apoptosis/drug effects* ; Rats, Sprague-Dawley ; Activating Transcription Factor 4/metabolism* ; Transcription Factor CHOP/metabolism* ; Rats ; Eukaryotic Initiation Factor-2/metabolism* ; Signal Transduction/drug effects* ; eIF-2 Kinase/metabolism* ; Cannabidiol/pharmacology* ; Neurons/metabolism* ; Brain Concussion/metabolism* ; Male ; Molecular Docking Simulation

Up-frameshift 1 (UPF1), as the most critical factor in nonsense-mediated messenger RNA (mRNA) decay (NMD), regulates tumor-associated molecular pathways in many cancers. However, the role of UPF1 in lung adenocarcinoma (LUAD) amino acid metabolism remains largely unknown. In this study, we found that UPF1 was significantly correlated with a portion of amino acid metabolic pathways in LUAD by integrating bioinformatics and metabolomics. We further confirmed that UPF1 knockdown inhibited activating transcription factor 4 (ATF4) and Ser51 phosphorylation of eukaryotic translation initiation factor 2α (eIF2α), the core proteins in amino acid metabolism reprogramming. In addition, UPF1 promotes cell proliferation by increasing the amino-acid levels of LUAD cells, which depends on the function of ATF4. Clinically, UPF1 mRNA expression is abnormal in LUAD tissues, and higher expression of UPF1 and ATF4 was significantly correlated with poor overall survival (OS) in LUAD patients. Our findings reveal that UPF1 is a potential regulator of tumor-associated amino acid metabolism and may be a therapeutic target for LUAD.
Activating Transcription Factor 4/genetics* ; Adenocarcinoma of Lung ; Amino Acids ; Cell Proliferation ; Eukaryotic Initiation Factor-2 ; Humans ; Lung Neoplasms ; RNA Helicases/metabolism* ; RNA, Messenger/metabolism* ; Trans-Activators/metabolism*

Animals ; Cell Line ; Diabetic Nephropathies ; metabolism ; Epithelial-Mesenchymal Transition ; drug effects ; Eukaryotic Initiation Factor-2 ; metabolism ; Glucose ; pharmacology ; Humans ; Kidney ; drug effects ; metabolism ; pathology ; Kidney Tubules, Proximal ; drug effects ; metabolism ; Rats ; Signal Transduction ; drug effects

A few clues about correlation between endoplasmic reticulum (ER) stress and mulberry (Morus alba) leaves were investigated in only the experimental autoimmune myocarditis and streptozotocin-induced diabetes. To investigate whether a novel extract of mulberry leaves fermented with Cordyceps militaris (EMfC) could suppress ER in fatty liver, alterations in the key parameters for ER stress response were measured in high fat diet (HFD)-induced obese C57L/6 mice treated with EMfC for 12 weeks. The area of adipocytes in the liver section were significantly decreased in the HFD+EMfC treated group as compared to the HFD+Vehicle treated group, while their level was higher in HFD+Vehicle treated group than No treated group. The level of the eukaryotic initiation factor 2 alpha (eIF2α) and inositol-requiring enzyme 1 beta (IRE1α) phosphorylation and CCAAT-enhancer-binding protein homologous protein (CHOP) expression were remarkably enhanced in the HFD+Vehicle treated group. However, their levels were restored in the HFD+EMfC treated group, although some differences were detected in the decrease rate. Similar recovery was observed on the ER stress-induced apoptosis. The level of Caspase-3, Bcl-2 and Bax were decreased in the HFD+EMfC and HFD+orlistat (OT) treated group compared to the HFD+Vehicle treated group. The results of the present study therefore provide first evidence that EMfC with the anti-obesity effects can be suppressed ER stress and ER stress-induced apoptosis in the hepatic steatosis of HFD-induced obesity model.
Adipocytes ; Animals ; Apoptosis ; Caspase 3 ; CCAAT-Enhancer-Binding Proteins ; Cordyceps* ; Diet, High-Fat ; Endoplasmic Reticulum Stress* ; Endoplasmic Reticulum* ; Eukaryotic Initiation Factor-2 ; Fatty Liver ; Liver ; Mice* ; Morus* ; Myocarditis ; Obesity ; Phosphorylation

Cyclooxygenase-2 (COX-2) is an enzyme induced by various proinflammatory and mitogenic stimuli. Celecoxib is a selective inhibitor of COX-2 that have been shown to affect cell growth and apoptosis. Lung cancer cells expressing COX-2 is able to be a target of celecoxib, this study focuses on investigating that celecoxib induces apoptosis via endoplasmic reticulum (ER) stress on lung cancer cells. We investigated whether celecoxib induced apoptosis on non-small cell lung cancer cell line, A549 and H460. The 50 µM of celecoxib increased apoptotic cells and 100 µM of celecoxib significantly induced apoptosis. To check involvement of caspase cascade, pretreatment of z-VAD-fmk blocked celecoxib-induced apoptosis. However, caspase-3, -8, and -9 were not activated, but cleavage of non-classical caspase-4 was detected using western blot. As checking ER stress associated molecules, celecoxib did not increase expressions of growth arrest and DNA damage inducible protein 34, activating transcription factor 4, and spliced X-box binding protiens-1, but increase of both glucose-regulated protein 78 (GRP78) and C/EBP homologous transcription factor were detected. Salubrinal, inhibitor of eIF2 and siRNA for IRE1 did not alter celecoxib-induced apoptosis. Instead, celecoxib-induced apoptosis might be deeply associated with ER stress depending on GRP78 because siRNA for GRP78 enhanced apoptosis. Taken together, celecoxib triggered ER stress on lung cancer cells and celecoxib-induced apoptosis might be involved in both non-classical caspase-4 and GRP78.
Activating Transcription Factor 4 ; Apoptosis ; Blotting, Western ; Carcinoma, Non-Small-Cell Lung* ; Caspase 3 ; Celecoxib* ; Cell Death* ; Cell Line ; Cyclooxygenase 2 ; DNA Damage ; Endoplasmic Reticulum Stress* ; Endoplasmic Reticulum* ; Eukaryotic Initiation Factor-2 ; Lung Neoplasms ; RNA, Small Interfering ; Transcription Factors

A correlation between endoplasmic reticulum (ER) stress and laxative effects was first reported in a constipation model treated with an aqueous extract of Liriope platyphylla (AEtLP) roots. To investigate the correlation between the laxative effect of uridine (Urd) and ER stress response, alterations in the key parameters for ER stress were measured in loperamide (Lop) induced constipation Sprague Dawley (SD) rats treated with Urd. The efficacy of the laxative effect of Urd was notable on the symptoms of chronic constipation, including alteration of stool parameters and structure of the transverse colon, in Lop induced constipated SD rats. In the PERK/eIF2-ATF4 pathway of ER stress response, the levels of eukaryotic initiation factor 2 alpha (eIF2α) phosphorylation and DNA damage-inducible protein (GADD34) transcripts were significantly enhanced in the Lop+Vehicle treated group. However, the levels were restored in the Lop+Urd treated group, although few differences were detected in the decrease rate. Similar changes were observed for levels of inositol-requiring enzyme 1 beta (IRE1β) phosphorylation and X-box binding protein 1 (XBP-1) transcript in the IRE1α/XBP pathway. Furthermore, the number of ER stress-induced apoptotic cells and Bax and Bcl-2 expression were recovered in the Lop+Urd treated group compared to the Lop+Vehicle treated group. The results of the present study therefore provide first evidence that the laxative effects of Urd may be tightly correlated with the recovery of ER stress response in constipation models.
Animals ; Apoptosis ; Carrier Proteins ; Colon, Transverse ; Constipation ; DNA ; Endoplasmic Reticulum ; Eukaryotic Initiation Factor-2 ; Loperamide* ; Phosphorylation ; Rats* ; Uridine*

OBJECTIVEPERK/eIF2α/CHOP is a major signaling pathway mediating endoplasmic reticulum (ER) stress related with atherosclerosis. Oxidized LDL (ox-LDL) also induces endothelial apoptosis and plays a vital role in the initiation and progression of atherosclerosis. The present study was conducted to explore the regulatory effect of ox-LDL on PERK/eIF2α/CHOP signaling pathway in vascular endothelial cells.

METHODSThe effects of ox-LDL on PERK and p-eIF2α protein expression of primary human umbilical vein endothelial cells (HUVECs) were investigated by Western blot analysis. PERK gene silencing and selective eIF2α phosphatase inhibitor, salubrinal were used to inhibit the process of ox-LDL induced endothelial cell apoptosis, caspase-3 activity, and CHOP mRNA level.

RESULTSOx-LDL treatment significantly increased the expression of PERK, PERK-mediated inactivation of eIF2α phosphorylation, and the expression of CHOP, as well as the caspase-3 activity and apoptosis. The effects of ox-LDL were markedly decreased by knocking down PERK with stable transduction of lentiviral shRNA or by selective eIF2α phosphatase inhibitor, salubrinal.

CONCLUSIONThis study provides the first evidence that ox-LDL induces apoptosis in vascular endothelial cells mediated largely via the PERK/eIF2α/CHOP ER-stress pathway. It adds new insights into the molecular mechanisms underlying the pathogenesis and progression of atherosclerosis.

Apoptosis ; Endoplasmic Reticulum Stress ; Eukaryotic Initiation Factor-2 ; genetics ; metabolism ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Lipoproteins, LDL ; genetics ; metabolism ; Signal Transduction ; Transcription Factor CHOP ; genetics ; metabolism ; eIF-2 Kinase ; genetics ; metabolism

A dysfunction of endoplasmic reticulum (ER) stress response can result in various diseases, including cancer, inflammation, diabetes and neurodegenerative disorders. To investigate whether ER stress response can play an essential role in the induction and treatment of chronic constipation, alterations in the key parameters for ER stress were measured in loperamide (Lop) induced constipation Sprague Dawley (SD) rats treated with aqueous extracts of Liriope platyphylla (AEtLP), which has been shown to have a laxative effect. Symptoms of chronic constipation including alteration of stool parameters and the transverse colon's structure were successfully induced by Lop treatment. Laxative effects such as enhancement of stools parameters, recovery of the mucosa thickness, increased muscle thickness and recovery of flat luminal surface were also observed in the Lop+AEtLP treated group. Furthermore, enhancement of eukaryotic initiation factor 2 alpha (eIF2α) phosphorylation and inositol-requiring enzyme 1 beta (IRE1β) expression, key indicators for ER stress, that were observed in the Lop+vehicle treated group were significantly recovered in the Lop+AEtLP treated group, although the phosphorylation level of c-Jun N-terminal protein kinase (JNK) remained constant. Moreover, alterations in the transcription level of the marker genes X-box binding protein 1 (XBP-1) and growth arrest and DNA damage-inducible protein (GADD34) were similar to those of eIF2α and IRE1β. However, their level was slightly or completely recovered after AEtLP treatment. Overall, this study provides the first evidence that ER stress response may be tightly correlated with chronic constipation induced by Lop treatment, as well as the laxative effects of AEtLP.
Animals ; Carrier Proteins ; Constipation* ; DNA ; Endoplasmic Reticulum Stress* ; Endoplasmic Reticulum* ; Eukaryotic Initiation Factor-2 ; Inflammation ; Loperamide ; Mucous Membrane ; Neurodegenerative Diseases ; Phenobarbital ; Phosphorylation ; Protein Kinases ; Rats*