Anticancer activities of chalcone flavokawain B from Alpinia pricei Hayata in human lung adenocarcinoma (A549) cells via induction of reactive oxygen species-mediated apoptotic and autophagic cell death

Chalcones found in fruits and vegetables have promising cancer chemopreventive properties. This study attempts to identify the anticancer efficacies of chalcone flavokawain B (FKB) in the rhizomes of Alpinia pricei Hayata by examining key molecular events in non-small-cell lung cancer (A549) cells. Our results indicated that in human A549 cells, FKB (0–15 μg/ml) decreases cell viability and colony formation, dysregulates the Bax:B-cell lymphoma 2 ratio and increases apoptotic DNA fragmentation. Mitochondrial (caspase-9/-3 and poly ADP ribose polymerase [PARP]) signaling was found to be involved in FKB-induced apoptosis. In addition, FKB-induced reactive oxygen species (ROS) generation, and N-acetylcysteine attenuated FKB-induced apoptotic cell death. Moreover, FKB triggered autophagy, as evidenced by the improved acidic vesicular organelle formation, lipidated light chain 3 (microtubule-related light chain 3) accumulation, and ATG7 expression and the decreased mammalian target of rapamycin phosphorylation. Furthermore, FKB suppressed ROS-mediated ATG4B expression. Inhibiting autophagy using 3-methyladenine/chloroquine diminished FKB-induced cell death, indicating that autophagy is triggered as a death mechanism by FKB. In summary, FKB has a crucial role in the execution and propagation of ROS-mediated apoptotic and autophagic cell death of lung adenocarcinoma cells.     

Cadmium results in accumulation of autophagosomes-dependent apoptosis through activating Akt-impaired autophagic flux in neuronal cells

Environmental exposure to cadmium (Cd) links to neurodegenerative disorders. Autophagy plays an important role in controlling cell survival/death. However, how autophagy contributes to Cd's neurotoxicity remains enigmatic. Here, we show that Cd induced significant increases in autophagosomes with a concomitant elevation of LC3-II and p62 in PC12 cells and primary neurons. Using autophagy inhibitor 3-MA, we demonstrated that Cd-increased autophagosomes contributed to neuronal apoptosis. Impairment of Cd on autophagic flux was evidenced by co-localization of mCherry and GFP tandem-tagged LC3 puncta in the cells. This is further supported by the findings that administration of chloroquine (CQ) potentiated the basic and Cd-elevated LC3-II and p62 levels, autophagosome accumulation and cell apoptosis, whereas rapamycin relieved the effects in the cells in response to Cd. Subsequently, we noticed that Cd evoked the phosphorylation of Akt and BECN1. Silencing BECN1 and especially expression of mutant BECN1 (Ser295A) attenuated Cd-increased autophagosomes and cell death. Of note, inhibition of Akt with Akt inhibitor X, or ectopic expression of dominant negative Akt (dn-Akt), in the presence or absence of 3-MA, significantly alleviated Cd-triggered phosphorylation of Akt and BECN1, autophagosomes, and apoptosis. Importantly, we found that Cd activation of Akt functioned in impairing autophagic flux. Collectively, these results indicate that Cd results in accumulation of autophagosomes-dependent apoptosis through activating Akt-impaired autophagic flux in neuronal cells. Our findings underscore that inhibition of Akt to improve autophagic flux is a promising strategy against Cd-induced neurotoxicity and neurodegeneration.     

Novel monofunctional platinum (II) complex Mono-Pt induces apoptosis-independent autophagic cell death in human ovarian carcinoma cells,distinct from cisplatin

Failure to engage apoptosis appears to be a leading mechanism of resistance to traditional platinum drugs in patients with ovarian cancer. Therefore, an alternative strategy to induce cell death is needed for the chemotherapy of this apoptosis-resistant cancer. Here we report that autophagic cell death, distinct from cisplatin-induced apoptosis, is triggered by a novel monofunctional platinum (II) complex named Mono-Pt in human ovarian carcinoma cells. Mono-Pt-induced cell death has the following features: cytoplasmic vacuolation, caspase-independent, no nuclear fragmentation or chromatin condensation, and no apoptotic bodies. These characteristics integrally indicated that Mono-Pt, rather than cisplatin, initiated a nonapoptotic cell death in Caov-3 ovarian carcinoma cells. Furthermore, incubation of the cells with Mono-Pt but not with cisplatin produced an increasing punctate distribution of microtubule-associated protein 1 light chain 3 (LC3), and an increasing ratio of LC3-II to LC3-I. Mono-Pt also caused the formation of autophagic vacuoles as revealed by monodansylcadaverine staining and transmission electron microscopy. In addition, Mono-Pt-induced cell death was significantly inhibited by the knockdown of either BECN1 or ATG7 gene expression, or by autophagy inhibitors 3-methyladenine, chloroquine and bafilomycin A₁. Moreover, the effect of Mono-Pt involved the AKT1-MTOR-RPS6KB1 pathway and MAPK1 (ERK2)/MAPK3 (ERK1) signaling, since the MTOR inhibitor rapamycin increased, while the MAPK1/3 inhibitor U0126 decreased Mono-Pt-induced autophagic cell death. Taken together, our results suggest that Mono-Pt exerts anticancer effect via autophagic cell death in apoptosis-resistant ovarian cancer. These findings lead to increased options for anticancer platinum drugs to induce cell death in cancer.     

Insulin receptor substrate-1 prevents autophagy-dependent cell death caused by oxidative stress in mouse NIH/3T3 cells

Background

Insulin receptor substrate (IRS)-1 is associated with tumorigenesis; its levels are elevated in several human cancers. IRS-1 protein binds to several oncogene proteins. Oxidative stress and reactive oxygen species (ROS) are involved in the initiation and progression of cancers. Cancer cells produce greater levels of ROS than normal cells do because of increased metabolic stresses. However, excessive production of ROS kills cancer cells. Autophagy usually serves as a survival mechanism in response to stress conditions, but excessive induction of autophagy results in cell death. In addition to inducing necrosis and apoptosis, ROS induces autophagic cell death. ROS inactivates IRS-1 mediated signaling and reduces intracellular IRS-1 concentrations. Thus, there is a complex relationship between IRS-1, ROS, autophagy, and cancer. It is not fully understood how cancer cells grow rapidly and survive in the presence of high ROS levels.

Methods and results

In this study, we established mouse NIH/3T3 cells that overexpressed IRS-1, so mimicking cancers with increased IRS-1 expression levels; we found that the IRS-1 overexpressing cells grow more rapidly than control cells do. Treatment of cells with glucose oxidase (GO) provided a continuous source of ROS; low dosages of GO promoted cell growth, while high doses induced cell death. Evidence for GO induced autophagy includes increased levels of isoform B-II microtubule-associated protein 1 light chain 3 (LC3), aggregation of green fluorescence protein-tagged LC3, and increased numbers of autophagic vacuoles in cells. Overexpression of IRS-1 resulted in inhibition of basal autophagy, and reduced oxidative stress-induced autophagy and cell death. ROS decreased the mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase signaling, while overexpression of IRS-1 attenuated this inhibition. Knockdown of autophagy-related gene 5 inhibited basal autophagy and diminished oxidative stress-induced autophagy and cell death.

Conclusion

Our results suggest that overexpression of IRS-1 promotes cells growth, inhibits basal autophagy, reduces oxidative stress-induced autophagy, and diminishes oxidative stress-mediated autophagy-dependent cell death. ROS-mediated autophagy may occur via inhibition of IRS-1/phosphatidylinositol 3-kinase/mTOR signaling. Our data afford a plausible explanation for IRS-1 involvement in tumor initiation and progression.     

Antrodia salmonea-induced oxidative stress abrogates HER-2 signaling cascade and enhanced apoptosis in ovarian carcinoma cells

Antrodia salmonea is well known in Taiwan as a traditional Chinese medicinal fungus and has demonstrated antioxidant, anti-inflammatory, and anticancer effects. However, the anticancer activity of A. salmonea against human ovarian cancer is still elusive. Therefore, we investigated the antiovarian tumor activity of a fermented culture broth of A. salmonea and exhibits its underlying molecular mechanism. A. salmonea shows a significant effect on cell viability in human ovarian carcinoma (SKOV-3 or A2780) cell lines with an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Increased terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells and annexin V–propidium iodide stained cells indicate that A. salmonea induces late apoptosis in SKOV-3 cells. Notably, treatment with A. salmonea induced the following events: Apoptosis; caspase-3, -8, -9 and poly(ADP-ribose) polymerase activation; first apoptosis signal (Fas) and Fas ligand activation; Bid cleavage; and Bax2–B-cell lymphoma 2 dysregulation. The results show that A. salmonea-induced apoptosis was mediated by both mitochondrial and death receptor pathways. An increase in intracellular reactive oxygen species (ROS) was also observed in A. salmonea-treated cells, whereas the antioxidant N-acetylcysteine (NAC) prevented A. salmonea-induced cell death and DNA fragmentation, indicating that A. salmonea-induced apoptosis was mediated by ROS generation. Interestingly, A. salmonea-induced apoptosis is associated with the suppression of human epidermal growth factor receptor-2 (HER-2/neu) and phosphoinositide 3-kinase (PI3K)–protein kinase B (AKT) expression in HER-2/neu overexpressing SKOV-3 cells. NAC significantly prevented A. salmonea-induced HER-2/neu depletion and PI3K/AKT inactivation, indicating that A. salmonea-triggered apoptosis is mediated by ROS-inhibited HER-2/neu signaling cascades. To our knowledge, this is the first report describing the anticancer activity of this potentially beneficial mushroom against human ovarian carcinoma.     

Inhibitory effect of hydnocarpin D on T-cell acute lymphoblastic leukemia via induction of autophagy-dependent ferroptosis

Hydnocarpin D (HD) is a bioactive flavonolignan compound that possesses promising anti-tumor activity, although the mechanism is not fully understood. Using T cell acute lymphoblastic leukemia (T-ALL) cell lines Jurkat and Molt-4 as model system, we found that HD suppressed T-ALL proliferation in vitro, via induction of cell cycle arrest and subsequent apoptosis. Furthermore, HD increased the LC3-II levels and the formation of autophagolysosome vacuoles, both of which are markers for autophagy. The inhibition of autophagy by either knockdown of ATG5/7 or pre-treatment of 3-MA partially rescued HD-induced apoptosis, thus suggesting that autophagy enhanced the efficacy of HD. Interestingly, this cytotoxic autophagy triggered ferroptosis, as evidenced by the accumulation of lipid ROS and decrease of GSH and GPX4, while inhibition of autophagy impeded ferroptotic cell death. Our study suggests that HD triggers multiple cell death processes and is an interesting compound that should be evaluated in future preclinical studies.     

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.     

Crosstalk between autophagy and apoptosis in the regulation of paclitaxel-induced cell death in v-Ha-ras-transformed fibroblasts

The previous studies by this author group has shown that paclitaxel, a mitotic inhibitor used in breast cancer chemotherapy, inhibits cell growth via induction of Raf-1-dependent apoptosis. In this article, the role of autophagy in paclitaxel anticancer action was investigated using v-Ha-ras-transformed NIH 3T3 cells. Paclitaxel induced a notable increase in the number of fluorescent particles labeled with monodansylcadaverine (MDC), a specific marker for autophagic vacuoles. MDC-labeled vacuoles clearly exhibited the fluorescent-tagged LC3 in cells transiently overexpressing GFP-LC3 (a protein that associates with autophagosome membranes). However, autophagy inhibition with 3-methyladenine (3-MA) failed to rescue v-Ha-ras-transformed NIH 3T3 cells from paclitaxel-induced cell death. More interestingly, the apoptosis inhibition by overexpression of the X-linked inhibitor of apoptosis (XIAP) did not fully block the cell death by paclitaxel, implying that apoptosis inhibition might accelerate the autophagic components of the paclitaxel response. Conversely, Raf-1 shRNA expression protected against paclitaxel-induced cell death through the simultaneous inhibition of both autophagy and apoptosis. These results suggest that both autophagy and apoptosis act as cooperative partners to induce cell death in v-Ha-ras-transformed NIH 3T3 cells treated with paclitaxel.     

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. In this study, 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 preautopghagosomal and 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 and the number of GFP-LC3-II-labeled autophagosome (punctuated pattern) positive cells and autophagic cell death (p     

Mitochondrial ROS generation for regulation of autophagic pathways in cancer

Mitochondria, the main source of reactive oxygen species (ROS), are required for cell survival; yet also orchestrate programmed cell death (PCD), referring to apoptosis and autophagy. Autophagy is an evolutionarily conserved lysosomal degradation process implicated in a wide range of pathological processes, most notably cancer. Accumulating evidence has recently revealed that mitochondria may generate massive ROS that play the essential role for autophagy regulation, and thus sealing the fate of cancer cell. In this review, we summarize mitochondrial function and ROS generation, and also highlight ROS-modulated core autophagic pathways involved in ATG4–ATG8/LC3, Beclin-1, p53, PTEN, PI3K–Akt–mTOR and MAPK signaling in cancer. Therefore, a better understanding of the intricate relationships between mitochondrial ROS and autophagy may ultimately allow cancer biologists to harness mitochondrial ROS-mediated autophagic pathways for cancer drug discovery.     

Short-chain fatty acids induced autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death

Short-chain fatty acids (SCFAs) are the major by-products of bacterial fermentation of undigested dietary fibers in the large intestine. SCFAs, mostly propionate and butyrate, inhibit proliferation and induce apoptosis in colon cancer cells, but clinical trials had mixed results regarding the anti-tumor activities of SCFAs. Herein we demonstrate that propionate and butyrate induced autophagy in human colon cancer cells to dampen apoptosis whereas inhibition of autophagy potentiated SCFA induced apoptosis. Colon cancer cells, after propionate treatment, exhibited extensive characteristics of autophagic proteolysis: increased LC3-I to LC3-II conversion, acidic vesicular organelle development, and reduced p62/SQSTM1 expression. Propionate-induced autophagy was associated with decreased mTOR activity and enhanced AMP kinase activity. The elevated AMPKα phosphorylation was associated with cellular ATP depletion and overproduction of reactive oxygen species due to mitochondrial dysfunction involving the induction of MPT and loss of Δψ. In this context, mitochondria biogenesis was initiated to recover cellular energy homeostasis. Importantly, when autophagy was prevented either pharmacologically (3-MA or chloroquine) or genetically (knockdown of ATG5 or ATG7), the colon cancer cells became sensitized toward propionate-induced apoptosis through activation of caspase-7 and caspase-3. The observations indicate that propionate-triggered autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death, whereas application of an autophagy inhibitor (Chloroquine) is expected to enhance the therapeutic efficacy of SCFAs in inducing colon tumor cell apoptosis.     

Blue light emitting diode induces apoptosis in lymphoid cells by stimulating autophagy

The present study was performed to examine the induction of apoptotic cell death and autophagy by blue LED irradiation, and the contribution of autophagy to apoptosis in B cell lymphoma A20 and RAMOS cells exposed to blue LED. Irradiation with blue LED reduced cell viability and induced apoptotic cell death, as indicated by exposure of phosphatidylserine on the plasma outside membrane and fragmentation of DNA. Furthermore, the mitochondrial membrane potential increased, and apoptotic proteins (PARP, caspase 3, Bax, and bcl-2) were observed. In addition, the level of intracellular superoxide anion (O₂⁻) gradually increased. Interestingly the formation of autophagosomes and level of LC3-II were increased in blue LED-irradiated A20 and RAMOS cells, but inhibited after pretreatment with 3-methyladenine (3-MA), widely used as an autophagy inhibitor. Inhibition of the autophagic process by pretreatment with 3-MA blocked blue LED irradiation-induced caspase-3 activation. Moreover, a significant reduction of both the early and late phases of apoptosis after transfection with ATG5 and beclin 1 siRNAs was shown by the annexin V/PI staining, indicating a crucial role of autophagy in blue LED-induced apoptosis in cells. Additionally, the survival rate of mice irradiated with blue LED after injection with A20 cells increased compared to the control group. Our data demonstrate that blue LED irradiation induces apoptosis via the mitochondrial-mediated pathway, in conjunction with autophagy. Further studies are needed to elucidate the precise mechanism of blue LED-induced immune cell death.     

小豆蔻明调控自噬抑制卵巢癌SKOV3细胞增殖的研究

目的：卵巢癌为女性常见病,死亡率高,分子靶向治疗药物较少,研发高效低毒的靶向新药意义重大。细胞自噬（autophagy）维持着细胞内稳态和生长,是抗癌药物作用的关键靶部位。本课题旨在明确小豆蔻明（Cardamonin,CAR）调控自噬对卵巢癌SKOV3细胞增殖的影响。方法：体外培养卵巢癌SKOV3细胞,不同药物分组处理,荧光显微镜下观察单黄酰戊二胺（MDC）染色后细胞自噬囊泡,WesternBlot法检测细胞自噬相关蛋白LC3的表达,四氮唑蓝（MTT）法观察SKOV3细胞增殖情况,流式细胞术检测SKOV3细胞凋亡的变化。结果：自噬抑制剂3-MA显著性降低SKOV3细胞内MDC染色的荧光颗粒数目、LC3II蛋白表达,抑制细胞增殖;而CAR（高、中剂量）、雷帕霉素和AZD8055与3-MA联用后细胞内MDC荧光颗粒数目增多、LC3II蛋白表达增加、细胞增殖抑制率及凋亡率明显升高;且高剂量CAR（10^-6mol·L^-1）的作用比低剂量CAR（10^-6mol·L^-1）明显。结论：CAR能够抑制SKOV3细胞增殖,诱导细胞自噬,促进细胞凋亡。CAR有望成为卵巢癌药物治疗的先导化合物。本研究为进一步研发此类化合物提供了实验依据及一定的理论基础。     

The reduced catalase expression in TrkA-induced cells leads to autophagic cell death via ROS accumulation

TrkA receptor activation is a pivotal process for neuronal cell differentiation and survival. However, its overactivation or removal of its ligand NGF tends to cause the cell death. Recently, we demonstrated that TrkA overexpression induces cell death via apoptosis. In this study we also show that the TrkA-mediated cell death is associated with autophagy. TrkA-induced cells revealed an increase of GFP-LC3 punctate formation, development of acidic vesicular organelles (AVO) and formation of autophagosomes, which were eventually blocked by the addition of some autophagy inhibitors such as 3-methyladenine, ammonium chloride or wortmannin. In addition, although expression of autophagy-related proteins such as LC3-II or Beclin-1 was subtly altered during the TrkA-mediated cell death, depletion of ATG5 or Beclin-1 substantially decreased cell death in TrkA-expressing cells. In particular, reactive oxygen species (ROS) were dramatically accumulated in TrkA-induced cells, and the high accumulation of ROS was released by treatment of autophagy inhibitors. Furthermore, addition of an antioxidant N-acetylcysteine promoted the survival of TrkA-expressing cells and suppressed AVO production in cells. We also showed that this ROS accumulation was closely associated with reduction of catalase expression. Taken together, TrkA overexpression causes ROS accumulation via reduced catalase expression, ultimately leading to autophagic cell death.     

Apoptosis and Autophagy in Photoreceptors Exposed to Oxidative Stress

Studies on human and animal models of retinal dystrophy have suggested that apoptosis may be the common pathway of photoreceptor cell death. Autophagy, the major cellular degradation process in animal cells, is important in normal development and tissue remodeling, as well as under pathological conditions. Previously we provided evidence that genes, whose products are involved in apoptosis and autophagy, may be co-expressed in photoreceptors undergoing degeneration. Here, we investigated autophagy in oxidative stress-mediated cell death in photoreceptors, analyzing the light-damage mouse model and 661W photoreceptor cells challenged with H₂O₂. In the in vivo model, we demonstrated a time-dependent increase in the number of TUNEL-positive cells, concomitant with the formation of autophagosomes. In vitro, oxidative stress increased mRNA levels of apoptotic and autophagic marker genes. H₂O₂ treatment resulted in the accumulation of TUNEL-positive cells, the majority of which contain autophagosomes. To determine whether autophagy and apoptosis might precede each other or co-occur, we performed inhibitor studies. The autophagy inhibitor 3-methyladenine (3-MA), silencing RNA (siRNA) against two genes whose products are required for autophagy (autophagy-related (ATG) gene 5 and beclin 1), as well as the pan-caspase-3 inhibitor, zVAD-fmk, were both found to partially block cell death. Blocking autophagy also significantly decreased caspase-3 activity, whereas blocking apoptosis increased the formation of autophagosomes. The survival effects of 3-MA and zVAD-fmk were not additive; rather treatment with both inhibitors lead to increased cell death by necrosis. In summary, the study first suggests that autophagy participates in photoreceptor cell death possibly by initiating apoptosis. Second, it confirms that cells that normally die by apoptosis will execute cell death by necrosis if the normal pathway is blocked. And third, these results argue that the up-stream regulators of autophagy need to be identified as potential therapeutic targets in photoreceptor degeneration.     

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.     

Raloxifene Induces Autophagy-Dependent Cell Death in Breast Cancer Cells via the Activation of AMP-Activated Protein Kinase

Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells.Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting BECN1 markedly reversed cell death and the level of LC3-II increased by raloxifene. Besides, raloxifene-induced cell death was not related to cleavage of caspases-7, -9, and PARP. These results indicate that raloxifene activates autophagy-dependent cell death but not apoptosis. Interestingly, raloxifene decreased the level of intracellular adenosine triphosphate (ATP) and activated the AMPK/ULK1 pathway. However it was not suppressed the AKT/mTOR pathway. Addition of ATP decreased the phosphorylation of AMPK as well as the accumulation of LC3-II, finally attenuating raloxifene-induced cell death.Our current study demonstrates that raloxifene induces autophagy via the activation of AMPK by sensing decreases in ATP, and that the overactivation of autophagy promotes cell death and thereby mediates the anti-cancer effects of raloxifene in breast cancer cells.     

Autophagy induced by ionizing radiation promotes cell death over survival in human colorectal cancer cells

Autophagy is commonly described as a cell survival mechanism and has been implicated in chemo- and radioresistance of cancer cells. Whether ionizing radiation induced autophagy triggers tumor cell survival or cell death still remains unclear. In this study the autophagy related proteins Beclin1 and ATG7 were tested as potential targets to sensitize colorectal carcinoma cells to ionizing radiation under normoxic, hypoxic and starvation conditions. Colony formation, apoptosis and cell cycle analysis revealed that knockdown of Beclin1 or ATG7 does not enhance radiosensitivity in HCT-116 cells. Furthermore, ATG7 knockdown led to an increased survival fraction under oxygen and glutamine starvation, indicating that ionizing radiation indeed induces autophagy which, however, leads to cell death finally. These results highlight that inhibition of autophagic pathways does not generally increase therapy success but may also lead to an unfavorable outcome especially under amino acid and oxygen restriction.     

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.     

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.