Solasonine induces apoptosis of the SGC-7901 human gastric cancer cell line in vitro via the mitochondria-mediated pathway.

Solasonine, a steroidal glycoalkaloid isolated from the herbal plant Solanum nigrum Linn., has shown active against multiple human cancers; however, there is little knowledge on the activity of solasonine against gastric cancer until now. This study aimed to examine the effect of solasonine on the biological behaviours of human gastric cancer SGC-7901 cells. The results showed that solasonine suppressed SGC-7901 cell proliferation in a dose-dependent manner. Solasonine treatment mainly induced the cell cycle arrest at G2 phase in SGC-7901 cells. Treatment with solasonine resulted in significant down-regulation of Bcl-2 and Caspase-3 protein expression and reduced Bax and Bcl-xL protein expression in SGC-7901 cells. Solasonine shows a comparable inhibitory effect on the proliferation of human gastric cancer SGC-7901 cells with cisplatin, and solasonine induces of SGC-7901 cell apoptosis through triggering the endoplasmic reticulum stress pathway and the mitochondrial pathway. Our data indicate that solasonine may be a promising agent for the treatment of gastric cancer.

Targeted therapy for intervertebral disc degeneration: inhibiting apoptosis is a promising treatment strategy.

Intervertebral disc (IVD) degeneration (IDD) is a multifactorial pathological process associated with low back pain (LBP). The pathogenesis is complicated, and the main pathological changes are IVD cell apoptosis and extracellular matrix (ECM) degradation. Apoptotic cell loss leads to ECM degradation, which plays an essential role in IDD pathogenesis. Apoptosis regulation may be a potential attractive therapeutic strategy for IDD. Previous studies have shown that IVD cell apoptosis is mainly induced by the death receptor pathway, mitochondrial pathway, and endoplasmic reticulum stress (ERS) pathway. This article mainly summarizes the factors that induce IDD and apoptosis, the relationship between the three apoptotic pathways and IDD, and potential therapeutic strategies. Preliminary animal and cell experiments show that targeting apoptotic pathway genes or drug inhibition can effectively inhibit IVD cell apoptosis and slow IDD progression. Targeted apoptotic pathway inhibition may be an effective strategy to alleviate IDD at the gene level. This manuscript provides new insights and ideas for IDD therapy.

A new tellurium-containing amphiphilic molecule induces apoptosis in HCT116 colon cancer cells.

BACKGROUND: Chalcogen-based redox modulators over the years have attracted considerable attention as anti-cancer agents. New selenium- and tellurium-containing compounds with a polar head group and aryl-groups of various lengths have recently been reported as biologically active in several organisms. In the present study, we used the most active of the tellurium compound DP41, and its selenium counterpart DP31 to investigate their effects on the human cancer cell line HCT116. METHODS: Cells were treated with DP41 or DP31 and the formation of superoxide radicals was determined using dihydroethidium. Cell cycle analysis and apoptosis was determined by cytofluorimetry. Proteins involved in ER signaling and apoptosis were determined by Western blot analysis and fluorescence microscopy. RESULTS: With 50µM of DP41, we observed an increase in O2(-) formation. There was, however, no such increase in O2(-) after treatment with the corresponding selenium compound under the same conditions. In the case of DP41, the production of O2(-) radicals was followed by an up-regulation of Nrf2, HO-1, phospho-eIF2α and ATF4. CHOP was also induced and cells entered apoptosis. Unlike the cancer cells, normal retinal epithelial ARPE-19 cells did not produce elevated levels of O2(-) radicals nor did they induce the ER signaling pathway or apoptosis. CONCLUSIONS: The tellurium-containing compound DP41, in contrast to the corresponding selenium compound, induces O2(-) radical formation and oxidative and ER stress responses, including CHOP activation and finally apoptosis. GENERAL SIGNIFICANCE: These results indicate that DP41 is a redox modulating agent with promising anti-cancer potentials.

[Estradiol inhibits differentiation of mouse macrophage into a pro-inflammatory phenotype by upregulating the IRE1α-XBP1 signaling axis].

OBJECTIVE: To explore the mechanism by which estradiol modulates the immunophenotype of macrophages through the endoplasmic reticulum stress pathway. METHODS: Peritoneal macrophages isolated from C57 mice were cultured in the presence of 60 ng/mL interferon-γ (IFN-γ) followed by treatment with estradiol (1.0 nmol/L) alone, estradiol with estrogen receptor antagonist (Acolbifene, 4 nmol/L), estradiol with IRE1α inhibitor (4 µ 8 C), or estradiol with IRE1α agonist. After the treatments, the expression levels of MHC-Ⅱ, iNOS and endoplasmic reticulum stress marker proteins IRE1α, eIF2α and ATF6 in the macrophages were detected with Western blotting, and the mRNA levels of TGF-ß, IL-6, IL-10 and TNF-α were detected with RT-PCR. RESULTS: Estrogen treatment of the macrophages significantly decreased the expressions of M1-related proteins MHC-Ⅱ (P=0.021) and iNOS (P < 0.001) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.004), increased the mRNA expression of TGF-ß (P=0.002) and IL-10 (P=0.008), and up-regulated the protein expressions of IRE1α (P < 0.001) and its downstream transcription factor XBP-1 (P < 0.001). Addition of the estrogen inhibitor obviously blocked the effect of estrogen. Compared with estrogen treatment alone, combined treatment of the macrophages with estrogen and the IRE1α inhibitor 4 µ 8 C significantly up-regulated the protein expressions of MHC-Ⅱ (P=0.002) and iNOS (P=0.003) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.024), and obviously down-regulated the mRNA expression of TGF-ß (P < 0.001) and IL-10 (P < 0.001); these changes were not observed in cells treated with estrogen and the IRE1α agonist. CONCLUSION: Estrogen can inhibit the differentiation of murine macrophages into a pro-inflammatory phenotype by up-regulating the IRE1α-XBP-1 signaling axis, thereby producing an inhibitory effect on inflammatory response.

Protective Effect of Nanoselenium on Renal Oxidative Damage Induced by Mercury in Laying Hens.

This study investigated the effects of dietary nanoselenium (nano-Se) supplementation protecting from renal oxidative damages induced by mercury (Hg) exposure in laying hens. Furthermore, endoplasmic reticulum (ER) stress pathway was explored to reveal the protective mechanism of nano-Se. A total of 576 40-week-old Hyline-White laying hens were randomly allocated to 4 groups with 6 pens per group and 24 hens per pen. The experimental groups were as follows: control (basal diet), control + 27.0 mg/kg Hg, control + 5.0 mg/kg nano-Se, and Hg27.0 + 5.0 mg/kg nano-Se. The results revealed that dietary Hg exposure significantly reduced laying performance (P < 0.05) and egg quality (P < 0.05), whereas nano-Se supplementation partially reversed the reductions. Besides, dietary Hg exposure could induce histopathology damages and apoptosis in kidney, whereas nano-Se addition could alleviate these toxicities effectively. After Hg exposure, the activities and gene expressions of superoxidative dismutase (SOD) (P < 0.05), catalase (CAT) (P < 0.01), glutathione reductase (GR) (P < 0.05) and glutathione peroxidase (GSH-Px) (P < 0.05), and glutathione (GSH) content (P < 0.05) were significantly decreased, while the malondialdehyde (MDA) level was significantly increased (P < 0.05) in kidney. However, nano-Se supplementation partially reversed the levels and gene expressions of these antioxidant biomarkers in kidney. Furthermore, dietary Hg exposure significantly increased the gene expressions of PERK (P < 0.05), ATF4 (P < 0.05), CHOP (P < 0.05), IRE1α (P < 0.05), TRAF2 (P < 0.05), ASK1 (P < 0.05), Caspase-9 (P < 0.05), Caspase-8 (P < 0.05), Caspase-3 (P < 0.05), and Bax/Bcl-2 (P < 0.05), whereas nano-Se supplementation partially reversed these increases of gene expressions. In summary, this study provides evidence that dietary Hg exposure can induce renal oxidative damages, and elucidates an important role of ER stress pathway in nano-Se alleviating renal apoptosis in laying hens.

Rapamycin regulates the balance between cardiomyocyte apoptosis and autophagy in chronic heart failure by inhibiting mTOR signaling.

The progressive loss of cardiomyocytes caused by cell death leads to cardiac dysfunction and heart failure (HF). Rapamycin has been shown to be cardioprotective in pressure­overloaded and ischemic heart diseases by regulating the mechanistic target of rapamycin (mTOR) signaling network. However, the impact of rapamycin on cardiomyocyte death in chronic HF remains undetermined. Therefore, in the current study we addressed this issue using a rat myocardial infarction (MI)­induced chronic HF model induced by ligating the coronary artery. Following surgery, rats were randomly divided into six groups, including the sham­, vehicle­ and rapamycin­operated groups, at 8 or 12 weeks post­MI. A period of 4 weeks after MI induction, the rats were treated with rapamycin (1.4 mg­kg­day) or vehicle for 4 weeks. Cardiac function was determined using echocardiography, the rats were subsequently euthanized and myocardial tissues were harvested for histological and biochemical analyses. In the cell culture experiments with H9c2 rat cardiomyocytes, apoptosis was induced using angiotensin II (100 nM; 24 h). Cardiomyocyte apoptosis and autophagy were assessed via measuring apoptosis­ and autophagy­associated proteins. The activities of mTOR complex 1 (mTORC1) and mTORC2 were evaluated using the phosphorylation states of ribosomal S6 protein and Akt, respectively. The activity of the endoplasmic reticulum (ER) stress pathway was determined using the levels of GRP78, caspase­12, phospho­JNK and DDIT3. Echocardiographic and histological measurements indicated that rapamycin treatment improved cardiac function and inhibited cardiac remodeling at 8 weeks post­MI. Additionally, rapamycin prevented cardiomyocyte apoptosis and promoted autophagy at 8 weeks post­MI. Rapamycin treatment for 4 weeks inhibited the mTOR and ER stress pathways. Furthermore, rapamycin prevented angiotensin II­induced H9c2 cell apoptosis and promoted autophagy by inhibiting the mTORC1 and ER stress pathways. These results demonstrated that rapamycin reduced cardiomyocyte apoptosis and promoted cardiomyocyte autophagy, by regulating the crosstalk between the mTOR and ER stress pathways in chronic HF.

An in vitro validation of the therapeutic effects of Tougu Xiaotong capsule on tunicamycin-treated chondrocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Tougu Xiaotong capsule (TXC) is a Chinese herbal compound that belongs to a range of Chinese herbs functioning as 'kidney invigorators and liver softeners' commonly used to treat osteoarthritis (OA) in China. AIMS OF THE STUDY: The aims of the present study are to confirm the therapeutic effects of TXC in an OA cell model and to determine the mechanisms involved in such effects. MATERIALS AND METHODS: A tunicamycin (Tm)-exposed OA cell model was employed, and the effects of TXC were confirmed by observing cell viability and apoptosis. The reduced cell viability and increased apoptosis caused by Tm were improved by TXC, confirming the cellular protection of TXC. We then investigated the expression of biomarkers related to the endoplasmic reticulum (ER) stress pathway, including microRNA-211 (miR-211), a regulator in the ER stress pathway. RESULTS: Downregulation of X-box binding protein 1 (Xbp-1) and miR-211 expression following Tm administration was reversed by TXC. Moreover, the upregulation by Tm of the expression levels of binding immunoglobulin protein, Xbp-1, activating transcription factor 4, C/EBP-homologous protein, Caspase-9 and Caspase-3 was downregulated by TXC. These results indicated that the ER stress pathway-related mechanism may play a potential role in the therapeutic effects of TXC. CONCLUSIONS: The present study provides evidence of the therapeutic effects of TXC at the cell level and describes a cellular model for establishing the mechanisms of the effects of TXC used in the treatment of OA.

Astragaloside attenuates the progression of prostate cancer cells through endoplasmic reticulum stress pathways.

Astragaloside (As) has been demonstrated extensively to serve roles in a variety of tumor types, including glioma, lung cancer, colorectal cancer, breast cancer and cervical cancer, and has therefore been widely used in Traditional Chinese Medicine. To the best of our knowledge, the present study was the first to investigate the efficacy of the Traditional Chinese Medicine astragaloside on tumor growth and the apoptosis of prostate cancer cells. In addition, further investigation into the underlying molecular mechanisms via the endoplasmic reticulum (ER) stress pathway was also performed. In the present study, the human prostate cancer DU-145 cell line was employed as an experimental model in vitro and cells were divided into five treatment groups: Dimethyl sulfoxide (DMSO) group (control), low-dose astragaloside group (L-As; 20 nmol/l), moderate-dose astragaloside group (M-As; 50 nmol/l), high-dose astragaloside group (H-As, 100 nmol/l) and ER stress suppressor group (tauroursodeoxycholic acid; TUDCA). The proliferative ability and apoptosis rate of the DU-145 cells were detected via Cell Counting kit-8 methods and flow cytometry, respectively. Furthermore, the ER stress factors [binding immunoglobulin protein (BiP), C/EBP homologous protein (CHOP) and caspase-12] were assessed through reverse transcription polymerase chain reaction. Additionally, the protein expression levels of inositol-requiring enzyme 1 (IRE1), phosphorylated protein kinase R-like ER kinase (p-PERK), iron-regulated transcriptional activator Aft (AFT)4 and AFT6 were measured detected by western blot analysis. Administration of As significantly reduced the cell viability and promoted apoptosis (P<0.05) in a dose-dependent manner. Expression of ER-stressed genes BiP, CHOP and caspase-12 mRNA was increased by As administration, while TUDCA treatment led to a lower mRNA expression of these genes, compared with the control group. Results of western blot analysis indicated that the protein expression of IRE1, AFT4 and AFT6 was upregulated in the H-As group, and that the ratio of p-PERK/PERK was also higher than in the other groups. The administration of As demonstrated significant therapeutic effects on the proliferation of prostate cancer cells, as well as the expression of related proteins and genes. The results of the present study suggested future clinical potential of As for the treatment of prostate cancer.

Estradiol inhibits differentiation of mouse macrophage into a pro-inflammatory phenotype by upregulating the IRE1α-XBP1 signaling axis / 南方医科大学学报

OBJECTIVE@#To explore the mechanism by which estradiol modulates the immunophenotype of macrophages through the endoplasmic reticulum stress pathway.@*METHODS@#Peritoneal macrophages isolated from C57 mice were cultured in the presence of 60 ng/mL interferon-γ (IFN-γ) followed by treatment with estradiol (1.0 nmol/L) alone, estradiol with estrogen receptor antagonist (Acolbifene, 4 nmol/L), estradiol with IRE1α inhibitor (4 μ 8 C), or estradiol with IRE1α agonist. After the treatments, the expression levels of MHC-Ⅱ, iNOS and endoplasmic reticulum stress marker proteins IRE1α, eIF2α and ATF6 in the macrophages were detected with Western blotting, and the mRNA levels of TGF-β, IL-6, IL-10 and TNF-α were detected with RT-PCR.@*RESULTS@#Estrogen treatment of the macrophages significantly decreased the expressions of M1-related proteins MHC-Ⅱ (P=0.021) and iNOS (P < 0.001) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.004), increased the mRNA expression of TGF-β (P=0.002) and IL-10 (P=0.008), and up-regulated the protein expressions of IRE1α (P < 0.001) and its downstream transcription factor XBP-1 (P < 0.001). Addition of the estrogen inhibitor obviously blocked the effect of estrogen. Compared with estrogen treatment alone, combined treatment of the macrophages with estrogen and the IRE1α inhibitor 4 μ 8 C significantly up-regulated the protein expressions of MHC-Ⅱ (P=0.002) and iNOS (P=0.003) and the mRNA expressions of TNF-α (P=0.003) and IL-6 (P=0.024), and obviously down-regulated the mRNA expression of TGF-β (P < 0.001) and IL-10 (P < 0.001); these changes were not observed in cells treated with estrogen and the IRE1α agonist.@*CONCLUSION@#Estrogen can inhibit the differentiation of murine macrophages into a pro-inflammatory phenotype by up-regulating the IRE1α-XBP-1 signaling axis, thereby producing an inhibitory effect on inflammatory response.

Apoptotic pathways of macrophages within osteolytic interface membrane in periprosthestic osteolysis after total hip replacement.

Macrophage apoptosis in interface membrane, which occurs through either death receptor, mitochondrion, or endoplasmic reticulum (ER) stress pathways, has been suggested to play an important role in promoting osteolysis. However, how and why macrophage apoptosis originates and the correlation among these apoptotic pathways is not yet clear. The objective of this study was to identify the apoptotic mechanism of macrophages, and to explore the relationship between the apoptotic pathways and progression of osteolysis. Transmission electron microscopy (TEM) was utilized to analyze the tissue ultrastructure of wear particles, and in situ apoptotic macrophage identification was performed by TUNEL staining. We analyzed the expression of the key biomarkers of apoptotic pathways via immunohistochemistry and Western blotting. Our results demonstrated that the majority of wear particles within osteolytic interface membrane was in the 30-60 nm range, and that macrophage apoptotic ratio increased along with osteolysis progression. Normal hip dysplasia and mechanical loosening of tissues showed low expression levels of biomarkers for ER stress (Ca2+ , JNK, cleaved Caspase-4, IRE1-α, Grp78/Bip, and CHOP), mitochondrion (Bcl-2, Bax, and Cytochrome c), and death receptor (Fas and cleaved Caspase-8) pathways, while osteolytic interface membrane tissues expressed high levels of these biomarkers. In addition, we found that the ER stress intensity was in complete conformity with mitochondrial dysfunction and was consistent with the results of death receptor activation. Thus, our findings suggested that wear particles generated at implant interface can accelerate macrophage apoptosis through changes in apoptotic pathways and ultimately aggravate the symptom of osteolysis. These data represent a preferential apoptotic signaling pathway of macrophages as specific target points for the prevention and therapeutic modulation of periprosthetic osteolysis.

Study on the Effect Mechanism of Diazoxide on the Proliferation and Apoptosis of Chondrocytes with Oxidative Injury / 中国药房

OBJECTIVE： To investigate the effect mechanism of diazoxide on the proliferation and apoptosis of chondrocytes with oxidative injury based on endoplasmic reticulum stress （ERS） pathway. METHODS： SD mice were selected for primary culture of articular chondrocytes. The 3rd generation chondrocytes were randomly divided into control group， injury model group and diazoxide group. Control group didn’t receive any treatment. The injury model group was incubated with 300 μmol/L hydrogen peroxide （H2O2） at 37 ℃ for 8 h. Diazoxide group was pretreated with 300 μmol/L diazoxide at 37 ℃ for 0.5 h，and then incubated with 300 μmol/L H2O2 for 8 h. The proliferation of chondrocytes was detected by CCK-8 assay. The apoptosis rate of chondrocytes was detected by flow cytometry. The expression of ERS-related proteins [Caspase-3， Bcl-2-associated X（Bax），C/EBP homologous protein （CHOP）] were detected by Western blotting assay. RESULTS： Compared with control group， the proliferation activity of chondrocytes in injury model group was significantly decreased， while apoptosis rate was increased significantly（P＜0.05）；the protein expression of Caspase-3， Bax and CHOP were increased significantly （P＜0.05）. Compared with injury model group， the proliferation activity of chondrocytes in diazoxide group was increased significantly， while the apoptosis rate was decreased significantly （P＜0.05）； the expression of above related proteins were decreased significantly （P＜0.05）. CONCLUSIONS： Diazoxide can improve the proliferation activity of chondrocytes with oxidative injury and inhibit their apoptosis by inhibiting ERS pathway.

Mechanism of endoplasmic reticulum stress pathway inhibitor salubrinal enhancing the apoptosis of head and neck squamous carcinoma cells / 中华放射肿瘤学杂志

Objective To explore the mechanism underlying the effect of endoplasmic reticulum stress pathway inhibitor Salubrinal on enhancing the apoptosis of head and neck squamous carcinoma cells.Methods Three types of head and neck squamous carcinoma cell lines (KB,Fadu,Detroit562) were divided into the control,Salburinal (sal),irradiation (IR) and sal combined with IR (IR+sal) groups.The expression levels of p-ATM/ATM,DNA-PK and cleaved Caspase-3 were quantitatively measured.The cell apoptosis rate was detected among four groups.The effect of Salburinal on cell viability was evaluated by MTT assay.Results Compared with the IR group,the expression level of p-ATM/ATM (t =3.5,8.43 and 9.42,all P＜0.05) was significantly up-regulated,whereas that of DNA-PK (t =9.19,17.44,16.67,all P＜ 0.05) was considerably down-regulated in the IR+sal group.The expression level of cleaved Caspase-3 in the IR+sal group was significantly higher compared with those in the other three groups (t=6.79,9.76 and 9.7g,all P＜0.05).Compared with the IR group,the cell apoptosis rate was significantly enhanced in the IR +sal group (t=5.67,6.95 and 7.28,all P＜0.05).Salubrinal exerted an effect upon the apoptosis of three cell lines in a concentration-and time-dependent manner.Conclusions As an endoplasmic reticulum stress pathway inhibitor,Salubrinal can enhance the apoptosis rate of head and neck squamous carcinoma cells.The underlying mechanism is probably correlated with irradiation-induced DNA double strand injury,suppressing the repairing of DNA damage and thereby increasing the apoptosis of tumor cells.

Endoplasmic reticulum stress inhibitor salubrinal enhanced radiosensitivity of head and neck carcinoma cells / 中华放射医学与防护杂志

Objective To explore the effect of salubrinal (sal,an endoplasmic reticulum stress inhibitor) on radiosensitivity of human head and neck squamous carcinoma cells (HNSCC).Methods Cells were divided into two groups of sal treatment and its control.For drug treatment group,cells were treated with 10 mmol/L sal for different time (12,24,36 h) and then irradiated.The levels of a core protein GRP78 of endoplasmic reticulum stress (ERS) in HNSCC (KB,Fadu,and Detroit 562 cells)were analyzed by Western blot assay at different time (0,20 min,1 h,3 h,6 h,12 h,24 h and 48 h) after irradiation.Cell survival was measured with colony formation assay.Results Western blot assay revealed that the protein levels of GRP78 in three kinds of HNSCC significantly increased from 20 min to 1 h and peaked at 3 h after radiation (t =12.72,13.37,18.31,P ＜ 0.05).Compared with the control group,treatment of cells with sal decreased GRP78 protein levels (t =14.25,5.34,3.12,P ＜ 0.05) in three cell lines and also significantly enhanced radiation damage and reduced cell viability.The sensitization enhancement ratios (SER) of sal in three cell lines were 1.16,1.05 and 1.06,respectively.Conclusions Rradiosensitivity of HNSCC could be effectively enhanced by sal treatment.