Heme Oxygenase-1 Promotes Survival of Renal Cancer Cells through Modulation of Apoptosis- and Autophagy-regulating Molecules

The cytoprotective enzyme heme oxygenase-1 (HO-1) is often overexpressed in different types of cancers and promotes cancer progression. We have recently shown that the Ras-Raf-ERK pathway induces HO-1 to promote survival of renal cancer cells. Here, we examined the possible mechanisms underlying HO-1-mediated cell survival. Considering the growing evidence about the significance of apoptosis and autophagy in cancer, we tried to investigate how HO-1 controls these events to regulate survival of cancer cells. Rapamycin (RAPA) and sorafenib, two commonly used drugs for renal cancer treatment, were found to induce HO-1 expression in renal cancer cells Caki-1 and 786-O; and the apoptotic effect of these drugs was markedly enhanced upon HO-1 knockdown. Overexpression of HO-1 protected the cells from RAPA- and sorafenib-induced apoptosis and also averted drug-mediated inhibition of cell proliferation. HO-1 induced the expression of anti-apoptotic Bcl-xL and decreased the expression of autophagic proteins Beclin-1 and LC3B-II; while knockdown of HO-1 down-regulated Bcl-xL and markedly increased LC3B-II. Moreover, HO-1 promoted the association of Beclin-1 with Bcl-xL and Rubicon, a novel negative regulator of autophagy. Drug-induced dissociation of Beclin-1 from Rubicon and the induction of autophagy were also inhibited by HO-1. Together, our data signify that HO-1 is up-regulated in renal cancer cells as a survival strategy against chemotherapeutic drugs and promotes growth of tumor cells by inhibiting both apoptosis and autophagy. Thus, application of chemotherapeutic drugs along with HO-1 inhibitor may elevate therapeutic efficiency by reducing the cytoprotective effects of HO-1 and by simultaneous induction of both apoptosis and autophagy.     

Silencing of Bcl-2 expression by small interfering RNA induces autophagic cell death in MCF-7 breast cancer cells

Apoptosis (programmed cell death type I) and autophagy (type II) are crucial mechanisms regulating cell death and homeostasis. The Bcl-2 proto-oncogene is overexpressed in 50-70% of breast cancers, potentially leading to resistance to chemotherapy, radiation and hormone therapy-induced apoptosis. Here, we investigated the role of Bcl-2 in autophagy in breast cancer cells. Silencing of Bcl-2 by siRNA in MCF-7 breast cancer cells downregulated Bcl-2 protein levels (>85%) and led to inhibition of cell growth (71%) colony formation (79%), and cell death (up to 55%) by autophagy but not apoptosis. Induction of autophagy was demonstrated by acridine orange staining, electron microscopy and an accumulation of GFP-LC3-II in autophagosomal membranes in MCF-7 cells transfected with GFP-LC-3(GFP-ATG8). Silencing of Bcl-2 by siRNA also led to induction of LC-3-II, a hallmark of autophagy, ATG5 and Beclin-1 autophagy promoting proteins. Knockdown of ATG5 significantly inhibited Bcl-2 siRNA-induced LC3-II expression, the number of GFP-LC3-II-labeled autophagosome positive cells and autophagic cell death (p < 0.05). Furthermore, doxorubicin at a high dose (IC(95), 1 microM) induced apoptosis but at a low dose (IC(50), 0.07 microM) induced only autophagy and Beclin-1 expression. When combined with Bcl-2 siRNA, doxorubicin (IC(50)) enhanced autophagy as indicated by the increased number cells with GFP-LC3-II-stained autophagosomes (punctuated pattern positive). These results provided the first evidence that targeted silencing of Bcl-2 induces autophagic cell death in MCF-7 breast cancer cells and that Bcl-2 siRNA may be used as a therapeutic strategy alone or in combination with chemotherapy in breast cancer cells that overexpress Bcl-2.     

Polyphyllin VII Induces an Autophagic Cell Death by Activation of the JNK Pathway and Inhibition of PI3K/AKT/mTOR Pathway in HepG2 Cells

Polyphyllin VII (PP7), a pennogenyl saponin isolated from Rhizoma Paridis, exhibited strong anticancer activities in various cancer types. Previous studies found that PP7 induced apoptotic cell death in human hepatoblastoma cancer (HepG2) cells. In the present study, we investigated whether PP7 could induce autophagy and its role in PP7-induced cell death, and elucidated its mechanisms. PP7 induced a robust autophagy in HepG2 cells as demonstrated by the conversion of LC3B-I to LC3B-II, degradation of P62, formation of punctate LC3-positive structures, and autophagic vacuoles tested by western blot analysis or InCell 2000 confocal microscope. Inhibition of autophagy by treating cells with autophagy inhibitor (chloroquine) abolished the cell death caused by PP7, indicating that PP7 induced an autophagic cell death in HepG2 cells. C-Jun N-terminal kinase (JNK) was activated after treatment with PP7 and pretreatment with SP600125, a JNK inhibitor, reversed PP7-induced autophagy and cell death, suggesting that JNK plays a critical role in autophagy caused by PP7. Furthermore, our study demonstrated that PP7 increased the phosphorylation of AMPK and Bcl-2, and inhibited the phosphorylation of PI3K, AKT and mTOR, suggesting their roles in the PP7-induced autophagy. This is the first report that PP7 induces an autophagic cell death in HepG2 cells via inhibition of PI3K/AKT/mTOR, and activation of JNK pathway, which induces phosphorylation of Bcl-2 and dissociation of Beclin-1 from Beclin-1/Bcl-2 complex, leading to induction of autophagy.     

Targeting the autophagy pathway using ectopic expression of Beclin 1 in combination with rapamycin in drug-resistant v-Ha-<Emphasis Type="Italic">ras</Emphasis>-transformed NIH 3T3 cells

The effectiveness of an apoptosis-targeting therapy may be limited in tumor cells with defects in apoptosis. Recently, considerable attention in the field of cancer therapy has been focused on the mammalian rapamycin target (mTOR), inhibition of which results in autophagic cell death. In our study using multidrug-resistant v-Ha-rastransformed NIH3T3 (Ras-NIH 3T3/Mdr) cells, we demonstrated that rapamycin-induced cell death may result from 2 different mechanisms. At high rapamycin concentrations (≥ 100 nM), cell death may occur via an autophagy-dependent pathway, whereas at lower concentrations (≤ 10 nM), cell death may occur after G1-phase cell cycle arrest. This effect was accompanied by upregulation of p21^Cip1 and p27^Kip1 expression via an autophagy-independent pathway. We also tested whether inhibition of mTOR with low concentrations of rapamycin and ectopic Beclin-1 expression would further sensitize multidrug resistance (MDR)-positive cancer cells by upregulating autophagy. Rapamycin at low concentrations might be insufficient to initiate autophagosome formation in autophagy but Beclin-1 overexpression triggered additional processes downstream of mTOR during G₁ cell cycle arrest by rapamycin. Our findings suggest that these combination strategies targeting autophagic cell death may yield significant benefits for cancer patients, because lowering rapamycin concentration for cancer treatment minimizes its side effects in patients undergoing chemotherapy.     

MCL-1 is a stress sensor that regulates autophagy in a developmentally regulated manner

Apoptosis has an important role during development to regulate cell number. In differentiated cells, however, activation of autophagy has a critical role by enabling cells to remain functional following stress. In this study, we show that the antiapoptotic BCL-2 homologue MCL-1 has a key role in controlling both processes in a developmentally regulated manner. Specifically, MCL-1 degradation is an early event not only following induction of apoptosis, but also under nutrient deprivation conditions where MCL-1 levels regulate activation of autophagy. Furthermore, deletion of MCL-1 in cortical neurons of transgenic mice activates a robust autophagic response. This autophagic response can, however, be converted to apoptosis by either reducing the levels of the autophagy regulator Beclin-1, or by a concomitant activation of BAX. Our results define a pathway whereby MCL-1 has a key role in determining cell fate, by coordinately regulating apoptosis and autophagy.     

Blocking autophagy enhances the apoptosis effect of bufalin on human hepatocellular carcinoma cells through endoplasmic reticulum stress and JNK activation

Bufalin extracts are a part of traditional Chinese medicine, Chansu. In the current study, we investigated the effect of bufalin on the proliferation of the human hepatocellular carcinoma (HCC) cell lines, Huh-7 and HepG-2, and explored the therapeutic potential of the drug. Our results demonstrated that bufalin markedly inhibited cell proliferation and promoted apoptosis in the Huh-7 and HepG-2 cells in vitro. The underlying mechanism of the bufalin-induced apoptosis was the induction of endoplasmic reticulum (ER) stress via the IRE1–JNK pathway. In addition, during the ER stress response, the autophagy pathway, characterized by the conversion of LC3-I to LC3-II, was activated, resulting in increased Beclin-1 protein levels, decreased p62 expression and stimulation of autophagic flux. Our data supported the pro-survival role of bufalin-induced autophagy when the autophagy pathway was blocked with specific chemical inhibitors; the involvement of the IRE1 pathway in the ER stress-induced autophagy was also demonstrated when the expression of IRE1 and CHOP was silenced using siRNA. These data indicate that combining bufalin with a specific autophagy inhibitor could be a promising therapeutic approach for the treatment of HCC.     

Necrostatin-1 Suppresses Autophagy and Apoptosis in Mice Traumatic Brain Injury Model

Traumatic brain injury (TBI) results in neuronal apoptosis, autophagic cell death and necroptosis. Necroptosis is a newly discovered caspases-independent programmed necrosis pathway which can be triggered by activation of death receptor. Previous works identified that necrostatin-1 (NEC-1), a specific necroptosis inhibitor, could reduce tissue damage and functional impairment through inhibiting of necroptosis process following TBI. However, the role of NEC-1 on apoptosis and autophagy after TBI is still not very clear. In this study, the amount of TBI-induced neural cell deaths were counted by PI labeling method as previously described. The expression of autophagic pathway associated proteins (Beclin-1, LC3-II, and P62) and apoptotic pathway associated proteins (Bcl-2 and caspase-3) were also respectively assessed by immunoblotting. The data showed that mice pretreated with NEC-1 reduced the amount of PI-positive cells from 12 to 48?h after TBI. Immunoblotting results showed that NEC-1 suppressed TBI-induced Beclin-1 and LC3-II activation which maintained p62 at high level. NEC-1 pretreatment also reversed TBI-induced Bcl-2 expression and caspase-3 activation, as well as the ratio of Beclin-1/Bcl-2. Both 3-MA and NEC-1 suppressed TBI-induced caspase-3 activation and LC3-II formation, Z-VAD only inhibited caspase-3 activation but increased LC3-II expression at 24?h post-TBI. All these results revealed that multiple cell death pathways participated in the development of TBI, and NEC-1 inhibited apoptosis and autophagy simultaneously. These coactions may further explain how can NEC-1 reduce TBI-induced tissue damage and functional deficits and reflect the interrelationship among necrosis, apoptosis and autophagy.     

HIF-1 Regulation of Chondrocyte Apoptosis: Induction of the Autophagic Pathway

The goal of our investigation was to explore the mechanism by which hypoxia regulates growth plate chondrocyte survival. At low O2 tension, chondrocytes were refractory to a staurosporine (i.e., apoptosis-inducing) challenge. To determine whether hypoxic survival was due to the expression of HIF-1, we evaluated the response of HIF silenced cells to staurosporine. Both, silenced cells and control chondrocytes were equally sensitive to the apoptogen challenge. To learn if resistance was mediated by the proteins of the autophagic pathway, we examined the expression of Beclin 1 and LC3. Both proteins were present in the growth plate as well as in N1511 chondrocytes. Moreover, silencing of Beclin 1 resulted in enhanced chondrocyte death. Thus, this gene served to maintain chondrocyte survival activity. Besides serving a cytoprotective role, it is known that autophagy can function in cell death. Accordingly, to ascertain if autophagy might also sensitize cells to apoptosis, we activated autophagy and examined viability following exposure to an apoptogen. Treatment with the autophagy inhibitor 3-methyladenine rendered the chondrocytes refractory to killing, suggesting that sustained autophagy promoted cell death. We next examined expression of BID and caspase-8. When autophagy was suppressed, chondrocytes promoted caspase-8 activation and activated BID. Finally, we explored the relationship between HIF-1 and Beclin 1. We noted a decrease in Beclin 1 expression and loss of caspase-8 activation in HIF silenced cells and Beclin 1-Bcl-2 association was maintained upon serum starvation. This study indicates that HIF-1 serves to regulate both autophagy and apoptosis.     

HIF-1 regulation of chondrocyte apoptosis: induction of the autophagic pathway

The goal of our investigation was to explore the mechanism by which hypoxia regulates growth plate chondrocyte survival. At low O2 tension, chondrocytes were refractory to a staurosporine (i.e., apoptosis-inducing) challenge. To determine whether hypoxic survival was due to the expression of HIF-1, we evaluated the response of HIF silenced cells to staurosporine. Both, silenced cells and control chondrocytes were equally sensitive to the apoptogen challenge. To learn if resistance was mediated by the proteins of the autophagic pathway, we examined the expression of Beclin 1 and LC3. Both proteins were present in the growth plate as well as in N1511 chondrocytes. Moreover, silencing of Beclin 1 resulted in enhanced chondrocyte death. Thus, this gene served to maintain chondrocyte survival activity. Besides serving a cytoprotective role, it is known that autophagy can function in cell death. Accordingly, to ascertain if autophagy might also sensitize cells to apoptosis, we activated autophagy and examined viability following exposure to an apoptogen. Treatment with the autophagy inhibitor 3-methyladenine rendered the chondrocytes refractory to killing, suggesting that sustained autophagy promoted cell death. We next examined expression of BID and caspase-8. When autophagy was suppressed, chondrocytes promoted caspase-8 activation and activated BID. Finally, we explored the relationship between HIF-1 and Beclin 1. We noted a decrease in Beclin 1 expression and loss of caspase-8 activation in HIF silenced cells and Beclin 1-Bcl-2 association was maintained upon serum starvation. This study indicates that HIF-1 serves to regulate both autophagy and apoptosis.     

Autophagic cell death unraveled: Pharmacological inhibition of apoptosis and autophagy enables necrosis

Apoptosis is a well-characterized pathway to cell death, yet how it is related to other forms of cell death such as necrosis, and possibly also autophagic cell death has not been entirely clear. Difficulties arise because necrotic cell death is poorly characterized at the molecular level, and also because autophagy is primarily a survival pathway that has been associated with cell death induction in some circumstances. A common theme appears to be now emerging where autophagy promotes survival of apoptosis-defective cells, and inhibition of the autophagy survival function in this setting represents a means to divert cells into a necrotic cell fate. In cells denied the ability to commit suicide by apoptosis, and that are also unable to access the autophagy survival mechanism to sustain homeostasis, necrosis is the default activity. This was most recently illustrated with the discovery that the caspase and apoptosis inhibitor, zVAD, also inhibits a lysosomal protease, and thereby autophagy, and it is this dual inhibition that is responsible for induction of necrotic cell death.(1) This radically alters the interpretation of earlier findings reporting induction of autophagic cell death by zVAD,(2) instead, suggests that autophagy functions to promote cell survival.     

Lapatinib induces autophagy, apoptosis and megakaryocytic differentiation in chronic myelogenous leukemia K562 cells

Lapatinib is an oral, small-molecule, dual tyrosine kinase inhibitor of epidermal growth factor receptors (EGFR, or ErbB/Her) in solid tumors. Little is known about the effect of lapatinib on leukemia. Using human chronic myelogenous leukemia (CML) K562 cells as an experimental model, we found that lapatinib simultaneously induced morphological changes resembling apoptosis, autophagy, and megakaryocytic differentiation. Lapatinib-induced apoptosis was accompanied by a decrease in mitochondrial transmembrane potential and was attenuated by the pancaspase inhibitor z-VAD-fmk, indicating a mitochondria-mediated and caspase-dependent pathway. Lapatinib-induced autophagic cell death was verified by LC3-II conversion, and upregulation of Beclin-1. Further, autophagy inhibitor 3-methyladenine as well as autophagy-related proteins Beclin-1 (ATG6), ATG7, and ATG5 shRNA knockdown rescued the cells from lapatinib-induced growth inhibition. A moderate number of lapatinib-treated K562 cells exhibited features of megakaryocytic differentiation. In summary, lapatinib inhibited viability and induced multiple cellular events including apoptosis, autophagic cell death, and megakaryocytic differentiation in human CML K562 cells. This distinct activity of lapatinib against CML cells suggests potential for lapatinib as a therapeutic agent for treatment of CML. Further validation of lapatinib activity in vivo is warranted.     

Downregulation of protein kinase CK2 activity facilitates tumor necrosis factor-α-mediated chondrocyte death through apoptosis and autophagy

Despite the numerous studies of protein kinase CK2, little progress has been made in understanding its function in chondrocyte death. Our previous study first demonstrated that CK2 is involved in apoptosis of rat articular chondrocytes. Recent studies have suggested that CK2 downregulation is associated with aging. Thus examining the involvement of CK2 downregulation in chondrocyte death is an urgently required task. We undertook this study to examine whether CK2 downregulation modulates chondrocyte death. We first measured CK2 activity in articular chondrocytes of 6-, 21- and 30-month-old rats. Noticeably, CK2 activity was downregulated in chondrocytes with advancing age. To build an in vitro experimental system for simulating tumor necrosis factor (TNF)-α-induced cell death in aged chondrocytes with decreased CK2 activity, chondrocytes were co-treated with CK2 inhibitors and TNF-α. Viability assay demonstrated that CK2 inhibitors facilitated TNF-α-mediated chondrocyte death. Pulsed-field gel electrophoresis, nuclear staining, flow cytometry, TUNEL staining, confocal microscopy, western blot and transmission electron microscopy were conducted to assess cell death modes. The results of multiple assays showed that this cell death was mediated by apoptosis. Importantly, autophagy was also involved in this process, as supported by the appearance of a punctuate LC3 pattern and autophagic vacuoles. The inhibition of autophagy by silencing of autophage-related genes 5 and 7 as well as by 3-methyladenine treatment protected chondrocytes against cell death and caspase activation, indicating that autophagy led to the induction of apoptosis. Autophagic cells were observed in cartilage obtained from osteoarthritis (OA) model rats and human OA patients. Our findings indicate that CK2 down regulation facilitates TNF-α-mediated chondrocyte death through apoptosis and autophagy. It should be clarified in the future if autophagy observed is a consequence versus a cause of the degeneration in vivo.     

Autophagy and apoptosis: where do they meet?

Autophagy and apoptosis are two important cellular processes with complex and intersecting protein networks; as such, they have been the subjects of intense investigation. Recent advances have elucidated the key players and their molecular circuitry. For instance, the discovery of Beclin-1’s interacting partners has resulted in the identification of Bcl-2 as a central regulator of autophagy and apoptosis, which functions by interacting with both Beclin-1 and Bax/Bak respectively. When localized to the endoplasmic reticulum and mitochondria, Bcl-2 inhibits autophagy. Cellular stress causes the displacement of Bcl-2 from Beclin-1 and Bax, thereby triggering autophagy and apoptosis, respectively. The induction of autophagy or apoptosis results in disruption of complexes by BH3-only proteins and through post-translational modification. The mechanisms linking autophagy and apoptosis are not fully defined; however, recent discoveries have revealed that several apoptotic proteins (e.g., PUMA, Noxa, Nix, Bax, XIAP, and Bim) modulate autophagy. Moreover, autophagic proteins that control nucleation and elongation regulate intrinsic apoptosis through calpain- and caspase-mediated cleavage of autophagy-related proteins, which switches the cellular program from autophagy to apoptosis. Similarly, several autophagic proteins are implicated in extrinsic apoptosis. This highlights a dual cellular role for autophagy. On one hand, autophagy degrades damaged mitochondria and caspases, and on the other hand, it provides a membrane-based intracellular platform for caspase processing in the regulation of apoptosis. In this review, we highlight the crucial factors governing the crosstalk between autophagy and apoptosis and describe the mechanisms controlling cell survival and cell death.     

Mimulone-Induced Autophagy through p53-Mediated AMPK/mTOR Pathway Increases Caspase-Mediated Apoptotic Cell Death in A549 Human Lung Cancer Cells

Anticancer properties and mechanisms of mimulone (MML), C-geranylflavonoid isolated from the Paulownia tomentosa fruits, were firstly elucidated in this study. MML prevented cell proliferation in a dose- and time-dependent way and triggered apoptosis through the extrinsic pathway in A549 human lung adenocarcinoma cells. Furthermore, MML-treated cells displayed autophagic features, such as the formation of autophagic vacuoles, a primary morphological feature of autophagy, and the accumulation of microtubule-associated protein 1 light chain 3 (LC3) puncta, another typical maker of autophagy, as determined by FITC-conjugated immunostaining and monodansylcadaverine (MDC) staining, respectively. The expression levels of LC3-I and LC3-II, specific markers of autophagy, were also augmented by MML treatment. Autophagy inhibition by 3-methyladenine (3-MA), pharmacological autophagy inhibitor, and shRNA knockdown of Beclin-1 reduced apoptotic cell death induced by MML. Autophagic flux was not significantly affected by MML treatment and lysosomal inhibitor, chloroquine (CQ) suppressed MML-induced autophagy and apoptosis. MML-induced autophagy was promoted by decreases in p53 and p-mTOR levels and increase of p-AMPK. Moreover, inhibition of p53 transactivation by pifithrin-α (PFT-α) and knockdown of p53 enhanced induction of autophagy and finally promoted apoptotic cell death. Overall, the results demonstrate that autophagy contributes to the cytotoxicity of MML in cancer cells harboring wild-type p53. This study strongly suggests that MML is a potential candidate for an anticancer agent targeting both autophagy and apoptotic cell death in human lung cancer. Moreover, co-treatment of MML and p53 inhibitor would be more effective in human lung cancer therapy.     

Autophagy inhibition enhances ursolic acid-induced apoptosis in PC3 cells

The phosphoinositol 3-kinase/Akt pathway plays a critical role in oncogenesis and the dysregulation of this pathway through loss of PTEN is a particularly common phenomenon in aggressive prostate cancers. Several recent studies have indicated that ursolic acid (UA), a pentacyclic triterpenoid, and its derivatives inhibit the growth of cancer cells by cell cycle arrest and the stimulation of apoptosis. In the present study, we report a novel autophagic response of UA in PTEN-deficient PC3 prostate cancer cells. As one of the major types of programmed cell death, autophagy has been observed in response to several anticancer drugs and demonstrated to be responsible for cell death. UA-induced autophagy in PC3 cells is associated with the reduced cell viability and the enhanced expression of LC3-II, an autophagosome marker in mammals, and monodansylcadaverine incorporation into autolysosomes. Furthermore, we found that UA exhibited anti-proliferative effects characterized by G1 phase arrest and autophagy at an early stage that precedes apoptosis. We also show that UA-induced autophagy in PC3 cells are mediated through the Beclin-1 and Akt/mTOR pathways. Inhibition of autophagy by either 3-methyladenine or Beclin-1/Atg5 small interfering RNA enhanced UA-induced apoptosis. Taken together, our data suggest that autophagy functions as a survival mechanism in PC3 cells against UA-induced apoptosis and a rational for the use of autophagy inhibitors in combination with UA as a novel modality of cancer therapy.     

Oncogenic Ras-induced expression of Noxa and Beclin-1 promotes autophagic cell death and limits clonogenic survival

Deregulated oncogenes such as MYC and RAS are typically insufficient to transform cells on their own due to the activation of pathways that restrain proliferation. Previous studies have shown that oncogenic H-Ras can induce proliferative arrest or senescence, depending on the cellular context. Here, we show that deregulated H-Ras activity can also lead to caspase-independent cell death with features of autophagy. Ras-induced autophagy was associated with upregulation of the BH3-only protein Noxa as well as the autophagy regulator Beclin-1. Silencing of Noxa or Beclin-1 expression reduced Ras-induced autophagy and increased clonogenic survival. Ras-induced cell death was also inhibited by coexpression of Bcl-2 family members that inhibit Beclin-1 function. Ras-induced autophagy was associated with Noxa-mediated displacement of the Bcl-2 family member, Mcl-1, from Beclin-1. Thus, Ras-induced expression of Noxa and Beclin-1 promotes autophagic cell death, which represents a mechanism to limit the oncogenic potential of deregulated Ras signals.     

Resveratrol depletes mitochondrial DNA and inhibition of autophagy enhances resveratrol-induced caspase activation

We recently demonstrated that resveratrol induces caspase-dependent apoptosis in multiple cancer cell types. Whether apoptosis is also regulated by other cell death mechanisms such as autophagy is not clearly defined. Here we show that inhibition of autophagy enhanced resveratrol-induced caspase activation and apoptosis. Resveratrol inhibited colony formation and cell proliferation in multiple cancer cell types. Resveratrol treatment induced accumulation of LC3-II, which is a key marker for autophagy. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased resveratrol-mediated caspase activation and cell death in breast and colon cancer cells. Inhibition of autophagy by silencing key autophagy regulators such as ATG5 and Beclin-1 enhanced resveratrol-induced caspase activation. Mechanistic analysis revealed that Beclin-1 did not interact with proapoptotic proteins Bax and Bak; however, Beclin-1 was found to interact with p53 in the cytosol and mitochondria upon resveratrol treatment. Importantly, resveratrol depleted ATPase 8 gene, and thus, reduced mitochondrial DNA (mtDNA) content, suggesting that resveratrol induces damage to mtDNA causing accumulation of dysfunctional mitochondria triggering autophagy induction. Together, our findings indicate that induction of autophagy during resveratrol-induced apoptosis is an adaptive response.     

TNF-α induces autophagy through ERK1/2 pathway to regulate apoptosis in neonatal necrotizing enterocolitis model cells IEC-6

Necrotizing enterocolitis (NEC) is a potentially fatal illness in premature neonates. Tumor necrosis factor-α (TNF-α) and autophagy are associated with the pathogenesis of NEC. This study aimed to explore whether TNF-α might regulate apoptosis in neonatal NEC model cells IEC-6 via regulation of autophagy. NEC rat model was induced by hand feeding and exposure to asphyxia/cold-stress for histologic examination. The NEC in vitro model (IEC-6/NEC cells) was established by stimulating the intestinal epithelial cell line IEC-6 with lipopolysaccharide (LPS, 100 μg/mL) for 3 h to investigate the effects of TNF-α on IEC-6 proliferation and apoptosis. In this study, NEC rats showed decreased proliferating cell nuclear antigen (PCNA) expression, increased TUNEL-positive cells, higher expression of TNF-α, p-ERK1/2, and autophagy-related proteins in rat small intestine compared with their controls. Additionally, the LPS-stimulated IEC-6/NEC cells showed a significantly decreased proliferation and increased apoptosis compared with the control cells. Furthermore, the LPS-stimulated IEC-6/NEC cells exhibited enhanced autophagy level, as evidenced by a dose-dependent increase in Beclin-1 protein expression, LC3II/LC3I ratio and accumulation of MDC-positive autophagic vacuoles. Moreover, inhibition of autophagy by wortmannin or LY294002 significantly abolished the LPS-mediated decreased proliferation and increased apoptosis of IEC-6/NEC cells. Results also showed that inhibition of ERK1/2 pathway using U0126 significantly inhibited TNF-α-induced autophagy. Furthermore, the TNF-α-mediated inhibition of IEC-6 proliferation and promotion of IEC-6 apoptosis was abolished by U0126. Our findings demonstrated that TNF-α might induce autophagy through ERK1/2 pathway to regulate apoptosis in neonatal NEC cells IEC-6. Our study enhances our understanding of neonatal NEC pathogenesis.     

Blue LED causes autophagic cell death in human osteosarcoma by increasing ROS generation and dephosphorylating EGFR

Osteosarcoma (OS) is the most common primary malignant bone tumour in adolescence. Lately, light-emitting diodes (LED)-based therapy has emerged as a new promising approach for several diseases. However, it remains unknown in human OS. Here, we found that the blue LED irradiation significantly suppressed the proliferation, migration and invasion of human OS cells, while we observed blue LED irradiation increased ROS production through increased NADPH oxidase enzymes NOX2 and NOX4, as well as decreased Catalase (CAT) expression levels. Furthermore, we revealed blue LED irradiation-induced autophagy characterized by alterations in autophagy protein markers including Beclin-1, LC3-II/LC3-I and P62. Moreover, we demonstrated an enhanced autophagic flux. The blockage of autophagy displayed a remarkable attenuation of anti-tumour activities of blue LED irradiation. Next, ROS scavenger N-acetyl-L-cysteine (NAC) and NOX inhibitor diphenyleneiodonium (DPI) blocked suppression of OS cell growth, indicating that ROS accumulation might play an essential role in blue LED-induced autophagic OS cell death. Additionally, we observed blue LED irradiation decreased EGFR activation (phosphorylation), which in turn led to Beclin-1 release and subsequent autophagy activation in OS cells. Analysis of EGFR colocalization with Beclin-1 and EGFR-immunoprecipitation (IP) assay further revealed the decreased interaction of EGFR and Beclin-1 upon blue LED irradiation in OS cells. In addition, Beclin-1 down-regulation abolished the effects of blue LED irradiation on OS cells. Collectively, we concluded that blue LED irradiation exhibited anti-tumour effects on OS by triggering ROS and EGFR/Beclin-1-mediated autophagy signalling pathway, representing a potential approach for human OS treatment.