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.     

Bel-7402细胞通过自噬逃避FAS/ActD诱导的凋亡

为了探究FAS抗体与放线菌素D（actinomycin D,ActD）诱导肝癌细胞Bel-7402凋亡的作用机制,通过自噬阻断剂3-MA的作用,来探讨自噬与凋亡的关系.利用电子显微镜和流式细胞仪观察细胞自噬及凋亡.结果表明,FAS/ActD在诱导细胞凋亡的同时伴有细胞自噬现象,在3-MA作用下,FAS/ActD所诱导的细胞自噬体减少,而凋亡现象严重.并且通过流式细胞仪分析表明,3-MA明显增高FAS/ActD所诱导的细胞凋亡率. Western印迹分析进一步显示,FAS/ActD能引起caspase-3激活产生断裂,同时刺激LC3和BECN1表达,而3-MA作用后自噬体减少,同时LC3和BECN1表达降低,但是caspase-3断裂带表达明显增加.以上结果提示,FAS/ActD诱导的Bel-7402细胞凋亡的同时伴有细胞自噬,Bel-7402细胞通过自噬逃避FAS/ActD诱导的凋亡.     

PERK-mediated Autophagy in Osteosarcoma Cells Resists ER Stress-induced Cell Apoptosis

Osteosarcoma is a bone cancer that develops commonly in children and adolescents. However, osteosarcoma treatments often fail by the development of chemoresistance to apoptosis, and the molecular mechanisms remain unclear. In this study, we propose that autophagy is responsible for osteosarcomatous resistance to apoptosis. We implicate PERK-mediated autophagy as a significant contributor to apoptosis resistance due to ER stress in osteosarcoma cells. By immunostainings and western blots, we identified that PERK activated osteosarcomatous autophagy via inhibiting mTORC1 pathway, thereby preventing cell apoptosis. While using RNAi, we knocked down PERK and found that autophagy was suppressed, result in osteosarcomatous apoptosis. Our results identify a novel role of PERK-mediated autophagy as a significant mechanism for osteosarcoma cell survival. These results will help to understand the mechanism of chemoresistance in osteosarcoma cells, and indicate a novel target for improving osteosarcoma therapy.     

p27 Protein Protects Metabolically Stressed Cardiomyocytes from Apoptosis by Promoting Autophagy

p27^Kip1 (p27), a key regulator of cell division, has been implicated in autophagy of cancer cells. However, its role in autophagy, the evolutionarily conserved catabolic process that enables cells to remove unwanted proteins and damaged organelles, had not been examined in the heart. Here we report that ectopic delivery of a p27 fusion protein (TAT-p27) was sufficient to induce autophagy in neonatal rat ventricular cardiomyocytes in vitro, under basal conditions and after glucose deprivation. Conversely, lentivirus-delivered shRNA against p27 successfully reduced p27 levels and suppressed basal and glucose-deprived levels of autophagy in cardiomyocytes in vitro. Glucose deprivation mimics myocardial ischemia and induces apoptosis in cardiomyocytes. During glucose deprivation, TAT-p27 inhibited apoptosis, whereas down-regulation of p27 decreased survival of cardiomyocytes. However, inhibition of autophagy by pharmacological (3-methyladenine, chloroquine, or bafilomycin A1) or genetic approaches (siRNA-mediated knockdown of Atg5) sensitized cardiomyocytes to glucose deprivation-induced apoptosis, even in the presence of TAT-p27. TAT-p27 was also able to provoke greater levels of autophagy in resting and fasting cardiomyocytes in vivo. Further, TAT-p27 enhanced autophagy and repressed cardiomyocytes apoptosis, improved cardiac function, and reduced infarct size following myocardial infarction. Again, these effects were lost when cardiac autophagy in vivo was blocked by chloroquine. Taken together, these data show that p27 positively regulates cardiac autophagy in vitro and in vivo, at rest and after metabolic stress, and that TAT-p27 inhibits apoptosis by promoting autophagy in glucose-deprived cardiomyocytes in vitro and in post-myocardial infarction hearts in vivo.     

Overexpression of Rcan1-1L Inhibits Hypoxia-Induced Cell Apoptosis through Induction of Mitophagy

Mitophagy, a cellular process that selectively targets dysfunctional mitochondria for degradation, is currently a hot topic in research into the pathogenesis and treatment of many human diseases. Considering that hypoxia causes mitochondrial dysfunction, which results in cell death, we speculated that selective activation of mitophagy might promote cell survival under hypoxic conditions. In the present study, we introduced the Regulator of calcineurin 1-1L (Rcan1-1L) to initiate the mitophagy pathway and aimed to evaluate the effect of Rcan1-1L-induced mitophagy on cell survival under hypoxic conditions. Recombinant adenovirus vectors carrying Rcan1-1L were transfected into human umbilical vein endothelial cells and human adult cardiac myocytes. Using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay and Trypan blue exclusion assay, Rcan1-1L overexpression was found to markedly reverse cell growth inhibition induced by hypoxia. Additionally, Rcan1-1L overexpression inhibited cell apoptosis under hypoxic conditions, as detected by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis assay. Meanwhile, the mitochondria-mediated cell apoptotic pathway was inhibited by Rcan1-1L. In contrast, knockdown of Rcan1-1L accelerated hypoxia-induced cell apoptosis. Moreover, Rcan1-1L overexpression significantly reduced mitochondrial mass, decreased depolarized mitochondria, and downregulated ATP and reactive oxygen species production. We further delineated that the loss of mitochondrial mass was due to the activation of mitophagy induced by Rcan1-1L. Rcan1-1L overexpression activated autophagy flux and promoted translocation of the specific mitophagy receptor Parkin into mitochondria from the cytosol, whereas inhibition of autophagy flux resulted in the accumulation of Parkin-loaded mitochondria. Finally, we demonstrated that mitochondrial permeability transition pore opening was significantly increased by Rcan1-1L overexpression, which suggested that Rcan1-1L might evoke mitophagy through regulating mitochondrial permeability transition pores. Taken together, we provide evidence that Rcan1-1L overexpression induces mitophagy, which in turn contributes to cell survival under hypoxic conditions, revealing for the first time that Rcan1-1L-induced mitophagy may be used for cardioprotection.     

Protection of Neuroblastoma Neuro2A Cells from Hypoxia-Induced Apoptosis by Cyclic Phosphatidic Acid (cPA)

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator with a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We have previously shown that cPA significantly suppresses ischemia-induced delayed neuronal death and the accumulation of glial fibrillary acidic protein in the CA1 region of the rat hippocampus. These results indicated that the systemic administration of cPA can protect hippocampal neurons against ischemia-induced delayed neuronal cell death. In the current study, we investigated the effects of cPA on neuronal cell death caused by hypoxia in vitro and the molecular mechanisms underlying these effects. We used cobalt chloride (CoCl₂) to expose cells to hypoxic conditions in vitro. Treating mouse neuroblastoma (Neuro2A) cells with CoCl₂ induced nuclear DNA condensation and phosphatidylserine exposure. However, adding cPA led to the suppression of CoCl₂-induced apoptosis in a cPA dose-dependent manner and attenuated the increase in the Bax/Bcl-2 ratio caused by CoCl₂. Quantitative PCR analysis showed that Neuro2A cells strongly express the LPA₁, LPA₂, and LPA₆, which are G-protein coupled receptors that can be activated by cPA. To date, LPA₁ and LPA₂ have been reported to exhibit antiapoptotic activity. Therefore, to assess the roles of LPA₁ and LPA₂ on cPA-induced neuroprotective functions, Ki16425, a selective LPA₁ and LPA₃ antagonist, was adopted to know the LPA₁ function and siRNA was used to knockdown the expression of LPA₂. On the basis of our results, we propose that cPA-induced protection of Neuro2A cells from CoCl₂-induced hypoxia damage is mediated via LPA₂.     

Nicotinate-Curcumin Impedes Foam Cell Formation from THP-1 Cells through Restoring Autophagy Flux

Our previous studies have indicated that a novel curcumin derivate nicotinate-curcumin (NC) has beneficial effects on the prevention of atherosclerosis, but the precise mechanisms are not fully understood. Given that autophagy regulates lipid metabolism, the present study was designed to investigate whether NC decreases foam cell formation through restoring autophagy flux in oxidized low-density lipoprotein (ox-LDL)-treated THP-1 cells. Our results showed that ox-LDL (100 μg/ml) was accumulated in THP-1 cells and impaired autophagy flux. Ox-LDL-induced impairment of autophagy was enhanced by treatment with the autophagy inhibitor chloroquine (CQ) and rescued by the autophagy inducer rapamycin. The aggregation of ox-LDL was increased by CQ, but decreased by rapamycin. In addition, colocalization of lipid droplets with LC3-II was remarkably reduced in ox-LDL group. In contrast, NC (10 μM) rescued the impaired autophagy flux by significantly increasing level of LC3-II, the number of autophagolysosomes, and the degradation of p62 in ox-LDL-treated THP-1 cells. Inhibition of the PI3K-Akt-mTOR signaling was required for NC-rescued autophagy flux. Notably, our results showed that NC remarkably promoted the colocalization of lipid droplets with autophagolysosomes, increased efflux of cholesterol, and reduced ox-LDL accumulation in THP-1 cells. However, treatment with 3-methyladenine (3-MA) or CQ reduced the protective effects of NC on lipid accumulation. Collectively, the findings suggest that NC decreases lipid accumulation in THP-1 cells through restoring autophagy flux, and further implicate that NC may be a potential therapeutic reagent to reverse atherosclerosis.     

Ceramide Prevents Neuronal Programmed Cell Death Induced by Nerve Growth Factor Deprivation

Abstract: We examined the ability of ceramide and sphingomyelinase (SMase) to prevent neuronal programmed cell death (PCD). We found that a cell-permeable ceramide analogue prevented neuronal PCD when applied to established sympathetic neuron primary cultures at the time of nerve growth factor (NGF) deprivation. Other amphiphilic lipids such as oleic acid failed to prevent cell death. Exogenous SMase also showed the same effect, probably by raising the intracellular ceramide level by sphingomyelin (SM) breakdown. Phosphocholine, another hydrolytic product of SM by SMase, did not prevent cell death. Other phospholipases, such as phospholipase C and phospholipase A₂, could not prevent cell death. Given the recent findings that the SM cycle is activated to increase the intracellular ceramide level on NGF binding to the low-affinity NGF receptor (LNGFR) and that NGF binding to LNGFR suppresses apoptosis in neural cell lines, our results suggest the possibility of the SM cycle as a signaling mechanism transducing the PCD-preventing activity of NGF.     

ASPP2过表达可增强DRAM介导的自噬对HepG2细胞的促调亡作用

p53凋亡刺激蛋白2（apoptosis stimulating protein 2 of p53, ASPP2）能特异性地与p53蛋白结合并增强其促凋亡功能,进而发挥抗肿瘤作用.最近文献提示,自噬对肿瘤发生、发展及肿瘤细胞对抗肿瘤药物的反应都具有重要作用.在本研究中,甲基磺酸(MMS)处理HepG2细胞24 h后,用calcein AM/PI和M30染色检测细胞凋亡,可引起早期（M30免疫组化阳性）和晚期细胞凋亡（PI染色阳性）. 给HepG2细胞转染GFP-LC3质粒后,发现MMS处理24 h可引起自噬的发生. ASPP2腺病毒（rAd-ASPP2）感染HepG2细胞引起ASPP2过表达后,再用MMS处理24 h,能引起更明显的早期、晚期细胞凋亡和自噬. 荧光定量PCR检测发现,rAd-ASPP2诱导了更高的BCL-2相关X蛋白基因（BAX）和p53蛋白的目的基因p53诱导的自噬调节蛋白（p53-induced modulator of autophagy,DRAM）的表达. 但仅用rAd-ASPP2处理HepG2细胞不能引起自噬和凋亡.利用2条DRAM特异性的siRNA下调DRAM的表达,发现rAd-ASPP2引起的自噬被完全抑制, 早期和晚期凋亡均部分被抑制,同时BAX 的mRNA水平也明显下降. 以上结果说明,ASPP2可通过上调BAX和DRAM基因的转录而促进MMS引起的HepG2细胞凋亡; 另外,DRAM介导的自噬是ASPP2促进MMS引起的肿瘤细胞凋亡的机制之一. 该研究可为肝癌的基因治疗提供新的思路.     

Dendropanoxide Induces Autophagy through ERK1/2 Activation in MG-63 Human Osteosarcoma Cells and Autophagy Inhibition Enhances Dendropanoxide-Induced Apoptosis

Anticancer effects of dendropanoxide (DP) newly isolated from leaves and stem of Dendropanax morbifera Leveille were firstly investigated in this study. DP inhibited cell proliferation and induced apoptosis in dose- and time-dependent manner in MG-63 human osteosarcoma cells, which was dependent on the release of cytochrome c to the cytosol and the activation of caspases. Moreover, the DP-treated cells exhibited autophagy, as characterized by the punctuate patterns of microtubule-associated protein 1 light chain 3 (LC3) by confocal microscopy and the appearance of autophagic vacuoles by MDC staining. The expression levels of ATG7, Beclin-1 and LC3-II were also increased by DP treatment. Inhibition of autophagy by 3-methyladenine (3-MA) and wortmannin (Wort) significantly enhanced DP-induced apoptosis. DP treatment also caused a time-dependent increase in protein levels of extracellular signal-regulated kinase 1 and 2 (ERK1/2), and inhibition of ERK1/2 phosphorylation with U0126 resulted in a decreased DP-induced autophagy that was accompanied by an increased apoptosis and a decreased cell viability. These results indicate a cytoprotective function of autophagy against DP-induced apoptosis and suggest that the combination of DP treatment with autophagy inhibition may be a promising strategy for human osteosarcoma control. Taken together, this study demonstrated for the first time that DP could induce autophagy through ERK1/2 activation in human osteosarcoma cells and autophagy inhibition enhanced DP-induced apoptosis.     

Andrographolide Analogue Induces Apoptosis and Autophagy Mediated Cell Death in U937 Cells by Inhibition of PI3K/Akt/mTOR Pathway

Background

Current chemotherapeutic agents based on apoptosis induction are lacking in desired efficacy. Therefore, there is continuous effort to bring about new dimension in control and gradual eradication of cancer by means of ever evolving therapeutic strategies. Various forms of PCD are being increasingly implicated in anti-cancer therapy and the complex interplay among them is vital for the ultimate fate of proliferating cells. We elaborated and illustrated the underlying mechanism of the most potent Andrographolide analogue (AG–4) mediated action that involved the induction of dual modes of cell death—apoptosis and autophagy in human leukemic U937 cells.

Principal Findings

AG–4 induced cytotoxicity was associated with redox imbalance and apoptosis which involved mitochondrial depolarisation, altered apoptotic protein expressions, activation of the caspase cascade leading to cell cycle arrest. Incubation with caspase inhibitor Z-VAD-fmk or Bax siRNA decreased cytotoxic efficacy of AG–4 emphasising critical roles of caspase and Bax. In addition, AG–4 induced autophagy as evident from LC3-II accumulation, increased Atg protein expressions and autophagosome formation. Pre-treatment with 3-MA or Atg 5 siRNA suppressed the cytotoxic effect of AG–4 implying the pro-death role of autophagy. Furthermore, incubation with Z-VAD-fmk or Bax siRNA subdued AG–4 induced autophagy and pre-treatment with 3-MA or Atg 5 siRNA curbed AG–4 induced apoptosis—implying that apoptosis and autophagy acted as partners in the context of AG–4 mediated action. AG–4 also inhibited PI3K/Akt/mTOR pathway. Inhibition of mTOR or Akt augmented AG–4 induced apoptosis and autophagy signifying its crucial role in its mechanism of action.

Conclusions

Thus, these findings prove the dual ability of AG–4 to induce apoptosis and autophagy which provide a new perspective to it as a potential molecule targeting PCD for future cancer therapeutics.     

Deguelin Induces Both Apoptosis and Autophagy in Cultured Head and Neck Squamous Cell Carcinoma Cells

Head and neck squamous cell carcinoma (HNSCC) represents more than 5% of all cancers diagnosed annually in United States and around the world. Despite advances in the management of patients with this disease, the survival has not been significantly improved, and the search for potential alternative therapies is encouraging. Here we demonstrate that deguelin administration causes a significant HNSCC cell death. Deguelin induces both cell apoptosis and autophagy by modulating multiple signaling pathways in cultured HNSCC cells. Deguelin inhibits Akt signaling, and down-regulates survivin and cyclin-dependent kinase 4 (Cdk4) expressions, by disrupting their association with heat shock protein-90 (Hsp-90). Deguelin induces ceramide production through de novo synthase pathway to promote HNSCC cell death. Importantly, increased ceramide level activates AMP-activated protein kinase (AMPK), which then directly phosphorylates Ulk1 and eventually leads to cell autophagy. We found that a low dose of deguelin sensitized HNSCC cells to 5-FU. Finally, using a nude mice Hep-2 xenograft model, we also showed a significant anti-tumor ability of deguelin in vivo. Together, we suggest that deguelin may represent a novel and effective chemo-agent against HNSCC.     

Nerve Growth Factor Rescues Pigment Cells from Ultraviolet-Induced Apoptosis by Upregulating BCL-2 Levels

Apoptosis plays an important role in eliminating dysfunctional damaged cells. For skin, the best characterized injurious environmental agent is ultraviolet (UV) irradiation. Most of the damaging UV irradiation is absorbed in the epidermis and leads to apoptosis of keratinocytes. However, epidermal melanocytes appear to be protected from UV-induced apoptosis. We now report that in pure cultures melanocytic cells undergo characteristic apoptosis after physiologic UV exposures. However, nerve growth factor (NGF) supplementation protects them from this programmed cell death. Furthermore, we show that NGF protects melanocytic cells from UV-induced apoptosis by upregulating BCL-2 protein in these cells and that prior downregulation of BCL-2 abrogates the NGF protective effect on melanocytes. Our data suggest that NGF, known to be constitutively produced by epidermal keratinocytes and induced in these cells after UV irradiation, may preserve the population of cutaneous melanocytes that would otherwise be depleted by casual sun exposure.     

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.     

Induction of Apoptosis and Autophagy Using Ectopic DSCR1 Expression in Breast Cancer Cells

Down syndrome critical region 1 gene (DSCR1) is an anti-angiogenesis gene that inhibits the growth of tumor cells. In this study, the role of autophagy and apoptosis in DSCR1-induced cytotoxicity were investigated in MDA-MB-468 breast cancer cells. Lentivirus vector harboring DSCR1 (LV-DSCR1⁺) was constructed in HEK 293 cells and the optimal dosage of lentivirus vector for infection was determined by the MTT assay. After infection of cells using LV-DSCR1⁺, acridine orange and ethidium bromide staining was performed to investigation of apoptosis and autophagy. Expression of DSCR1 and marker genes for angiogenesis (VEGF), apoptosis (Bax and Bcl2) and autophagy (LC3 and Beclin) were determined by Real time PCR. The cellular morphological changes related to apoptosis and autophagy was happened after 48 hours of viral infection. Fragmented bright orange nucleuses and vacuoles were observed due to the cell apoptosis and autophagy after acridine orange and ethidium bromide staining. Upregulation of Bax, Lc3, DSCR1 and Beclin1 and downregulation of Bcl2 and VEGF was detected due to treatment with LV-DSCR1⁺. These results demonstrated that LV-DSCR1⁺ can induce apoptosis and autophagy, therefore suggesting that it may serves as an efficient tool to breast cancer treatment.     

Induction of Endothelial Cell Apoptosis by Solid Tumor Cells

The mechanisms by which tumor cells extravasate to form metastasis remain controversial. Previous studies performedin vivoandin vitrodemonstrate that the contact between tumor cells and the vascular wall impairs endothelium integrity. Here, we investigated the effect of breast adenocarcinoma MCF-7 cells on the apoptosis of human umbilical vein endothelial cells (HUVEC). TUNEL labeling, nuclear morphology, and DNA electrophoresis indicated that MCF-7 cells induced a two- to fourfold increase in HUVEC apoptosis. Caspase-3 activity was significantly enhanced. Neither normal cells tested (mammary epithelial cells, fibroblasts, leukocytes) nor transformed hematopoietic cells tested (HL60, Jurkat) induced HUVEC apoptosis. On the contrary, cells derived from solid tumors (breast adenocarcinoma, MDA-MB-231 and T47D; fibrosarcoma, HT 1080) had an effect similar to that of MCF-7 cells. The induction of apoptosis requires cell-to-cell contact, since it could not be reproduced by media conditioned by MCF-7 cells cultured alone or cocultured with HUVEC. Our results suggest that cells derived from solid tumors may alter the endothelium integrity by inducing endothelial cell apoptosis. On the contrary, normal or malignant leukocytes appear to extravasate by distinct mechanisms and do not damage the endothelium. Our data may lead to a better understanding of the steps involved in tumor cell extravasation.