Reactive Oxygen Species Scavenger NAC Research Articles

Low shear stress induced vascular endothelial cell pyroptosis by TET2/SDHB/ROS pathway

Vascular endothelial cell (VEC) inflammation induced by low shear stress plays key roles in the initiation and progression of atherosclerosis (As). Pyroptosis is a form of inflammatory programmed cell death that is critical for As. However, the effect of low shear stress on VEC pyroptosis and the underlying mechanisms were not clear. Here we show that low shear stress promoted VEC pyroptosis and reduced the expression of Ten-Eleven Translocation 2 (TET2) methylcytosine dioxygenase. Loss of TET2 resulted in the upregulation of the expression and activity of mitochondrial respiratory complex II subunit succinate dehydrogenase B (SDHB) by decreasing the recruitment of histone deacetylase 2, independent of DNA demethylation modification. The overexpression of SDHB mediated mitochondrial injury and increased the production of reactive oxygen species (ROS). The administration of ROS scavenger NAC alleviated VEC pyroptosis induced by SDHB overexpression and TET2 shRNA. These findings show that low shear stress induced endothelial cell pyroptosis through the TET2/SDHB/ROS pathway and offer new insights into As.

Antrodia camphorata extract (ACE)-induced apoptosis is associated with BMP4 expression and p53-dependent ROS generation in human colon cancer cells

Ethnopharmacological relevanceAntrodia camphorata (AC) is a rare functional fungus in Taiwan and is known as traditional Chinese medicine. It has been reported to inhibit proliferation and promote apoptosis in human cancer cells.Aim of the study: To investigate the potential mechanism of apoptosis induced in colon cancer cells by Antrodia camphorata extract (ACE). Materials and methodsThe MTT assay and crystal violet staining were used to determine relative cell viability in vitro at 24 and 48 h. The effects of ACE on apoptosis were determined by Hoechst 33342 staining and flow cytometric analysis following Annexin V-FITC/PI staining. The gene expression profile of HCT116 cells was assessed by the RNA sequencing system. In combination with RNA-seq data and qRT-PCR, Western blot analysis was used to evaluate expression of proteins. The intracellular ROS of HCT116 cells were determined using a DCFH-DA fluorescence probe. ResultsACE significantly reduces cell viability in a dose-dependent manner and triggers apoptosis. To explore the underlying mechanism, we performed transcriptome analysis of ACE-treated colon cancer HCT116 cells. Bioinformatics analyses showed that ACE treatment is associated with pathways in cancer. We further used Cytoscape to analyze hub genes in this network. Among them, BMP4, which is associated with cancer cell death through regulation of the tumor suppressor p53, was significantly decreased at both mRNA and protein levels in ACE treatment groups. We found that cell death is reversible via inactivation or knockdown of p53 gene and reduction of reactive oxygen species (ROS) generation in response to ACE exposure, indicating that p53 plays an important role in ROS generation induced by ACE. Meanwhile, ROS scavenger NAC was used to verify that cell death is reversible via reduction of ROS. ConclusionOur findings demonstrate that ACE has potential as an anticancer agent that induces apoptosis through BMP4 and p53-dependent response to ROS in human colon cancer.

A novel Diels-Alder adduct of mulberry leaves exerts anticancer effect through autophagy-mediated cell death.

Guangsangon E (GSE) is a novel Diels-Alder adduct isolated from leaves of Morus alba L, a traditional Chinese medicine widely applied in respiratory diseases. It is reported that GSE has cytotoxic effect on cancer cells. In our research, we investigated its anticancer effect on respiratory cancer and revealed that GSE induces autophagy and apoptosis in lung and nasopharyngeal cancer cells. We first observed that GSE inhibits cell proliferation and induces apoptosis in A549 and CNE1 cells. Meanwhile, the upregulation of autophagosome marker LC3 and increased formation of GFP-LC3 puncta demonstrates the induction of autophagy in GSE-treated cells. Moreover, GSE increases the autophagy flux by enhancing lysosomal activity and the fusion of autophagosomes and lysosomes. Next, we investigated that endoplasmic reticulum (ER) stress is involved in autophagy induction by GSE. GSE activates the ER stress through reactive oxygen species (ROS) accumulation, which can be blocked by ROS scavenger NAC. Finally, inhibition of autophagy attenuates GSE-caused cell death, termed as "autophagy-mediated cell death." Taken together, we revealed the molecular mechanism of GSE against respiratory cancer, which demonstrates great potential of GSE in the treatment of representative cancer.

Erastin induces apoptotic and ferroptotic cell death by inducing ROS accumulation by causing mitochondrial dysfunction in gastric cancer cell HGC‑27.

Erastin, a classical inducer of non-apoptotic cell death, exerts cytotoxicity in several types of cancer cells, including gastric cancer cells, by depleting glutathione, which is a primary cellular antioxidant, thus causing reactive oxygen species (ROS) accumulation. Although numerous studies have focused on the non-apoptotic cell death induced by erastin, whether erastin induces apoptosis remains unknown. The present study confirmed the cytotoxicity of erastin in HGC-27 cells and used a 30% inhibitory concentration (IC30, approximately 6.23 µM) for further analysis. The cell cycle analysis revealed that 6.23 µM of erastin inhibited proliferation by blocking the cell cycle at the G1/G0 phase. Further analysis also showed that 6.23 µM of erastin clearly inhibited HGC-27 malignant behaviors, including migration, invasion, colony formation and tumor formation in soft agar. The observation of ROS accumulation due to erastin treatment led to determination of the effects of erastin on mitochondrial function and, as expected, erastin treatment decreased transcriptional activity and ATP production in mitochondria and disrupted the mitochondrial potential; these effects were reversed by the addition of the ROS scavenger NAC. To evaluate the effect of erastin in inducing apoptosis, HGC-27 cells were treated with 6.23 µM of erastin for 7 days and then analyzed. Evident apoptotic cell death was induced by erastin and this apoptosis was reversed by the addition of an apoptosis inhibitor (zVAD) or NAC but not by the addition of a ferroptosis inhibitor (ferrostatin-1). Furthermore, the detection of caspase-3 and poly (adenosine diphosphate-ribose) polymerase (PARP) also confirmed that treatment with erastin promoted the cleavage of caspase-3 and PARP, which are hallmarks of apoptosis. Taken together, the present study revealed that a low dose of erastin inhibited malignant behavior and induced apoptosis by causing mitochondrial dysfunction.

A TOR2A Gene Product: Salusin-β Contributes to Attenuated Vasodilatation of Spontaneously Hypertensive Rats.

Attenuated vasodilatation of small arteries is a hallmark feature of hypertension. Salusin-β, which is a TOR2A gene product and an important vasoactive peptide, has a close relationship with cardiovascular disease. This study aimed to determinate the roles of salusin-β in vasodilatation, and its signal pathways in Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Isometric tension experiments were performed. Vasodilatation was induced by acetylcholine (ACh) or sodium nitroprusside (SNP). Plasma salusin-β levels and their protein expressions in coronary artery (CA), mesenteric artery (MA), and pulmonary artery (PA) of SHR were much higher than that of WKY. Intravenous injection of salusin-β increased arterial blood pressure in SHR, while anti-salusin-β IgG decreased it. Salusin-β further deteriorated, while anti-salusin-β IgG improved, the attenuated ACh-induced relaxation, the decreased nitric oxide (NO) level, and endothelial nitric oxide synthase (eNOS) activity in arteries of SHR, and salusin-β had no significant effect on SNP-induced relaxation. The NAD(P)H oxidase activity and reactive oxygen species (ROS) level in arteries of SHR were much higher than that of WKY, which was further increased by salusin-β but reduced by anti-salusin-β IgG. ROS scavenger NAC or antioxidant apocynin significantly inhibited, while SOD inhibitor DETC aggravated, the effects of salusin-β, and the eNOS inhibitor L-NAME inhibited the effects of anti-salusin-β IgG. These results indicated that enhanced salusin-β activity is involved in attenuated endothelium-dependent vasodilatation pathogenesis in SHR by activating NAD(P)H oxidase derived ROS generation and inhibiting eNOS activation and NO release.

High cholesterol induces apoptosis and autophagy through the ROS-activated AKT/FOXO1 pathway in tendon-derived stem cells

BackgroundHypercholesterolemia increases the risk of tendon pain and tendon rupture. Tendon-derived stem cells (TDSCs) play a vital role in the development of tendinopathy. Our previous research found that high cholesterol inhibits tendon-related gene expression in TDSCs. Whether high cholesterol has other biological effects on TDSCs remains unknown.MethodsTDSCs isolated from female SD rats were exposed to 10 mg/dL cholesterol for 24 h. Then, cell apoptosis was assessed using flow cytometry and fluorescence microscope. RFP-GFP-LC3 adenovirus transfection was used for measuring autophagy. Signaling transduction was measured by immunofluorescence and immunoblotting. In addition, Achilles tendons from ApoE −/− mice fed with a high-fat diet were histologically assessed using HE staining and immunohistochemistry.ResultsIn this work, we verified that 10 mg/dL cholesterol suppressed cell proliferation and migration and induced G0/G1 phase arrest. Additionally, cholesterol induced apoptosis and autophagy simultaneously in TDSCs. Apoptosis induction was related to increased expression of cleaved caspase-3 and BAX and decreased expression of Bcl-xL. The occurrence of autophagic flux and accumulation of LC3-II demonstrated the induction of autophagy by cholesterol. Compared with the effects of cholesterol treatment alone, the autophagy inhibitor 3-methyladenine (3-MA) enhanced apoptosis, while the apoptosis inhibitor Z-VAD-FMK diminished cholesterol-induced autophagy. Moreover, cholesterol triggered reactive oxygen species (ROS) generation and activated the AKT/FOXO1 pathway, while the ROS scavenger NAC blocked cholesterol-induced activation of the AKT/FOXO1 pathway. NAC and the FOXO1 inhibitor AS1842856 rescued the apoptosis and autophagy induced by cholesterol. Finally, high cholesterol elevated the expression of cleaved caspase-3, Bax, LC3-II, and FOXO1 in vivo.ConclusionThe present study indicated that high cholesterol induced apoptosis and autophagy through ROS-activated AKT/FOXO1 signaling in TDSCs, providing new insights into the mechanism of hypercholesterolemia-induced tendinopathy.Graphical abstractHigh cholesterol induces apoptosis and autophagy through the ROS-activated AKT/FOXO1 pathway in tendon-derived stem cells.

Ginkgolic acid (GA) suppresses gastric cancer growth by inducing apoptosis and suppressing STAT3/JAK2 signaling regulated by ROS

Gastric cancer is a frequently occurring cancer with high mortality each year worldwide. Finding new and effective therapeutic strategy against human gastric cancer is still urgently required. Ginkgolic acid (GA), a botanical drug, is extracted from the seed coat of Ginkgo biloba L. with various bioactive properties, including anti-tumor. Unfortunately, if GA has antitumor effect on human gastric cancer and the underlying molecular mechanisms have yet to be investigated. In the present study, we found that GA markedly reduced the gastric cancer cell viability. Furthermore, GA treatment led to the reduced migration ability of gastric cancer cells, which was associated with the decreased protein expression levels of Rho-associated protein kinase 1 (ROCK1), matrix metalloproteinase-2 (MMP-2), MMP-9 and α-smooth muscle actin (α-SMA). In addition, GA dose-dependently induced apoptosis in gastric cancer cells through activating Caspase-9/-3 and poly(ADP-Ribose) polymerase (PARP), which was along with the reduced Bcl-2 and Bcl-xl expression levels, and the elevated Bax and Bad levels. Consistently, Cyto-c protein expression in cytoplasm was also up-regulated by GA. Moreover, the production of reactive oxygen species (ROS) was significantly induced by GA. The activation of signal transducer and activator of transcription 3/janus kinase 2 (Stat3/JAK2) signaling pathway was inhibited by GA treatment. Intriguingly, blocking Stat3/JAK2 activation could further promote apoptosis and reduce cell viability induced by GA. However, GA-induced cell death was clearly abolished by ROS scavenger NAC, while the activation of Stat3/JAK2 signaling was restored by NAC. In vivo, GA showed effective role in reducing gastric tumor growth. Together, the findings here indicated that GA could be considered as an effective therapeutic candidate against human gastric cancer progression in future.

Trimethylamine N-oxide promotes apoE-/- mice atherosclerosis by inducing vascular endothelial cell pyroptosis via the SDHB/ROS pathway.

Trimethylamine N-oxide (TMAO) is produced from the phosphatidylcholine metabolism of gut flora and acts as a risk factor of cardiovascular disease. However, the underlying mechanisms for its proatherogenic action remain unclear. This study aimed to observe the effect of TMAO on endothelial cell pyroptosis and explore the underlying mechanisms. Our results showed that TMAO promoted the progression of atherosclerotic lesions in apolipoprotein E-deficient (apoE-/- ) mice fed a high-fat diet. Pyroptosis and succinate dehydrogenase complex subunit B (SDHB) upregulation were detected in the vascular endothelial cells of apoE-/- mice and in cultured human umbilical vein endothelial cells (HUVECs) treated with TMAO. Overexpression of SDHB in HUVECs enhanced pyroptosis and impaired mitochondria and high reactive oxygen species (ROS) level. Pyroptosis in the SDHB overexpression of endothelial cells was inhibited by the ROS scavenger NAC. In summary, TMAO promotes vascular endothelial cell pyroptosis via ROS induced through SDHB upregulation, thereby contributing to the progression of atherosclerotic lesions.

MPPa-PDT suppresses breast tumor migration/invasion by inhibiting Akt-NF-\u03baB-dependent MMP-9 expression via ROS

BackgroundBreast cancer is one of the most commonly diagnosed cancers in women, with high morbidity and mortality. Tumor metastasis is implicated in most breast cancer deaths; thus, inhibiting metastasis may provide a therapeutic direction for breast cancer. In the present study, pyropheophorbide-α methyl ester-mediated photodynamic therapy (MPPa-PDT) was used to inhibit metastasis in MCF-7 breast cancer cells.MethodsUptake of MPPa was detected by fluorescence microscopy. Cell viability was evaluated by the Cell Counting Kit-8 (CCK-8). ROS generation was detected by 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). The migration of cells was assessed by wound healing assay, and invasion ability was assessed by Matrigel invasion assay. Levels of MMP2 and MMP9 were measured by PCR. Akt, phospho-Akt (Ser473), phospho-NF-κB p65 (Ser536) and NF-κB p65 were measured by western blotting. The F-actin cytoskeleton was observed by immunofluorescence. Lung tissue was visualized by hematoxylin and eosin staining.ResultsFollowing MPPa-PDT, migration and invasion were decreased in the MCF-7 cells. MPPa-PDT downregulated the expression of MMP2 and MMP9, which are responsible for the initiation of metastasis. MPPa-PDT reduced the phosphorylation of Akt and NF-κB. MPPa-PDT also reduced the expression of F-actin in cytoskeleton in MCF-7 cells. These effects were blocked by the reactive oxygen species scavenger NAC or the Akt activator SC79, while the PI3K inhibitor LY294002 or the Akt inhibitor triciribine enhanced these effects. Moreover, MPPa-PDT inhibited tumor metastasis and destroyed F-actin in vivo.ConclusionTaken together, these results demonstrate that MPPa-PDT inhibits the metastasis of MCF-7 cells both in vitro and in vivo and may be involved in the Akt/NF-κB-dependent MMP-9 signaling pathway. Thus, MPPa-PDT may be a promising treatment to inhibit metastasis.

Polychlorinated biphenyl quinone promotes macrophage polarization to CD163+ cells through Nrf2 signaling pathway

Polychlorinated biphenyls (PCBs) are notorious environmental pollutants. For their hydrophobic and lipophilic capability, they are wildly spread to environment to threat human health thus attracts more attention. In this study, we observed increasing numbers of CD163 positive (CD163+) macrophages in aortic valve of ApoE-/- mice after 2,3,5-trichloro-6-phenyl-[1,4]-benzoquinone (PCB29-pQ) treatment, the metabolite of polychlorinated biphenyl. In addition, in vitro studies identified that PCB29-pQ exposure significantly provoked the shifting of RAW264.7 macrophages and bone marrow derived monocytes (BMDMs) to CD163+ macrophages. Upon PCB29-pQ administration, CD163 and CD206 levels were enhanced in RAW264.7 cells as well as in BMDMs. However, the concentration of iron and total cholesterol (TC) were reduced due to the boosting of ferroportin (Fpn) and ATP binding cassette transporter, subfamily A, member 1 (ABCA1) which are efflux transporters of iron and cholesterol individually. Further investigation on mechanism indicated that PCB29-pQ exposure induced reactive oxygen species (ROS), which may result in activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a protein responsible for macrophage polarization. After that, we blocked Nrf2 through Nrf2 shRNA and ROS scavenger NAC, which significantly reversed the shifting of macrophage to CD163+ sub-population. These results confirmed the importance of Nrf2 in inducing macrophage polarization. In short, our study uncovered that PCB29-pQ could promote macrophage/monocyte polarization to CD163+ macrophage which would be a potential incentive to accelerate atherosclerosis through Nrf2 signaling pathway.

Ubenimex induces apoptotic and autophagic cell death in rat GH3 and MMQ cells through the ROS/ERK pathway.

PurposeUbenimex, an aminopeptidase N (APN) inhibitor, is widely known for its use as an adjunct therapy for cancer therapy. However, in recent studies, it has also conferred antitumour effects in many cancers, but its anticancer mechanism is largely unknown. This study aims to investigate the specific anticancer activities and mechanisms of ubenimex in GH3 and MMQ cells.Materials and methodsIn this study, we investigated the anticancer effects of ubenimex in GH3 and MMQ cells. Cell viability and cell death were assessed by the Cell Counting Kit-8 kit (CCK-8) and a LIVE/DEAD cell imaging kit. Apoptosis and intracellular reactive oxygen species (ROS) generation were assessed by flow cytometry and fluorescence microscopy. Autophagosome formation was detected by transmission electron microscopy, and autophagic flux was measured with mRFP-GFP-LC3 adenoviral transfection. The protein expression level was detected by Western blotting.ResultsThe results revealed that treatment with ubenimex induced apoptotic and autophagic cell death in GH3 and MMQ cells, which resulted in decreased viability, an increased proportion of apoptotic cells, and autophagosome formation. Further experiments showed that ubenimex induced ROS generation and activated the ROS/ERK pathway. The ROS scavenger NAC could attenuate ubenimex-induced apoptosis and autophagy.ConclusionOur studies revealed that ubenimex exerted anticancer effects by inducing apoptotic and autophagic cell death in GH3 and MMQ cells, rendering it a possible effective adjunctive therapy for pituitary treatment.

TMP21 regulates autophagy by modulating ROS production and mTOR activation

Autophagy is a catabolic cellular response to stress that has been liked to various human diseases. However, the precise involvement of autophagy in health and disease remains unclear. To explore the molecular mechanisms of autophagy, we investigated the effect of TMP21. We found that the down-regulation of TMP21 induced autophagy in SH-SY5Y cells. In addition, the enhanced autophagy observed upon TMP21 depletion was almost completely blocked in ATG5 knockout (KO) or ATG7-KO HeLa cells. Silencing of TMP21 in SH-SY5Y cells also increased the production of cellular reactive oxygen species (ROS). Accordingly, treatment with the ROS scavenger NAC suppressed autophagy activation as well as ROS production in TMP21-depleted cells. In addition, the inhibition of mTOR by treatment with Torin1 was mitigated in TMP21 overexpressing cells compared with that in control cells. Taken together, these results indicated that TMP21 could regulate autophagy by modulating ROS production and mTOR activation.

Quinalizarin induces cycle arrest and apoptosis via reactive oxygen species-mediated signaling pathways in human melanoma A375 cells.

Quinalizarin, a bioactive and highly selective compound, is known to promote apoptosis in colon and lung cancer cells. However, studies evaluating quinalizarin-induced apoptosis in melanoma cells have not been conducted. In the present study, we investigated the underlying mechanisms of antimelanoma activity of quinalizarin in human melanoma A375 cells. The MTT assay and Trypan blue staining were used to evaluate the cell viability. The flow cytometry was used to detect cell cycle, apoptosis and reactive oxygen species (ROS). Western blot was used to detect the expression of cell cycle and apoptosis-related proteins, MAPK, and STAT3. The results revealed a significant dose and time dependent effect of quinalizarin on inhibiting proliferation in three kinds of human melanoma cells, and had no significant toxic effects on normal cells. Moreover, quinalizarin triggered G2/M phase cell arrest by modulating the protein expression levels of CDK 1/2, cyclin A, cyclin B, p21 and p27, and induced apoptosis by down-regulating the antiapoptotic protein Bcl-2 and upregulating the proapoptotic protein BAD, leading to the activation of caspase-3 and PARP in the caspase cascade in A375 cells. Quinalizarin treatment led to apoptosis of A375 cells via activation of MAPK and inhibition of STAT3 signaling pathways. In addition, quinalizarin increased the level of ROS, but ROS scavenger NAC inhibited quinalizarin-induced apoptosis by regulating MAPK and STAT3 signaling pathways. In summary, quinalizarin induces cell cycle arrest and apoptosis via ROS-mediated MAPK and STAT3 signaling pathways in human melanoma A375 cells, and quinalizarin may be used as a novel and effective antimelanoma therapeutic.

Two novel 1,4‑naphthoquinone derivatives induce human gastric cancer cell apoptosis and cell cycle arrest by regulating reactive oxygen species‑mediated MAPK/Akt/STAT3 signaling pathways.

1,4-Naphthoquinone derivatives have superior anticancer effects, but their use has been severely limited in clinical practice due to adverse side effects. To reduce the side effects and extend the anticancer effects of 1,4-naphthoquinone derivatives, 2-(butane-1-sulfinyl)-1,4-naphthoquinone (BQ) and 2-(octane-1-sulfinyl)-1,4-naphthoquinone (OQ) were synthesized, and their anticancer activities were investigated. The anti-proliferation effects, determined by MTT assays, showed that BQ and OQ significantly inhibited the viability of gastric cancer cells and had no significant cytotoxic effect on normal cell lines. The apoptotic effect was determined by flow cytometry, and the results showed that BQ and OQ induced cell apoptosis by regulating the mitochondrial pathway and cell cycle arrest at the G2/M phase via inhibition of the Akt signaling pathway in AGS cells. Furthermore, BQ and OQ significantly increased the levels of reactive oxygen species (ROS) and this effect was blocked by the ROS scavenger NAC in AGS cells. BQ and OQ induced apoptosis by upregulating the protein expression of p38 and JNK and downregulating the levels of ERK and STAT3. Furthermore, expression levels of these proteins were also blocked after NAC treatment. These results demonstrated that BQ and OQ induced apoptosis and cell cycle arrest at the G2/M phase in AGS cells by stimulating ROS generation, which caused subsequent activation of MAPK, Akt and STAT3 signaling pathways. Thus, BQ and OQ may serve as potential therapeutic agents for the treatment of human gastric cancer.

Transmural pressure drives proliferation of human arterial smooth muscle cells via mechanism associated with NADPH oxidase and Survivin

Proliferation of vascular smooth muscle cells (VSMCs) plays an important role in various vascular diseases. Abnormal hemodynamic factors are important stimulus for promoting proliferation of VSMCs. In this study, we show that transmural pressure (TP) promotes the proliferation of human arterial smooth muscle cells (HASMCs) and its related mechanism. HASMCs were treated with different TPs (0,100,120,140,160,180 and 200 mmHg) in a custom-made pressure loading apparatus for 6 h. Results showed that proliferation of HASMCs was significantly promoted when the TP was over 160 mmHg compared with 0 mmHg (atmosphere pressure). In like manner, the expressions of NADPH oxidase 2(Nox2) and Survivin (SVV) and production of intracellular reactive oxygen species (ROS) were all elevated distinctly when TP exceeded 160 mmHg. Moreover, ROS scavenger NAC reduced TP-induced proliferation of HASMCs and expression of SVV largely, and slightly down-regulated expression of NOX2. NOX inhibitor apocynin (Apo) also significantly reduced TP-induced proliferation of HASMCs and expression of SVV and almost completely eliminated TP-induced production of ROS. These results demonstrate that TP drives proliferation of HASMCs via mechanism associated with NOX and SVV.

Hsp90 Inhibitor SNX-2112 Enhances TRAIL-Induced Apoptosis of Human Cervical Cancer Cells via the ROS-Mediated JNK-p53-Autophagy-DR5 Pathway.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent cancer cell apoptosis-inducing factor that can induce apoptosis in a variety of cancer cells. However, resistance to TRAIL in cancer cells is a huge obstacle in creating effective TRAIL-targeted clinical therapies. Thus, agents that can either enhance the effect of TRAIL or overcome its resistance are needed. In this study, we combined TRAIL with SNX-2112, an Hsp90 inhibitor we previously developed, to explore the effect and mechanism that SNX-2112 enhanced TRAIL-induced apoptosis in cervical cancer cells. Our results showed that SNX-2112 markedly enhanced TRAIL-induced cytotoxicity in HeLa cells, and this combination was found to be synergistic. Additionally, we found that SNX-2112 sensitized TRAIL-mediated apoptosis caspase-dependently in TRAIL-resistant HeLa cells. Mechanismly, SNX-2112 downregulated antiapoptosis proteins, including Bcl-2, Bcl-XL, and FLIP, promoted the accumulation of reactive oxygen species (ROS), and increased the expression levels of p-JNK and p53. ROS scavenger NAC rescued SNX-2112/TRAIL-induced apoptosis and suppressed SNX-2112-induced p-JNK and p53. Moreover, SNX-2112 induced the upregulation of death-receptor DR5 in HeLa cells. The silencing of DR5 by siRNA significantly decreased cell apoptosis by the combined effect of SNX-2112 and TRAIL. In addition, SNX-2112 inhibited the Akt/mTOR signaling pathway and induced autophagy in HeLa cells. The blockage of autophagy by bafilomycin A1 or Atg7 siRNA abolished SNX-2112-induced upregulation of DR5. Meanwhile, ROS scavenger NAC, JNK inhibitor SP600125, and p53 inhibitor PFTα were used to verify that autophagy-mediated upregulation of DR5 was regulated by the SNX-2112-stimulated activation of the ROS-JNK-p53 signaling pathway. Thus, the combination of SNX-2112 and TRAIL may provide a novel strategy for the treatment of human cervical cancer by overcoming cellular mechanisms of apoptosis resistance.

2-Bromopalmitate sensitizes osteosarcoma cells to adriamycin-induced apoptosis via the modulation of CHOP

Osteosarcoma is the most common primary malignant bone tumour, but the survival rate of patients has plateaued since the mid-1980s. Adriamycin is an integral component of the current first-line chemotherapies used for osteosarcoma, but dose-dependent severe side effects often limit its clinical application. Here, we propose a potential combination regimen in which adriamycin plus 2-bromopalmitate, a palmitoylation inhibitor, exhibited powerful therapeutic effects on osteosarcoma. First, 2-bromopalmitate strongly increased the proliferation inhibition of adriamycin in both human osteosarcoma cell lines and primary osteosarcoma cells. Adriamycin-induced apoptosis in osteosarcoma cells was enhanced when synergized with 2-bromopalmitate. Our study indicated that the reactive oxygen species scavenger NAC and GSH could largely reverse the apoptosis induced by adriamycin combined with 2-bromopalmitate, demonstrating that reactive oxygen species played an essential role in this combination therapy. Moreover, CHOP was remarkably elevated in the combination group, and silencing of CHOP almost completely blocked the apoptosis induced by the combination of 2-bromopalmitate and adriamycin. Taken together, our study provides a prospective therapeutic strategy to eliminate osteosarcoma, which is propitious to clinical combination therapy development.

Apoptosis and autophagy induced by DVDMs-PDT on human esophageal cancer Eca-109 cells.

Esophageal cancer is a common gastrointestinal cancer. About 300,000 people die from esophageal cancer every year in the world. Photodynamic therapy (PDT) has attracted attention as a feasible cancer therap for this diagnosis. Sinoporphyrin sodium (DVDMs) is a novel sensitizer isolated from photofrin. In this study, we aimed to investigate the effects of DVDMs mediated photodynamic therapy and the possible mechanism on human esophageal cancer Eca-109 cells. Cell viability was measured by MTT assay and cell apoptosis was determined by Annexin V-PE/7-AAD and western blot. MDC staining and western blot were used to evaluate cell autophagy. The production of intracellular reactive oxygen species (ROS) was detected by flow cytometry. The expression of MAPK and HO-1 were detected by western blot. DVDMs-PDT decreased cell viability and induced cell apoptosis and autophagy. Autophagy inhibition reduced cell apoptosis triggered by DVDMs-PDT in Eca-109 cells. Generation of ROS was detected in DVDMs-PDT group. p38MAPK, JNK and HO-1 were activated after PDT treatment and the activation were reversed by adding ROS scavenger NAC. Our studies demonstrated that DVDMs-PDT induced apoptosis and autophagy in Eca-109 cells. DVDMs-PDT induced ROS generation in Eca-109 cells, and the generation of ROS activated p38MAPK and JNK. Activation of p38MAPK and JNK may be involved in PDT-induced apoptosis.

Hypoxia Induces Inflammatory Response and Cell Death by Promoting NLRP3 Inflammsome Activation via ROS-TXNIP Axis in Pancreatic β cells

Introduction Hypoxia is the major obstruction in improving the efficiency of islet transplantation and it is involved in the pathogenesis of type 2 diabetes. Activation of inflammasomes in ischemic condition is identified in various tissues. Here we investigated whether hypoxia activates the inflammasome in β cells and the role of inflammsome plays in hypoxic stress. Methods Neonatal porcine islet cell cluster (NICC) and mouse insulinoma cell line 6 (MIN6) cells were exposed to hypoxia(1% O2) for 8-48hrs. MTS and Annexin V/PI FCA was performed to assess the β cell viability and apoptosis. NICC cell death was measured by AO/PI staining. IL-1β and HMGB1 releasing was detected by ELISA. Protein level of inflammsome component NLRP3, ASC, cleaved caspase-1, TXNIP and precursor/mature form of IL-1β and IL-18 was measured by Western blot. NLRP3, ASC and TXNIP were knocked down using shRNA. NAC were used as a ROS scavenger to investigate the underlying mechanism. Results Both in NICC and MIN6, hypoxia(1% O2) time dependently increased NLRP3 and ASC protein level associated with up-regulation of NF-κB signaling. In NICC, hypoxia directly activated NLRP3 inflammasome characterized by increasing protein level of cleaved caspase-1 and mature form of IL-1β and IL-18, while in MIN6, it worked only in the presence of a small dose of LPS(10ng/ml). Besides, hypoxia increased extracellular level of IL-1β and HMGB1 in MIN6. Knockdown of NLRP3 and ASC markedly reduced mature form of IL-1β and decreased HMGB1 releasing after exposure to hypoxia, and it partially protected against hypoxia-induced decrease of β cell viability. Additionally, NLRP3 inflammasome inhibitor parthenolide significantly increased β cell viability in hypoxia. Finally, in response to hypoxia reactive oxygen species (ROS) and the thioredoxin-interacting protein (TXNIP) were up-regulated. Pretreated with ROS scavenger NAC significantly decreased TXNIP and inhibited NLRP3 inflammasome activation. The important role of TXNIP was highlighted by the rescue of hypoxic MIN6 from apoptosis in TXNIP-knockdown MIN6 cells. Conclusion Our data indicate for the first time that inflammasome is involved in hypoxia-induced β cell death, and suggest that ROS-TXNIP-NLRP3 axis could be a potential target for improving islet transplantation. NSFC 81201171. NSFC 81471715. NSFC 81671752.

Corilagin inhibits breast cancer growth via reactive oxygen species-dependent apoptosis and autophagy.

Corilagin is a component of Phyllanthus urinaria extract and has been found of possessing anti‐inflammatory, anti‐oxidative, and anti‐tumour properties in clinic treatments. However, the underlying mechanisms in anti‐cancer particularly of its induction of cell death in human breast cancer remain undefined. Our research found that corilagin‐induced apoptotic and autophagic cell death depending on reactive oxygen species (ROS) in human breast cancer cell, and it occurred in human breast cancer cell (MCF‐7) only comparing with normal cells. The expression of procaspase‐8, procaspase‐3, PARP, Bcl‐2 and procaspase‐9 was down‐regulated while caspase‐8, cleaved PARP, caspase‐9 and Bax were up‐regulated after corilagin treatment, indicating apoptosis mediated by extrinsic and mitochondrial pathways occurred in MCF‐7 cell. Meanwhile, autophagy mediated by suppressing Akt/mTOR/p70S6K pathway was detected with an increase in autophagic vacuoles and LC3‐II conversion. More significantly, inhibition of autophagy by chloroquine diphosphate salt (CQ) remarkably enhanced apoptosis, while the caspase inhibitor z‐VAD‐fmk failed in affecting autophagy, suggesting that corilagin‐induced autophagy functioned as a survival mechanism in MCF‐7 cells. In addition, corilagin induced intracellular reactive oxygen species (ROS) generation, when reduced by ROS scavenger NAC, apoptosis and autophagy were both down‐regulated. Nevertheless, in SK‐BR3 cell which expressed RIP3, necroptosis inhibitor Nec‐1 could not alleviate cell death induced by corilagin, indicating necroptosis was not triggered. Subcutaneous tumour growth in nude mice was attenuated by corilagin, consisting with the results in vitro. These results imply that corilagin inhibits cancer cell proliferation through inducing apoptosis and autophagy which regulated by ROS release.