Resveratrol Enhances Palmitate-Induced ER Stress and Apoptosis in Cancer Cells

Background

Palmitate, a saturated fatty acid (FA), is known to induce toxicity and cell death in various types of cells. Resveratrol (RSV) is able to prevent pathogenesis and/or decelerate the progression of a variety of diseases. Several in vitro and in vivo studies have also shown a protective effect of RSV on fat accumulation induced by FAs. Additionally, endoplasmic reticulum (ER) stress has recently been linked to cellular adipogenic responses. To address the hypothesis that the RSV effect on excessive fat accumulation promoted by elevated saturated FAs could be partially mediated by a reduction of ER stress, we studied the RSV action on experimentally induced ER stress using palmitate in several cancer cell lines.

Principal Findings

We show that, unexpectedly, RSV promotes an amplification of palmitate toxicity and cell death and that this mechanism is likely due to a perturbation of palmitate accumulation in the triglyceride form and to a less important membrane fluidity variation. Additionally, RSV decreases radical oxygen species (ROS) generation in palmitate-treated cells but leads to enhanced X-box binding protein-1 (XBP1) splicing and C/EBP homologous protein (CHOP) expression. These molecular effects are induced simultaneously to caspase-3 cleavage, suggesting that RSV promotes palmitate lipoapoptosis primarily through an ER stress-dependent mechanism. Moreover, the lipotoxicity reversion induced by eicosapentaenoic acid (EPA) or by a liver X receptor (LXR) agonist reinforces the hypothesis that RSV-mediated inhibition of palmitate channeling into triglyceride pools could be a key factor in the aggravation of palmitate-induced cytotoxicity.

Conclusions

Our results suggest that RSV exerts its cytotoxic role in cancer cells exposed to a saturated FA context primarily by triglyceride accumulation inhibition, probably leading to an intracellular palmitate accumulation that triggers a lipid-mediated cell death. Additionally, this cell death is promoted by ER stress through a CHOP-mediated apoptotic process and may represent a potential anticancer strategy.     

原花色素对HepS细胞增殖抑制诱导凋亡的研究

本文通过体外培养肝癌HepS细胞,以不同浓度原花色素处理12—72h后,MTT法测定细胞生长抑制作用,采用DNA片断分析、DNA琼脂糖凝胶电泳、荧光染色以及流式细胞技术等方法来探讨原花色素体外抑制肝癌HepS细胞及诱导其凋亡的作用。实验结果显示原花色素能抑制HepS细胞的生长,并且呈现出明显的时效和量效关系,DNA电泳出现典型的凋亡DNA梯形带,在荧光显微镜下,凋亡细胞呈亮绿色,H和AnnexinV．FIFC双染后,经流式细胞仪检测、分析显示凋亡细胞明显增多。因此原花色素能抑制肝癌HepS细胞株的生长,可能与诱导其细胞凋亡有关。     

Ulk1/FUNDC1 Prevents Nerve Cells from Hypoxia-Induced Apoptosis by Promoting Cell Autophagy

Cell autophagy and cell apoptosis are both observed in the process of hypoxia-induced ischemic cerebral infarction (ICI). Unc-51 like autophagy activating kinase 1 (Ulk1) and FUN14 Domain-containing Protein 1 (FUNDC1) are both involved in the regulation of cell autophagy. This study aimed to investigate the regulatory effects of Ulk1 and FUNDC1 on hypoxia-induced nerve cell autophagy and apoptosis. Cell viability was measured using cell counting kit-8 (CCK-8) assay. Cell apoptosis was detected using Annexin V-PE/7-ADD staining assay. qRT-PCR was used to quantify the mRNA levels of Ulk1 and FUNDC1 in PC-12 cells. Cell transfection was performed to up-regulate the expression of Ulk1. 3-Methyladenine (3-MA) was used as autophagy inhibitor and rapamycin was used as autophagy activator in our experiments. SP600125 was used as c-Jun N-terminal kinase (JNK) inhibitor. Western blotting was performed to analyze the expression levels of key factors that are related to cell autophagy, apoptosis and JNK pathway. We found that hypoxia simultaneously induced apoptosis and autophagy of PC-12 cells. The activation of Ulk1 and FUNDC1 were also found in PC-12 cells after hypoxia induction. Overexpression of Ulk1 promoted the activation of FUNDC1 and prevented PC-12 cells from hypoxia-induced apoptosis. Suppression of Ulk1 had opposite effects. Furthermore, we also found that JNK pathway participated in the effects of Ulk1 overexpression on PC-12 cell apoptosis reduction. To conclude, Ulk1/FUNDC1 played critical regulatory roles in hypoxia-induced nerve cell autophagy and apoptosis. Overexpression of Ulk1 prevented nerve cells from hypoxia-induced apoptosis by promoting cell autophagy.     

Inhibition of pRB Pathway Differentially Modulates Apoptosis in Esophageal Cancer Cells

Esophageal cancer is the sixth most common cause of cancer-related death worldwide. Current chemotherapy regimens include a combination of 5-fluorouracil (5-FU) and cisplatin, but more efficient therapy strategies are needed to increase 5-year survival. Alterations in the signaling pathway of the tumor suppressor gene Rb-1, which encodes a phosphoprotein (pRB) that negatively regulates the G1/S transition of the cell cycle, are present in 70% of all tumors, but its role in esophageal cancer is still unclear. Most of these are alterations leading to up-regulation of the activity of cyclin-dependent kinases (CDKs) to phosphorylate pRB, which suggests that keeping the wild type pRB phosphorylated might be advantageous. Besides proliferation, pRB also regulates apoptosis induced by tumor necrosis factor-alpha (TNF-α) and DNA-damage. We investigated the status of phosphorylation of pRB along esophageal tumorigenesis stages, as well as whether hyperphosphorylation of pRB could suppress apoptosis induced by cisplatin, 5-FU, or TNF-α in esophageal cancer cells. pRB phosphorylation increased progressively from normal esophageal tissue to metaplasia and adenocarcinoma, suggesting that pRB phosphorylation increases along esophageal tumor stages. When RB-1 was knocked down or CDK inhibitors reduced the levels of phosphorylated pRB, opposite apoptotic effects were observed, depending on the combination of drugs tested: whereas TNF-α- and cisplatin-induced apoptosis increased, 5-FU-induced apoptosis decreased. Taken together, these data suggest that pRB plays a role in esophageal adenocarcinoma and that, depending on the type of anti-cancer treatment, combining CDK inhibitors and chemotherapy has the potential to increase the sensitivity of esophageal cancer cells to cell death.     

10-羟基喜树碱对人膀胱癌细胞增殖、侵袭的抑制作用及诱导凋亡的研究

探讨10-羟基喜树碱(10-HCPT)对人膀胱癌细胞T24增殖、侵袭和诱导凋亡的作用.0.25μmol/L和0.50μmol/L10-HCPT处理T24细胞4d后,用细胞增殖抑制试验、软琼脂集落形成试验、侵袭试验、趋化运动试验、黏附试验和组织蛋白酶B活性测定细胞增殖和侵袭能力的变化;药物处理2d后,采用吖啶橙/溴乙啶荧光双染法和末端脱氧核苷酸转移酶介导dUTP-地高辛缺口末端标记法检测细胞凋亡.结果0.25μmol/L和0.50μmol/L10-HCPT分别使T24细胞增殖下降66.1%和74.8%;对细胞侵袭、运动、黏附及组织蛋白酶B分泌均有明显的抑制作用,并使T24细胞凋亡率显著升高,与对照组比较,差异有显著性意义(P<0.05或0.01).提示10-HCPT有抗T24增殖和侵袭作用,并有诱导凋亡的作用.其抗侵袭机制是对侵袭的多个基本环节起抑制作用.     

Poly(ADP-Ribose) Polymerase Inhibition in Oxidant-Stressed Endothelial Cells Prevents Oncosis and Permits Caspase Activation and Apoptosis

Endothelial cells (EC) are subject to oxidative-induced cell death. Activation of poly(ADP-ribose) polymerase (PARP) occurs early in oxidant-induced EC injury and putatively mediates cell death by depleting its substrate, NAD⁺. In this study, the role of PARP in H₂O₂-induced EC death was investigated. EC were exposed to oxidant stress and viability continuously monitored using fluorescent dye exclusion. Inhibition of PARP with 1,5-dihydroxyisoquinoline (DIQ) delayed the time course of oxidant-induced EC death. Concurrent addition of the protein synthesis inhibitor, cycloheximide, or the endonuclease inhibitor, aurintricarboxylic acid, to PARP-inhibited cells further delayed the onset and attenuated the extent of H₂O₂-induced cell lysis, consistent with an active mode of cell death. Caspase-3-like activity, a hallmark of apoptosis, was negligible in oxidant-treated EC alone, however, inhibition of PARP by 3-aminobenzamide or DIQ dramatically increased caspase-3-like activity. Morphological assessment confirmed that the primary mode of death in oxidant-stressed EC was oncosis. However, following PARP inhibition, the cells switched to apoptosis. Since inflammation is associated with oncosis and not apoptosis, the results presented here could explain the beneficial effects seen with PARP inhibition in various in vivo models of oxidant injury and provide a mechanism to manipulate this injury into a state of cell death that could ultimately be controlled.     

Morroniside Prevents Peroxide-induced Apoptosis by Induction of Endogenous Glutathione in Human Neuroblastoma Cells

(1) Morroniside belongs to an extensive group of natural iridorid glycosides. In the present study, using human neuroblastoma SH-SY5Y cells, we have investigated the protective effects of this compound on modifications in endogenous reduced glutathione (GSH), intracellular oxygen species (ROS) and apoptotic death on H₂O₂-mediated cytoxicity. (2) Incubation of cells with morroniside led to a significant dose-dependent elevation of cellular GSH accompanied by a marked protection against H₂O₂-mediated toxicity. Morroniside at 1–100 μM inhibited the formation of ROS and the activation of caspase-3 and 9, and the upregulation of Bcl-2, whereas no significant change occurred in Bax levels. (3) The results indicated that the anti-oxidative and anti-apoptotic properties render this natural compound potentially protective against H₂O₂-induced cytotoxicity. (4) This study suggested that intracellular GSH appeared to be an important factor in morroniside-mediated cytoprotection against H₂O₂-toxicity in SH-SY5Y cells.     

Stress Granules Inhibit Apoptosis by Reducing Reactive Oxygen Species Production

Cells can undergo two alternative fates following exposure to environmental stress: they either induce apoptosis or inhibit apoptosis and then repair the stress-induced alterations. These processes minimize cell loss and prevent the survival of cells with aberrant DNA and protein alterations. These two alternative fates are partly controlled by stress granules (SGs). While arsenite, hypoxia, and heat shock induce the formation of SGs that inhibit apoptosis, X-ray irradiation and genotoxic drugs do not induce SGs, and they are more prone to trigger apoptosis. However, it is unclear precisely how SGs control apoptosis. This study found that SGs suppress the elevation of reactive oxygen species (ROS), and this suppression is essential for inhibiting ROS-dependent apoptosis. This antioxidant activity of SGs is controlled by two SG components, GTPase-activating protein SH3 domain binding protein 1 (G3BP1) and ubiquitin-specific protease 10 (USP10). G3BP1 elevates the steady-state ROS level by inhibiting the antioxidant activity of USP10. However, following exposure to arsenite, G3BP1 and USP10 induce the formation of SGs, which uncovers the antioxidant activity of USP10. We also found that the antioxidant activity of USP10 requires the protein kinase activity of ataxia telangiectasia mutated (ATM). This work reveals that SGs are critical redox regulators that control cell fate under stress conditions.     

无花果果浆对肿瘤细胞增殖抑制和诱导凋亡作用

为了探讨无花果果浆(Fig fruit latex,FFL)对人肿瘤细胞的生长抑制作用及其机制,用无花果果浆处理体外培养的人肿瘤细胞,细胞增殖试验(MTT法)、克隆形成试验研究FFL对人肿瘤细胞的增殖抑制作用,Brdu掺人试验、吖啶橙/溴乙啶(AO/EB)染色、流式细胞术检测FFL对肿瘤细胞DNA合成、凋亡和细胞周期的影响.结果,用FFL处理后,肿瘤细胞增殖活性降低(P＜0.05),克隆形成下降(P＜0.05),Brdu标记指数降低(P＜0.05),吖啶橙染色凋亡细胞增多(P＜0.05);细胞周期分布改变,凋亡指数升高(P＜0.01),G0/G1期细胞数增加(P＜0.01),S期细胞数减少(P＜0.01),在一定剂量内对正常细胞无明显影响.试验结果提示,FFL对所试肿瘤细胞的增殖有显著地抑制作用,其作用机理可能与抑制肿瘤细胞DNA合成,诱导肿瘤细胞凋亡及细胞周期阻滞有关.     

Prion Protein Protects Cancer Cells against Endoplasmic Reticulum Stress Induced Apoptosis

Unfolded protein response(UPR) is an adaptive reaction for cells to reduce endoplasmic reticulum(ER) stress. In many types of cancers, such as lung cancer and pancreatic cancer, cancer cells may harness ER stress to facilitate their survival and growth. Prion protein(PrP) is a glycosylated cell surface protein that has been shown to be up-regulated in many cancer cells. Since PrP is a protein prone to misfolding, ER stress can result in under-glycosylated PrP, which in turn may activate ER stress. To assess whether ER stress leads to the production of under-glycosylated PrP and whether underglycosylated PrP may contribute to ER stress thus leading to cancer cell apoptosis, we treated different cancer cells with brefeldin A(BFA), thapsigargin(Thps), and tunicamycin(TM). We found that although BFA, Thps, and TM treatment activated UPR, only ATF4 was consistently activated by these reagents, but not other branches of ER stress. However, the canonical PERK-eIF2α-ATF4 did not account for the observed activation of ATF4 in lung cancer cells. In addition, BFA,but neither Thps nor TM, significantly stimulated the expression of cytosolic PrP. Finally, we found that the levels of PrP contributed to anti-apoptosis activity of BFA-induced cancer cell death. Thus, the pathway of BFA-induced persistent ER stress may be targeted for lung and pancreatic cancer treatment.     

Long-term Heat Exposure Prevents Hypoxia-Induced Apoptosis in Mouse Fibroblast Cells

Long-term continuous exposure to high ambient temperatures induces complete heat acclimation in humans and animals. However, to date, the effects of long-term exposure to heat stress on cells have not been fully evaluated. In this study, we investigated an adaptive physiological process induced in culture cells by continuous exposure to mild heat stress for 60 days. The results of this investigation provide evidence that after long-term heat acclimation in cells, (1) heat shock protein levels are increased, (2) hypoxia inducible factor-1α (HIF-1α) expression is upregulated, and (3) heat shock-induced and hypoxia-induced apoptoses are attenuated. These results suggest that the hypoxia response pathway is an intrinsic part of the heat acclimation repertoire and that the HIF-1 pathway following long-term heat acclimation induces cells with cross tolerance against hypoxia.     

Clusterin抑制人肾近曲小管上皮HK-2细胞的凋亡

Clusterin是一种硫酸糖蛋白.最近研究发现,clusterin具有抗凋亡作用,同时对肾细胞具有保护作用,但抗凋亡的具体机制仍不清楚.为研究clusterin及其不同功能区域在人肾近曲小管上皮HK-2细胞中的抗凋亡作用,构建了含有全长及缺失前导序列的clusterin重组质粒（分别命名为pIRES2-EGFP/cluac和pIRES2-EGFP/clubc）.将重组质粒转染人肾近曲小管上皮HK-2细胞后,检测转染细胞中clusterin的表达及其抗Na₂SeO₃（10μmol/L）诱导的凋亡作用.Western印迹显示,转染pIRES2-EGFP/cluac的HK-2细胞培养上清及细胞裂解液中均可检测到clusterin蛋白表达,但转染pIRES2-EGFP/clubc的HK-2细胞仅在裂解液中检测到clusterin,在培养上清液中未检测到该蛋白表达.流式细胞术检验显示,HK-2 /clubc细胞实验组出现明显凋亡峰,而 HK-2 /cluac细胞组则未见凋亡;两组的凋亡百分率之间也存在显著性差异（P<0.05）.以Cy3标记的Annexin V染色后于荧光显微镜下观察细胞凋亡情况与FCM检测结果基本一致.上述结果证明,clusterin有明显的抑制人肾近曲小管上皮HK-2细胞凋亡的作用;clusterin前导序列是其发挥抗凋亡作用的必需功能区域,提示clusterin抗凋亡作用是通过细胞外途径产生的.     

RNA Interference-Mediated Inhibition of Wild-Type Torsin A Expression Increases Apoptosis Caused by Oxidative Stress in Cultured Cells

To assess RNAi mediated inhibition of the expression of wt-DYT1 on H₂O₂-induced toxicity in NIH 3T3 cells and primary cortical neurons. To detect the function of wild-type Torsin A and the effect of SiRNA on the wt-DYT1 gene. The shRNA expression vector was constructed by ligating annealed complementary shRNA oligonucleotides into the down-stream of the human U6 promoter (PU6) of the RNAi-ready pSIREN-Shuttle vector. Then, the pSIREN-Shuttle-DYT1-shRNA cassette was ligated to Adeno-X Viral DNA to construct the recombinant adenoviral vector pAd-DYT1-shRNA. Cultured cerebral cortical neurons and NIH 3T3 cells were transfected with pAd-DYT1-shRNA and pSIREN-Shuttle-DYT1-shRNA. We evaluated NIH 3T3 cells and neurons in the presence of oxidative stress using a TUNEL assay under different conditions. The knockdown efficacy of the DYT1 was confirmed by real-time RT-PCR and Western Blot analysis. After exposure to H₂O_2, the quantity of NIH 3T3 cells transfected with pSIREN-Shuttle-DYT1-shRNA, which stained positively in the TUNEL assay, was significantly higher than the cells transfected with pSIREN-Shuttle-negative control-shRNA. (44.85 ± 1.81% vs. 8.98 ± 2.73%, t = 26.168). There were significantly more apoptotic neurons infected with pAd-DYT1-shRNA (45.63 ± 7.53%) than neurons infected with pAd-X-negative control-shRNA (17.33 ± 2.43%) (t = 9.816). The observed silencing of wild-type Torsin A expression by DYT1-shRNA was sequence-specific. RNAi-mediated inhibition of the expression of wild-type Torsin A increases apoptosis caused by oxidative stress. It is reasonable to consider that wild-type Torsin A has the capacity to protect cortical neurons against oxidative stress, and in the development of DYT1-delta GAG-dystonia the neuroprotective function of wild-type Torsin A may be compromised.     

Integration of Endothelial Cells in Multicellular Spheroids Prevents Apoptosis and Induces Differentiation

Single endothelial cells (EC) seeded in suspension culture rapidly undergo apoptosis. Addition of survival factors, such as VEGF and FGF-2, does not prevent apoptosis of suspended EC. However, when cells are allowed to establish cell–cell contacts, they become responsive to the activities of survival factors. These observations have led to the development of a three-dimensional spheroid model of EC differentiation. EC spheroids remodel over time to establish a differentiated surface layer of EC and a center of unorganized EC that subsequently undergo apoptosis. Surface EC become quiescent, establish firm cell–cell contacts, and can be induced to express differentiation antigens (e.g., induction of CD34 expression by VEGF). In contrast, the unorganized center spheroid cells undergo apoptosis if they are not rescued by survival factors. The responsiveness to the survival factor activities of VEGF and FGF-2 was not dependent on cell shape changes since it was retained after cytochalasin D treatment. Taken together, these findings characterize survival factor requirements of unorganized EC and indicate that polarized surface EC differentiate to become independent of exogenous survival factors. Furthermore, they demonstrate that spheroid cell culture systems are useful not just for the study of tumor cells and embryonic stem cells but also for the analysis of differentiated functions of nontransformed cells.     

Dose-Dependent AMPK-Dependent and Independent Mechanisms of Berberine and Metformin Inhibition of mTORC1, ERK,DNA Synthesis and Proliferation in Pancreatic Cancer Cells

Natural products represent a rich reservoir of potential small chemical molecules exhibiting anti-proliferative and chemopreventive properties. Here, we show that treatment of pancreatic ductal adenocarcinoma (PDAC) cells (PANC-1, MiaPaCa-2) with the isoquinoline alkaloid berberine (0.3–6 µM) inhibited DNA synthesis and proliferation of these cells and delay the progression of their cell cycle in G1. Berberine treatment also reduced (by 70%) the growth of MiaPaCa-2 cell growth when implanted into the flanks of nu/nu mice. Mechanistic studies revealed that berberine decreased mitochondrial membrane potential and intracellular ATP levels and induced potent AMPK activation, as shown by phosphorylation of AMPK α subunit at Thr-172 and acetyl-CoA carboxylase (ACC) at Ser⁷⁹. Furthermore, berberine dose-dependently inhibited mTORC1 (phosphorylation of S6K at Thr³⁸⁹ and S6 at Ser^240/244) and ERK activation in PDAC cells stimulated by insulin and neurotensin or fetal bovine serum. Knockdown of α₁ and α₂ catalytic subunit expression of AMPK reversed the inhibitory effect produced by treatment with low concentrations of berberine on mTORC1, ERK and DNA synthesis in PDAC cells. However, at higher concentrations, berberine inhibited mitogenic signaling (mTORC1 and ERK) and DNA synthesis through an AMPK-independent mechanism. Similar results were obtained with metformin used at doses that induced either modest or pronounced reductions in intracellular ATP levels, which were virtually identical to the decreases in ATP levels obtained in response to berberine. We propose that berberine and metformin inhibit mitogenic signaling in PDAC cells through dose-dependent AMPK-dependent and independent pathways.     

Casticin Potentiates TRAIL-Induced Apoptosis of Gastric Cancer Cells through Endoplasmic Reticulum Stress

Background

Casticin is one of the main active components obtained from Fructus Viticis and has been reported to exert anti-carcinogenic activity on a variety of cancer cells but the precise mechanism underlying this activity remains unclear.

Materials and Methods

Apoptotic activities of casticin (1.0 µmol/l) and TRAIL (25, 50 ng/ml) alone or in combination in the gastric cancer cell lines BGC-823, SGC-7901 and MGC-803 were detected by the use of a cell apoptosis ELISA detection kit, flow cytometry (FCM) with propidium iodide (PI) staining and activities of caspase-3, -8 and -9 by ELISA and cleavage of polyADP-ribose polymerase (PARP) protein using western blot analysis. Death receptors (DR) expression levels were evaluated using FCM analysis and western blotting. 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) was used as a probe to measure the increase in reactive oxygen species (ROS) levels in cells. Multiple interventions, such as siRNA transfection and pharmacological inhibitors were used to explore the mechanisms of these actions.

Results

Subtoxic concentrations of casticin significantly potentiated TRAIL-induced cytotoxicity and apoptosis in BGC-823, SGC-7901 and MGC-803 cells. Casticin dramatically upregulated DR5 receptor expression but had no effects on DR4 or decoy receptors. Deletion of DR5 by siRNA significantly reduced the apoptosis induced by the co-application of TRAIL and casticin. Gene silencing of the CCAAT/enhancer binding protein homologous protein (CHOP) and pretreatment with salubrinal, an endoplasmic reticulum (ER) stress inhibitor, attenuated casticin-induced DR5 receptor expression, and apoptosis and ROS production. Casticin downregulated the expression levels of the cell survival proteins cFLIP, Bcl-2, XIAP, and survivin. In addition, casticin also induced the expressions of DR5 protein in other gastric cancer cells (SGC-7901 and MGC-803).

Conclusion/Significance

Casticin enhances TRAIL-induced apoptosis through the downregulation of cell survival proteins and the upregulation of DR5 receptors through actions on the ROS-ER stress-CHOP pathway.