cytochrome-c-t and 3-methyladenine

Endothelial dysfunction induced by bubbles plays an important role in decompression sickness (DCS), but the mechanism of which has not been clear. The present study was to investigate the role of autophagy in bubble-induced endothelial injury. Human umbilical vein endothelial cells (HUVECs) were treated with bubbles, autophagy markers and endothelial injury indices were determined, and relationship strengths were quantified. Effects of autophagy inhibitor 3-methyladenine (3-MA) were observed. Bubble contact for 1, 5, 10, 20 or 30 minutes induced significant autophagy with increases in LC3-II/I ratio and Beclin-1, and a decrease in P62, which correlated with bubble contact duration. Apoptosis rate, cytochrome C and cleaved caspase-3 increased, and cell viability decreased following bubble contact for 10, 20 or 30 minutes, but not for 1 or 5 minutes. Injuries in HUVECs were correlated with LC3-II/I ratio and partially reversed by 3-MA in 10, 20 or 30 minutes contact, but worsened in 1 or 5 minutes. Bubble pre-conditioning for 1 minutes resulted in increased cell viability and decreased apoptosis rate compared with no pre-conditioning, and 30-minutes pre-conditioning induced opposing changes, all of which were inhibited by 3-MA. In conclusion, autophagy was involved and played a biphasic role in bubble-induced endothelial injury.

Topics: Adenine; Apoptosis; Autophagosomes; Autophagy; Beclin-1; Caspase 3; Cell Survival; Cytochromes c; Decompression Sickness; Endothelium; Human Umbilical Vein Endothelial Cells; Humans; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; RNA-Binding Proteins; Time Factors

Autophagy alterations have been observed in a variety of neurological disorders, however, very few studies have focused on autophagy alterations in epilepsy. The ketogenic diet (KD) likely ameliorates neuronal loss in several seizure models. However, whether this neuroprotective function occurs via starvation-induced autophagy and its prevalence in chronic kindled seizures remains unknown. The aim of this study was to determine the role of autophagy following seizure under KD, and the potential mechanism involved. Pentylenetetrazol (PTZ)-kindled rats, which were fed a Normal diet (ND) or KD, were pretreated with intraventricular infusions of saline, autophagy inducer rapamycin (RAP), or inhibitor 3-methyladenine (3-MA). KD alleviated seizure severity, decreased the number of Fluoro-jade B (FJB)-positive cells in the hippocampus of kindled rats. These effects were abolished by 3-MA pretreatment. RAP pretreatment did not affect seizure severity, but decreased the number of FJB-positive cells in ND group. KD decreased the percentage of damaged mitochondria in kindled group. Hippocampal Beclin-1 was increased by KD in vehicle group. The autophagy proteins Atg5, Beclin-1 and the ratio of microtubule-associated protein 1 light chain 3 (LC3) II to LC3-I in kindled KD-fed rats were higher, and the autophagy substrate P62 was lower than those in the kindled ND-fed rats, indicating an increase in autophagy following KD. Pretreatment with RAP increased the level of LC3-II/LC3-I, and pretreatment with 3-MA increased the level of P62 in KD-fed rats. To further clarify the mechanism of autophagy protection, the levels of key mitochondria related molecules were examed. The results showed that mitochondrial cytochrome c was up-regulated, cytosolic cytochrome c and the downstream cleaved caspase-3 was down-regulated in KD-fed rats, indicating a decrease in mitochondrial apoptosis. Taken together, our results indicated that KD activates autophagic pathways and reduces brain injury during PTZ-kindled seizures. The neuroprotective effect of KD is likely exerted via a reduction of mitochondrial cytochrome c release.

Topics: Adenine; Animals; Apoptosis; Autophagy; Caspase 3; Cytochromes c; Diet, Ketogenic; Epilepsy; Hippocampus; Male; Mitochondria; Neurons; Neuroprotective Agents; Pentylenetetrazole; Rats; Rats, Sprague-Dawley; Seizures; Sirolimus

Epidermal growth factor receptor (EGFR) is over-expressed in about 50% of Triple negative breast cancers (TNBCs), but EGFR inhibitors have not been effective in treating TNBC patients. Increasing evidence supports that autophagy was related to drug resistance at present. However, the role and the mechanism of autophagy to the treatment of TNBC remain unknown. In the current study, we investigated the effect of autophagy inhibitor to gefitinib (Ge) in TNBC cells in vitro and in nude mice vivo. Our study demonstrated that inhibition of autophagy by 3-Methyladenine or bafilomycin A1 improved Ge's sensitivity to MDA-MB-231 and MDA-MB-468 cells, as evidence from stronger inhibition of cell vitality and colony formation, higher level of G0/G1 arrest and DNA damage, and these effects were verified in nude mice vivo. Our data showed that the mitochondrial-dependent apoptosis pathway was activated in favor of promoting apoptosis in the therapy of Ge combined autophagy inhibitor, as the elevation of BAX/Bcl-2, Cytochrome C, and CASP3. These results demonstrated that targeting autophagy should be considered as an effective therapeutic strategy to enhance the sensitivity of EGFR inhibitors on TNBC.

Topics: Adenine; Animals; Autophagy; Caspase 3; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Survival; Cytochromes c; Drug Synergism; Female; Gefitinib; Gene Expression Regulation, Neoplastic; Humans; In Vitro Techniques; Macrolides; Mice; Mice, Nude; Mitochondria; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Quinazolines; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays

Breast cancer, the most commonly diagnosed cancer in women worldwide, is treated in various ways. Ramalin is a chemical compound derived from the Antarctic lichen Ramalina terebrata and is known to exhibit antioxidant and antiinflammatory activities. However, its effect on breast cancer cells remains unknown. We examined the ability of ramalin to induce apoptosis and its mechanisms in MCF-7 and MDA-MB-231 human breast cancer cell lines. Ramalin inhibited cell growth and induced apoptosis in both cell lines in a concentration-dependent manner. By upregulating Bax and downregulating Bcl-2, ramalin caused cytochrome c and apoptosis-inducing factor to be released from the mitochondria into the cytosol, thus activating the mitochondrial apoptotic pathway. In addition, activated caspase-8 and caspase-9 were detected in both types of cells exposed to ramalin, whereas ramalin activated caspase-3 only in the MDA-MB-231 cells. Ramalin treatment also increased the levels of LC3-II and p62. Moreover, the inhibition of autophagy by 3-methyladenine or Atg5 siRNA significantly enhanced ramalin-induced apoptosis, which was accompanied by a decrease in Bcl-2 levels and an increase in Bax levels. Therefore, autophagy appears to be activated as a protective mechanism against apoptosis in cancer cells exposed to ramalin. These findings suggest that ramalin is a potential anticancer agent for the treatment of patients with non-invasive or invasive breast cancer.

Topics: Adenine; Antineoplastic Agents; Antioxidants; Apoptosis; Apoptosis Inducing Factor; Autophagy; Autophagy-Related Protein 5; bcl-2-Associated X Protein; Biological Products; Breast Neoplasms; Caspases; Cell Cycle; Cell Proliferation; Cytochromes c; Female; Glutamates; Humans; Lichens; Microtubule-Associated Proteins; Mitochondria; Proto-Oncogene Proteins c-bcl-2

Longitudinal bone growth takes place in epiphyseal growth plates located in the ends of long bones. The growth plate consists of chondrocytes traversing from the undifferentiated (resting zone) to the terminally differentiated (hypertrophic zone) stage. Autophagy is an intracellular catabolic process of lysosome-dependent recycling of intracellular organelles and protein complexes. Autophagy is activated during nutritionally depleted or hypoxic conditions in order to facilitate cell survival. Chondrocytes in the middle of the growth plate are hypoxic and nutritionally depleted owing to the avascular nature of the growth plate. Accordingly, autophagy may facilitate their survival. To explore the role of autophagy in chondrocyte survival and constitutional bone growth, we generated mice with cartilage-specific ablation of either Atg5 (Atg5cKO) or Atg7 (Atg7cKO) by crossing Atg5 or Atg7 floxed mice with cartilage-specific collagen type 2 promoter-driven Cre. Both Atg5cKO and Atg7cKO mice showed growth retardation associated with enhanced chondrocyte cell death and decreased cell proliferation. Similarly, inhibition of autophagy by Bafilomycin A1 (Baf) or 3-methyladenine (3MA) promoted cell death in cultured slices of human growth plate tissue. To delineate the underlying mechanisms we employed ex vivo cultures of mouse metatarsal bones and RCJ3.IC5.18 rat chondrogenic cell line. Baf or 3MA impaired metatarsal bone growth associated with processing of caspase-3 and massive cell death. Similarly, treatment of RCJ3.IC5.18 chondrogenic cells by Baf also showed massive cell death and caspase-3 cleavage. This was associated with activation of caspase-9 and cytochrome C release. Altogether, our data suggest that autophagy is important for chondrocyte survival, and inhibition of this process leads to stunted growth and caspase-dependent death of chondrocytes.

Topics: Adenine; Animals; Apoptosis; Autophagy; Autophagy-Related Protein 5; Autophagy-Related Protein 7; Caspases; Cell Death; Cell Line; Cell Proliferation; Cell Survival; Chondrocytes; Collagen Type II; Cytochromes c; Gene Deletion; Growth Plate; Humans; Hypoxia; Immunohistochemistry; In Situ Hybridization; Macrolides; Metatarsal Bones; Mice; Mice, Knockout; Microtubule-Associated Proteins; Proteins; Rats; Ubiquitin-Activating Enzymes

Isorhamnetin (ISO) is a flavonoid from plants of the Polygonaceae family and is also an immediate metabolite of quercetin in mammals. To date, the anti‑tumor effects of ISO and the underlying mechanisms have not been elucidated in lung cancer cells. The present study investigated the inhibitory effects of ISO on the growth of human lung cancer A549 cells. Treatment of the lung cancer cells with ISO significantly suppressed cell proliferation and colony formation. ISO treatment also resulted in a significant increase in apoptotic cell death of A549 cells in a time- and dose-dependent manner. Further investigation showed that the apoptosis proceeded via the mitochondria‑dependent pathway as indicated by alteration of the mitochondrial membrane potential, the release of cytochrome C and caspase activation. Of note, treatment with ISO also induced the formation of autophagosomes and light chain 3‑II protein in A549 cells. Furthermore, co‑treatment with autophagy inhibitors 3‑methyladenine and hydroxychloroquine significantly inhibited the ISO‑induced autophagy and enhanced the ISO‑induced apoptotic cell death in vitro as well as in vivo. Thus, the results of the present study suggested that ISO is a potential anti‑lung cancer agent. In addition, the results indicated that the inhibition of autophagy may be a useful strategy for enhancing the chemotherapeutic effect of ISO on lung cancer cells.

Topics: Adenine; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Dose-Response Relationship, Drug; Drug Synergism; Epithelial Cells; Gene Expression; Humans; Hydroxychloroquine; Lung Neoplasms; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Microtubule-Associated Proteins; Mitochondria; Quercetin; Xenograft Model Antitumor Assays

Melatonin has been proven to possess neuroprotection property against various neurological diseases by decreasing cerebral oxidative stress and inhibiting inflammatory process. However, whether administration of melatonin influences the autophagy pathway, which has recently been reported playing a pivotal role in traumatic brain injury, is yet not fully understood. We supposed that treatment of melatonin enhances the autophagy pathway after traumatic brain injury (TBI) in mice and subsequently inhibited the mitochondrion apoptotic pathway. Firstly, we investigated the neurological severity score, brain water content and neuronal apoptosis in mice cortex to demonstrate the neuroprotection of melatonin. Then we determined the autophagy markers, namely Beclin1 and LC3-II, using western blot and immunofluorescence. Next, we evaluated the mitochondrial apoptotic pathway in the presence or absence of melatonin. More significantly, we employed 3-methyladenine (3-MA) to inhibit the autophagy pathway, to further confirm our hypothesis. The results showed that melatonin significantly ameliorated secondary brain injury induced by TBI. In addition, melatonin enhanced autophagy after TBI, which was accompanied by a decrease in both the translocation of Bax to mitochondria and the release of cytochrome C to cytoplasm. Furthermore, simultaneous treatment of 3-MA reversed the beneficial effects of melatonin on mitochondrial apoptotic pathway. Taken together, we conclude that melatonin enhances autophagy, which inhibits mitochondrial apoptotic pathway, thus protecting mice from secondary brain injury after TBI.

Topics: Adenine; Animals; Apoptosis; Autophagy; bcl-2-Associated X Protein; Body Water; Brain Chemistry; Brain Injuries; Cerebral Cortex; Cytochromes c; Male; Melatonin; Mice; Mitochondria; Neuroprotective Agents; Signal Transduction

Osteosarcoma (OS) is the most common primary malignant bone cancer in children and adolescents. Although paclitaxel (PCX) has been considered one of the most important cancer chemotherapeutic drugs, the current protocols for OS treatment do not incorporate this agent. Therefore, the purpose of this study was to evaluate the induction of cell death in OS cells after exposure to PCX, to identify the cell death mechanism(s) activated by PCX and to investigate whether autophagy is associated with PCX-induced apoptosis. The results of the present study confirmed that exposure to low PCX concentrations can induce apoptotic cell death in Saos-2 cells; furthermore, caspase-3 activation, PARP degradation and XIAP downregulation were observed in combination with PCX-induced apoptosis. The potential involvement of mitochondrial events (intrinsic apoptotic pathway) in PCX-induced apoptosis in OS cells was verified by the alteration (depolarization) of mitochondrial membrane potential. In addition, pretreatment with 3-methyladenine (3-MA), a specific inhibitor of autophagy, significantly increased PCX-induced apoptotic cell death in Saos-2 cells. The augmentation of PCX-induced apoptosis by 3-MA was accompanied by increase in the cytochrome c release from the mitochondria, caspase-3 activity and XIAP downregulation, which suggests that inhibiting autophagy further stimulates the PCX-induced mitochondrion-related (intrinsic) apoptotic pathway by provoking caspase-3 activation. Thus, autophagy observed during PCX-induced apoptosis in Saos-2 OS cells represents the role of cytoprotection in cellular homeostatic processes. In conclusion, the results of this study revealed that PCX exposure effectively induces OS cell death by apoptosis associated with the mitochondrial-mediated caspase-dependent pathway. PCX can increase autophagic activity and suppressing autophagy enhances PCX-induced apoptosis in OS cells. Therefore, it is suggested that combination treatment involving low-dose PCX therapy and autophagy inhibitor therapy could be an effective and potent strategy for improved chemotherapy for OS in the near future.

Topics: Adenine; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Bone Neoplasms; Caspase 3; Cell Death; Cell Line, Tumor; Cytochromes c; Cytoprotection; Humans; Membrane Potential, Mitochondrial; Mitochondria; Osteosarcoma; Paclitaxel; X-Linked Inhibitor of Apoptosis Protein

Cerebral ischemia-reperfusion (I-R) is a complex pathological process. Although autophagy can be evoked by ischemia, its involvement in the reperfusion phase after ischemia and its contribution to the fate of neurons remains largely unknown. In the present investigation, we found that autophagy was activated in the reperfusion phase, as revealed in both mice with middle cerebral artery occlusion and oxygen-glucose deprived cortical neurons in culture. Interestingly, in contrast to that in permanent ischemia, inhibition of autophagy (by 3-methyladenine, bafilomycin A 1, Atg7 knockdown or in atg5(-/-) MEF cells) in the reperfusion phase reinforced, rather than reduced, the brain and cell injury induced by I-R. Inhibition of autophagy either with 3-methyladenine or Atg7 knockdown enhanced the I-R-induced release of cytochrome c and the downstream activation of apoptosis. Moreover, MitoTracker Red-labeled neuronal mitochondria increasingly overlapped with GFP-LC3-labeled autophagosomes during reperfusion, suggesting the presence of mitophagy. The mitochondrial clearance in I-R was reversed by 3-methyladenine and Atg7 silencing, further suggesting that mitophagy underlies the neuroprotection by autophagy. In support, administration of the mitophagy inhibitor mdivi-1 in the reperfusion phase aggravated the ischemia-induced neuronal injury both in vivo and in vitro. PARK2 translocated to mitochondria during reperfusion and Park2 knockdown aggravated ischemia-induced neuronal cell death. In conclusion, the results indicated that autophagy plays different roles in cerebral ischemia and subsequent reperfusion. The protective role of autophagy during reperfusion may be attributable to mitophagy-related mitochondrial clearance and inhibition of downstream apoptosis. PARK2 may be involved in the mitophagy process.

Topics: Adenine; Animals; Apoptosis; Autophagy; Autophagy-Related Protein 5; Autophagy-Related Protein 7; Brain Ischemia; Cytochromes c; Cytoprotection; Glucose; Hypoxia; Male; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Mitochondria; Mitophagy; Neurons; Quinazolinones; Rats; Reperfusion Injury; Ubiquitin-Protein Ligases

Herein, inhibition of hepatocyte growth factor receptor, c-Met, significantly increased cytochrome c release and Bax/Bcl-2 ratio, indicating that c-Met played an anti-apoptotic role. The following experiments are to elucidate this anti-apoptotic mechanism, then the effect of c-Met on autophagy has also been discussed.. Investigated was the influence of c-Met on apoptosis, autophagy and loss of mitochondrial transmembrane potential (Δψm), and the relevant proteins were examined.. First, we found that activation of extracellular signal-regulated kinase (ERK), p53 was promoted by c-Met interference. Subsequent studies indicated that ERK was the upstream effector of p53, and this ERK-p53 pathway mediated release of cytochrome c and up-regulation of Bax/Bcl-2 ratio. Secondly, the inhibition of c-Met augmented oridonin-induced loss of mitochondrial transmembrane potential (Δψm), resulting apoptosis. Finally, the inhibition of c-Met increased oridonin-induced A549 cell autophagy accompanied by Beclin-1 activation and conversion from microtubule-associated protein light chain 3 (LC3)-I to LC3-II. Activation of ERK-p53 was also detected in autophagy process and could be augmented by inhibition of c-Met. Moreover, suppression of autophagy by 3-methyladenine (3-MA) or small interfering RNA against Beclin-1 or Atg5 decreased oridonin-induced apoptosis. Inhibition of apoptosis by pan-caspase inhibitor (z-VAD-fmk) decreased oridonin-induced autophagy as well and Loss of Δψm also occurred during autophagic process.. Thus, inhibiting c-Met enhanced oridonin-induced apoptosis, autophagy and loss of Δψm in A549 cells.

Topics: Adenine; Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Protein 5; bcl-2-Associated X Protein; Beclin-1; Cytochromes c; Diterpenes, Kaurane; Extracellular Signal-Regulated MAP Kinases; Humans; Isodon; Lung Neoplasms; Membrane Potential, Mitochondrial; Membrane Proteins; Microtubule-Associated Proteins; Mitochondria; Phytotherapy; Plant Extracts; Proto-Oncogene Proteins c-met; RNA, Small Interfering; Tumor Suppressor Protein p53

Angiogenesis plays an important role in many pathological processes. Identification of novel anti-angiogenic agents will provide new insights into the mechanisms for angiogenesis as well as potential lead compounds for developing new drugs. In the present study, a series of resveratrol methylated derivatives have been synthesized and screened. We found trans-3,4-dimethoxystilbene (3,4-DMS) with the fullest potential to develop as an anti-angiogenic agent. In vitro and in vivo analyses suggested that 3,4-DMS could effectively inhibit endothelial cell proliferation, migration, tube formation, and endogenous neovascularization. Our results showed that 3,4-DMS exerted its anti-angiogenic effect likely through induction of endothelial cell apoptosis via a pathway involving p53, Bax, cytochrome c, and caspase proteases. Moreover, 3,4-DMS also induced macroautophagy in endothelial cells through activation of AMPK and the downstream inhibition of mTOR signaling pathway. Further studies indicated that intracellular calcium ([Ca(2+)](i)) might bridge the 3,4-DMS-induced apoptosis and macroautophagy through modulating reactive oxygen species (ROS) levels in endothelial cells. Combination of 3,4-DMS with inhibitor of autophagy, such as 3-methyladenine (3-MA) and autophagy-related gene (ATG) 5 small interfering RNA (siRNA), potentiated the pro-apoptotic and anti-angiogenic effects of 3,4-DMS. Our study provides a novel angiogenic inhibitor and a useful tool in exploring the molecular mechanisms for the crosstalk between apoptosis and macroautophagy in endothelial cells. 3,4-DMS could be served as a potential lead compound for developing a class of new drugs targeting angiogenesis-related diseases.

Topics: Adenine; AMP-Activated Protein Kinases; Angiogenesis Inhibitors; Apoptosis; Autophagy; Autophagy-Related Protein 5; bcl-2-Associated X Protein; Calcium; Caspase 3; Caspase 9; Cell Line; Cell Movement; Cell Proliferation; Cell Survival; Cytochromes c; Human Umbilical Vein Endothelial Cells; Humans; Microtubule-Associated Proteins; Neovascularization, Physiologic; Reactive Oxygen Species; Resveratrol; RNA Interference; RNA, Small Interfering; Stilbenes; TOR Serine-Threonine Kinases; Tumor Suppressor Protein p53

Apoptosis induction is often associated with increased autophagy, indicating interplay between these two important cellular events in cell death and survival. In this study, the programmed cell death and autophagy induced by two nitrostyrene derivative compounds (NTS1 and NTS2) was studied using the tumorigenic Ehrlich ascitic tumor (EAT) cells. EAT cells were highly sensitive to NTS1 and NTS2 cytotoxicity in a dose-dependent manner. NTS1 and NTS2 IC(50) was less than 15.0μM post 12h incubation. Apoptosis was primarily induced by both compounds, as demonstrated by an increase in Annexin-V positive cells, concurrently with cytochrome c release from mitochondria to cytosol and caspase-3 activation. Although cytosolic Ca(2+) mobilization is involved in autophagy as well as apoptosis in response to cellular stress in many cancer cell types, from the two nitrostyrene derivative compounds studied, mainly NTS1 mobilized this ion and disparate autophagy in EAT cells. These results suggest that EAT induced cell death by NTS1 and NTS2 involved a Ca(2+)-dependent and a Ca(2+)-independent pathways, respectively. In accordance with these results, the treatment of EAT cells with 3 methyladenine (3-MA), an autophagy inhibitor; significantly increased the number of apoptotic cells after NTS1 treatment, suggesting that pharmacological modulation of autophagy augments the NTS1 efficacy. Thus, we denote the importance of studies involving autophagy and apoptosis during pre-clinical studies of new drugs with anticancer properties.

Topics: Adenine; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Calcium; Carcinoma, Ehrlich Tumor; Caspase 3; Cell Line, Tumor; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Enzyme Inhibitors; Nitro Compounds; Styrenes

Sirtuins (SIRTs), NAD+-dependent class III histone deacetylases (HDACs), play an important role in the regulation of cell division, survival and senescence. Although a number of effective SIRT inhibitors have been developed, little is known about the specific mechanisms of their anticancer activity. In this study, we investigated the anticancer effects of sirtinol, a SIRT inhibitor, on MCF-7 human breast cancer cells. Apoptotic and autophagic cell death were measured. Sirtinol significantly inhibited the proliferation of MCF-7 cells in a concentration-dependent manner. The IC50 values of sirtinol were 48.6 µM (24 h) and 43.5 µM (48 h) in MCF-7 cells. As expected, sirtinol significantly increased the acetylation of p53, which has been reported to be a target of SIRT1/2. Flow cyto-metry analysis revealed that sirtinol significantly increased the G1 phase of the cell cycle. The upregulation of Bax, downregulation of Bcl-2 and cytochrome c release into the cytoplasm, which are considered as mechanisms of apoptotic cell death, were observed in the MCF-7 cells treated with sirtinol. The annexin V-FITC assay was used to confirm sirtinol-induced apoptotic cell death. Furthermore, the expression of LC3-II, an autophagy-related molecule, was significantly increased in MCF-7 cells after sirtinol treatment. Autophagic cell death was confirmed by acridine orange and monodansylcadaverine (MDC) staining. Of note, pre-treatment with 3-methyladenine (3-MA) increased the sirtinol-induced MCF-7 cell cytotoxicity, which is associated with blocking autophagic cell death and increasing apoptotic cell death. Based on our results, the downregulation of SIRT1/2 expression may play an important role in the regulation of breast cancer cell death; thus, SIRT1/2 may be a novel molecular target for cancer therapy and these findings may provide a molecular basis for targeting SIRT1/2 in future cancer therapy.

Topics: Acetylation; Adenine; Apoptosis; Autophagy; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Benzamides; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Down-Regulation; Female; G1 Phase Cell Cycle Checkpoints; Histone Deacetylase Inhibitors; Humans; Microtubule-Associated Proteins; Naphthols; Sirtuins; Tumor Suppressor Protein p53; Up-Regulation

Azadirachta indica, commonly known as neem, has a wide range of medicinal properties. Neem extracts and its purified products have been examined for induction of apoptosis in multiple cancer cell types; however, its underlying mechanisms remain undefined. We show that neem oil (i.e., neem), which contains majority of neem limonoids including azadirachtin, induced apoptotic and autophagic cell death. Gene silencing demonstrated that caspase cascade was initiated by the activation of caspase-9, whereas caspase-8 was also activated late during neem-induced apoptosis. Pretreatment of cancer cells with pan caspase inhibitor, z-VAD inhibited activities of both initiator caspases (e.g., caspase-8 and -9) and executioner caspase-3. Neem induced the release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria, suggesting the involvement of both caspase-dependent and AIF-mediated apoptosis. p21 deficiency caused an increase in caspase activities at lower doses of neem, whereas p53 deficiency did not modulate neem-induced caspase activation. Additionally, neem treatment resulted in the accumulation of LC3-II in cancer cells, suggesting the involvement of autophagy in neem-induced cancer cell death. Low doses of autophagy inhibitors (i.e., 3-methyladenine and LY294002) did not prevent accumulation of neem-induced LC3-II in cancer cells. Silencing of ATG5 or Beclin-1 further enhanced neem-induced cell death. Phosphoinositide 3-kinase (PI3K) or autophagy inhibitors increased neem-induced caspase-3 activation and inhibition of caspases enhanced neem-induced autophagy. Together, for the first time, we demonstrate that neem induces caspase-dependent and AIF-mediated apoptosis, and autophagy in cancer cells.

Topics: Adenine; Apoptosis; Apoptosis Inducing Factor; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Blotting, Western; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Fluorescent Antibody Technique; Glycerides; Humans; Insecticides; Limonins; Membrane Proteins; Mitochondria; Terpenes; Tumor Suppressor Protein p53

Cisplatin (DDP)-based adjuvant chemotherapy is widely used for the treatment of esophageal cancer. However, DDP resistance has become more common and thus new approaches are required to be explored. Cisplatin was used to induce autophagy in the human esophageal cancer cell line, EC9706 cells, and the effect of autophagy on the survival of EC9706 cells was investigated using an autophagy inhibitor 3-MA. Cell viability was measured by CCK8 assay. Apoptosis and cell cycle were detected by flow cytometry. Monodansylcadaverine (MDC) was used to detect autophagy. Western blotting assay was used to investigate the molecular changes that occurred in the course of treatment. DDP inhibited cell proliferation, induced cell death and cell cycle arrest at S phage. Moreover, autophagy was activated through class III PI3K pathway. The expression of autophagy-related Beclin1 and LC3-I was up-regulated and part of LC3-I was converted into LC3-II. However, after the combination treatment of 3-MA and DDP, the cell inhibitory rate increased; the apoptosis rate and the numbers of cells in S phase also increased. Furthermore, the accumulation of autophagic vacuoles was decreased; the expression of Beclin1 and LC3 was significantly down-regulated and the release of cytochrome c was decreased. DDP-induced apoptosis in EC9706 cells can be enhanced by the inhibitor of autophagy, 3-MA. Autophagy might play a role as a self-protective mechanism in DDP-treated esophageal cancer cells, and its inhibition could be a novel strategy for the adjuvant chemotherapy of esophageal cancer.

Topics: Adenine; Antineoplastic Agents; Apoptosis; Autophagy; Blotting, Western; Carcinoma, Squamous Cell; Cell Cycle; Cell Proliferation; Cisplatin; Cytochromes c; Drug Synergism; Esophageal Neoplasms; Flow Cytometry; Humans; Tumor Cells, Cultured

Sulforaphane (SUL), an isothiocyanate naturally present in widely consumed vegetables, particularly broccoli, has recently attracted attention due to its inhibitory effects on tumor cell growth by inducing apoptosis. We investigated the ability of SUL to induce autophagy in human colon cancer cells and whether inhibition of autophagy could potentiate the proapoptotic effect of SUL.. The proliferation of cells treated with SUL was assessed by MTS assay and colony-forming assay. Apoptosis and caspases activity were investigated by flow cytometry. The formation of acidic vesicular organelles (AVOs) was detected in acridine-orange-stained cells by flow cytometry. Western blotting was used for the detection of light chain 3 (LC3). Localizations of LC3 and cytochrome c were analyzed by immunocytochemistry.. The proapoptotic effect was observed by treatment of cells with relatively high concentrations of SUL for long periods of time. After 16 h of treatment, evident formation of AVOs and recruitment of LC3 to autophagosomes, features of autophagy, were observed. Treatment of cells with a specific autophagy inhibitor (3-methyladenine) potentiated the proapoptotic effect of SUL, which was dependent on the activation of caspases and the release of cytochrome c to the cytosol.. The present results demonstrate induction of autophagy in colon cancer cells as a protective reaction against the proapoptotic effect of SUL, and consequently, the potentiation of the proapoptotic effect by autophagy inhibition. These findings provide a premise for use of autophagy inhibitors in combination with chemotherapeutic agents for treatment of colorectal cancer.

Topics: Adenine; Anticarcinogenic Agents; Apoptosis; Autophagy; Blotting, Western; Caspases; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Colony-Forming Units Assay; Cytochromes c; Flow Cytometry; Humans; Isothiocyanates; Sulfoxides; Thiocyanates

The etiology of Parkinson's disease (PD) is not completely defined, although environmental factors (for example, exposure to the herbicide paraquat [PQ]) and genetic susceptibility (such as DJ-1 mutations that have been associated with an autosomal-recessive form of early-onset PD) have been demonstrated to contribute. Alterations in macroautophagy have been described in the pathogenesis of this neurodegenerative disease. We have established a model system to study the involvement of the DJ-1 protein in PQ-induced autophagy. When we transfected cells exposed to PQ with DJ-1-specific siRNA, we observed an inhibition of the autophagic events induced by the herbicide, as well as sensitization additive with PQ-induced apoptotic cell death and exacerbation of this cell death in the presence of the autophagy inhibitor 3-methyladenine. These results suggest, for the first time, an active role for DJ-1 in the autophagic response produced by PQ, opening the door to new strategies for PD therapy.

Topics: Adenine; Apoptosis; Autophagy; Caspase 3; Cell Line, Tumor; Cytochromes c; Enzyme Activation; Herbicides; Humans; Intracellular Signaling Peptides and Proteins; Membrane Potential, Mitochondrial; Models, Biological; Neuroblastoma; Oncogene Proteins; Paraquat; Protein Deglycase DJ-1; Reactive Oxygen Species; RNA Interference

5-fluorouracil-(5-FU)-based adjuvant chemotherapy is widely used for the treatment of colorectal cancer. However, 5-FU resistance in the course of treatment has become more common. Therefore, new therapeutic strategies and/or new adjuvant drugs still need to be explored.. Two colon-cancer-derived cell lines, colon26 and HT29, were used to investigate the effect of 5-FU, 3-methyladenine (3-MA, an autophagy inhibitor), or their combination on apoptotic cell death and autophagy. MTT assay, Hochest plus propidium iodide (PI) staining, and DNA fragmentation assay were used to observe apoptosis. Meanwhile, monodansylcadaverine (MDC) was used to detect autophagy. Finally, immunoblotting assay was used to explore the molecular change that occurred.. We observed the apoptosis induced by 5-FU in colon cancer cells. Meanwhile, autophagy was also stimulated. The combination treatment of 3-MA and 5-FU significantly increased the apoptotic cell death. By isolating the subcellular fractions of mitochondria and cytosol, we observed that the release of cytochrome c was increased in combination-treated cells. Cytochrome c resulted in the activation of caspase-3, thus activating PARP. Moreover, the anti-apoptotic protein, Bcl-xL, was significantly downregulated by 3-MA.. Our results suggest that 5-FU-induced apoptosis in colon cancer cells can be enhanced by the inhibitor of autophagy, 3-MA. Autophagy might play a role as a self-defense mechanism in 5-FU-treated colon cancer cells, and its inhibition could be a promising strategy for the adjuvant chemotherapy of colon cancer.

Topics: Adenine; Antimetabolites, Antineoplastic; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Blotting, Western; Caspase 3; Cell Line, Tumor; Cell Proliferation; Colonic Neoplasms; Cytochromes c; Drug Synergism; Drug Therapy, Combination; Fluorouracil; Humans

In vivo administration of the mitochondrial inhibitor 3-nitropropionic acid (3-NP) produces striatal pathology mimicking Huntington's disease (HD). However, the mechanisms of cell death induced by metabolic impairment are not fully understood. Previous studies showed that 3-NP triggered p53-depedent autophagy activation and cell death. The present study investigated the contribution of the Bcl-2 signaling pathway to autophagy activation and cell death induced by 3-NP. Rat striatum was intoxicated with 3-NP by stereotaxic injection. 3-NP up-regulated the expression of the autophagic protein beclin 1 but down-regulated the expression of the antiapoptotic protein Bcl-2. Pretreatment with the autophagy inhibitor 3-methyladenine (3-MA) significantly inhibited the 3-NP-induced alterations in beclin 1 and Bcl-2 protein levels. Similarly, the 3-NP-induced decline in Bcl-2 was also prevented by the lysosomal inhibitor E64, indicating degradation of Bcl-2 by lysosomes. In agreement with the time course of 3-NP-induced cell death, an increase in the release of cytochrome c from mitochondria was observed. 3-MA also attenuated the 3-NP-induced release of cytochrome c. On the other hand, 3-NP-induced elevations in proapoptotic protein Bax and autophagic protein beclin 1 and LC3-II were significantly enhanced by the Bcl-2-specific inhibitor HA14-1. Furthermore, HA14-1 increased the release of cytochrome c and 3-NP-induced striatal damage. These results suggest that induction of autophagy leads to degradation of Bcl-2. Meanwhile, down-regulation of Bcl-2 amplifies autophagy activation and apoptotic signaling. Bcl-2 thus plays important roles in mitochondria dysfunction-induced apoptotic death of stritatal neurons by modulating both autophagic and apoptotic processes.

Topics: Adenine; Analysis of Variance; Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; bcl-2-Associated X Protein; Beclin-1; Benzopyrans; Blotting, Western; Cell Death; Corpus Striatum; Cytochromes c; Dose-Response Relationship, Drug; Down-Regulation; Enzyme Inhibitors; Mitochondria; Neurotoxins; Nitriles; Nitro Compounds; Propionates; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Staining and Labeling; Time Factors; Up-Regulation

In primary culture, hepatocytes dedifferentiate, and their cytoplasm undergoes remodeling. Here, our aim was to characterize changes of mitochondria during remodeling. Hepatocytes were cultured one to five days in complete serumcontaining Waymouth's medium. In rat hepatocytes loaded with MitoTracker Green (MTG), tetramethylrhodamine methylester (TMRM), and/or LysoTracker Red (LTR), confocal microscopy revealed that mitochondria number and mass decreased by approximately 50% between Day 1 and Day 3 of culture. As mitochondria disappeared, lysosomes/autophagosomes proliferated five-fold. Decreased mitochondrial content correlated with (a) decreased cytochrome c oxidase activity and mitochondrial number observed by electron microscopy and (b) a profound decrease of PGC-1alpha mRNA expression. By contrast, mtDNA content per cell remained constant from the first to the third day of culture, although ethidium bromide (de novo mtDNA synthesis inhibitor) caused mtDNA to decrease by half from the first to the third culture day. As mitochondria disappeared, their MTG label moved into LTR-labeled lysosomes, which was indicative of autophagic degradation. A multiwell fluorescence assay revealed a 2.5-fold increase of autophagy on Day 3 of culture, which was decreased by 3-methyladenine, an inhibitor of autophagy, and also by cyclosporin A and NIM811, both selective inhibitors of the mitochondrial permeability transition (MPT). These findings indicate that mitochondrial autophagy (mitophagy) and the MPT underlie mitochondrial remodeling in cultured hepatocytes.

Topics: Adenine; Amines; Animals; Autophagy; Biological Transport; Cell Compartmentation; Cells, Cultured; Cyclosporine; Cytochromes c; DNA, Mitochondrial; Electron Transport Complex IV; Gene Expression Regulation; Hepatocytes; Lysosomes; Male; Mitochondria, Liver; Permeability; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Phagosomes; Rats; Rats, Sprague-Dawley; RNA-Binding Proteins; RNA, Messenger; Transcription Factors

Previous studies reported that the neurotoxin, Crotoxin, isolated from the venom of South American rattlesnake had potent anti-tumor activity. Here, we investigated the involvement of autophagy and apoptosis in the Crotoxin-induced death of chronic myeloid leukemia cell line K562 cells. The neurotoxin dose dependently inhibited the viability of K562 cells. Crotoxin stimulated the autophagic activity as evidenced by the appearance of punctuate monodansylcadaverine (MDC) fluorescence staining in the cytoplasm and increased the formation of autophagosomes. Crotoxin caused the collapse of the mitochondrial membrane potential, release of cytochrome c and activation of caspase-3. Caspase inhibitors attenuated Crotoxin-induced K562 cell death, while blockage of autophagy maturation with 3-methyladenine (3-MA) and NH4Cl potentiated the neurotoxin's cytotoxicity. These results suggest that an apoptotic mechanism contributes to the Crotoxin-induced death of K562 cells, while the activation of autophagy delays neurotoxin-induced apoptosis.

Topics: Adenine; Amino Acid Chloromethyl Ketones; Ammonium Chloride; Apoptosis; Autophagy; Caspase 3; Caspase Inhibitors; Caspases; Cell Survival; Crotoxin; Cytochromes c; Enzyme Activation; Humans; K562 Cells; Lysosomes; Mitochondria; Oligopeptides; Vacuoles

The mechanisms of mitochondrial alterations in aged post-mitotic cells, including formation of so-called 'giant' mitochondria, are poorly understood. To test whether these large mitochondria might appear due to imperfect autophagic mitochondrial turnover, we inhibited autophagocytosis in cultured neonatal rat cardiac myocytes with 3-methyladenine. This resulted in abnormal accumulation of mitochondria within myocytes, loss of contractility, and reduced survival time in culture. Unlike normal aging, which is associated with slow accumulation of predominantly large defective mitochondria, pharmacological inhibition of autophagy caused only moderate accumulation of large (senescent-like) mitochondria but dramatically enhanced the numbers of small mitochondria, probably reflecting their normally more rapid turnover. Furthermore, the 3-methyladenine-induced accumulation of large mitochondria was irreversible, while small mitochondria gradually decreased in number after withdrawal of the drug. We, therefore, tentatively conclude that large mitochondria selectively accumulate in aging post-mitotic cells because they are poorly autophagocytosed. Mitochondrial enlargement may result from impaired fission, a possibility supported by depressed DNA synthesis in large mitochondria. Nevertheless, enlarged mitochondria retained immunoreactivity for cytochrome c oxidase subunit 1, implying that mitochondrial genes remain active in defective mitochondria. Our findings suggest that imperfect autophagic recycling of these critical organelles may underlie the progressive mitochondrial damage, which characterizes aging post-mitotic cells.

Topics: Adenine; Animals; Autophagy; Cell Survival; Cellular Senescence; Cytochromes c; DNA, Mitochondrial; Electron Transport Complex IV; Immunohistochemistry; Lysosomes; Microscopy, Electron; Mitochondria, Heart; Myocytes, Cardiac; Oxidative Stress; Rats